Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.     

Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots

The presence of the glycolytic enzymes from hexokinase to pyruvate kinase in plastids of seedling pea (Pisum sativum L.) roots was investigated. The recoveries, latencies and specific activities of each enzyme in different fractions was compared with those of organelle marker enzymes. Tryptic-digestion experiments were performed on each enzyme to determine whether activities were bound within membranes. The results indicate that hexokinase (EC 2.7.1.2) and phosphoglyceromutase (EC 5.4.2.1) are absent from pea root plastids. The possible function of the remaining enzymes is considered.Abbreviations GADPH glyceraldehyde 3-phosphate dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate: fructose 6-phosphate 1-phosphotransferase Bronwen A. Trimming gratefully acknowledges the award of a studentship from the Science and Engineering Research Council     

Alterations in glucose metabolism in chick embryo cells transformed by Rous sarcoma virus. Transformation-specific changes in the activities of key enzymes of the glycolytic and hexose monophosphate shunt pathways

Effects of transformation by Rous sarcoma virus of Schmidt-Ruppin strain on the activities of key enzymes of the glycolytic and the hexose monophosphate shunt pathways in chick-embryo cells were investigated. Activities of hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, and glucose-6-P dehydrogenase were increased about twofold in the transformed cells, but that of 6-P-gluconate dehydrogenase remained unaltered. The transformation-mediated increase in the activity of hexokinase was confined entirely to the bound form of the enzyme. Cells infected with a temperature-sensitive mutant (Ts-68) of Schmidt-Ruppin strain of Rous sarcoma virus showed the typical increase in the rate of 2-deoxyglucose uptake and the activities of hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-P dehydrogenase at the permissive temperature (37 °C), but when the infected cells were grown at the nonpermissive temperature (41 °C), the increases in the sugar uptake and activities of these enzymes were abolished. Unlike the regulatory enzymes, lactate dehydrogenase activity was increased at both the permissive and the nonpermissive temperatures.     

Glycolytic activity of rat aorta after exercise

Activities of glycolytic enzymes in the aorta were investigated in female Wistar rats. There were two groups of rats; one served as the control (sedentary rats), while the other group was forced to run on a treadmill for 10 weeks. In the control animals, the activities of hexokinase, phosphofructokinase and aldolase were relatively lower than those of the other glycolytic enzymes (phosphoglucose isomerase, lactate dehydrogenase and pyruvate kinase). After exercise, the activity of phosphofructokinase increased by 15%, whereas the other enzymatic activities were much the same as in the controls. Within the limits of the experiments, the increased percentage of phosphofructokinase was statistically significant (p less than 0.05). Since phosphofructokinase is a putative rate limiting enzyme, this enzymatic activation may indicate that glycolytic activity in the rat aorta is enhanced during and after running exercise.     

Enzymes of Energy Metabolism in the Mudpuppy Retina

Abstract: The distributions of glycogen phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, citrate synthase, malate dehydrogenase, β-hydroxyacyl CoA dehydrogenase, and adenylokinase were determined in the mudpuppy retina. Distinct differences were found in regard to the glycolytic and oxidative capacities of the various layers. In the outer retina, citric acid cycle enzymes were high while glycolytic enzymes were low. Synaptic zones were distinctly enriched in all energy-producing enzymes. Mudpuppy photoreceptors were found to be rich in phosphorylase but poor in glucose-6-phosphate dehydrogenase, suggestive of some evolutionary divergence from mammals in the metabolic machinery which is used to support the visual process.     

Rat brain glycolysis regulation by estradiol-17 beta.

The effect of estradiol-17 beta on the activities of glycolytic enzymes from female rat brain was studied. The following enzymes were examined: hexokinase (HK, EC 2.7.1.1), phosphofructokinase (PFK, EC 2.7.1.11), aldolase (EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 2.7.5.3), enolase (EC 4.2.1.11) and pyruvate kinase (PK, EC 2.7.1.40). The activities of HK (soluble and membrane-bound), PFK and PK were increased after 4 h of hormone treatment, while the others remained constant. The changes in activity were not seen in the presence of actinomycin D. The significant rise of the activities of the key glycolytic enzymes was also observed in the cell culture of mouse neuroblastoma C1300 treated with hormone. Only three of the studied isozymes, namely, HKII, B4 and K4 were found to be estradiol-sensitive for HK, PFK and PK, respectively. The results obtained suggest that rat brain glycolysis regulation by estradiol is carried out in neurons due to definite isozymes induction.     

