Regional enzyme development in rat brain. Enzymes of energy metabolism.

The development of several key enzymes of pyruvate and 3-hydroxybutyrate metabolism and of the tricarboxylic acid cycle was studied in six regions (cerebellum, medulla oblongata and pons, hypothalamus, striatum, mid-brain and cortex) of the neonatal, suckling and adult rat brain (2 days before birth to 60 days after birth). The enzymes whose developmental patterns were studied were: pyruvate dehydrogenase (EC 1.2.4.1), 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and fumarase (EC 4.2.1.2). Citrate synthase, isocitrate dehydrogenase and pyruvate dehydrogenase develop as a cluster in each region, although the pyruvate dehydrogenase appears to lag slightly behind the others. As with the glycolytic-enzyme cluster [Leong & Clark (1984) Biochem. J. 218, 131-138] the timing of the development of the activity of this group of enzymes varies from region to region; 50% of the adult activity developed first in the medulla oblongata, followed by the hypothalamus, striatum and mid-brain, and then in the cortex and cerebellum respectively. The 3-hydroxybutyrate dehydrogenase activity also develops earlier in the medulla oblongata than in the other regions. The results are discussed with respect to the neurophylogenetic development of the brain regions studied and the importance of the development of the enzymes of aerobic glycolysis in relationship to the development of neurological maturation.     

Enzyme Activities in Regions of the Hypothalamus

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].     

The Development of Enzymes of Energy Metabolism in the Brain of a Precocial (Guinea Pig) and Non-Precocial (Rat) Species

Abstract: Key enzymes of ketone body metabolism (3-hydroxybutyrate de-hydrogenase, 3-oxo-acid: CoA transferase, acetoacetyl-CoA thiolase) and glucose metabolism (hexokinase, lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase) have been measured in the brains of foetal, neonatal and adult guinea pigs and compared to those in the brains of neonatal and adult rats. The activities of the guinea pig brain ketone-body-metabolising enzymes remain relatively low in activity throughout the foetal and neonatal periods, with only slight increases occurring at birth. This contrasts with the rat brain, where three- to fourfold increases in activity occur during the suckling period (0–21 days post partum), followed by a corresponding decrease in the adult. The activities of the hexokinase (mitochondrial and cytosolic), pyruvate dehydrogenase, lactate dehydrogenase and citrate synthase of guinea pig brain show marked increases in the last 10–15 days before birth, so that at birth the guinea pig possesses activities of these enzymes similar to the adult state. This contrasts with the rat brain where these enzymes develop during the late suckling period (10–15 days after birth). The development of the enzymes of aerobic glycolytic metabolism correlate with the onset of neurological competence in the two species, the guinea pig being a "precocial" species born neurologically competent and the rat being a "non-precocial" species born neurologically immature. The results are discussed with respect to the enzymatic activities required for the energy metabolism of a fully developed, neurologically competent mammalian brain and its relative sensitivity to hypoxia.     

The Activities of Some Energy-Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.     

Correlation of enzymatic,metabolic, and behavioral deficits in thiamin deficiency and its reversal

To clarify the enzymatic mechanisms of brain damage inthiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced¹⁴CO₂ and¹⁴C-acetylcholine from [U-¹⁴C]glucose at rates similar to controls under resting conditions, their K⁺-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60–65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3.) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30–32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.Thus, changes in the neurological signs during pyrithiamin-induced thiamin deficiency and in recovery paralleled the reversible damage to a mitochondrial enzyme and impairment of glucose oxidation and acetylcholine synthesis. A more sustained deficit in the pentose pathway enzyme, transketolase, may relate to the anatomical abnormalities that accompany thiamin deficiency.Dedicated to Henry McIlwain.     

Factors involved in changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of acetyl-CoA carboxylase (EC 6.4.1.2), phosphofructokinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), extramitochondrial aconitate hydratase (EC 4.2.1.3) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) have been measured in the livers of developing rats from late foetal life to maturity. 2. The effect of altering the weaning time on some enzymes associated with lipogenesis has been studied. Weaning rats at 15 days of age instead of 21 days results in an immediate increase in the activity of ;malic' enzyme (EC 1.1.1.40) whereas the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and ATP citrate lyase (EC 4.1.3.8) did not increase until 4-5 days and acetyl-CoA carboxylase 2-3 days after early weaning. Weaning rats on to an artificial-milk diet led to complete repression of the rise in activity of hepatic enzymes associated with lipogenesis normally found on weaning, except for ;malic' enzyme, which increased in activity after 20 days of age. 3. The effect of intraperitoneal injections of glucagon, cortisol, growth hormone and thyroxine on the same hepatic enzymes has been investigated. Only thyroxine had any effect on enzyme activities and caused a 20-fold increase in ;malic' enzyme activity and a twofold increase in ATP citrate lyase activity. 4. The activities of hepatic glucose 6-phosphate dehydrogenase and ;malic' enzyme are higher in adult female than in adult male rats and it has been shown that this sex difference in enzyme activities is due to both male and female sex hormones. 5. Hepatic malate, citrate, pyruvate, glucose 6-phosphate and phosphoenolpyruvate concentrations have been measured throughout development. 6. The results are discussed in relation to the dietary and hormonal control of hepatic enzyme activities during development.     

