搜档网
当前位置:搜档网 › 糖酵解抑制剂

糖酵解抑制剂

糖酵解抑制剂
糖酵解抑制剂

12The Open Andrology Journal, 2012, 4, 12-20

1876-827X/12 2012 Bentham Open Open Access

Effects of Some Respiratory and Glycolytic Inhibitors on Mitochondrial Functionality in Bovine Semen

S.K. Shahani1, S.G. Revell2,C.McG. Argo3 and R.D. Murray*,1

1University of Liverpool Institute of Translational Medicine, Leahurst Campus, Chester High Road, Neston. CH64 7TE,

UK; 2Genus Breeding Limited, Freezing Unit, Llanrhydd, Ruthin, Denbighshire LL15 2UP, UK; 3University of Liverpool Institute of Ageing and Chronic Disease, Leahurst Campus, Chester High Road, Neston. CH64 7TE, UK.

Abstract: Fertility potential of spermatozoa depends on maintenance of the mitochondrial membrane potential ( m)

that provides energy for sperm hyperactivation immediately prior to successful fertilization. Mitochondrial structure and

integrity are associated with sperm motility and reduced fertility, and measurement of m may be suitable for

determining bull semen quality. Mitochondrial membrane potential in four commercial AI bulls was assessed using JC-1

and propidium iodide in the presence of the glycolytic inhibitors 2-deoxy-D-glucose (DOG) and iodoacetamide (IAM)

and the respiratory inhibitor valinomycin (VAL) to determine the maximum m at minimum incubation. Flow

cytometry recorded m within a unified population for all treatments that represented sperm with low and high m

respectively. Maximum m was seen at 40 min incubation. Mean high fluorescence intensity (MFI) (orange) was

significantly greater for untreated compared to the treated sperm, at 40 and 80 min incubation, in both fresh and frozen-

thawed semen. In sperm treated with VAL and IAM, m was lowered significantly, and the proportion of sperm with

high: low m ratio was higher in control and DOG- treated samples representing more active mitochondria. In samples

treated with VAL and IAM, the ratio was reversed, representing loss in activity. Cryopreservation increased the high:low

m ratio only in bull 1 by 30% and lowered it in bulls 2, 3 and 4 by 20% - 70% compared to fresh semen. The rise in

bull 1 may relate to be the product of sperm demonstrating a capacitation- like effect of the freeze-thaw process which

stimulated sperm hyperactivity.

We conclude that mitochondrial function was affected adversely on freeze-thaw process. The 40 min incubation is

satisfactory for future studies. Furthermore, IAM, a known glycolytic inhibitor, has a similar effect to VAL, a recognized

respiratory inhibitor, which should provide a basis for further research.

Keywords: Mitochondria, cryo-preservation, bull semen, flow cytometry.

INTRODUCTION

Cryopreservation alters the integrity and functionality of some sperm through damage to the plasma and acrosomal membranes and associated disruption of mitochondrial function [1, 2]. Consequently, the percentage of fully functional sperm with intact cell and organelle membranes is reduced after the freeze-thawing process [3]. On freezing, ice crystals form in the extra-cellular medium, increasing the osmolarity of the unfrozen solution. As intra-cellular water diffuses out in response to this change in osmotic gradient, the cell and plasma membranes become dehydrated [4]. At thawing, this phenomenon is reversed as the extra-cellular ice crystals melt and water re-enters the sperm. The ionic permeability and enzyme activity within the plasma membrane are disrupted creating lipid phase transitions at around 17-36°C [5-7]. Working with boar semen Guthrie et al., [8] increased the extender osmolarity from 300 to 600mOsm/kg, and found a 50% reduction in mitochondrial membrane potential. After freeze/thaw, Bilodeau et al., [9] found that sperm glutathione (GSH) concentration was *Address correspondence to this author at the University of Liverpool Institute of Translational Medicine, Leahurst Campus, Chester High Road, Neston. CH64 7TE, UK; Tel.: +44-151- 794- 6056;

E-mail: richmu@https://www.sodocs.net/doc/b710713846.html, reduced by 78% and superoxide dismutase activity by 50%, suggesting that oxidative stress had occurred. Premature ageing and capacitation have been associated with impaired acrosomal and/or mitochondrial membrane integrity, lowering ATP synthesis and reducing motility [10], as does bacterial contamination within an ejaculate [11].

Mitochondrial respiratory activity has been investigated in vitro using drugs that uncouple oxidative phosphorylation (OXPHOS), either by inhibiting electron transport chain (ETC) or blocking the essential link between the respiratory pathways and phosphorylation. One such drug, carbonyl cyanide m-chlorophenylhydrazone, markedly reduced mitochondrial membrane potential ( m) in both mouse and boar spermatozoa [8, 12]. A similar reduction has been observed in human sperm, treated with potassium cyanide that blocks cytochrome C oxidase within complex IV of the ETC, or p-trifluoromethoxy carbonyl cyanide phenylhydra-zone, and valinomycin that both uncouple OXPHOS [13].

ATP synthesis pathways may be investigated in sperm using a number of specific enzyme inhibitors. One example is 2-deoxy-D-glucose (DOG), a competitive inhibitor of glycolysis that targets hexokinase within the reaction glucose glucose 6-phosphate by competing with glucose during phosphorylation: the resulting product is DOG-6-phosphate

Interruption of Metabolic Pathways and Mitochondrial Function The Open Andrology Journal, 2012, Volume 4 13

which cannot be metabolized further [14, 15]. When bovine sperm are re-suspended in a medium containing DOG and pyruvate and incubated at 37°C for 10 minutes, at least 70% of sperm cells remain motile, demonstrating that spermatozoa maintain motility utilizing mitochondrial ATP without employing the glycolytic pathway at this level [16] . By comparison, mouse sperm incubated similarly show a significant reduction in their motility, even in the presence of pyruvate and lactate [12, 15]: this suggests that glycolytic ATP is crucial for sperm motility, or the decrease in motility might be due to utilization of mitochondrial ATP by DOG when phosphorylated with hexokinase. Investigating mitochondrial membrane potential in mouse sperm labelled with JC-1, Mukai and Okuno [12] found no change in m in the presence of DOG and either glucose or pyruvate substrates, suggesting that DOG had no effect on mitochondrial respiration.

Another glycolytic inhibitor is iodoacetamide (IAM) which inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by binding irreversibly to cysteine residues [15]. IAM can deprive rat astrocytes of glutathione (GSH), inhibit cellular GAPDH activity, and reduce cell lactate synthesis. It may cause cell death after a long incubation period [17] and should not be used to inactivate GAPDH and interrupt glycolysis because it lowers the GSH content of cultured cells. IAM has no effect on sperm ATP concentrations and motility if oxidizable substrate is provided but it does lower ATP concentrations and motility in the presence of glucose suggested that IAM had no effect on mitochondrial ATP that sperm utilize to maintain their motility, but it interrupted glycolysis, preventing an adequate supply of respiratory substrate for mitochondria, thereby decreasing ATP synthesis and motility [15].

Another group of drugs that can be used to investigate mitochondrial function are the K+ ionophores. One of these is valinomycin (VAL), a dodecadepsipeptide that induces mitochondrial swelling especially in the presence of acetate [18] through an increased uptake of K+. Inhibition of ETC in mitochondria occurs, due to loss of inner membrane potential, oxidation of pyridine nucleotides, and induction of apoptosis [19]. In nature, dodecadepsipeptides are found as a cellular toxins produced by several Streptomyces species of bacteria and have a similar effect on mitochondrial activity as VAL [20]. VAL is a passive carrier for K+, when it encounters the cell membrane surface: it transports these ions across membranes, thereby inhibiting oxidative phosphorylation by antagonizing the proton motive force. To give a true measure of mitochondrial activity sperm incubated with VAL should produce a base fluorescence value, which can be subtracted from, or compared with, that for the same sample incubated without VAL.

In our preliminary experiments to obtain maximum uptake of JC-1 in sperm population at minimum incubation time, variable results were observed with same bull examined on different days as well as fresh and post thaw semen. Selection of motile sperm by Bovipure showed maximum m at 30 minutes in fresh and post-thaw sperm labelled with JC-1, while maximum m was observed at 150 and 90min corresponding fresh and post-thaw semen washed with Ruthin extender, a glycolysis supporting medium (data not shown). Uprating of m in motile sperm of 3/4 bulls was seen in post-thaw semen. This was consistent with the results of Garrett et al., [21] who observed higher oxygen consumption rate in post-thaw semen. Whereas, semen washed in Ruthin medium: m has been increased with increase in incubation time in all bulls of fresh sperm and 2/6 bulls of post-thaw sperm respectively. Uprating of m in bulls might be sperm that initially were dependent on ATP generated by glycolysis accumulating its end product pyruvate/lactic acid, later with increase in incubation time the sperm demonstrated capacitation like changes and became hyperactive [22], which result in increased demand for ATP and relied on mitochondrial ATP for long time.

This paper investigated the effect of freeze/thawing on sperm m obtained from bulls incubated in the presence of respiratory and glycolysis inhibitors. The use of these inhibitors may help to determine the maximum mitochondrial potential at a minimum incubation time after restricting glycolysis activity, thus forcing the sperm to derive their energy from respiration and also the extent of background fluorescence by comparing fluorescence when incubated with and without inhibitors.

MATERIALS AND METHODS

Four treatments were examined using JC-1 and propidium iodide (PI): control and 90 nM valinomycin (VAL) providing 44 mM sorbitol as a glycolytic substrate and 5 mM 2-deoxy-D-glucose (DOG) and 0.5 mM iodoacetamide (IAM) providing 9 mM pyruvic acid as a respiratory substrate.

Preparation of Fresh and Frozen/Thawed Semen Ejaculates were collected from four mature commercial AI bulls using an AV. From each bull, an aliquot of 1-2 ml

of raw semen after first dilution in 20% egg yolk-tris-glycerol (EYTG) extender was taken according to semen concentration range 400-800x106/ml in a centrifuge tube. Phosphate-buffered saline (PBS) was added to give a volume

of 10ml. This suspension was centrifuged at 600 x g for 10 minutes at room temperature. The supernatant was removed and the resulting pellet transferred to a fresh test tube in a water bath at 39°C. PBS extender was added to the pellet to give a suspension volume of 2ml and the resulting concentration of sperm was estimated using a nucleocounter (Chemometec A/S, Denmark). The concentration was adjusted to 24 x106 per ml by adding warmed Ruthin extender to Control and VAL and PBS extender to DOG and IAM treatments (Fig. 1). ‘Ruthin’ extender is the same as the

‘Reading’ extender [23] except for the substitution of sorbitol for glucose and the removal of trehalose [24]. From this suspension, 250 l from each in duplicate for each treatment was removed for investigation that contained 6x106 sperm. 250 l of Ruthin extender and DOG and IAM suspension were added to semen as per treatment. Samples were labelled as Control (C1, C2), Valinomycin (VAL1, VAL2), 2-deoxy-D-glucose (DOG1, DOG2) and Iodoacetamide (IAM1, IAM2). The labelled samples were left for 15 minutes before staining.

