Fermentation enzymes in strains of Paracoccus denitrificans

Various strains of Paracoccus denitrificans grown under conditions of unrestricted oxygen supply contained low but measurable activities of fermentation enzymes such as ethanol dehydrogenase and 2,3-butanediol dehydrogenase. However, when the bacteria were subsequently incubated for up to 22 h under restricted aeration conditions permitting respiration rates of only 10 or 6% of the maximum value to occur, the above enzymes increased in specific activities by 5- or 10-fold to 0.14 mol/min·mg protein. Lactate dehydrogenase was not detected. Six strains tested reacted almost alike.Cells grown anaerobically on fructose in the presence of limiting concentrations of KNO₃ contained specific activities of up to 0.41 (in case of ethanol dehydrogenase) and 0.56 (butanediol dehydrogenase) mol/min·mg protein. Lactate dehydrogenase was only formed at low activity (0.012 mol/min·mg protein) after a long period of incubation.Cells of P. denitrificans strain Stanier 381 grown anaerobically in the chemostat on fructose+KNO₃ with either fructose or nitrate as the limiting factor differed with respect to the specific enzyme activities, too. Ethanol dehydrogenase was high under conditions of nitrate limitation and low under fructose limitation. 2,3-Butanediol dehydrogenase, but not lactate dehydrogenase, was formed in moderate activities.     

Stimulated murine peritoneal macrophages in suspension and cell culture: enzyme determinations by biochemical and histochemical means

Summary Peritoneal exudate cells of mice were studied up to 14 days after i.p. injection of thioglycollate broth medium by means of conventional enzyme determinations and quantitative histochemical measurements of individual cells. Cells from the peritoneal cavity were either investigated immediately after harvesting or after culturing periods of up to six days for enzymic activities of aminopeptidase, esterase, lactate dehydrogenase and -galactosidase. A fairly good correlation exists between biochemical determinations of aminopeptidase and esterase activity and the mean of histochemical data. Following a sharp increase in the number of cells after stimulation, aminopeptidase, esterase and lactate dehydrogenase activities per cell were found to be increased. Moreover, the cells taken three or five days after stimulation synthesized large amounts of aminopeptidase and esterase as shown by culturing experiments. This capacity of the cells was subsequently lost in cells harvested seven day after stimulation but -galactosidase increased and lactate dehydrogenase was more readily released into culture supernatants. The increase in aminopeptidase and esterase was dependent on protein synthesis since it was abolished by cycloheximide. Thioglycollate broth medium provokes immigration of cells into the peritoneal cavity where cells apparently differentiate by increasing their aminopeptidase and esterase concentrations, and by raising their intracellular catabolism rates, which leads eventually to the decay of the cells. The different enzymic phenotypes and the large heterogeneity at any time point after stimulation presumably also reflect different functional properties during the inflammatory process.     

Further enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the male rat

Summary The segmentation of the proximal tubules of the male rat kidney was studied by means of enzyme histochemical reactions. Soluble oxidoreductases (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, NAD- and NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase) were demonstrated using methods which reduce enzyme diffusion (incubating in presence of polyvinyl alcohol) and eliminate interference from tissue tetrazolium reductases. Less soluble or insoluble enzymes (glucose 6-phosphatase, -hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases) were demonstrated by incubation in conventional watery media.Segmental differences were observed in respect to all enzymes studied, and most reactions clearly visualized the three segments known to exist from ultrastructural as well as previous histochemical studies: The pars convoluta includes the first (P₁) and most of the second (P₂) segment. The transition to the third segment (P₃) is in the beginning of the pars recta. Also these reactions revealed a difference between the first part of the P₃, which runs through the cortex in the medullary rays, and the terminal part transversing the outer stripe of the medulla. In most instances intensity of reaction decreased in the last portion of the P₃.A number of the enzymes studied were mainly or solely localized to the P₃ (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenases, -hydroxybutyrate dehydrogenase, 3-hydroxysteroid dehydrogenase, decarboxylating malate dehydrogenase and uridine diphosphate glucose dehydrogenase). Some possible functional implications of the findings are discussed.Supported by grants from Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — Mr. Kaj L. Pedersen is thanked for valuable photographic assistance.     

A histoenzymatic study of rat intrafusal muscle fibres

Summary The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K₂-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkalipreincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K₂-EDTA preincubation. Following either acetic acid solution or cold and room temperature K₂-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K₂-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing -GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

A comparative histochemical evaluation of various dehydrogenases in the oral squamous epithelium

Summary A histochemical observation was made of various dehydrogenase activities in oral squamous epithelia. The localization of dehydrogenases showed a relatively similiarity except for the intensity of the dehydrogenase activity. Succinic dehydrogenase activity was generally confined to the basal cell layer and adjacent cell layers; superficial layers did not show any enzymatic activity. Lactic, and malic dehydrogenase activities were localized in the basal cells to st. granulosum, and the activity of lactic dehydrogenase was the highest. -Glycerophosphate, glutamic, glucose-6-phosphate and TPN-isocitric dehydrogenase activities were observed in all the epithelial cells with the exception for the hornified layer, and they were found generally low. -Hydroxybutyric dehydrogenase was low and contained in both of st. germinativum and st. granulosum, the keratohyalin in st. granulosum being occasionally found reactive to this enzymatic activity.In connective tissue cells and collagen bundels, activities of lactic, and malic dehydrogenase were intense, while other dehydrogenases were low or trace amount.In the oral squamous epithelium under normal conditions, the dehydrogenase localization concerning the glucose metabolism and TCA cycle member and other close pathways was not similar. Nor were their activities found likewise. Those findings lead to a conclusion that the epithelial cells of the same layer many show a selective metabolic activity.With 21 Figures in the Text     

