Homogeneous Dehydrogenase Research Articles

Analysis of heteroplasmy in mitochondrial mutation m.C5178A in gene of subunit 2 of NADH dehydrogenase in homogenates of affected by atherosclerosis aortic intima

Analysis of heteroplasmy in mitochondrial mutation m.C5178A in gene of subunit 2 of NADH dehydrogenase in homogenates of affected by atherosclerosis aortic intima

The tricarboxylic acid cycle in L3Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L3Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD+ and NADP+ specific) and α-ketoglutarate dehydrogenase in homogenates of L3T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD+ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD+] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L3T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L3T. circumcincta are probably similar to those in the tissues of their host species.

Troglitazone is a competitive inhibitor of 3β-hydroxysteroid dehydrogenase enzyme in the ovary

Objective: Troglitazone is a potent inhibitor of progesterone release from porcine granulosa cells. This is associated with a marked increase in pregnenolone secretion, implicating inhibition of the 3β-hydroxysteroid dehydrogenase enzyme. This study determined whether troglitazone is a direct inhibitor of 3β-hydroxysteroid dehydrogenase activity. Study Design: Homogenates of porcine granulosa cells underwent classic enzyme kinetic analysis through Lineweaver-Burke and Dixon plotting. Human ovarian homogenates were also assayed for the effects of troglitazone on 3β-hydroxysteroid dehydrogenase enzyme activity. Enzyme kinetics data were analyzed by the HyperKinetics software program. Analysis of variance was used to determine statistical significance for human ovarian homogenate experiments. Results: In porcine granulosa cells Lineweaver-Burke analysis found that troglitazone inhibition of 3β-hydroxysteroid dehydrogenase enzyme activity was competitive in nature, with 5 μg/mL troglitazone increasing the apparent Michaelis constant from 1.3 to 4.3 μmol/L (no change in maximum velocity). Dixon plot analysis demonstrated that the inhibition constant for troglitazone of 3β-hydroxysteroid dehydrogenase is approximately 6.5 μg/mL, which is in the same order of magnitude as its therapeutic concentration in blood. Troglitazone also significantly decreased the activity of 3β-hydroxysteroid dehydrogenase in homogenates of human ovarian tissue. Conclusion: We conclude that troglitazone can inhibit steroidogenesis in the ovary by direct competitive inhibition of 3β-hydroxysteroid dehydrogenase. (Am J Obstet Gynecol 2001;184:575-9.)

Cofactor Identification of Threonine-Serine Dehydratase from Sheep Liver

L-Threonine-Serine dehydratase catalyzes the conversion of L-threonine and L-Serine to α-ketobutyric acid and pyruvate, respectively. The enzyme has been purified to homogeneity from extracts of sheep liver. In the past, various cofactors have been suggested for threonine dehydratase from both prokaryotic and eukaryotic tissue. While some direct evidence for the presence of pyriodoxal 5′-phosphate in impure preparations is present in the literature no direct evidence for the cofactor in homogeneous dehydrogenase from mammalian tissue has been reported. The threonine dehydratase of sheep liver has been obtained in a homogeneous form and a spectral study provides clear evidence for the presence of pyridoxal 5′-phosphate. Both the physical properties of homogeneous threonine dehydratase and a study of spectral properties of its cofactor are reported in this communication.

Distribution of glucuronidation capacity (1-naphthol and morphine) along the rat intestine

The distribution of glucuronidation capacity along the rat intestine was investigated using mucosal cells, isolated from the small intestine, the caecum, and the colon plus rectum. The glucuronidation capacity for 1-naphthol decreases from 787 ± 75 (duodenum) to 128 ± 13 (colon plus rectum) pmoles/min.mg cell protein. The ratio between 1-naphthol and morphine glucuronidation was constant throughout the intestine (7.15 ± 0.37). The distribution of maximal activity of UDP-glucuronosyltransferase in intestinal cell homogenates follows the same pattern. The maximal activity of UDPglucose dehydrogenase in homogenates corresponds closely to the glucuronidation rate in mucosal cells. The activity of β-glucuronidase in intestinal cell homogenates is constant along the duodenum and jejunum but increases throughout the terminal ileum, caecum, colon and rectum. Subcellular fractionation studies using marker enzymes indicate that UDPglucose dehydrogenase and β-glucuronidase are cytosolic enzymes in intestinal mucosal cells. Although UDP-glucuronosyltransferase activity is found in both the mitochondrial and the microsomal fractions, no indications for a mitochondrial localization of this enzyme can be found. Activity in the mitochondrial fraction appears to be due to endoplasmic reticulum, associated with the mitochondrial fraction.