Thirty straws, each containing 0.25ml, of frozen bull semen were received in liquid nitrogen and thawed by immersing them in a water bath at 39°C for 30 secs. The

14 The Open Andrology Journal, 2012, Volume 4 Shahani et al.

contents of each straw were transferred into a centrifuge tube in water bath at 39°C and thereafter processed as for the fresh semen (Fig. 1).

Fluorescence Staining of Sperm and Flow Cytometry After semen preparation, 360 l was taken from 500 l semen into fresh test tube and 40 l of JC-1 were added, together with 4 l VAL as appropriate (Fig. 1), approximately 2 minutes apart to accommodate running time on the flow cytometer. Each sample was incubated at 39°C for 40 and 80min and 40 l of JC-1 incubated stirred semen from each treatment was added to 995 l of warmed Ruthin/PBS extender as per treatment and 5 l PI in flow tube (Fig. 1). The readings were then taken at each incubation time. The final stain concentrations of JC-I and PI were 0.1 M and 12 M respectively. Samples were analyzed on a Partec Cyflow Space Flow Cytometer (Partec GmbH, Gorlitz, Germany). Regional and logical gates were set to select live sperm population and exclude non-sperm specific events and dead cells.

Statistical Analysis

The real time data acquisition, analysis, and display was

performed using FloMax?, the PC based FCM software.

Mean fluorescence intensity measured in Relative Fluorescence Units (RFU) of green (low m) and orange/red (high m) appeared in log-X and log-Y respectively, stored in an Excel spreadsheet. After cleaning

the data, differences between fresh and frozen-thawed samples, within and between bulls after incubation with or

without inhibitors were investigated using ANOVA, applying Tukey and Dunnett’s comparison test within a

general linear model procedure in Minitab16. Significant

differences was accepted when P <0.05.

RESULTS

Flow cytometry recording of mitochondrial membrane potential in fresh and frozen-thawed sperm incubated with JC-1, presented all dot plots within one single population in CON, VAL, DOG and IAM treatments. This one parameter fluorescence display included both green and orange fluorescence but to varying degrees representing sperm with low and high m respectively (Fig. 2). The orange and green populations in dot plots were difficult to separate. Therefore orange:green (high m: low m) fluorescence ratio was performed to integrate green and orange fluorescence value in fresh and freeze-thaw semen. Comparison of Control (CON) with Treatment

To compare m of untreated spermatozoa in a medium supporting glycolysis (CON) with the treated spermatozoa after suspending their anaerobic (DOG, IAM) and aerobic (VAL) metabolic pathways: mean fluorescence intensity (MFI) of monomers was significantly higher in IAM and VAL and DOG and VAL as compare to CON at 40 min incubation in spermatozoa of fresh and freeze-thaw semen respectively (Figs. 3a & 4a ). Similar trend at 80 minutes

incubation was seen in all treatments of fresh and freeze-thaw (except IAM) semen (Figs. 3b & 4b ). In contrast MFI of aggregates appeared to be significantly higher in CON as compared to IAM and VAL treatments at 40 min incubation in fresh (excluding VAL) and freeze-thaw semen and only against IAM at 80 min incubation in sperm of fresh and

freeze-thaw semen (Figs. 3ab & 4ab ). A shift in m from high to low was found in samples treated with VAL and

IAM. The high: low m (h m:l m) ratio was higher in CON and DOG treated samples representing more active

mitochondria, while in samples treated with VAL and IAM the h m:l m fluorescence values were reversed to distinctly lower representing less active mitochondria at both incubation times in fresh and freeze-thaw semen (Figs. 3ab & 4ab ). High: low m ratio was significantly higher in

Fig. (1). Flow chart for JC-I and PI protocol after treatment with control (CON), valinomycin (VAL), 2-deoxy-D-glucose (DOG) and

iodoacetamide (IAM).

250μl Ruthin 250μl semen

AFTER CENTRIFUGATION CON

Flow tubes

Incubation

Interruption of Metabolic Pathways and Mitochondrial Function

The Open Andrology Journal, 2012, Volume 4 15

CON than other treatments in all incubation times and semen. To get a true measure of JC-1, after subtracting the h m:l m ratio of VAL from the h m:l m ratio of CON at both incubation times (data not presented), it was

revealed that even though VAL causes a significant reduction in sperm mitochondrial activity, this has not been abolished completely.

Fig. (2). Dot plots resulting from flow cytometric analyses of spermatozoa stained with JC-1. Dot plot of population with highly functional mitochondria displayed oval outline (A, C) and elongated in less functional mitochondria (B, D). A) population in presence of glycolytic substrate. B) population treated with IAM in presence of pyruvate. C) population treated with DOG in presence of pyruvate. D) population treated with VAL in presence of glycolytic substrate.

Fig. (3). (a & b) Comparison of control with treatments of fresh for 40 and 80 minutes incubation times of low and high mitochondrial membrane potential (l m and h m) and h m:l m ratio. Bars with the superscripts are significantly different to control; * = p<0.05; ** = p<0.01 and *** = p<0.001.

rg:grn

I A fo r the i n *

16 The Open Andrology Journal, 2012, Volume 4

Shahani et al.

Comparison Between Incubation Times Within Bull Although no significant differences were observed in h m:l m ratio between 40 and 80 min incubation times within each treatment of fresh and freeze-thaw semen except h m:l m ratio of Con freeze-thaw semen and DOG fresh and freeze-thaw semen (Fig. 5). However, maximum h m:l m ratio was seen at 40 min in all treatments and semen, but slightly lowered with VAL, fresh semen. When incubation time was compared within bulls, there was a reduction of h m:l m ratio over time in untreated sperm (CON) of fresh and fresh-thaw semen of all bulls excluding bull 3 fresh semen (Fig. 6). These variations in h m:l m values were only significant within bull 2 fresh semen and bull 3 and bull 4 freeze-thaw semen.

Comparison Between Fresh and Frozen-Thawed Semen Within Bulls

In this section the results are presented at 40 min incubation because the maximum h m:l m ratio was observed at this point. The effect of cryopreservation on the proportion of h m:l m in identical samples of sperm

before and after cryopreservation in the presence of glycolysis supported medium (CON), glycolysis (DOG, IAM) and respiratory (VAL) inhibitors stained with JC-1 observed variable results (Fig. 7a & b ). Cryopreservation increased h m:l m ratio only in bull 1 (30%) and decreased in bulls 2 (70%), 3 (20%) and 4 (30%) compared to fresh semen. The rise in bull 1 may be the product of sperm demonstrated capacitation like changes and became hyperactive [22], which result in increase in demand for ATP and relied on mitochondrial ATP for long time. Whereas decrease in h m:l m ratio in remaining bulls may be the involvement of an oxidative stress due to significant drop in antioxidant level during a freeze/thaw cycle [9]. Freeze-thaw also caused drop in h m:l m ratio in all bulls’ sperm in presence of DOG that forced sperm to generate their energy exclusively via mitochondria (Fig. 7a & b ). In contrast h m:l m ratio of all bulls seemed to be higher in freeze-thaw sperm treated with VAL but comparable in bull 4 (Fig. 7a & b ). Interestingly, similar change in h m:l m close to VAL observed with glycolysis intermediate inhibitor IAM (Fig. 7a & b ). This variation in h m:l m ratio likely to

Fig. (4). (a & b) Comparison of control with treatments of frozen-thawed semen for 40 and 80 minutes incubation times of low and high mitochondrial membrane potential (l m and h m) and h m:l m ratio. Bars with the superscripts are significantly different to control; * = p<0.05; ** = p<0.01 and *** = p<0.001.

Interruption of Metabolic Pathways and Mitochondrial Function The Open Andrology Journal, 2012, Volume 4 17

be the IAM effect on mitochondria or freeze/thaw process that changed mitochondrial status, which lessen the JC-1 accumulation in sperm mitochondria, increase green fluorescence and ultimately reduced h m:l m ratio. Comparison of High: Low m Ratio Between Bulls In spermatozoa of fresh semen at 40 min incubation: a higher h m:l m ratio was observed in bull 4 for all treatments except DOG, while a lower ratio was seen in bull 1 and bull 2, at all treatment (Fig. 7a & b). This ratio was only significant between bull one and four, bull four and all bulls and bull one, three and four for CON, IAM and VAL respectively (Fig. 7a & b). Inconsistency in h m:l m ratio was observed between the bulls in freeze-thaw semen at each treatment and incubation times. At 40 min incubation h m:l m ratio of freeze-thaw spermatozoa was higher in bull 1 treated with CON and IAM, this was significant with bull 2 at CON level; and lower in bull 2 at CON, DOG and VAL treatments, which was only significant with bull 3 at DOG level (Fig. 7a & b). It seemed that inconsistency in h m:l m ratio between the bulls is a characteristic of individual bulls rather than the effect of treatment or incubation time or sperm may switch energy demand from respiration to glycolysis for a while.

Fig. (5). Comparison of high: low m (h m:l m) ratio of fresh and freeze-thaw semen between 40 minutes (0) and 80 minutes (1) incubation within bulls. Bars with the superscripts are significantly different between incubation times; a= P<0.05; aa= P<0.01.

Fig. (6). Comparison of high: low m (h m:l m) ratio of fresh and freeze-thaw semen between 40 minutes (0) and 80 minutes (1) incubation within bulls. Bars with the superscripts are significantly different between incubation times; **= P<0.01.

18 The Open Andrology Journal, 2012, Volume 4

Shahani et al.

DISCUSSION

In this investigation, we demonstrated for the first time bovine sperm mitochondrial function following inhibition of glycolytic pathways in the presence of respiratory substrate in fresh and frozen-thawed semen. Each dot plot of treatments showed a single cell population of membrane intact spermatozoa and each population displayed both green and orange fluorescence but to varying degrees. The orange and green populations in dot plots were difficult to separate. Therefore examination of orange:green (high:low m) fluorescence ratio was performed to integrate green and orange fluorescence value in fresh and post-thaw semen. Bovine spermatozoa incubated with JC-1 exhibited significant differences between treatments at low and high m and incubation times. An increase in the population with active mitochondria has been observed in samples either provided with only glycolytic substrate (sorbitol) or

with glycolytic inhibitor (DOG) and pyruvate. Similar results have been observed by Mukai and Okuno [12] when mouse sperm were incubated with JC-1 in presence of either glucose or DOG and pyruvate. Consistency in m between treatments Control and DOG might be due to presence of sorbitol in the former treatment, which is reduced to fructose by the polyol pathway and fructose further reduced into pyruvate/lactate, providing respiratory substrate to sperm like the DOG treated sample. In addition the increase in m in DOG treated sperm may be due to inhibition of glycolysis which in turn may cause the sperm to force mitochondria to work at a higher level to generate energy and the reduction in m at 80 min incubation might be due to sperm having utilized all the respiratory substrate pyruvate as a marked reduction in seminal pyruvic acid has been observed after 60 min [25].

Addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) reduced the mitochondrial inner-membrane

Fig. (7). (a & b) Comparison of high: low m (h m:l m) ratio between bulls and fresh (A) and freeze-thaw (B) sperm within bulls at 40 minutes incubation. Bars with the superscripts are significantly different to freeze-thaw semen; *= P<0.05 and **= P<0.01. Bars that do not share a superscript letter are significantly different between bulls.