Determination of enzyme activities of energy metabolism in the maturing rat oocyte.

Enzyme activities were determined quantitatively in individual rat oocytes to study their energy metabolism during maturation. Low hexokinase activity and high activities of lactate dehydrogenase and enzymes in the phosphate pathway, i.e., glucose 6-P and 6-P gluconate dehydrogenases, were characteristic of immature oocytes. Hexokinase may be a rate-limiting enzyme that enables oocytes to use glucose as an energy source. During maturation, the activities of hexokinase, phosphofructokinase, and malate dehydrogenase increased significantly, suggesting that the glycolytic pathway, as well as the tricarboxylic acid cycle, developed as the first meiotic division proceeded. In contrast, the activities of glucose 6-P and 6-P gluconate dehydrogenases decreased in maturing oocytes. The observation that the enzyme pattern in mature oocytes resembles more closely that in somatic cells appears to be significant, especially in light of previous studies showing this developmental trend in preimplantation embryos.     

Activity and androgenic control of glycolytic enzymes in the epididymis and epididymal spermatozoa of the rat.

1. Procedures were developed for the extraction and assay of glycolytic enzymes from the epididymis and epididymal spermatozoa of the rat. 2. The epididymis was separated into four segments for analysis. When rendered free of spermatozoa by efferent duct ligation, regional differences in enzyme activity were apparent. Phosphofructokinase, glycerol phosphate dehydrogenase and glucose 6-phosphate dehydrogenase were more active in the proximal regions of the epididymis, whereas hexokinase, lactate dehydrogenase and phosphorylase were more active in the distal segment. These enzymes were less active in the epididymis of castrated animals and less difference was apparent between the proximal and distal segments. However, the corpus epididymidis from castrated rats had lower activities of almost all enzymes compared with other epididymal segments. 3. Spermatozoa required sonication to obtain satisfactory enzyme release. Glycolytic enzymes were more active in spermatozoa than in epididymal tissue, being more than 10 times as active in the case of hexokinase, phosphoglycerate kinase and phosphoglycerate mutase. 4. The specific activities of a number of enzymes in the epididymis were dependent on the androgen status of the animal. These included hexokinase, phosphofructokinase, aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, glycerol phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphorylase. 5. The caput and cauda epididymidis differed in the extent to which enzyme activities changed in response to an altered androgen status. The most notable examples were hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, 6-phosphogluconate dehydrogenase and phosphorylase.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

The effects of calcium ions on the activities of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from various muscles

1. The effects of Ca²⁺ on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca²⁺ in the control of contraction in the two types of insect muscle. An increase in Ca²⁺ concentration from 0.001μm to 10μm did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca²⁺ above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca²⁺ from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca²⁺ does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as `substrate-cycling'.     

Changes in the metabolome of Saccharomyces cerevisiae associated with evolution in aerobic glucose-limited chemostats

The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h(-1). It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5-10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5-10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.     

Cell-density-dependent Changes in the Metabolism of Chloronema Cell Cultures: I. Relationship between Cell Density and Enzymic Activities

In the growing chloronema cell suspension cultures of the moss Funaria hygrometrica Hedw., activities of several enzymes have been found to be cell-density-dependent. Cyclic nucleotide phosphodiesterase (cNPDE), nitrate reductase (NR), and protein kinase showed highest activity at a low cell density (1 to 2 milligrams per milliliter) while indoleacetic acid (IAA) oxidase and peroxidase were highest at a high cell density (>10 milligrams per milliliter). 3′-Nucleotidase and the glycolytic enzymes (aldolase, hexokinase, phosphofructokinase, phosphoglucoisomerase, pyruvate kinase, and triose phosphate isomerase) showed no significant dependence on the cell density. Alternatively, if the NR and peroxidase activities were determined as a function of time in batch cultures, their levels were maximal 60 to 70 and 320 hours after subculture, respectively, the corresponding cell densities being 1 to 2 and 23 milligrams per milliliter. The relationship between cell density and NR and peroxidase activities is the same, whether these enzymes are measured in batch cultures during a growth cycle or in the cells cultured at different initial inoculum densities for a constant time. Conventionally enzymic changes have been correlated with growth phases; however, it is felt that the pattern of enzymic activities can also be interpreted as cell-density-dependent.     