Oxidation of formaldehyde and acetaldehyde by NAD +-dependent dehydrogenases in rat nasal mucosal homogenates

Homogenates of respiratory and olfactory tissue from the rat nasal cavity were examined for their capacity to catalyze the NAD +-dependent oxidation of formaldehyde (in the presence and absence of glutathione) and of acetaldehyde. Both aldehydes were oxidized efficiently by nasal mucosal homogenates, and formaldehyde dehydrogenase (FDH) and aldehyde dehydrogenase (AldDH) were tentatively identified in both tissue samples. At least two isozymes of AldDH, differing with respect to their apparent K m and V max values with acetaldehyde as substrate, were found in the nasal mucosa, one of which may catalyze the oxidation of both formaldehyde and acetaldehyde. The specific activity of FDH in the olfactory mucosa was twice that in the respiratory mucosa, whereas the specific activity of the higher K m isozyme of AldDH was five to eight times greater in respiratory than in olfactory tissue. The specific activity of the lower K m isozyme of AldDH was similar in respiratory and olfactory homogenates. Repeated exposures of rats to formaldehyde (15 ppm, 6 hr/day, 10 days) or to acetaldehyde (1500 ppm, 6 hr/day, 5 days) did not substantially affect the specific activities of FDG and AldDH in nasal mucosal homogenates. Glutathione is a cofactor for FDH; the concentration of nonprotein sulfhydryls in respiratory mucosal homogenates was approximately 2.8 μmoles/g and was not changed significantly by repeated exposures to formaldehyde (15 ppm, 6 hr/day, 9 days). These data indicate that the rat nasal mucosa, which is the major target site for both aldehydes in inhalation toxicity studies, can metabolize both formaldehyde and acetyldehyde, and that the specific activities of formaldehyde and aldehyde dehydrogenase in homogenates of the nasal mucosa are essentially unchanged following repeated exposures to toxic concentrations of either compound.

Characterization of succinate dehydrogenase and α-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of α-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected α-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that α-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.

Effect of l-thyroxine on metabolism in Japanese quails ( Cotournix cotournix japonica)—II. Activity of got and GPT in liver, LDH and ICDH in heart and kidney after multiple injections of l-thyroxine

1. 1. Quails hatched from eggs incubated at physiological temperature (37.5°C—normal quails) and elevated (39.3°C—warm quails) were injected with l-thyroxine (T 4) at the dose of 600 μg/kg of body weight, every 48 hr for 17 days. 2. 2. Twenty-four hours after the last injection activity of aspartate aminotransferase (GOT), alanine aminotransferase (GPT) was determined in liver homogenates and lactate dehydrogenase and isocitrate dehydrogenase in homogenates of heart and kidney. 3. 3. Significant increase of the activity of GPT in liver homogenates was observed in normal and warm quails (up to 252.9 and 186.8% of control, respectively). 4. 4. The activity of aspartate aminotransferase increased significantly in liver homogenates of T 4-treated normal quails, while such changes in the warm quails were not observed. 5. 5. Activities of lactate dehydrogenase (LDH) and isocitrate dehydrogenase (ICDH) in heart and kidney homogenates in both T 4-treated groups of birds did not change.

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 m m) stimulated the conversion of [1- 14C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial NADH NAD + ratio is elevated, dichloroacetate inhibited the conversion of [1- 14C]lactate to glucose. Dichloroacetate stimulated 14CO 2 production from [1- 14C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial NADH NAD + ratio, changed the metabolism of [1- 14C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 n m) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 m m [1- 14C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA 70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 m m pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.