Interruption of Metabolic Pathways and Mitochondrial Function The Open Andrology Journal, 2012, Volume 4 19

potential as an uncoupler demonstrated as green emission from mitochondria [12]. Change in state of JC-1 from aggregates to monomers has been observed in human sperm [26] and stallion sperm [27], using VAL to validate the JC-1 probe. In the present study, VAL reduced m in bull spermatozoa resulting in a shift in fluorescence from orange (high m) to green (low m). When subtracting the aggregate of valinomycin from control, the true value for mitochondrial potential was lower as compared to control (data not shown) indicating that valinomycin has not completely abolished mitochondrial activity and or the presence of aggregates fluorescence could be background fluorescence of JC-1 due to the sensitivity of the fluorescence detectors; it is unlikely that sperm populations would ever show zero fluorescence. Increase in VAL concentration may help to differentiate the mitochondrial and fluorochrome activity.

Hathaway [28] observed that IAM completely reduced respiration in sea urchin spermatozoa within 45min. IAM, an inhibitor of GAPDH, did not reduce motility in presence of pyruvate and lactate but inhibited ATP produced from glycolysis with no effect on ATP produced by mitochondrial respiration in mouse sperm [15]. Recently Schmidt and Dringen [17] found that IAM markedly reduced glutathione (GSH), cellular GAPDH and lowered cellular lactate production in astrocytes and reduction in GSH level resulted

in loss of mitochondrial membrane potential in mice sperm [29]. It has been reported that the sperm mitochondrial cysteine-rich protein (SMCP) and phospholipid hydroperoxide glutathione peroxidise are the proteins localized in the mitochondrial capsule and enhances sperm motility [30]. Like GAPDH, IAM may alter SMCP, GSH level and decrease m. SMCP gene knockout in mice sperm reduced sperm motility and fertility [31]. In our study, IAM disrupted the mitochondrial activity in the presence of pyruvate and also shifted fluorescence from orange (high m) to green (low m). It reveals that IAM might be involved in the reduction of mitochondrial activity or restricting pyruvate oxidation, therefore depriving mitochondria of respiratory substrate.

Freeze-thaw cycle is the basis of major changes in spermatozoa such as increases in osmotic and oxidative stress, and membrane permeability, thereby significant decrease in sperm viability and mitochondrial membrane potential [32, 33]. Using JC-1 probe to evaluate the functional potential of mitochondria of bovine sperm, Celeghini et al., [34], observed that for the percentage of spermatozoa with greater mitochondrial function, potential was approximately five fold less in frozen-thawed semen than in fresh semen. Similar results were found by Arruda and his colleagues [35] in cattle semen after cryopreservation with Tris–egg-yolk extender and another extender containing glycerol. Disappearance and significant reduction in aggregates, shift to monomers and marked change in aggregates:monomers ratio was also observed in frozen-thawed bull sperm [33, 36] suggesting that the bulls had low cryopreservation tolerance. In this study washed frozen-thawed spermatozoa diluted in extender supporting glycolysis showed significant increase (30%) in

h m:l m ratio than that of the fresh spermatozoa in bull

1 but decrease (20 to 70%) in remaining three bulls. Changes in sperm membrane occur during the freezing process, similar to those that occur during capacitation and may induce damage to the mitochondria [22, 37]. Oxidative stress is another factor that influences sperm mitochondrial function during freeze-thaw process [9]. Motility, mitochondrial function, and viability are the most likely all interrelated aspects of the overall physiological status of the spermatozoa [38, 39]. Therefore high level of h m:l m in bull 1 might be due to the capacitation- like effect of freeze-thaw, which moved sperm towards hyperactivation and consequently a rise in mitochondrial activity, whereas low level in other three bulls perhaps be the sperm mitochondria encountered to oxidative stress produced on decrease in antioxidant level during a freeze/thaw cycle.

On comparing incubation times using h m:l m ratio as factor of variation: maximum h m:l m ratio observed at 40 min incubation in all treatments and slightly lowered with VAL. Within the bulls at control level, decrease in h m:l m ratio from 40 to 80 min has been observed in all bulls of fresh and freeze-thawed sperm but increased in bull 3 in fresh semen, suggesting that variation in h m:l m is a characteristic of individual bulls or sperm of bull 3 may initially fulfil their energy demand via glycolysis for a while. Similarly among the bulls the ratio of h m:l m, was higher in bull 4 followed by bull 3 and comparable in bull 1 and 2 of fresh semen; and for frozen-thawed semen, this ratio was comparable in bull 1, 2 and 4 but markedly reduced in bull 2. Generally the commercial testing bull semen is based on post-thawed progressive motility, which has never correlated well with fertility [40, 41]; therefore highly functional mitochondria after prolonged incubation of any bull pre or post freeze/thaw cycle may be a good indicator of bull selection and fertility prediction. CONCLUSION

Mitochondrial function was significantly decreased on freeze-thaw process. Maximum mitochondrial potential has been found at 40 min incubation in fresh and frozen-thawed semen, suggesting that the 40 min incubation will be satisfactory for assessment of mitochondrial membrane potential in future. Mitochondrial activity was markedly reduced in samples treated with IAM and VAL indicating that IAM which is a known glycolytic inhibitor has a similar effect to VAL, recognized as a respiratory inhibitor, which can be validated by looking at either sperm oxygen consumption or ATP production or motility in the presence of IAM.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS

The author is grateful to the Livestock and Fisheries Department, Government of Sindh, Pakistan for financial support. The author is also thankful to Mr. Abdul Qadir Junejo, Director, Animal Breeding Sindh, for their constructive advice.

20 The Open Andrology Journal, 2012, Volume 4 Shahani et al.

REFERENCES

[1]Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of

mammalian sperm: what we ask them to survive. J Androl 1990;

11: 73-88.

[2]Parks JE, Graham JK. Effects of cryopreservation procedures on

sperm membranes. Theriogenology 1992; 38: 209-22.

[3]Holt WV. Alternative strategies for the long-term preservation of

spermatozoa. Reprod Fertil Dev 1997; 9: 309-19.

[4]Mazur P. Freezing of living cells: mechanisms and implications.

Am J Physiol 1984; 247: C125-C42.

[5]Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet

JW, Crowe JH. Cold shock damage is due to lipid phase-transitions

in cell-membranes - a demonstration using sperm as a model. J Exp

Zool 1993; 265: 432-7.

[6]Holt WV. Fundamental aspects of sperm cryobiology: the

importance of species and individual differences. Theriogenology

2000; 53: 47-58.

[7]Holt WV, North RD. Partially irreversible cold-induced lipid phase

transitions in mammalian sperm plasma membrane domains:

freeze-fracture study. J Exp Zool 1984; 230: 473-83.

[8]Guthrie HD, Welch GR, Long JA. Mitochondrial function and

reactive oxygen species action in relation to boar motility.

Theriogenology 2008; 70: 1209-15.

[9]Bilodeau JF, Chaterjee S, Sirard MA, Gagnon C. Levels of

antioxidant defenses are decreased in bovine spermatozoa after a

cycle of freezing and thawing. Mol Reprod Dev 2000; 55: 282-8. [10]Lindemann CB, Fisher M, Lipton M. A comparative study of the

effects of freezing and frozen storage on intact and demembranated

bull spermatozoa. Cryobiology 1982; 19: 20-8.

[11]Fraczek M, Piasecka M, Gaczarzewicz D, et al. Membrane stability

and mitochondrial activity of human ejaculated spermatozoa during

in vitro experimental infection with Escherichia coli,

Staphylococcus haemoly and Bacteroides ureolyticus. Andrologia

2012; 20: 1-15.

[12]Mukai C, Okuno M. Glycolysis plays a major role for adenosine

triphosphate supplementation in mouse sperm flagellar movement.

Biol Reprod 2004; 71: 540-7.

[13]Amaral A, Ramalho-Santos. J. Assessment of mitochondrial

potential: implications for the correct monitoring of human sperm

function. Int J Androl 2010; 33: e180-e6.

[14]Hiipakka RA, Hammerstedt RH. 2-Deoxyglucose transport and

phosphorylation by bovine sperm. Biol Reprod 1978; 19: 368-79. [15]Pasupuleti V. Role of glycolysis and respiration in sperm

metabolism and motility. MS thesis. Kent State University, Kent,

Ohio, 2007.

[16]Krzyzosiak J, Molan P, Vishwanath R. Measurements of bovine

sperm velocities under true anaerobic and aerobic conditions. Anim

Reprod Sci 1999; 55: 163-73.

[17]Schmidt MM, Dringen R. Differential effects of iodoacetamide and

iodoacetate on glycolysis and glutathione metabolism of cultured

astrocytes. Front Neuro 2009; 1: 1-10.

[18]Harris EJ, Cockrell R, Pressman BC. Induced and spontaneous

movements of potassium ions into mitochondria. J Biochem 1966;

99: 200-13.

[19]Saris NEL, Andersson MA, Mikkola R, et al. Microbial toxin’s

effect on mitochondrial survival by increasing K+ uptake. Toxicol

Ind Health 2009; 25: 441-6.

[20]Andersson MA, Mikkola R, Kroppenstedt RM, et al. The

mitochondrial toxin produced by Streptomyces griseaus strains

isolated from an indoor environment is valinomycin. Appl Environ

Microbiol 1998; 64: 4767-73.

[21]Garrett JAL, Revell SG, Leese HJ. ATP production by bovine

spermatozoa and its relationship to semen fertilising ability. J

Androl 2008; 29: 449-58. [22]Watson PF. Recent developments and concepts in the

cryopreservation of spermatozoa and the assessment of their post-

thawing function. Reprod Fertil Dev 1995; 7: 871-91.

[23]Revell SG, Glossop CE. A long-life ambient temperature diluent

for boar semen. Anim Prod 1989; 48: 579-84.

[24]Woolley DM, Crockett RF, Groom WDI, Revell SG. A study of

synchronisation between the flagella of bull spermatozoa, with

related observations. J Exp Biol2009; 212: 2215-23.

[25]Marden WGR. Source of endogenous pyruvic acid in bovine

seminal fluid and utilization. J Dairy Sci 1961; 44: 1688-97.

[26]Troiano L, Granata AR, Cossarizza A, et al. Mitochondrial

membrane potential and DNA stainability in human sperm cells: a

flow cytometry analysis with implications for male infertility. Exp

Cell Res 1998; 241: 384-93.

[27]Love CC, Thompson JA, Brinsko SP, et al. Relationship between

stallion sperm motility and viability as detected by two

fluorescence staining techniques using flow cytometry.

Theriogenology 2003; 60: 1127-38.

[28]Hathaway RR. Activation of respiration in sea urchin spermatozoa

by egg water. Biol Bull 1963; 125: 486-98.

[29]Perl A, Yueming Q, Kazim RC, et al. Transaldolase is essential for

maintenance of the mitochondrial transmembrane potential and

fertility of spermatozoa. Proc Natl Acad Sci USA 2006; 103:

14813-8.

[30]Hawthorne SK, Goodarzi G, Bagarova J, et al. Comparative

genomics of the sperm mitochondria-associated cysteine-rich

protein gene. Genomics 2006; 87: 382-91.

[31]Nayernia K, Ibrahim MA, Elke BG, et al. Asthenozoospermia in

mice with targeted deletion of the sperm mitochondrion-associated

cysteine-rich protein (SMCP) gene. Mol Cell Biol 2002; 22: 3046-

52.

