Oxidative enzymes in the pancreatic islets of normal and obese-hyperglycemic mice

Summary Histochemical data are presented concerning distributions of succinic dehydrogenase (SD), lactic dehydrogenase (LD), diphosphopyridine nucleotide diaphorase (DPND), triphosphopyridine nucleotide diaphorase (TPND) and glucose-6-phosphate dehydrogenase (G-6-PD) in the pancreas from the American variety of obese-hyperglycemic mice (AO-mice) and their lean litter mates (AN-mice).A high LD activity was found in the exocrine parenchyma, while the reaction in the islet tissue and the duct epithelium was only weak. A considerable reaction for DPND was noted throughout the pancreas. SD activity was slightly more pronounced in the acinar tissue and duct epithelium as compared to the islet tissue, where only a moderate activity appeared. Strong reactions for TPND and G-6-PD were found in the islet cells and duct epithelium, while the activity in the exocrine parenchyma was less pronounced. The hyperactive islet B cells in the AO-mice showed no obvious differences in enzyme activity and distribution compared to that of the AN-mice. The enzyme pattern of the A cells could not be clearly distinguished from that in the B cells.The results suggest the existence at least in the B cells of the mice islet tissue of an active hexosemonophosphate shunt. The probable significance of the hexosemonophosphate shunt for insulin synthesis is briefly discussed.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - EM Embden-Meyerhof - G-6-PD Glucose-6-phosphate dehydrogenase - HMP Hexose monophosphate - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethyl-thiazol-2-yl) tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - PVP Polyvinyl pyrrolidone (M. W. 11 000) - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form     

Ultrastructure and enzyme histochemistry of the pancreatic islets in the horse

Summary The characteristic localization of the silver-negative A₂ cells in the central part of the pancreatic islets in the horse offers a good opportunity to study the ultrastructure and histochemistry of this type of islet cell. Electron microscopical analyses revealed that the A₂ cells contained dense spherical granules varying considerably in size. Light and dark A₂ cells were identified. The presence of numerous secretory granules of very low density was the most conspicous feature of the B cells. These cells also showed considerable differences in density. A second type of peripheral islet cell was characterized by a very high content of mitochondria and ribosomes. These small islet cells contained tiny granules and are probably identical with the A₁ cells.Negative reactions for alkaline and acid phosphatases were obtained throughout the islet tissue, while a strong glucose-6-phosphatase activity was displayed by the peripheral cells. The diphosphopyridine and triphosphopyridine nucleotide diaphorase activities were high in the peripheral cells, considerably weaker reactions being noted in the A₂ cells. On the whole there was a low succinic dehydrogenase activity in the islet tissue with a somewhat weaker enzyme staining in the A₂ than in the peripheral cells. The reactions for glucose-6-phosphate dehydrogenase and lactic dehydrogenase were also less pronounced in the A₂ cells than in the intensely reacting peripheral cells.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - G-6-PD Glucose-6-phosphate dehydrogenase - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethylthiazol-2-yl)-tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

Histochemical study of enzyme patterns in the human submaxillary gland

Summary The histochemical distribution of various enzymes, such as alkaline phosphatase, acid phosphatase, esterase, -glycosidase, aminopeptidase, succinic dehydrogenese and TPN diaphorase, in human submaxillary glands has been determined.Acini and ducts of human submaxillary gland were devoid of alkaline phosphatase activity, but this enzyme was observed in capillaries and somewhat in myoepithelium.Activities of acid phosphatase, esterase, -glucuronidase and -galactosidase were generally observed in the entire cytoplasm of serous acini; but the cytoplasm of mucous acini was either negative or showed only trace amounts.Aminopeptidase reaction of both acini and ducts was generally negative.Succinic dehydrogenase and TPN diaphorase activities were strongly active in intralobuler ducts. Serous acini exhibited less activity with these enzymes; and mucous cells showed still less and were almost negative. In serous acini, there was much greater activity of TPN diaphorase than of succinic dehydrogenase.With 7 Figures in the Text     

