Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

A quantitative histochemical study of lactate dehydrogenase and succinate dehydrogenase activities in the membrana granulosa of the ovulatory follicle of the rat

Using a microdensitometer, lactate dehydrogenase and succinate dehydrogenase activities were measured in the membrana granulosa of the rat ovulatory follicle. Ovaries were removed on each day of the oestrous cycle; oestrus, dioestrus-1, dioestrus-2, and proestrus; and enzyme activities measured in the membrana granulosa as a whole and in four regions within it: peripheral (PR), antral (AR), cumulus oophorus (CO) and corona radiata (CR). Throughout the cycle, lactate dehydrogenase activity was greatest in PR. On oestrus, lactate dehydrogenase activity was progressively less in AR, CO and CR. On dioestrus-1, activity was identical in AR and CO and less in CR. On dioestrus-2, activity was greater in AR than in CO or CR. By proestrus, activity was equal in AR, CO and CR. In the membrana granulosa as a whole, and in each region, lactate dehydrogenase activity declined as ovulation approached. In contrast, succinate dehydrogenase activity in the membrana granulosa as a whole and in PR was constant throughout the cycle. Activity fluctuated in the other regions. Succinate dehydrogenase activity on oestrus was greatest in PR, less in AR and CO and least in CR. On the remaining days, succinate dehydrogenase activity was greatest in PR and less but equal in the remainder of the membrana granulosa.     

Investigation of the presence of branched-chain alpha-keto acid dehydrogenase in mammalian hepatic peroxisomes.

1. Rat liver was fractionated into peroxisomes and mitochondria and branched-chain keto acid (BCKA) dehydrogenase activity was measured. 2. All BCKA dehydrogenase activity was associated with the mitochondrial fraction and none with the peroxisomal fraction. 3. BCKA dehydrogenase was also not detected in hepatic peroxisomes of rats treated with clofibrate which induces several peroxisomal enzymes. 4. Hepatic peroxisomes from rabbit, hamster and dog also did not show any BCKA dehydrogenase activity. 5. We conclude that mammalian hepatic peroxisomes do not contain BCKA dehydrogenase.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.     

Subcellular distribution of delta 5-3 beta-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus).

1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties.     

Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.     

Fibre-optic spectrophotometry of immature bovine skeletal muscles and the cellular distribution of myoglobin and succinate dehydrogenase

Summary Samples of diaphragm and pectoralis profundus were taken from nine calves with a range of blood haemoglobin levels of 4 to 8.5 g/100 ml. In both muscles, fibres with strong succinate dehydrogenase activity contained myoglobin, but in the pectoralis there were many fibres with strong alkaline ATPase activity and weak succinate dehydrogenase activity that had low or undetected levels of myoglobin. The whole cross-sectional area of individual fibres was scanned to map the distribution of succinate dehydrogenase activity. Among fibres with similar levels of ATPase activity, those from the diaphragm had greater succinate dehydrogenase activity than those from the pectoralis. Subsarcolemmal succinate dehydrogenase activity was greater than the axial succinate dehydrogenase activity, and radial gradients of succinate dehydrogenase activity were steepest in the diaphragm. For pectoralis fibres with weak ATPase, the mean and the axial succinate dehydrogenase activities were correlated with blood haemoglobin levels (r=0.62 and r=0.61, respectively;P<0.05 with a Student'st-test). Muscle colour was measured directly by fibre-optic spectrophotometry and correlations of absorbance with succinate dehydrogenase activity were obtained. Absorbance at 620 nm 24 h post-mortem was correlated with succinate dehydrogenase activity in pectoralis fibres with weak ATPase (r=0.81;P<0.005).     

Changes in the activity of selected enzymes during starvation of Physarum polycephalum

The levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and cyclic phosphodiesterase activities were examined in growing and starving plasmodia of Physarum polycephalum. The activities of lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase decreased whereas that of cyclic phosphodiesterase increased. The change in activity of lactate dehydrogenase was the result of the variation of the activity of a single enzyme quite similar to the lactate dehydrogenases of higher animals.     

Histochemistry of the glycogen body of the turkey spinal cord

Summary Enzyme histochemical studies of the glycogen body of the turkey showed very little activity of suocinic dehydrogenase and cytochrome oxidase in the glycogen body cells, and marked activity of lactic dehydrogenase, NAD-diaphorase and the hexosemonophosphate shunt enzymes. Gradients of histochemical staining intensity for lactic and succinic dehydrogenase in the glycogen body and spinal cord were confirmed by biochemical assays of homogenates of these tissues. It was concluded that glycogen body metabolism is predominantly glycolytic. Alkaline phosphatase activity was weak; acid phosphatase activity was moderate. There was no acetyl cholinesterase or nonspecific cholinesterase activity in the glycogen body.This investigation was supported by U. S. Public Health Grant B 3250.Submitted in partial fulfillment of Masters' degree requirements.     