[32]Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, et al.

Detection of apoptosis-like changes during the cryopreservation

process in equine sperm. J Androl 2008; 29: 213-21.

[33]Garner DL, Thomas AC, Gravance CG. The effect of glycerol on

the viability, mitochondrial function and acrosomal integrity of

bovine spermatozoa. Reprod Dom Anim 1999; 34: 399-404.

[34]Celeghini ECC, Arruda RP, Andrade AFC, Nascimento J, Raphael

CF, Rodrigues PHM. Effects that bovine sperm cryopreservation

using two different extenders has on sperm membranes and

chromatin. Anim Reprod Sci 2008; 104: 119-31.

[35]Arruda RP, Gonzalez FRA, Celeghini ECC, Raphael CF. Effects of

cryopreservation using different freezing techniques and

cryoprotectants on plasmatic, acrosomal and mitochondrial

membranes of bovine spermatozoa. Acta Sci Vet 2005; 33: S329. [36]Thomas CA, Garner DL, Dejarnette JM, Marshall CE. Effect of

cryopreservation of bovine sperm organelle function and viability

as determined by flow cytometry. Biol Reprod 1998; 58: 786-93. [37]Nagy S, Jansen J, Topper EK, Gadella BM. A triple stain flow

cytometric method to assess plasma and acrosome membrane

integrity of cryopreserved bovine sperm immediately after thawing

in presence of egg-yolk particles. Biol Reprod 2003; 68: 1828-35. [38]Anderson MJ, Chapman SJ, Videan EN, et al. Functional evidence

for differences in sperm competition in humans and chimpanzees.

Am J Phys Anthrop 2007; 134: 274-80.

[39]Garner DL, Thomas AC, Joerg HW, Dejarnette JM, Marshall CE.

Fluorometric assessments of mitochondrial function and viability in

cryopreserved bovine spermatozoa. Biol Reprod 1997; 57: 1401-6. [40]Holt WV, Van Look KJW. Concepts in sperm heterogeneity, sperm

selection and sperm competition as biological foundations for

laboratory tests of semen quality. Reproduction 2004; 127: 527-35. [41]Rodriguez-Martinez H. Laboratory semen assessment and

prediction of fertility: still utopia? Reprod Domest Anim 2003; 38:

312-8.

Received: October 29, 2011 Revised: April 21, 2012 Accepted: April 25, 2012

? Shahani et al.; Licensee Bentham Open.

This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (https://www.sodocs.net/doc/b710713846.html,/-licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

糖酵解途径

糖酵解途径(glycolytic pathway)是指细胞在胞浆中分解葡萄糖生成丙酮酸(pyruvate)的过程,此过程中伴有少量ATP的生成.在缺氧条件下丙酮酸被还原为乳酸(lactate)称为糖酵解.有氧条件下丙酮酸可进一步氧化分解生成乙酰CoA进入三羧酸循环,生成CO2和H2O. 葡萄糖不能直接扩散进入细胞内,其通过两种方式转运入细胞:一种是在前一节提到的与Na+共转运方式,它是一个耗能逆浓度梯度转运,主要发生在小肠粘膜细胞、肾小管上皮细胞等部位;另一种方式是通过细胞膜上特定转运载体将葡萄糖转运入细胞内(图4-1),它是一个不耗能顺浓度梯度的转运过程.目前已知转运载体有5种,其具有组织特异性如转运载体-1(GLUT-1)主要存在于红细胞,而转运载体-4(GLUT-4)主要存在于脂肪组织和肌肉组织. 糖酵解过程 糖酵解分为两个阶段共10个反应,每个分子葡萄糖经第一阶段共5个反应,消耗2个分子ATP为耗能过程,第二阶段5个反应生成4个分子ATP为释能过程. 1.第一阶段 (1)葡萄糖的磷酸化(phosphorylation of glucose) 进入细胞内的葡萄糖首先在第6位碳上被磷酸化生成6-磷酸葡萄糖(glucose 6 phophate,G-6-P),磷酸根由ATP供给,这一过程不仅活化了葡萄糖,有利于它进一步参与合成与分解代谢,同时还能使进入细胞的葡萄糖不再逸出细胞.催化此反应的酶是己糖激酶(hexokinase,HK).己糖激酶催化的反应不可逆,反应需要消耗能量

ATP,Mg2+是反应的激活剂,它能催化葡萄糖、甘露糖、氨基葡萄糖、果糖进行不可逆的磷酸化反应,生成相应的6-磷酸酯,6-磷酸葡萄糖是HK的反馈抑制物,此酶是糖氧化反应过程的限速酶(rate limiting enzyme)或称关键酶(key enzyme)它有同工酶Ⅰ-Ⅳ型,Ⅰ、Ⅱ、Ⅲ型主要存在于肝外组织,其对葡萄糖Km值为10-5~10-6M Ⅳ型主要存在于肝脏,特称葡萄糖激酶(glucokinase,GK),对葡萄糖的Km值1~10-2M,正常血糖浓度为5mmol/L,当血糖浓度升高时,GK 活性增加,葡萄糖和胰岛素能诱导肝脏合成GK,GK能催化葡萄糖、甘露糖生成其6-磷酸酯,6-磷酸葡萄糖对此酶无抑制作用. (2)6-磷酸葡萄糖的异构反应(isomerization of glucose-6-phosphate) 这是由磷酸己糖异构酶(phosphohexose isomerase)催化6-磷酸葡萄糖(醛糖aldose sugar)转变为6-磷酸果糖(fructose-6-phosphate,F-6-P)的过程,此反应是可逆的. (3)6-磷酸果糖的磷酸化(phosphorylation of fructose-6-phosphate) 此反应是6磷酸果糖第一位上的C进一步磷酸化生成1,6-二磷酸果糖,磷酸根由ATP供给,催化此反应的酶是磷酸果糖激酶1(phosphofructokinase l,PFK1). PFK1催化的反应是不可逆反应,它是糖的有氧氧化过程中最重要的限速酶,它也是变构酶,柠檬酸、ATP等是变构抑制剂,ADP、AMP、Pi、1,6-二磷酸果糖等是变构激活剂,胰岛素可诱导它的生成. (4)1.6 二磷酸果糖裂解反应(cleavage of fructose 1,6 di/bis phosphate)

生物化学糖代谢知识点总结

各种组织细胞 体循环小肠肠腔 第六章糖代谢 糖(carbohydrates)即碳水化合物,是指多羟基醛或多羟基酮及其衍生物或多聚物。 根据其水解产物的情况,糖主要可分为以下四大类: 单糖:葡萄糖(G )、果糖(F ),半乳糖(Gal ),核糖 双糖:麦芽糖(G-G ),蔗糖(G-F ),乳糖(G-Gal ) 多糖:淀粉,糖原(Gn ),纤维素 结合糖: 糖脂 ,糖蛋白 其中一些多糖的生理功能如下: 淀粉:植物中养分的储存形式 糖原:动物体内葡萄糖的储存形式 纤维素:作为植物的骨架 一、糖的生理功能 1. 氧化供能 2. 机体重要的碳源 3. 参与组成机体组织结构,调节细胞信息传递,形成生物活性物质,构成具有生理功能的糖蛋白。 二、糖代谢概况——分解、储存、合成 三、糖的消化吸收 食物中糖的存在形式以淀粉为主。 1.消化 消化部位:主要在小肠,少量在口腔。 消化过程:口腔 胃 肠腔 肠黏膜上皮细胞刷状缘 吸收部位:小肠上段 吸收形式:单糖 吸收机制:依赖Na+依赖型葡萄糖转运体(SGLT )转运。 2.吸收 吸收途径:

过程 2 H 2 四、糖的无氧分解 第一阶段:糖酵解 第二阶段:乳酸生成 反应部位:胞液 产能方式:底物水平磷酸化 净生成ATP 数量:2×2-2= 2ATP E1 E2 E3 调节:糖无氧酵解代谢途径的调节主要是通过各种变构剂对三个关键酶进行变 构调节。 生理意义: 五、糖的有氧氧化 E1:己糖激酶 E2: 6-磷酸果糖激酶-1 E3: 丙酮酸激酶 NAD + 乳 酸 NADH+H + 关键酶 ① 己糖激酶 ② 6-磷酸果糖激酶-1 ③ 丙酮酸激酶 调节方式 ① 别构调节 ② 共价修饰调节 糖无氧氧化最主要的生理意义在于迅速提供能量,这对肌收缩更为重要。 是某些细胞在氧供应正常情况下的重要供能途径。 ① 无线粒体的细胞,如:红细胞 ② 第一阶段:糖酵解途径 G (Gn ) 丙酮酸胞液

糖酵解反应过程

有氧氧化的步骤简介 有氧氧化包括三个大的阶段,分别为糖的酵解、乙酰COA的 形成和三羧酸循环。 糖酵解反应过程 步骤名称底物酶产物能量 1 葡萄糖磷 酸化葡萄糖己糖激酶 HK 6-磷酸 葡萄糖 消耗ATP 一个 2 6-磷酸葡 萄糖异构6-磷酸葡 萄糖 葡萄糖己 糖异构酶 6-磷酸果 糖 3 6-磷酸果 糖磷酸化6-磷酸果 糖 磷酸果糖 激酶1 (PFK1) 1,6-二磷 酸果糖 消耗ATP 一个 4 磷酸丙糖 的生成1,6-二磷 酸果糖 缩醛酶磷酸二羟 丙酮,3- 磷酸甘油 醛 5 丙糖的转 化磷酸二羟 丙酮 磷酸丙糖 异构酶 3-磷酸甘 油醛 6 3-磷酸甘 油醛氧化 脱氢3-磷酸甘 油醛 3-磷酸甘 油脱氢酶 1,3-二磷 酸甘油酸 7 底物水平1,3-二磷磷酸甘油3-磷酸甘产生两分

磷酸化酸甘油酸酸激酶油酸子ATP 8 3-磷酸甘 油酸异构3-磷酸甘 油醛 磷酸甘油 酸变位酶 2-磷酸甘 油酸 9 2-磷酸甘 油酸烯醇 化2-磷酸甘 油酸 烯醇化酶磷酸烯醇 式甘油酸 10 底物水平 磷酸化磷酸烯醇 式甘油酸 丙酮酸激 酶 丙酮酸产生两分 子ATP 糖酵解过程简述 糖酵解在胞浆内进行,分为两阶段,第一阶段为3-磷酸甘油醛的生成,第二阶段为丙酮酸的生成。 第一阶段包括五个步骤 第一步为葡萄糖的磷酸化,葡萄糖在己糖激酶的催化下,消耗一个ATP,在6号c原子上挂上一个磷酸基,生成6-磷酸葡萄糖。第二步为6-磷酸葡萄糖的异构,在葡糖糖异构酶的催化下,6-磷酸葡萄糖异构为6-磷酸果糖。 第三步为6-磷酸果糖的磷酸化,在6-磷酸果糖激酶1的催化下,消耗一分子ATP,生成1,6-二磷酸果糖。 第四步为磷酸丙糖的生成,1,6-二磷酸果糖在缩醛酶的催化下生成一分子磷酸二羟丙酮和一分子3-磷酸甘油醛。 第五步为磷酸二羟丙酮的异构,磷酸二羟丙酮在丙糖异构酶的作用下生成3-磷酸甘油醛。