Changes in activity of the regulatory glycolytic enzymes and of the pyruvate-dehydrogenase complex during the development of Xenopus laevis

In this study we investigated the variations of the maximal activities of the rate-controlling glycolytic enzymes (i.e., hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK) and of the pyruvate-dehydrogenase complex (PDHc) during the early embryogenesis of Xenopus laevis (from cleavage through hatching). All the enzymatic assays, using different coupled reactions, were performed spectrophotometrically on cytosolic and mitochondrial fractions. The maximal HK activity increases markedly from neurulation onwards, PFK activity presents a peak around gastrulation, PK activity remains relatively constant throughout the period studied and the highest PDHc activity is observed during cleavage. The specific activities display the same temporal pattern. Furthermore, in the sequence of reactions by which glucose is degraded to form acetyl-CoA, the maximal activities of PFK and PK are not limiting while those of HK and PDHc could be rate-limiting at relatively late developmental stages (hatching).     

METABOLIC CONTROL MECHANISMS IN MAMMALIAN SYSTEMS

Abstract— The regulation by thyroid hormone of the activities of hexokinase (ATP: D-hexose 6-phosphotransferase; EC 2.7.1.1), phosphofructokinase (ATP: D-fructose-6- phosphate 1-phosphotransferase; EC 2.7.1.11) and pyruvate kinase (ATP: pyruvate phosphotransferase; EC 2.7.1.40) has been investigated in the soluble fractions of the cerebral cortex and cerebellum of the rat. Ontogenetic studies on these key glycolytic enzymes demonstrated marked increases in the normal cerebral cortex between 1 day and 1 yr of age; less pronounced increases in enzyme activities were noted in the normal cerebellum. Neonatal thyroidectomy, induced by treatment of 1-day-old rats with 100 μCi of ¹³¹I, ied to an impairment of body and brain growth and inhibited the developmental increases in hexokinase, phosphofructokinase and pyruvate kinase in both the cerebral cortex and cerebellum. Whereas 50 μCi of ¹³¹I had little or no effect on these brain enzymes, 200 μCi of the radioisotope markedly inhibited (35–65 per cent) the developmental increases of the various enzyme activities investigated. When administration of the radioisotope was delayed for 20 days after birth, little or no inhibition of the development of brain glycolytic enzymes was observed. Whereas treatment of normal neonatal animals with L-tri-iodothyronine had no significant effect on the activities of cerebro-cortical and cerebellar glycolytic enzymes, the hormone increased their activities in young cretinous rats. However, when the initiation of tri-iodothyronine treatment was delayed until neonatally thyroidectomized rats had reached adulthood, this hormone failed to produce any appreciable change in enzyme activity. Our results indicate that thyroid hormone exerts an important regulatory influence on the activities of hexokinase, phosphofructokinase and pyruvate kinase in the developing cerebral cortex and cerebellum.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Glucose metabolism and template synthesis in the mitotic cycle of human diploid fibroblasts

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.     

Enhancement in the activities of mouse epidermal glucose-6-phosphate dehydrogenase, hexokinase, phosphofructokinase, and pyruvate kinase by 12-O-tetradecanoyl-phorbol-13-acetate

The active ingredient in the tumor-promoting croton oil, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was shown to increase the activity of mouse skin epidermal glucose-6-phosphate dehydrogenase (+84%), hexokinase (+100%), phosphofructokinase (+158%), and pyruvate kinase (+101%). This increase in activity of these key enzymes of glucose metabolism occurred 2-8 h after TPA application and was due to a net increase in the enzyme content. This increase in the activity of the glycolytic enzymes, as well as the reported TPA-induced increase in the synthesis of RNA and DNA and cell proliferation, suggest that activation of the glycolytic pathway may be involved in the carcinogenic effects of tumor promoters.     

BRAIN MITOCHONDRIA : II. The Relationship of Brain Mitochondria to Glycolysis

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.     

Activities of glucose metabolic enzymes in human preantral follicles: in vitro modulation by follicle-stimulating hormone, luteinizing hormone, epidermal growth factor, insulin-like growth factor I, and transforming growth factor beta1

Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor beta1 (TGFbeta1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFbeta1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFbeta1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities.     

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.