β-Amyloid inhibits integrated mitochondrial respiration and key enzyme activities

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.     

Nicotinamide–adenine dinucleotide phosphate enzymes in the mosquito during growth and aging

1. Optimum conditions were established for determining the activities of the NADP(+)-linked enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase, in mosquito tissues. 2. The activity of each dehydrogenase was determined in samples of mosquitoes of different ages throughout the life-span. The specific-activity curves attained maximal values in the pupal or early adult period. From these maxima an 81% decrease in glucose 6-phosphate-dehydrogenase and 67% decrease in 6-phosphogluconate-dehydrogenase activities occurred after the tenth day of adult life; a 77% decrease in isocitrate-dehydrogenase activity occurred before the fifth day. 3. The activity differences were found in different body regions as well as in whole organisms. 4. Starvation of the larva or adult did not result in decreases in enzyme activity. 5. These findings support the hypothesis that the activities of enzymes that form NADPH are related to the biosynthetic activity, for the enzyme activities increased during the period of cellular growth and decreased during the aging period.     

Effect of thyroid hormones on the activity of pyruvate kinase and lactate dehydrogenase in mature rat brain

The enzymatic activities of two "key" enzymes of the glycolytic pathway, pyruvate kinase and lactic dehydrogenase, were studied in seven areas of the brain in male adult rats in states of pharmacologically induced hyper and hypothyroidism. The brain areas were: anterior cortex, adenohypophysis, hypothalamus, amygdaline nucleus, septum, hippocampus and cerebellum. In T3 treated animals, pyruvate kinase activity showed significant increase in all the areas studied while lactic dehydrogenase activity decreased. In propyl-thiouracil treated animals these enzyme activities showed no significant variations from those in animals of the control group.     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.     

Metabolic adaptation of MDCK cells to different growth conditions: effects on catalytic activities of central metabolic enzymes

Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.     

Differences in the distribution of energy-metabolizing enzymes in rat brain regions

The activities of several enzymes of glucose metabolism (glycolytic and tricarboxylic acid pathways) in four different regions of rat brain (cerebellum, medulla oblongata and pons, cerebral cortex and diencephalon) have been studied. Statistical differences were found in the activities of all the enzymes analyzed in the four regions, except in the case of the soluble hexokinase and pyruvate kinase. The changes observed in citrate synthase activity may account for physiological differences in those areas related to myelin formation and energy metabolism. Cerebral cortex and diencephalon showed enzyme activities which were generally greater than those of the cerebellum and medulla oblongata and pons. The results obtained lend support to the concept of a differential energy metabolism in brain regions.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

Age-related quantitative changes in enzyme activities of rat brain

The patterns of brain enzymes linked to energy metabolism have been determined in rats aged between 3 and 21 months and compared to those of the developing brain as an estimate of the senescent energy capacity of this organ. During aging, pyruvate kinase increases, pointing towards an enhancement of the glucose-dependence of this organ. However, NAD-isocitrate dehydrogenase declines, suggesting a reduction of Krebs cycle activity in the aged rat brain. An increase in cytoplasmic NAD-malate dehydrogenase found during aging could provide an alternative mechanism of NAD recovery.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Development of mitochondrial energy metabolism in rat brain

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70μm-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg²⁺+glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.     

Changes in glucose metabolism from discrete regions of rat brain and its relationship to reproductive failure during experimental diabetes

This study reports the effects of alloxan induced diabetes on glucose metabolism enzymes viz. Hexokinase, Lactate dehydrogenase, and Glucose-6-phosphate dehydrogenase from discrete brain regions. Enzymes activity was assayed from hypothalamic areas such as medial preoptic area and median eminence-arcuate region which have gonadotropin releasing hormone cell bodies and their terminals, respectively and other brain regions like septum, amygdala, hippocampus, and thalamus. In all the areas studied, induction of diabetes resulted in a significant decrease in particulate bound HK activity, whereas soluble HK, LDH and G6PDH activity showed increase at 3, 8, 15 and 28 days intervals. Insulin treatment of diabetic rats led to recovery in enzyme activity. Blood glucose levels increased significantly after induction of diabetes and recovery was seen after insulin treatment. The present results suggest that altered cerebral glucose metabolism may also be responsible for reproductive failure observed in diabetic rats. (Mol Cell Biochem141: 97–102, 1994)     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.