糖酵解 三羧酸循环最全总结

在高等植物中存在着多条呼吸代谢的生化途径,这是植物在长期进化过程中,对多变环境条件适应的体现。在缺氧条件下进行酒精发酵和乳酸发酵,在有氧条件下进行三羧酸循环和戊糖磷酸途径,还有脂肪酸氧化分解的乙醛酸循环以及乙醇酸氧化途径等(图5-2)。 图5-2 植物体内主要呼吸代谢途径相互关系示意图 一、糖酵解 己糖在细胞质中分解成丙酮酸的过程,称为糖酵解(glycolysis)。整个糖酵解化学过程于1940年得到阐明。为纪念在研究这一途径中有突出贡献的三位生物化学家:G.Embden,O.Meyerhof和J.K.Parnas,又把糖酵解途径称为EmbdenMeyerhofParnas途径,简称EMP途径(EMP pathway)。糖酵解普遍存在于动物、植物、微生物的细胞中。 (一)糖酵解的化学历程 糖酵解途径(图5-3)可分为下列几个阶段:

图5-3糖酵解途径 1.己糖的活化(1~9)是糖酵解的起始阶段。己糖在己糖激酶作用下,消耗两个ATP逐步转化成果糖-1,6二磷酸(F-1,6-BP)。 如以淀粉作为底物,首先淀粉被降解为葡萄糖。淀粉降解涉及到多种酶的催化作用,其中,除淀粉磷酸化酶(starch phosphorylase)是一种葡萄糖基转移酶外,其余都是水解酶类,如α-淀粉酶(α-amylase)、β-淀粉酶(β-amylase)、脱支酶(debranching enzyme)、麦芽糖酶(maltase)等。 2.己糖裂解(10~11)即F-1,6-BP在醛缩酶作用下形成甘油醛-3-磷酸和二羟丙酮磷酸,后者在异构酶(isomerase)作用下可变为甘油醛-3-磷酸。 3.丙糖氧化(12~16)甘油醛-3-磷酸氧化脱氢形成磷酸甘油酸,产生1个ATP和1个NADH,同时释放能量。然后,磷酸甘油酸经脱水、脱磷酸形成丙酮酸,并产生1个ATP,这一过程分步完成,有烯醇化酶和丙酮酸激酶参与反应。

糖酵解的过程

糖酵解的過程EMP途徑分為兩個階段,第一 個階段是磷酸丙糖的生成過 程(耗能過程),第二和階段是丙酮酸生成過程(產能過程)。 下面讓我們來慢慢分解反應過程 第一階段第一步 △磷酸化:G→G6P Extracellular fluid:胞外液 Cytoplasm:細胞質 Glucose:葡萄糖 Phosphorylation:磷酸化作用 Plasma:等離子;血漿 Membrane:膜;薄膜第一階段 第二階段 己糖激酶 EMP 途 徑 中 第 一 個 限 速 酶激酶:一类从高能供体分子(如ATP)转移磷酸基团到特定靶分子(底物)的酶;这一过程谓之磷酸化。 已糖激酶:催化从ATP转移磷酸基团至各种六碳糖上去的酶。 激酶都需要Mg2+作为辅助因子。 首先我們來看一下糖酵解的第一階段

第一階段第二步△G6P F6P 第一階段第三步 ③磷酸化:F6P → FDP 磷酸葡萄糖异构酶 PFK是第二个限速酶,也是 EMP途径的关键酶,其活性 大小控制着整个途径的进 程。 磷酸果糖激酶是一种别构 酶,是糖酵解三个限速酶中 催化效率最低的酶,因此被 认为是糖酵解作用最重要 的限速酶。

第一階段第四步 ④裂解 (FBP → DHAP + G3P) 第一階段第⑤步 ⑤异构化(DHAP → G3P) 1,6-二磷酸果糖 2×3-磷酸甘油醛

第二階段第六步 ⑥氧化(G3P → 1,3-BPG) 第二階段第七步 ⑦转化(1,3-BPG → 3PG) 再來,我們來看糖 酵解的第二階段。 高能磷酸鍵 3-磷酸甘 ◎EMP第一次产生高能磷酸键; ◎EMP中唯一的脱氢反应,并产生了还原剂NADH。 ◎该酶是巯基酶,所以它可被碘乙酸不可逆地抑制,所以碘乙酸能抑制糖酵解。 ◎底物水平磷酸化:直接利用代谢 中间物氧化释放的能量产生ATP的 磷酸化类型。

生物化学原理- 糖酵解

第十五章糖酵解 本章主线: 糖酵解 丙酮酸代谢命运 (乙醇发酵乳酸发酵) 糖酵解调控 巴斯德效应 3种单糖代谢 (果糖、半乳糖、甘露糖) 一、糖酵解 糖酵解概述: ●位置:细胞质 ●生物种类:动物、植物以及微生物共有 ●作用:葡萄糖分解产生能量 ●总反应:葡萄糖+2ADP+2 NAD++2Pi →2 丙酮酸+2ATP+2NADH+2H++2H2O 具体过程: 第一阶段(投入A TP阶段): 1分子葡萄糖转换为2分子甘油醛-3-磷酸;投入2分子ATP。 ○1 反应式:葡萄糖+ ATP→葡萄糖-6-磷酸+ADP 酶:己糖激酶(需Mg2+参与) 是否可逆:否 说明: ●保糖机制——磷酸化的葡萄糖被限制在细胞内,磷酸化的糖带有负电荷的磷酰基,可防 止糖分子再次通过质膜。(应用:解释输液时不直接输葡萄糖-6-磷酸的原因) ●己糖激酶以六碳糖为底物,专一性不强。 ●同功酶——葡萄糖激酶,是诱导酶。葡萄糖浓度高时才起作用。 ○2 反应式:葡萄糖-6-磷酸→果糖-6-磷酸 酶:葡萄糖-6-磷酸异构酶 是否可逆:是 说明:

●是一个醛糖-酮糖转换的同分异构化反应(开链?异构?环化) ●葡萄糖-6-磷酸异构酶表现出绝对的立体专一性 ●产物为α-D-呋喃果糖-6-磷酸 ○3 反应式:果糖-6-磷酸+ATP→果糖-1,6-二磷酸+ADP 酶:磷酸果糖激酶-I 是否可逆:否 说明: ●磷酸果糖激酶-I的底物是β-D-果糖-6-磷酸与其α异头物在水溶液中处于非酶催化的快 速平衡中。 ●是大多数细胞糖酵解中的主要调节步骤。 ○4 反应式:果糖-1,6-二磷酸→磷酸二羟丙酮+甘油醛-3-磷酸 酶:醛缩酶 是否可逆:是 说明: ●平衡有利于逆反应方向,但在生理条件下,甘油醛-3-磷酸不断地转化成丙酮酸,大大 地降低了甘油醛-3-磷酸的浓度,从而驱动反应向裂解方向进行。 ●注意断键位置:C3-C4 ○5 反应式:磷酸二羟丙酮→甘油醛-3-磷酸 酶:丙糖磷酸异构酶 是否可逆:是 说明: ●葡萄糖分子中的C-4和C-3 →甘油醛-3-磷酸的C-1; 葡萄糖分子中的C-5和C-2 →甘油醛-3-磷酸的C-2; 葡萄糖分子中的C-6和C-1 →甘油醛-3-磷酸的C-3。 ●缺少丙糖磷酸异构酶,将只有一半丙糖磷酸酵解,磷酸二羟丙酮堆积。 第二阶段(产出A TP阶段):此阶段各物质的量均加倍 2分子甘油醛-3-磷酸转换为2分子丙酮酸;产出4分子ATP ○6 反应式:甘油醛-3-磷酸+NAD++Pi→1,3-二磷酸甘油酸+NADH+H+ 酶:甘油醛-3-磷酸脱氢酶 是否可逆:是 说明: ●酵解中唯一一步氧化反应。是一步吸能反应,与第7步反应耦联有利于反应进行。 ●NAD+是甘油醛-3-磷酸脱氢酶的辅酶 ●1,3-二磷酸甘油酸中形成一个高能酸酐键。 ●无机砷酸(AsO43-)可取代无机磷酸作为甘油酸- 3-磷酸脱氢酶的底物,生成一个不稳

糖酵解习题

糖代谢习题 1、写出在糖酵解反应过程中 A.在标准条件下耗能的反应 B.在标准条件下产能的反应 C.消耗ATP的反应 D.产生ATP的反应 E.在红细胞中处于或接近平衡的反应。 2、经过糖酵解途径后,如果丙酮酸分子中的某个碳原子被同 位素14C标记的话,请问这个碳原子在葡萄糖分子碳骨架中的哪个位置? 3、在红细胞中进行的糖酵解反应,如果下列物质浓度突然上 升会出现什么现象? A.ATP B. AMP C. 1,6-二磷酸果糖 D. 2,6-二磷酸果糖 E. 柠檬酸 F. 6-磷酸葡萄糖。 4、 1.3-二磷酸甘油酸水解生成3-磷酸甘油酸和无机磷酸(Pi) 的标准自由能(?G0’)是-49.6 kj/mol: 1.3-BPG + H20 →3-PG + Pi ATP水解的标准自由能是-30.5 kj/mol: ATP + H20 →ADP + Pi 1). 磷酸甘油酸激酶所催化的反应得标准自由能是多少? ADP + 1,3-BPG →ATP + 3-PG 2). 该反应的平衡常数是多少?

3). 如果红细胞中[1,3-BPG]和[3-PG]的浓度是1 μM,和120 μ M,假设磷酸甘油酸激酶催化的反应在平衡状态,请问[ATP]/[ADP] 是多少? 5、若进行柠檬酸循环的细胞中包含有磷酸烯醇式丙酮酸羧激酶,那么怎样的过程能使1分子的α-酮戊二酸氧化成5分子的CO2? 6、每进行一次三羧酸循环,1分子的乙酰CoA被氧化,即产生2分子的CO2。那么乙酰CoA的碳原子在其第一轮的循环中会被转变成CO2吗? 7、柠檬酸是对称分子,在柠檬酸循环的第二步中,乌头酸酶催化了半个柠檬酸分子脱水,却不催化相同的另一半脱水。这如何解释? 8、一系列的反应中,细胞需要NADPH的量远远超过5-p- 核糖的量。那么(a)细胞是如何获得NADPH的?(b) 过量的5-p-核糖有何去路? 9、丙二酸是琥珀酸脱氢酶催化反应的的一种竞争性抑制 剂。解释为什么增高草酰乙酸的浓度能够克服丙二酸的抑 制作用。假定这一反应发生在肝脏制剂中。 10、①丙氨酸降解产生丙酮酸,亮氨酸降解产生乙酰CoA。 这两种氨基酸的降解能为柠檬酸循环的中间物库作出补充 吗?②储存在动物脂肪组织的三酰甘油是能量的重要来 源。脂肪酸降解产生乙酰CoA,后者能激活丙酮酸羧化酶。