Inhibitors of human placental C 19 and C 21 3β-hydroxysteroid dehydrogenases

The effect of several natural and synthetic steroids on the activity of Δ 5,3β- hydroxysteroid dehydrogenase in homogenates of human placenta has been measured by a method which determines the conversion of labelled dehydroepiandrosterone to androstenedione, testosterone, estradiol-17β, and estrone and of labelled pregnenolone to progesterone and 5α-pregnane-3,20-dione. The method utilizes thin-layer chromatography systems and radio-gas-liquid chromatography which separate each steroidal product from each substrate. Enzymatic activity can be determined rapidly and efficiently in multiple samples of very small amounts of tissue. The present report demonstrates that nucleophilic substituents on, adjacent to, or at some distance from the site on the steroid molecule catalyzed by the enzyme may increase the inhibitory capacity of the parent steroid or confer inhibitory capacity to an inactive parent steroid. Selective inhibition of the conversion of pregnenolone by several steroids demonstrates substrate specificity of the C 19- and C 21-3β-hydroxysteroid dehydrogenases. The most potent of these selective inhibitors are, in descending order of inhibitory potency: 2α-bromo-17β-hydroxy-5α-androstane-3-one-17β-acetate; 3β,17α-dihydroxy-5-pregnene-3,20-dione-16α-nitrile; 3β-hydroxy-5α-pregnane-20-one-16α-nitrile; and 2α-bromo-5a-androstane-3,17-dione. The most potent inhibitors of both enzymes are 2α-cyano-4,4-dimethyl-2,3α-tetrahydrofuran-2-spiro-17,5-androstene-3-one and 6,16β-dimethyl-3β-hydroxy-5-pregnene-16α-nitrile. The usual form of cyanoketone (2α-cyano-17β-hydroxy-4,4,17α-trimethyl-5-androstene-3-one) does not inhibit either enzyme.

Effects of dibutyryl cyclic adenosine 3', 5'-monophosphate on pyruvate metabolism in rat adipose tissue

The effects of the dibutyryl derivative of adenosine 3',5'-monophosphate (cyclic AMP) on pyruvate metabolism in segments of rat epididymal adipose tissue were evaluated. The oxidation of [1- 14C]pyruvate and [1- 14C]lactate to 14CO 2 was accelerated by dibutyryl cyclic AMP at a concentration too low to activate lipolysis (0.30 mM). Higher, lipolytic concentrations further augmented oxidation of [1- 14C]pyruvate. Oxidation of [3- 14C]pyruvate was progressively increased by dibutyryl cyclic AMP while conversion to fatty acid was depressed. Total recovery of radioactivity from [3- 14C]pyruvate in CO 2 and triglyceride was increased by dibutyryl cyclic AMP. Cyclic AMP (10 −5–10 −3 M) increased the activity of pyruvate dehydrogenase in homogenates of adipose tissue. These data suggest that dibutyryl cyclic AMP increases the entry of pyruvate into the citric acid cycle, perhaps by activating pyruvate dehydrogenase. The data further suggest that dibutyryl cyclic AMP must exert some other direct or indirect action later in the metabolic scheme to curtail lipogenesis.

Differences in levels of xanthine dehydrogenase activity between inbred and outbred strains of Drosophila melanogaster.

Levels of activity of xanthine dehydrogenase in homogenates of individual flies were measured by fluorometry for three inbred strains and two large, randomly breeding, laboratory populations of Drosophila melanogaster. The patterns of activity during the first five days following eclosion differed significantly between the inbred strains and the randomly breeding populations. In the latter, the levels rise from day 0 to day 2, then fall through day 4; in the former, the levels rise continuously throughout the period. The variances within days calculated on the logarithms of the observations are constant for the large populations, but show significant variability within and between the inbred strains. These differences parallel earlier observations on time of development and probability of embryonic success in the same strains. All these results suggest more effective regulatory mechanisms in the randomly breeding populations.

Effect of diet and triiodothyronine on the activity of sn-glycerol-3-phosphate dehydrogenase and on the metabolism of glucose and pyruvate by adipose tissue of obese patients.

Lipogenesis and the metabolism of sn-glycerol-3-phosphate were studied in 23 fat biopsies from eight grossly obese patients. The first biopsy was obtained after a minimum of 12 days on a 3500 cal diet, the second biopsy after 2 wk on a 900 cal diet, and the third biopsy after an additional 2 wk on 900 cal supplemented with thiiodothyronine, 250 mug/day.Oxygen consumption and respiratory quotient declined during caloric restriction. Oxygen consumption was restored to the initial level during treatment with triiodothyronine, and the respiratory quotient rose somewhat.Lipogenesis from glucose and pyruvate was demonstrated in fat obtained from the first biopsy but could not be detected in the other biopsies. The incorporation of radioactivity from pyruvate into fatty acids was stimulated by the addition of glucose. Insulin stimulated lipogenesis in pieces of fat from the first biopsy, but isolated fat cells were unaffected by insulin. After caloric restriction no effects of insulin could be detected. The activity of both the cytoplasmic and mitochondrial sn-glycerol-3-phosphate dehydrogenase in homogenates of adipose tissue declined with caloric restriction. Treatment with triiodothyronine enhanced the activity of the mitochondrial sn-glycerol-3-phosphate dehydrogenase but did not affect the cytoplasmic enzyme.