The Histochemical Localization of Triphosphopyridine Nucleotide Diaphorase

A histochemical method is described for the localization of triphosphopyridine nucleotide diaphorase using a recently synthesized tetrazolium salt (Nitro-BT). By virtue of the favorable histochemical properties of this reagent, it has been possible to demonstrate that whereas DPN diaphorase is usually restricted to the mitochondria, the TPN diaphorase activity of corresponding cells was distributed throughout the cytoplasm in granules too fine to be considered mitochondria. Furthermore, although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed. For example, whereas TPN diaphorase and isocitric dehydrogenase have an extensive distribution in the kidney cortex, 6-phosphogluconic dehydrogenase is limited to the cells of the macula densa.     

Multifunctionality of lipoamide dehydrogenase: activities of chemically trapped monomeric and dimeric enzymes

Lipoamide dehydrogenase from pig heart exists in monomer-dimer equilibrium. The effect of the state of subunit aggregation on the multifunctionality of lipoamide dehydrogenase was investigated by the use of chemically trapped monomeric and dimeric enzymes. Reductive carboxymethylation with 2-mercaptoethanol-iodoacetate yields the stable monomeric enzyme which has been isolated for structural and kinetic studies. The chemically induced monomerization is accompanied by conformational changes resulting in an increased mobility of flavin-adenine dinucleotide. The chemically trapped monomer shows an enhanced diaphorase activity, a reduced electron transferase activity, and a complete loss in dehydrogenase as well as transhydrogenase activities. The enhanced diaphorase activity is associated with increased catalytic efficiencies and the reversal of an inhibitory NADH effect at high concentrations. Treatment of lipoamide dehydrogenase with dimethyl suberimidate gives amidinated samples containing crosslinked dimer. The crosslinked enzyme exhibits a higher dehydrogenase catalytic efficiency than the noncrosslinked enzyme with different kinetic mechanisms without significantly affecting the kinetic parameters of diaphorase reaction. Although the dimeric structure is intimately associated with the dehydrogenase activity, it does not preclude the diaphorase activity. An altered flavin-adenine dinucleotide environment accompanying monomerization is likely responsible for the enhanced diaphorase activity.     

Histochemical comparison of oxidative enzymes in adrenal glands of mammals

Summary Succinic dehydrogenase, five DPN-linked dehydrogenases-lactic dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, -glycerophosphate dehydrogenase, -hydroxybutyric dehydrogenase, two TPN linked dehydrogenases — glucose-6-phosphate dehydrogenase, isocitric dehydrogenase and 3-ol steroid dehydrogenase were studied in mouse, rat, guinea pig, rabbit, dog, cat, cow, monkey and human adrenal glands. Histochemical studies were made of a characteristic distribution of different level of enzyme activity. In mammals adrenal glands, glucose-6-phosphate dehydrogenase showed the highest activity and its localization was divided into the following two groups: 1) High enzymatic activity was demonstrated in the zona fasciculata and reticularis of the rat, guinea pig, rabbit, cat and 2) high enzymatic activity was demonstrated in the zona glomerulosa and reticularis of the dog, cow and monkey. A precise relationship between the localization and endocrinological function remains abscure.     

Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.     

The post-mortem stability of certain oxidative enzymes in brain and spinal cord

Summary An investigation has been carried out on the stability of several enzymes in portions of rabbit brain and spinal cord kept at controlled temperatures between 22 and 37° C for periods up to 24 hours before processing for enzyme activity. The enzymes studied were NAD diaphorase, succinate, lactate, glutamate and glucose-6-phosphate dehydrogenases, and monoamine oxidase. One-wavelength plug cytophotometric measurements of enzyme activity were carried out on Purkinje cells, neuropil of the granular layer of the cerebellar cortex and on anterior horn cells.Succinate dehydrogenase activity proved to be stable after 24 hours post-mortem exposure at 37°C. Lactate dehydrogenase, NAD diaphorase and monoamine oxidase activities were less stable at the higher temperatures but were stable at 22°C. Glutamate and glucose-6-phosphate dehydrogenase activities fell significantly with exposure at 22°C. It thus appears possible to make valid histochemical measurements of the activities of certain oxidative enzymes in selected post-mortem brain material.This research was aided by a grant from the National Health and Medical Research Council of Australia.     