糖酵解过程详解

葡萄糖糖酵解详解 作者为了大家的方便,在网上搜集了资料,请交流,请提意见! 1,名称解析:在供氧不足时,体内组织细胞中的葡萄糖或糖元,分解为乳酸的过程称为无氧分解,由于此过程与与酵母菌使糖生醇发酵的过程基本相似,故称为糖酵解。 2,代谢位置:糖酵解是在细胞液中进行的。 3,过程可以分为两个阶段来理解: 第一阶段叫活化裂解阶段:由葡萄糖或糖元变成两分子磷酸丙糖(3-磷酸甘油醛和磷酸二羟丙酮),下面分别叙述: ○1如下图所示,为第一阶段的第○1小段。这一小段分两种情况:一个是从葡萄糖开始,一个是从糖元开始。 上图就表示从葡萄糖开始,葡萄糖首先在磷酸化酶催化下进行磷酸解,由ATP提供磷酸基生成6-磷酸葡萄糖,ATP本身变成ADP。大家注意代谢反应方程式的写法就是上面这个简化的表示式,相当于我们通常使用的下面的意思: 葡萄糖已糖激酶磷酸葡萄糖+ADP+H2O, 在这一阶段请注意: ▲从能量的角度来看,就消耗了一个ATP。但如果是从糖元开始,则因糖元在磷酸化酶催化下进行磷酸解是已变成了1-磷酸葡萄糖,下一步在变化酶作用下变成6-磷酸葡萄糖时就不消耗能量了,所以从糖元开始的糖酵解就少消耗这个ATP了。或者说因为糖原缩合时已经挂上了一分子磷酸,糖原一水解就是6磷酸葡萄糖,所以葡萄糖就不用再磷酸化了,就少消耗了一个atp。 ▲这阶段的已糖激酶是限速酶,决定反应的速度。 下面这图表示催化剂已糖酶的催化过程是把已糖酶把葡萄糖结合在一起形成1-磷酸葡萄糖(和6-磷酸葡萄糖是异构体)。 ○2第二小阶段是6-磷酸葡萄糖在已糖异构化酶催化下生成6-磷酸果糖,下面是这个反应的开链式和哈沃斯式的反应式:

糖酵解特点

四、糖代谢概况 有氧 无氧 H 2O 及CO 2 乳酸 乳酸、氨基酸、甘油 糖原 核糖 + NADPH+H+ 磷酸戊糖途径 淀粉 消化与吸收 无氧分解(糖酵解) 糖酵解(glycolysis)是指葡萄糖在无氧条件下 分解生成乳酸并释放出能量的过程。 糖酵解的全部反应过程在胞液(cytoplasm)中进行,代谢的终产物为乳酸(lactate),一分子葡萄糖经无氧酵解可净生成两分子ATP 。 无氧酵解的反应过程可分为活化、裂解、放能 和还原四个阶段。

酸的生醇发酵及葡萄糖的无氧分解 葡萄糖EMP + NAD CH2OH CH3 乙醇 NADH+H+ NAD+ CO2 乳酸 COOH CH(O H) C H3 CHO CH3 COOH C==O CH3 丙酮酸 1.活化(a c t i v a t i o n)-己糖磷酸酯的生成 活化阶段是指葡萄糖经磷酸化和异构反应生成1,6-二磷酸果糖(FDP)的反应过程。该过程共由三步化学反应组成。 (一)糖酵解途径 葡萄糖磷酸化 磷酸葡萄糖(G-6-P) G-6-P异构为(F-6-P) F-6-P再磷酸化为( F-1,6-BP )......(1)......(2) (3)

ADP ATP ADP * (2 无氧酵解的活化阶段 第一阶段总结: 消耗ATP 不生成ATP 从葡萄糖开始→ 2分子ATP 从糖原开始→ 1分子ATP

.裂解(lysis)——磷酸丙糖的生 一分子F-1,6-BP 裂解为两分子可以互变的磷酸丙糖 (triose phosphate),包括两步反应: F-1,6-BP 裂解为3-磷酸甘油醛和磷酸二羟丙酮 磷酸二羟丙酮异构为3-磷酸甘油醛 (5) (4) 第二阶段总结: 1、一分子六碳糖分解为2分子能够互变的磷酸丙糖。 2、既不消耗ATP ,也不生成ATP 。 3.放能(r e l e a s i n g e n e r g y )——丙酮酸的生成 3-磷酸甘油醛经脱氢、磷酸化、脱水及放能等 反应生成丙酮酸,包括六步反应。 3- 磷酸甘油醛脱氢并磷酸化生成1,3- 二磷酸甘油酸 1,3-,将其交给ADP 生成ATP 3-磷酸甘油酸异构为2-磷酸甘油酸 (6) ......(7) (8)

糖酵解途径中的关键酶

糖酵解途径中的关键酶: 丙酮酸脱氢酶系: 三羧酸循环中的关键酶: ①三羧酸循环的概念:指乙酰CoA和草酰乙酸缩合生成含三个羧基的柠檬酸,反复的进行脱氢脱羧,又生成草酰乙酸,再重复循环反应的过程。 ②TAC过程的反应部位是线粒体。 TCA的生理意义: 1.为生物体提供大量的生物能,完成生物物质的完全降解 2.通过TCA可为蛋白质、核酸的合成提供重要的中间产物,如a-酮戊二酸、草酰乙酸 3.各类有机物质相互转变的枢纽 磷酸戊糖途径(HMP途径)的生理意义: 1.生成了大量核糖-5-P,为合成核苷酸衍生物(如辅酶等)、合成核酸准备了原料,修复再生组织中,次条途径比较旺盛。 2.提供了大量的NADPH,它在脂类、固醇类等物质的生物合成和羟化转化过程中是十分重要的电子供体;与解毒药物有关的肝脏约有30%的G走次途径;它还是GSH还原酶的辅酶。 3.光合作用的暗反应密切相关。 4.产生大量的能量 糖异生: 部位:肝脏 提问:哪些物质可以通过糖异生途径形成糖元? 凡能转变成糖代谢中间产物的物质。 脂质消化的主要部位:十二指肠 β-氧化: β-氧化发生在肝及其它细胞的线粒体内。 β-氧化包括四个步骤: 终止子:DNA分子上有终止转录的特殊信号,也是特定的核苷酸序列,称为终止子。 氨基酸:体内不能合成,必须由食物蛋白质供给的氨基酸称为必需氨基酸 必需氨基酸一共有八种或十种:Lys、Trp、Phe、Met、Thr、Leu、Ile、V al、(婴幼儿能合成部分His和Arg)。 体内氨的主要代谢去路是用于合成无毒的尿素。合成尿素的主要器官是肝脏 催化这些反应的酶存在于胞液和线粒体中。 高等植物,以谷氨酰胺或天冬酰胺形式储存氨,不排氨。 翻译:将DNA传递给mRNA的遗传信息,根据核酸链上每三个核苷酸决定一个氨基酸的三联体密码规则,合成出具有特定氨基酸顺序蛋白质肽链的过程,这一过程被称为翻译 遗传密码具有以下特点: ①连续性:密码子无标点符号 ②简并性:氨基酸可以有几组不同的密码子 ③通用性:高等和低等生物共用同一套密码; ④方向性:即解读方向为5′→ 3′; ⑤摆动性:密码子专一性由头两位碱基决定 ⑥起始密码: AUG;

生物化学 第22章 糖酵解

第22章糖酵解

?能源和碳源 1一 1.切生物都有使糖类化合物在体内分解为二氧化碳(或有机小分子)和水,放出能量的共同的代谢的化学途径,即无氧代谢和有氧代谢氧化分解糖类,糖作为生物的能源物质。 2.糖类代谢的中间产物可转化或合成其它化合物(提供碳源和碳链骨架),以构成组织细胞。 )

、吸 糖的消化、吸收 糖的消化 ?糖类的消化 1.淀粉在口腔和小肠内转变为葡萄糖 2.双糖的水解-----膜消化 33.纤维素的水解 4.淀粉和糖原的磷酸解:1-p-G 糖类的吸收 ?糖类的吸收 1. 2. 主动转运被动转运

主动转运 小肠中葡萄糖 的吸收示意图 返回

被动转运 载体蛋白运 转的方向总 是从糖浓度 高处向低处 高处向低处, 因此不需耗 能 返回

糖酵解途径发现历史 ?1875年法国科学家巴斯得(L.Pasteur)就发现葡萄糖在无氧条件下被酵母菌分解生成乙醇的现象。 糖在无氧条件下被酵母菌分解生成乙醇的现象?1897年德国的汉斯·巴克纳兄弟(Hans buchner和Edward buchner)发现发酵作用可以在不含细胞的酵母抽提液中进行。 1905年哈登,A.(Arthur Harden)和扬,?A(Arthur W.(William Young)实验中证明了无机磷酸的作用。 恩,( ?1940年前德国的生物化学家恩伯顿,G(Gustar Embden)和迈耶霍夫,O(Otto Meyerhof)等人的努力完全阐明了糖酵解的整个途径,揭示了生物化学的普遍性。因此糖酵解途径又称Embden-Meyerhof途径(简称EMP)。

糖酵解的过程

糖酵解的過程 EMP 途徑分為兩個階段,第一個階段是磷酸丙糖的生成過程(耗能過程),第二和階段是丙酮酸生成過程(產能過程)。 下面讓我們來慢慢分解反應過程 第一階段 第一步 △一 磷酸化:G →G6P Extracellular fluid :胞外液 Cytoplasm :細胞質 Glucose :葡萄糖 Phosphorylation :磷酸化作用 Plasma :等離子;血漿 Membrane :膜;薄膜 第一階段 第二階段 己糖激酶 EMP 途 徑中第一個限速酶 激酶:一类从高能供体分子(如ATP )转移磷酸基团到特定靶分子(底物)的酶;这一过程谓之磷酸化。 已糖激酶:催化从ATP 转移磷酸基团至各种六碳糖上去的酶。 激酶都需要Mg2+作为辅助因子。 首先我們來看一下糖酵解的第一階段

第一階段第二步△二 G6P F6P 磷酸葡萄糖异构酶 第一階段第三步 ③磷酸化:F6P → FDP PFK是第二个限速酶,也是 EMP途径的关键酶,其活 性大小控制着整个途径的 进程。 磷酸果糖激酶是一种别构 酶,是糖酵解三个限速酶中 催化效率最低的酶,因此被 认为是糖酵解作用最重要 的限速酶。

第一階段 第四步 ④ 裂解 (FBP → DHAP + G3P ) 第一階段 第⑤步 ⑤ 异构化(DHAP → G3P ) 1,6-二磷酸果糖 2×3-磷酸甘油醛 ◎上述5步反应完成了糖酵解的准备阶段。 ◎包括两个磷酸化步骤,由六碳糖裂解为两分子三碳糖,最后都转变为3-磷酸甘油醛。 ◎在准备阶段中,并没有从中获得任何能量,与此相反,却消耗了两个ATP 分子。 ◎以下的5步反应包括氧化-还原反应、磷酸化反应。这些反应正是从3-磷酸甘油醛提取能量形成ATP 分子。