Hormonal control of glycerolphosphate dehydrogenase in the rat brain

—Following hypophysectomy or adrenalectomy, glycerolphosphate dehydrogenase (GPDH) (EC 1.1.1.8) activity decreased exponentially in the cerebral hemispheres and brain stem of adult male rats. The latter region was more affected than the former. Malate dehydrogenase (EC 1.1.1.40), isocitrate dehydrogenase (EC 1.1.1.42), lactate dehydrogenase (EC 1.1.1.27) and mitochondrial glycerolphosphate dehydrogenase (EC 1.1.95.5) activities remained unchanged. Injection of adrenocorticotrophic hormone or cortisol in hypophysectomized rats or cortisol in adrenalectomized rats restored GPDH activity. Thyroidectomy and gonadectomy had no effect on GPDH activity. Liver GPDH was not decreased by hypophysectomy or adrenalectomy. Muscle GPDH was diminished slightly by adrenalectomy and as much as brain GPDH by hypophysectomy. In young rats GPDH developmental increase in activity was inhibited by hypophysectomy. These results clearly show that brain GPDH activity is specifically regulated by cortisol (and probably closely related corticosteroids).     

Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase.

Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.     

Effects of thiamine deprivation on the growth and metabolism of the phytoflagellatePolytomella agilis

The effects of thiamine deprivation on the growth, respiration, and activity of several enzymes of the phytoflagellate protozoanPolytomella agilis were studied. Vitamin deprivation had no effect on the exponential growth rate; the peak population of cultures grown without thiamine was 50% of the control level. The rates of oxygen consumption in control and thiamine-deprived cultures were not significantly different from each other. The activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in vitamin-deprived cells were 14% and 30%, respectively, of the control values. In these cells, the succinic dehydrogenase activity was 10% and mitochondrial ATPase activity was twice that of control cells. Vitamin deprivation had no effect on the activities of malate dehydrogenase and isocitrate lyase, but pyruvic carboxylase activity increased fourfold. These results indicate a complex role for thiamine in the regulation of growth, respiration, and metabolism in this organism.     

Effect of amphotericin B on the energy metabolism of tissue forms of Candida albicans]

Dehydrogenase activity of the tissue form cells of C. albicans during the infection process in albino mice with and without amphotericin B treatment was studied. The strength of the metabolic reactions resulting in accumulation of ATP was evident from the activity of 4 main enzymes, i.e. succinate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase. The enzymatic activity was determined by the tetrasol method based on formation of diphormazan. Investigation of the fungal cells 10 minutes after the infection showed that preliminary intravenous or intraperitoneal administration of amphotericin B did not change the activity of the tissue forms. The cytochemical characteristics of the fungal cells remained the same as that in the untreated animals. Six hours after infection of the animals treated with amphotericin B administered intravenously the fungus vegetation decreased from 52 to 38 per cent, while in the animals treated with amphotericin B administered intraperitoneally it was suppressed completely. Simultaneously the energy metabolism was also suppressed, the activity of alcohol dehydrogenase being suppressed most significantly. The activity of this enzyme in the cells of C. albicans isolated from the animals treated with the antibiotic administered intraperitoneally was 14 times lower than that in the cells of the culture isolated from the control animals.     

Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.     

Acetaldehyde dehydrogenase activity of the AdhE protein of Escherichia coli is inhibited by intermediates in ubiquinone synthesis

Defects in the acd gene (which may be allelic to ubiH) result in the inactivation of the coenzyme A-linked acetaldehyde dehydrogenase activity of the multifunctional AdhE protein of Escherichia coli. This activity is restored by addition of ubiquinone-0 to cell extracts. However, the alcohol dehydrogenase activity of the AdhE protein is not decreased by an acd mutation. Abolition of ubiquinone biosynthesis by mutation of ubiA or ubiF does not affect either the acetaldehyde dehydrogenase or the alcohol dehydrogenase activity of AdhE. Guaiacol (2-methoxyphenol), which resembles the intermediate that builds up in ubiH mutants, except in lacking the octaprenyl side-chain, was found to inhibit ethanol metabolism in vivo, presumably via inhibition of acetaldehyde dehydrogenase. In vitro assays confirmed that guaiacol inhibited acetaldehyde dehydrogenase. This suggests that the acetaldehyde dehydrogenase activity of AdhE is specifically inhibited by intermediates of ubiquinone synthesis that accumulate in acd mutants and that this inhibition may be relieved by ubiquinone.     