Oxidative enzyme histochemistry of the chick esophagus and proventriculus

Succinic dehydrogenase, NAD and NADP diaphorases, choline oxidase, d-amino acid oxidase, α-glycerophosphate dehydrogenase, glutamic dehydrogenase, lactic dehydrogenase and aldolase were identified histochemically in the esophagus and proventriculus of the developing chick embryo. In general, the deep glands of the proventriculus reacted more strongly than the epithelium and mucous glands of the esophagus to the tests. The intensity of the enzymatic activity seems correlated with the number of mitochondria and metabolic activity of the cells.     

Human brain "high Km" aldehyde dehydrogenase: purification, characterization, and identification as NAD+ -dependent succinic semialdehyde dehydrogenase

NAD-dependent succinic semialdehyde dehydrogenase (EC 1.2.1.24) has been purified to homogeneity from human brain via ion-exchange chromatography and affinity chromatography employing Blue Sepharose and 5'-AMP Sepharose. Succinic semialdehyde dehydrogenase was never previously purified to homogeneity from any species; this preparation therefore allows the determination of its molecular weight, subunit molecular weight, subunit composition, isoelectric points, and substrate specificity for the first time. The enzyme is a tetramer of Mr230,000 to 245,000 and consists of weight-nonidentical subunits (Mr 61,000 and 63,000). On isoelectric focusing the enzyme separates into five bands with the following isoelectric points: 6.3, 6.6, 6.8, 6.95, and 7.15. Its substrates include glutaric semialdehyde, nitrobenzaldehyde, and short chain aliphatic aldehydes in addition to succinic semialdehyde which is the best substrate. The Km values for succinic semialdehyde, acetaldehyde, and propionaldehyde are 1,875, and 580 microM, respectively. The enzyme is inactive with 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde as substrates. Its subcellular localization is in the mitochondrial fraction. Succinic semialdehyde dehydrogenase is sensitive to inhibition by disulfiram (a drug used therapeutically to produce alcohol aversion) resembling, in this respect, aldehyde dehydrogenase (EC 1.2.1.3). It does not, however, interact with the antibody developed in the rabbit vs aldehyde dehydrogenase, suggesting that the two enzymes are structurally distinct.     

Malic acid production by an electrochemical reduction system combined with the use of diaphorase and methylviologen

A regenerating reaction combined with the use of native malate dehydrogenase, native diaphorase, methylviologen, NAD, oxalacetic acid as the substrate and lipoamide as a stabilizer was carried out in the presence of electrolysis. Consequently, malic acid was efficiently produced from oxalacetic acid in the regenerating reaction. A glassy carbon bead electrode was used as a cathode. Twenty four milliamperes were passed at a rotation speed of 500 rpm, 29.8 +/- 0.3 degrees C and -1.0 V. It was found that lipoamide has a stabilizing effect on malate dehydrogenase and diaphorase. Low concentration (50 muM) of NAD was also effective for the stabilization of malate dehydrogenase. NADH regeneration activity based on malic acid production rate was 4.7 U/mg of the enzyme protein of the commercial diaphorase preparation. The current efficiency was more than 74%, compared with the theoretical yield, in the presence of enough oxalacetic acid.     

Phenazine Methosulfate; its Use in Evaluating Activity of Dehydrogenase Systems of Avian Liver

Lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and suecinate dehydrogenase were demonstrated in livers of 15-day chick embryos. The addition of phenazine methosulfate (PMS) to the LDH and MDH incubation mixtures reduced diformazan deposition in the liver epithelium but not in connective tissue. A 30 sec formalin fixation, absence of PMS, or the addition of sodium azide or potassium cyanide to the PMS-containing incubation mixtures facilitated formazan deposition. These results are explained by assuming that, in the absence of PMS, dehydrogenase activity is demonstrated via endogenous diaphorase. When PMS is present, Nitro BT reduction occurs within the incubation mixture. A side effect of the azide or cyanide is an interference with, the action of PMS, thus allowing diformazan deposition via the endogenous diaphorase when this is present in the tissue.     