糖酵解、TCA途径

糖酵解途径(EMP途径) 定义:葡萄糖经过一系列步骤降解成丙酮酸并生成ATP过程,被认为是微生物最古老原始的获能方式。指在O2不足情况下,葡萄糖或糖原分解为丙酮酸或乳酸,并伴随少量ATP生成。在细胞质中进行。 两个阶段: 一:活化阶段 a:葡萄糖磷酸化:活化葡萄糖,消耗1ATP,使葡萄糖和磷酸结合成葡萄糖-6-磷酸(己糖激酶) b:葡萄糖-6-磷酸重排成果糖-6-磷酸(葡萄糖磷酸异构酶) c:生成果糖-1、6-二磷酸(6-磷酸果糖激酶-1),消耗1ATP d:果糖-1、6-二磷酸断裂为3-磷酸甘油醛和磷酸二羟丙酮(醛缩酶)e:磷酸二羟丙酮很快转变为3-磷酸甘油醛。(丙糖磷酸异构酶)二:放能阶段 a:3-磷酸甘油醛氧化生成1、3-二磷酸甘油酸,释出2电子和1H+,生成NADH+ H+,且将能量转移至高能磷酸键中。 b:不稳定的1、3-二磷酸甘油酸失去高能磷酸键,生成3-磷酸甘油酸,能量转移至ATP中,生成1ATP(发生第一次底物水平磷酸化)c:3-磷酸甘油酸重排生成2-磷酸甘油酸 d:2-磷酸甘油酸脱水生成磷酸烯醇式丙酮酸 e:磷酸烯醇式丙酮酸将磷酸基团转移给ADP生成ATP,同时形成丙酮酸(发生第一次底物水平磷酸化)

附图:

总反应式: 一.糖无氧氧化反应(分为糖酵解途径和乳酸生成两个阶段)(一)糖酵解的反应过程(不是限速酶的反应均是可逆的) 1.葡萄糖磷酸化为6-磷酸葡萄糖 [1] 己糖激酶(hexokinase)催化,I-IV型,肝细胞中为IV型,又称葡萄糖激酶 区别:前者Km值小、特异性差。 意义:浓度较低时,肝细胞不能利用Glc。 [2]需要Mg++参与,消耗1分子ATP [3] 关键酶(限速酶):己糖激酶。 [4]反应不可逆,受激素调控。 [5]磷酸化后的葡萄糖不能透过细胞膜而逸出细胞。

糖酵解途径

第六章糖代谢 第一节糖酵解途径** 糖酵解途径中,葡萄糖在一系列酶的催化下,经10步反应降解为2分子丙酮酸,同时产生2分子NADH+H+和2分子ATP。 主要步骤为(1)葡萄糖磷酸化形成二磷酸果糖;(2)二磷酸果糖分解成为磷酸甘油醛和磷酸二羟丙酮,二者可以互变;(3)磷酸甘油醛脱去2H及磷酸变成丙酮酸,脱去的2H被NAD+所接受,形成NADH+H+。 丙酮酸的去路: (1)有氧条件下,丙酮酸进入线粒体氧化脱羧转变为乙酰辅酶A,同时产生1分子NADH+H+。乙酰辅酶A进入三羧酸循环,最后氧化为CO2和H2O。 (2)在厌氧条件下,可生成乳酸和乙醇。同时NAD+得到再生,使酵解过程持续进行。 第二节三羧酸循环*** 在线粒体基质中,丙酮酸氧化脱羧生成的乙酰辅酶A,再与草酰乙酸缩合成柠檬酸,进入三羧酸循环。柠檬酸经脱水加水转变成异柠檬酸,异柠檬酸经连续两次脱羧和脱羧生成琥珀酰CoA;琥珀酰CoA发生底物水平磷酸化产生1分子GTP和琥珀酸;琥珀酸再脱氢,加水及再脱氢作用依次变成延胡索酸,苹果酸及循环开始的草酰乙酸。三羧酸循环每循环一次放出2分子CO2,产生3分子NADH+H+,和一分子FADH2。 第三节磷酸戊糖途径** 在胞质中,在磷酸戊糖途径中磷酸葡萄糖经氧化阶段和非氧化阶段被氧化分解为CO2,同时产生NADPH + H+。 其主要过程是G-6-P脱氧生成6-磷酸葡萄糖酸,再脱氢,脱羧生成核酮糖-5-磷酸。6分子核酮糖-5-磷酸经转酮反应和转醛反应生成5分子6-磷酸葡萄糖。中间产物甘油醛-3-磷酸,果糖-6-磷酸与糖酵解相衔接;核糖-5-磷酸是合成核酸的原料,4-磷酸赤藓糖参与芳香族氨基酸的合成;NADPH+H+提供各种合成代谢所需要的还原力。 第四节糖异生作用** 非糖物质如丙酮酸,草酰乙酸和乳酸等在一系列酶的作用下合成糖的过程,

糖酵解三羧酸循环总结归纳

精心整理 在高等植物中存在着多条呼吸代谢的生化途径,这是植物在长期进化过程中,对多 变环境条件适应的体现。在缺氧条件下进行酒精发酵和乳酸发酵,在有氧条件下进行三 羧酸循环和戊糖磷酸途径,还有脂肪酸氧化分解的乙醛酸循环以及乙醇酸氧化途径等 (图5-2)。 图5-2植物体内主要呼吸代谢途径相互关系示意图 一、糖酵解 己糖在细胞质中分解成丙酮酸的过程,称为糖酵解(glycolysis)。整个糖酵解化学 1.糖酵解普遍存在于生物体中,是有氧呼吸和无氧呼吸途径的共同部分。 2.糖酵解的产物丙酮酸的化学性质十分活跃,可以通过各种代谢途径,生成不同的 物质(图5-4)。 图5-4丙酮酸在呼吸和物质转化中的作用 3.通过糖酵解,生物体可获得生命活动所需的部分能量。对于厌氧生物来说,糖酵 解是糖分解和获取能量的主要方式。 4.糖酵解途径中,除了由己糖激酶、磷酸果糖激酶、丙酮酸激酶等所催化的反应以 外,多数反应均可逆转,这就为糖异生作用提供了基本途径。 二、发酵作用 生物体中重要的发酵作用有酒精发酵和乳酸发酵。在酒精发酵(alcoholfermentation) 过程中,糖类经过糖酵解生成丙酮酸。然后,丙酮酸先在丙酮酸脱羧酶

(pyruvicaciddecarboxylase)作用下脱羧生成乙醛。 CH3COCOOH→CO2+CH3CHO(5-5) 乙醛再在乙醇脱氢酶(alcoholdehydrogenase)的作用下,被还原为乙醇。 CH3CHO+NADH+H+→CH3CH2OH+NAD+(5-6) 在缺少丙酮酸脱羧酶而含有乳酸脱氢酶(lacticaciddehydrogenase)的组织里,丙酮酸便被NADH还原为乳酸,即乳酸发酵(lactatefermentation)。 CH3COCOOH+NADH+H+→CH3CHOHCOOH+NAD+(5-7) 在无氧条件下,通过酒精发酵或乳酸发酵,实现了NAD+的再生,这就使糖酵解得以继续进行。 无氧呼吸过程中形成乙醇或乳酸所需的NADH+H+,一般来自于糖酵解。因此,当植物进行无氧呼吸时,糖酵解过程中形成的2分子NADH+H+就会被消耗掉(图5-5), (thiaminepyrophosphate,TPP)、辅酶A(coenzymeA)、硫辛酸(lipoicacid)、 FAD(flavinadeninedinucleotide)、NAD+(nicotinamideadeninedinucleotide)和Mg2+。 图5-6三羧酸循环的反应过程 上述反应中从底物上脱下的氢是经FAD→FADH2传到NAD+再生成NADH+H+。 2.反应(2)乙酰CoA在柠檬酸合成酶催化下与草酰乙酸缩合为柠檬酸,并释放CoASH,此反应为放能反应(△G°,=-32.22kJ·mol-1)。 3.反应(3)由顺乌头酸酶催化柠檬酸脱水生成顺乌头酸,然后加水生成异柠檬酸。 4.反应(4)在异柠檬酸脱氢酶催化下,异柠檬酸脱氢生成NADH,其中间产物草酰琥珀酸是一个不稳定的β-酮酸,与酶结合即脱羧形成α-酮戊二酸。 5.反应(5)α酮戊二酸在α酮戊二酸脱氢酶复合体催化下形成琥珀酰辅酶A和NADH,并释放CO2。