Sex-linked differences in activity of enzymes in the blood of the urodele amphibian Pleurodeles waltl.

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at -18 or +4 degrees C.     

Circadian variations in the activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in the liver of control and streptozotocin-induced diabetic rats

The aim of this study was to examine: the 24 h variation of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin-induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light-dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6-phosphogluconate dehydrogenase activity was measured by substituting 6-phosphogluconate as substrate. Glucose-6-phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two-way ANOVA revealed that 6-phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6-phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose-6-phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6-phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one-way ANOVA). Time-dependent variation in glucose-6-phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ-treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH-generating enzymes exhibit 24 h variation, which is not influenced by diabetes.     

A histochemical study on the correlation between secretory activity and the TCA cycle in the gland cell

Summary The activity of succinic dehydrogenase and malic dehydrogenase was observed histochemically in the gland stomach of rats, and also the relationship between the secretory activity of the gastric gland cells and the process of the TCA cycle in the cells was studied.Histochemically, enzyme activity is plainly visible in the gastric parietal cells but in the gastric chief cells and mucous neck cells.The secretory activity of the cells was promoted by the administration of food, the sub-cutaneous injection of histamine, histidine, acetylcholine or eserin.The activity of succinic dehydrogenase appears to be constant regardless of secretory activity except in a few cases. The activity of malic dehydrogenase increases as secretory activity is promoted. It seems very unlikely that one step in the cycle (the transformation of malic acid into oxalacetic acid) would be accelerated while the other step (the transformation of succinic acid into fumaric acid) is not. This inconsistency of activity may be attributed to the histochemical reaction. Thus the increase of malic dehydrogenase activity is seen as an acceleration of the whole TCA cycle. It is our conclusion, therefore, that the source of energy within the cell, i.e. the TCA cycle, is a process which parallels secretory activity.     

The effect of cryptorchidism on the quantitative histology, histochemistry and hydrolytic enzyme activity of the rat testis.

Cryptorchidism of the mature rat testis led to degeneration of the seminiferous tubules and changes in enzyme patterns and activities. Spermatogenic stages 1-4, containing pachytene primary spermatocytes in late meiotic prophase, and stage 5, containing recently formed round spermatids, were damaged by 48 h. Within 96 h stages showed a loss of germinal cells into the lumen and this was almost complete by 192 h. Acid phosphatase showed increased histochemical activity in the basal area of the seminiferous tubule up to 96 h of cryptorchidism, and at 192 h much of the activity was located in large lipidcontaining bodies within the remaining seminiferous epithelium. Total and free biochemical acid phosphatase decreased during cryptorchidism in parallel with cell loss; there were no significant changes in total cathepsin D activity but free enzyme activity was increased throughout the experimental period indicating increased lability of lysosomes in the Sertoli cell. Lactate dehydrogenase activity was mainly tubular but succinate dehydrogenase also showed interstitial activity. Lipoamide dehydrogenase (NADH) was found mainly in the interstitium. During cryptorchidism both lactate and succinate dehydrogenase activity decreased in the tubules parallel to the loss of germinal cells, whereas lipoamide dehydrogenase (NADH) activity increased in both interstitial and tubular areas. It is suggested that the initial lesion in the seminiferous epithelium, produced by cryptorchidism is in the Sertoli cell and that germ cell damage may result from reduced function of the Sertoli cell.     

A correlation of the intensity of the gas discharge luminescence of the skin and the activity of succinate dehydrogenase in blood lymphocytes in different states of the body

The integral area of gas discharge luminescence of the hand finger skin and the activity of succinate dehydrogenase in lymphocytes in blood smears from patients with food allergia and attendant hypertension has been measured. Succinate dehydrogenase in these patients was hyperactivated or inhibited as compared with healthy persons. At more substantial deviations of activity, a more clearly pronounced hypertension was observed. The area of luminescence of the skin in some patients was near the upper boundary of norm or was beyond its limits. A high degree of correlation (r = 0.85) between the two parameters has been revealed. At moderate and high succinate dehydrogenase activity, the parameters of gas-discharge visualization decreased; the inhibition of succinate dehydrogenase activity enhances, the gas-discharge visualization increases. This probably indicates the contribution to the irradiation from hand fingers of the superoxide formed in the respiratory chain of mitochondria in the region of coenzyme Q upon its incomplete reduction caused by the inhibition of succinate dehydrogenase.