The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 micro in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

The Cytochemical Localization of Oxidative Enzymes : I. Diphosphopyridine Nucleotide Diaphorase and Triphosphopyridine Nucleotide Diaphorase

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.     

Activity of some dehydrogenase enzymes in mitochondria from Physarum polycephalum.

1. Coupled mitochondria were isolated from exponentially growing Physarum polycephalum. 2. Activity of malate dehydrogenase (oxalacetate reduction) was 10.9 mumol/min/mg protein; the apparent Km was 64 microM. 3. The activity of NADP-isocitric dehydrogenase (IDH) was 110 nmol/min/mg with apparent Km of 35 microM. 4. NAD-IDH showed allosteric properties with AMP as a positive modulator. The apparent Km for the unmodulated activity, 2 mM, was decreased to 0.95 mM by 0.13 mM AMP. 5. Succinic dehydrogenase activity was estimated as three times higher than that of alpha-glycerophosphate dehydrogenase. 6. Mitochondria contained significant amounts of phenolic compounds. Protein estimation by the Bradford method is recommended.     

Cyclic succinic dehydrogenase activity in rat liver mitochondria

Succinic dehydrogenase activity was assayed in isolated rat liver mitochondria. Rats had been exposed for at least two weeks to a 24 hour feeding-fasting schedule, 5 hours feeding, 19 hours fasting. Enzyme activity was determined at nine specific hours over a 24 hour period. Lowest enzyme activity occured six hours after the feeding cages had been closed. The highest activity, which was 158 percent greater than the lowest activity, occurred during the next feeding period. It is concluded that time of sacrifice is an important consideration when determining succinic dehydrogenase activity.     

Tri-iodothyronine enhances the formation of light mitochondria during cold exposure

The effect of cold exposure and of PTU and PTU + T3 administration on the protein content and succinic dehydrogenase activity of three mitochondrial populations obtained from rat liver was examined. Our results indicated the following: Succinic dehydrogenase activity increases mainly in the light mitochondrial fraction of cold-exposed rats. PTU administration of cold-exposed animals does not affect the increment in enzyme activity of the heavy fraction but blocks the increment of the light fraction. PTU + T3 administration restores succinic dehydrogenase activity to the values prevalent in normal cold-exposed rats. These findings suggest that thyroid hormone may stimulate the formation of light mitochondria during cold exposure.     

Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system.

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular location of nitrate reductase and nitrite reductase. II. Wheat roots

Approximately 15% of the total nitrite reductase of crude homogenates of wheat roots applied to sucrose gradients was separated with an organelle whose isopycnic density was about 1.22 g·cm⁻³. The activity recovered in the supernatant was thought to be particulate in origin, because similar ratios of activity of isoenzyme 1 and 2 of nitrite reductase were found in both particulate and supernatant fractions. The particle with nitrite reductase activity also contained glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triose phosphate isomerase and NADPH diaphorase. This root particle and whole chloroplasts from leaves had a similar isopycnic density as well as these enzymes, and thus the data suggest that the root particle may be a proplastid.

Nitrate reductase was found only in the supernatant and it was not associated with any of the root organelles.

Mitochondria from wheat roots had an equilibrium density of 1.18 g·cm₋₃ and contained both NAD and NADP glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triosephosphate isomerase and NADPH diaphorase but not nitrite reductase. Microbodies of wheat roots had an equilibrium density of about 1.20 g·cm⁻³ on the sucrose gradient and contained catalase and glycollate oxidase.     

Age and the succinate dehydrogenase activity of human cardiac neurons]

Under study was the SDG activity in human heart neurons in postnatal ontogensis. Succinic dehydrogenase was revealed with nitrablue tetrazole after Nachlas, quantitative measurements in preparations were made in a cytospectrophotometer. A regular developmental increase in the SDG activity in human heart neurons was established to have certain fluctuations in different age periods. The most intensive growth of the enzyme activity was found in early childhood, youth and senile age which is related to physiological state of the organism and the cardio-vascular activity.