糖酵解的生理意义

糖酵解的生理意义:产生ATP;提供生物合成的原料;糖酵解与肿瘤:缺氧与缺氧诱导的转录因子HIF-1,DNA上的缺氧应答元件;参与糖酵解途径的一些酶的兼职功能。C6H12O6+2Pi +2ADP+2NAD→2CH3COCOOH+2ATP + 2H2O+2NADH +2H;;TCA 循环乙酰-CoA+3NAD+FAD+GDP+Pi+2H2O→2CO2+3NADH+FADH2+GTP+2H++CoA;;TCA循环的功能:产生更多的ATP;提供多种生物分子合成的原料;是糖、氨基酸和脂肪酸最后的共同分解途径;某些代谢中间物作为其他代谢途径的别构效应物;产生CO2;乙醛酸循与三羧酸循环区别:在每一轮循环中,有两分子乙酰-CoA进入,净生一分子琥珀酸;只产生NADH,但不产生FADH2;无底物水平磷酸化反应,因此不产生ATP;不生成CO2,无碳单位的损失;具有此途径的生物能够以乙酸作为唯一碳源。磷酸戊糖途径的功能:与NADPH 有关的功能1. 提供生物合成的还原剂NADPH2. 解毒——细胞色素P450单加氧酶解毒系统需要NADPH参与对毒物的羟基化反应。3. 免疫4. 维持红细胞膜的完整5. 间接进入呼吸链;提供核苷酸及其衍生物合成的前体5-磷酸核糖;提供芳香族氨基酸和维生素B6的合成需要的赤藓糖。糖异生的生理功能在饥饿或碳水化合物摄入不足的情况下,可以补充血糖,维持血糖浓度的稳定,为那些特别依赖葡萄糖氧化放能的细胞和组织提供燃料;植物和某些微生物使用乙酸作为糖异生的前体,使得它们能以乙酸作为唯一碳源;减轻或消除代谢性酸中毒。机体使用糖原作为能量储备的理由首先,糖原动员起来更为容易,因为它是高度分支的分子,糖原的磷酸解反应可以在各非还原端同时展开;其次,糖原的分解以及后面的糖酵解既可以在有氧又可以在无氧的条件下进行;动物体内偶数脂肪酸无法转化为葡萄糖,当饥饿的时候,肝糖原可迅速分解并转化为血糖,为脑组织等提供燃料。糖原合成 1.需要活化的葡萄糖单位——UDPG(动物和酵母)细菌(ADPG) 2.需要引物——一是没有完全降解的糖原分子;二是糖原素或糖原蛋白 3.糖原合成方向:从还原端向非还原端进行 4.分支需要分支酶。β-氧化的功能:产生ATP,其产生ATP的效率要高于葡萄糖。产生大量的H2O。这对于某些生活在干燥缺水环境的生物十分重要,像骆驼已将β-氧化作为获取水的一种特殊手段。脂肪酸合成的反应历程:1.引发反应:作为引物的乙酰基从乙酰辅酶A转移到脂肪酸合酶的一个亚基上。2.活化的“二碳单位”的装载。3.缩合是一步碳链延伸的反应。 4.还原以KR催化NADPH为还原剂的氧化还原反应 5.脱水 6.再还原 7.软脂酸的释放8乙酰-CoA+14NADPH+7H++7ATP→软脂酸+14NADP++8HSCoA+6H2O+7ADP+7Pi联合脱氨反应:由转氨酶和谷氨酸脱氢酶组合在一起的脱氨基反应。作用次序:先在转氨酶的催化下,一种氨基酸的氨基被转移到α-酮戊二酸的羰基上形成Glu,然后Glu在谷氨酸脱氢酶的催化下发生氧化脱氨反应,产生α-酮戊二酸、NH4+和NAD(P)H。氨基酸“X”+α-酮戊二酸→谷氨酸+α-酮酸“X”→α-酮戊二酸+NH4++NAD(P)H。从头合成:从最简单的小分子,如CO2和氨基酸等开始,经过多步反应,消耗更多的能量,最后生成核苷酸的过程。补救途径:指核苷酸降解的中间产物(包括核苷和碱基)被循环利用,重新转变成核苷酸的过程。嘌呤环上各原子来源:1N天冬氨酸,2C8C N10-甲酰-四氢叶酸,3N9N 谷氨酰胺,4C5C7N甘氨酸,6CCO2嘧啶核苷酸的从头合成前体:Gln、CO2、Asp等。与嘌呤核苷酸从头合成的区别:嘌呤核苷酸是先形成β-N-糖苷键,然后再逐步形成嘌呤环。而嘧啶核苷酸是先形成嘧啶环,然后再与PRPP形成β-N-糖苷键;嘌呤核苷酸从头合成所有的反应都是在细胞质基质内发生的,而嘧啶核苷酸从头合成的反应则发生在线粒体。DNA复制的一般特征①以原来的DNA两条链作为模板,四种dNTP为前体,还需要Mg2+②作为模板的DNA需要解链③半保留复制④需要引物——主要是RNA,少数是蛋白质⑤复制的方向始终是5′→3′⑥具有固定的起点⑦多为双向复制,少数为单向复制⑧半不连续性⑨具有高度的忠实性和进行性DNA聚合酶的全名是依赖于DNA的DNA聚合酶,就是以DNA为模板,催化DNA合成的聚合酶。该酶不能催化DNA的从头合成决定了DNA复制需要引物,而它只能从5'→3'催化聚合决定了DNA复制的单向性。DNA聚合酶Ⅰ具有5'→3'的DNA聚合酶活性还具有5'-外切酶和3'-外切酶活性。复制体:由DNA和多种蛋白质组装而成的催化DNA 复制的复合体。DNA 转录一般特征(与DNA复制的比较):与DNA复制的共同性质1.需要模板、解链和解除转录过程中形成的正超螺旋2.只能按照5′→3′的方向进行与DNA复制不同的性质1.不需要引物2.NTPs 代替dNTPs; UTP代谢dTTP3.缺乏校对活性4.发生在特定的区域(不是所有的DNA序列)5.对于一个特定的基因而言,只有一条链转录1.转录具有选择性和不对称性,只发生在DNA分子上的某些特定区域。2.以四种NTP--ATP、GTP、CTP和UTP为前体还需要Mg2+ 。3.转录需要模板、解链,但不需要引物。4.最先转录出来的核苷酸通常为嘌呤核苷酸。5.转录方向总是从5’→3’。6.转录也具有高度的忠实性,但是比DNA复制低。 7.有高度的进行性。8..转录受到严格的调控。细菌RNA聚合酶抑制剂:利福霉素和利链霉素启动子在转录起始点的上游存在着一些特殊的高度保守性的碱基序列。帽子结构功能:提高mRNA的稳定性;参与识别起始密码子的过程,提高mRNA的可翻译性;有助于mRNA通过核孔从细胞核运输到细胞质;提高剪接反应的效率。核糖体:核糖体是一种复杂的核糖核蛋白颗粒,由大小两个亚基组成,存在于原核细胞和真核细胞的细胞质中,以及真核细胞的线粒体和叶绿体中。核糖体的功能位点:1. A部位—氨酰tRNA结合部位,也称为受体部位;2. P 部位—肽酰tRNA结合部位;3. E部位—空载tRNA临时结合的部位;4. 肽酰转移酶活性部位——催化肽键形成的部位(大亚基);5. mRNA结合部位;6. 多肽链离开通道——正在延伸的多肽链离开核糖体的通道;7. 一些可溶性蛋白质因子(起始因子、延伸因子和终止因子)的结合部位。mRNA作为翻译的模板,至少含有一个ORF,即以起始密码子开始、以终止密码子结束的一段连续的核苷酸序列。tRNA:在翻译中,tRNA 是一种双功能接头分子,一头与氨基酸结合,一头通过反密码环的反密码子与mRNA结合,对mRNA进行解码。翻译的一般特征:1. mRNA、tRNA和核糖体起相同的作用2. 翻译的极性:阅读mRNA的方向都是从5′端→3′端,多肽链生长的方向总是从N-端→C-端。3. 遗传密码是三联体密码4. 正确的氨基酸的参入取决于mRNA上的密码子与tRNA上的反密码子之间的相互作用,与tRNA所携带的氨基酸无关5. 密码子与反密码子的相互识别遵守摆动规则6. 在核糖体上同源tRNA的识别是诱导契合的过程三联体密码性质:1.简并与兼职2.密码子的摆动性3.通用和例外4.不重叠,无标点5.方向性6.同一种氨基酸的不同密码子使用的频率不尽相同。因子结构与功能:IF2(GTP)-促进起始tRNA与核糖体小亚基结合;IF3-核糖体的解离和mRNA的结合;EF-G-结合核糖体和GTP,促进核糖体移位;RF1识别UAA或UAG,RF2识别UAA或UGA;RRF-翻译终止后,促进核糖体解体。操纵子模型认为:一些功能相关的结构基因成簇存在,构成所谓的多顺反子,它们的表达作为一个整体受到控制元件的调节。控制元件由启动子、操纵基因)和调节基因组成。调节基因编码调节蛋白,与操纵基因结合而调节结构基因的表达。

糖酵解中间产物的测定

华南农业大学实验报告 专业班次 11农学一班组别201130010110 题目糖酵解中间产物的测定姓名梁志雄日期 【实验原理】 利用碘乙酸对糖酵解过程中3-磷酸甘油醛脱氢酶的抑制作用,使3-磷激甘油醛不再向前变化而积累。硫酸肼作为稳定剂,用来保护3-磷酸甘油醛使不自发分解。然后用2,4—二硝基苯肼与3-磷酸甘油醛在碱性条件下形成2,4—二硝基苯肼—丙糖的棕色复 合物,其棕色程度与3-磷酸甘油醛含量成正比 【实验材料和试剂】 实验仪器:恒温水浴锅、离心机、电子天平 实验用品:试管、移液管、玻璃棒 实验材料与试剂:酵母、10%三氯乙酸、5%葡萄糖、0.75mol/L NaOH、0.002mol/L 碘乙酸、2,4-二硝基苯肼溶液、0.56mol/L硫酸肼 【实验步骤】 1、发酵过程观察取干燥试管3支,编号1~3,分别加入0.2g酵母,再按下表所示加入试剂 37摄氏度保温时用留在试管中的玻璃棒间断搅拌1~2次。 2、终止发酵和补加试剂 37摄氏度保温45min后,按下表所示添加试剂 表二

3、发酵液过滤上述3支试管分别过滤,取滤液用于显色鉴定 4、显色鉴定取3支试管,按下表所列顺序加入试剂,观察各管颜色的深浅,记录 【实验记录】 实验记录与上述表格中 【实验结论】 1、在步骤1中,三氯乙酸是一种蛋白酶的变性剂,可以是酵母中的所有的酶失活, 使糖酵解反应无法或只可很小程度上进行。在表一的1号试管中,由于加入了 三氯乙酸,使酵母中的酶失活,不能分解葡萄糖,所以不能产生气泡;至于2 号试管,由于加入了碘乙酸和硫酸肼,使糖酵解反应过程中的3-磷酸甘油醛累 积,但是它门不能使反应完全停留在生成3-磷酸甘油醛这个阶段,碘乙酸和硫 酸肼只是起到了延缓的作用,所以还是有部分葡萄糖被酵母菌分解生成CO2和 H2O,3号试管中做为空白对照,葡萄糖被酵母菌分解,产生大量CO2和H2O 2、步骤2中,在表二的2号和3号试管中分别加入三氯乙酸是让两试管中的葡萄 糖分解反应终止,加入试剂后立刻用玻璃棒搅拌是因为酶的作用很迅速; 3、步骤4的表四中,两次加入氢氧化钠主要都是为了使反应环境为碱性,使目的 反应可以发生

(完整版)生物化学糖代谢知识点总结

肠粘膜上皮细胞 体循环 小肠肠腔 第六章糖代谢 糖(carbohydrates)即碳水化合物,是指多羟基醛或多羟基酮及其衍生物或多聚物。 根据其水解产物的情况,糖主要可分为以下四大类: 单糖:葡萄糖(G)、果糖(F),半乳糖(Gal),核糖 双糖:麦芽糖(G-G),蔗糖(G-F),乳糖(G-Gal) 多糖:淀粉,糖原(Gn),纤维素 结合糖: 糖脂,糖蛋白 其中一些多糖的生理功能如下: 淀粉:植物中养分的储存形式 糖原:动物体内葡萄糖的储存形式 纤维素:作为植物的骨架 一、糖的生理功能 1. 氧化供能 2. 机体重要的碳源 3. 参与组成机体组织结构,调节细胞信息传递,形成生物活性物质,构成具有生理功能的糖蛋白。 二、糖代谢概况——分解、储存、合成 三、糖的消化吸收 食物中糖的存在形式以淀粉为主。 1.消化消化部位:主要在小肠,少量在口腔。 消化过程:口腔胃肠腔肠黏膜上皮细胞刷状缘 吸收部位:小肠上段 吸收形式:单糖 吸收机制:依赖Na+依赖型葡萄糖转运体(SGLT)转运。 2.吸收吸收途径:SGLT 肝脏 各种组织细胞门静脉

过程 第二阶段:丙酮酸的氧化脱羧 第三阶段:三羧酸循环 第四阶段:氧化磷酸化 CO 2 NADH+H + FADH 2 H 2 O [O] TAC 循环 ATP ADP 四、糖的无氧分解 第一阶段:糖酵解 第二阶段:乳酸生成 反应部位:胞液 产能方式:底物水平磷酸化 净生成ATP 数量:2×2-2= 2ATP E1 E2 E3 调节:糖无氧酵解代谢途径的调节主要是通过各种变构剂对三个关键酶进行变 构调节。 生理意义: 五、糖的有氧氧化 1、反应过程 E1:己糖激酶 E2: 6-磷酸果糖激酶-1 E3: 丙酮酸激酶 NAD + 乳 酸 NADH+H + 关键酶 ① 己糖激酶 ② 6-磷酸果糖激酶-1 ③ 丙酮酸激酶 调节方式 ① 别构调节 ② 共价修饰调节 ? 糖无氧氧化最主要的生理意义在于迅速提供能量,这对肌收缩更为重要。 ? 是某些细胞在氧供应正常情况下的重要供能途径。 ① 无线粒体的细胞,如:红细胞 ② 代谢活跃的细胞,如:白细胞、骨髓细胞 第一阶段:糖酵解途径 G (Gn ) 乙酰CoA 胞液 线粒体

相关主题