Carbohydrate metabolism in the thyroid gland.

ABSTRACT The presence of the 7 principal tricarboxylic acid cycle enzymes—aconitasc, fumarase, condensing enzyme, and isocitric, α-ketoglutaric, succinic, and malic dehydrogenase—was demonstrated in sheep and human thyroid tissue. Quantitative assays were adapted for the measurement of aconitase, fumarase, isocitric, succinic, and malic dehydrogenase in homogenates of this tissue. It was found that methimazole, potassium perchlorate and thiocyanate did not inhibit significantly any of these 5 enzymatic activities in sheep thyroid homogenates. These 5 enzymes were not found exclusively in mitochondria. The sheep thyroid content of aconitase, fumarase, and isocitric, malic, and succinic dehydrogenase is much lower than the liver content of these enzymes. This finding in a metabolically highly active tissue suggests the existence in this tissue of an alternate pathway of glucose catabolism. The content of 5 of the aforementioned enzymes in thyroids from treated thyrotoxic patients was correlated with the deg...

The hexose monophosphate pathway in arterial tissue

The metabolism of carbohydrate by normal and abnormal arterial tissue impinges on every phase of basic and medical physiology. Kirk, Wang, and Brands&up (l) have demonstrated the presence of glucose-&phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in homogenates of human aortic, pulmonary artery, and coronary artery tissue. This significant finding indicates the functioning in these tissues of the hexose monophosphate @IMP) pathway, which may be either the principle or an alternate pathway of glucose dissimilation. The presence of a pathway that appears to have a major role in supplying TPNH for reductive synthesis by arterial tissue may be highly important in the elucidation of the role of lipids in arterial disease such as arteriosclerosis We have confirmed and extended this observation tn experiments employing glucose-l -C 14 and glucose-6-C14. Using intact thoracic aorta from guinea pigs, we have been able to show that this tissue will produce, under different conditions, a greater amount of CO2 from carbon one of glucose relative to glucose carbon six. Table I shows the results obtained from a typical experiment in which intact thoracic aorta, stripped of adventitia, was allowed to respire in the presence of glucose-l-Cl4 or glucose-6-C14. From the table it can be seen that there is an increased appearance of C-l of glucose as CO2 relative to C-6 with a C-1$2-6 ratio of 6.6. The addition of autolo~us sera produced an increase in the C-l:C-6 ratio from 6.6 to 17.3, almost a threefold enhancement, In the presence of yeast extract,

Malic dehydrogenase and related enzymes in the culture form of Trypanosoma cruzi

1. 1. A very active malic dehydrogenase has been found in homogenates of T. cruzi. 2. 2. Conditions for maximum activity of diphosphopyridine nucleotide reduction have been determined. 3. 3. Malic dehydrogenase is linked to cytochrome c reduction, indophenol reduction, and fumarate reduction presumably through flavoproteins. 4. 4. Fumarase having an optimum pH 7.8 is present. The activity is equivalent to 60.8 μl fumarate per mg dry weight per hour. This may not be maximal. 5. 5. Oxalacetate and iodoacetate inhibit malic dehydrogenase. 6. 6. Homogenates may be stored in air at 5 °C for several days. Only about 2% activity is lost per day. 7. 7. The activity of malic dehydrogenase in homogenates parallels the extent of cell rupture when isotonic KCl is used as suspending medium.

Succinic Dehydrogenase in Protozoa

THERE is, as yet, relatively little information about the occurrence of individual enzymes in Protozoa. We have been successful in demonstrating the presence of a succinic dehydrogenase in homogenates of Paramecium caudatum. This has been done using a modified Cartesian diver respirometer of macro dimensions (40–60 µ1. total volume).