Glucose-6-phosphate dehydrogenase is enriched in oligodendrocytes of the rat spinal cord

Glucose-6-phosphate dehydrogenase (G6PD) was localized in rat spinal cord by catalytic enzyme histochemistry and immunocytochemistry. G6PD detected by either method was shown to be strongly enriched in cell bodies and processes of oligodendrocytes, whereas in the compact myelin G6PD was not detected. The enzyme histochemical procedure for the demonstration of G6PD was also adapted for microphotometric measurements of G6PD activity in the spinal cord white matter. There was a linear relationship between G6PD activity and section thickness up to 14 m and between G6PD activity and reaction time up to 5–6 min as demonstrated by kinetic and end-point measurements. Significantly lower activities were measured in endpoint measurements than in kinetic measurements because of formazan loss during rinsing. Methoxyphenazine methosulphate as an exogenous electron carrier and sodium azide as a blocker of the respiratory chain significantly increased the demonstrable G6PD activity. The K _m was 0.62 mM and the V _max 3 mol glucose-6-phosphate/cm³ wet tissue and per min at 25°C. It is concluded that G6PD in oligodendrocytes may be important for the generation of NADPH required for lipid biosynthesis related to myelogenesis, and reduction of glutathione required for protection of membrane sulphydryl groups.     

Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system

Summary The initial reaction kinetics of succinate dehydrogenase in situ were investigated in sections of mouse unfixed liver using an ARGUS-100 image analyser system. The sections were incubated on substrate-containing agarose gel films. Images of a section, illuminated with monochromatic light (584 nm), were captured with the image analyser in real time at intervals of 10 s during the incubation. The absorbances of selected hepatocytes in the successive images were determined as a function of time. In every cell, the absorbance increased non-linearly after the first minute of incubation. The initial velocity of the dehydrogenase was calculated from the linear activities during the first 20 s of incubation. Hanes plots of the initial velocities and succinate concentration yielded the following mean kinetic constants. For periportal hepatocytes, the apparentK _m=1.2±0.8 mM andV _max=29±2 mol hydrogen equivalents formed/cm³ hepatocyte cytoplasm per min. For pericentral hepatocytes,K _m=1.4±1.0 mM andV _max=21±2 mol hydrogen equivalents/cm³ per min. TheK _m values are very similar to those determined previously from biochemical assays. These results, and the observed dependence of the initial velocity on the enzyme concentration, suggest that the technique reported here is valid for the histochemical assay of succinate dehydrogenase.     

Effect of phosphoros nutrition in peat on tomato plant growth and fruit development

Summary The effect of P nutrition on the growth of tomato plants in peat was examined. Initially, plants received an adequate supply of P and then received either nil, 0.78 or 2.34 kg superphosphate per m³ in combination with either 50 g N/ml (N₁) or 300 g N/ml (N₂) as ammonium nitrate in a liquid feed. Vegetative growth was restricted in the lower P treatmentsi.e. inhibited shoot growth, reduced duration of leaf expansion phase, thinner stems and reduced vegetative dry wt. Plants receiving N₂ showed a greater restriction in growth compared with N₁ plants when the P supply was limiting. P deficiency disrupted protein metabolism in the leaves, in that soluble leaf protein was reduced and trichloroacetic acid-soluble N accumulated. Flower development was accelerated by low P applications but the final numbers of flowers and the fruit-setting efficiency were reduced. Lowering the N supply reduced the fruit yield by 36 per cent while an intermediate P level reduced yields by about 15 per cent. Maximum fruit yields and good vegetative growth occurred when plants contained 0.4 per cent P or above in the mature leaves, and this value was achieved by adding the highest level (2.34 kg/m³) of superphosphate to the peat.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

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.     

FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q₁₀, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.     

Enzyme,protein, and nucleic acid content of two morphological forms of Trypanosoma (Schizotrypanum) cruzi

Summary Trypanosoma (Schizotrypanum) cruzi (Corpus Christi strain) was cultivated at 28°C over a monolayer of African Green Monkey kidney cells (Vero line). The epimastigote was converted into the trypomastigote by serial passage of the organisms at 33°C in a modified culture system. This resulted in preparations containing better than 90% trypomastigotes.The biochemical composition of the epimastigotes and trypomastigotes was determined in whole cells and cell-free homogenates of organisms grown in these systems. The epimastigote contained 50±2 g of protein, 2.0±0.1 g of RNA, and 1.7±0.2 g of DNA per 10⁷ organisms; while the trypomastigote contained: 24±1 g of protein, 1.4±0.1 g of RNA, and 2.4±0.3 g of DNA per 10⁷ organisms. Data was also obtained on the specific activities of certain metabolically important enzymes. The following (in nmoles min^-1mg^-1 of protein) are given in the order; enzyme, epimastigote activity, trypomastigote activity: aspartic aminotransferase, 1058±139, 466±16; alanine aminotransferase, 1076±131, 474±27; aldolase, 172±8, 11±0.6; isocitric dehydrogenase (NADP-linked), 137±7, 58±4; malic dehydrogenase, 2270±116, 1073±93; glucose-6-phosphate dehydrogenase, 50±3, 115±16; 6-phosphogluconic acid dehydrogenase, 101±7, 30±2.These results were consistent with the hypothesis that the trypomastigote is an intermediate, resting form of T. (S.) cruzi possessing a reduced level of metabolic activity, especially with respect to carbohydrates. Differentation of the epimastigote into the trypomastigote form is accompanied by an adjustment of enzyme concentrations which reflect this reduction in metabolic activity.     

Effect of fluridone on free sugar level in heat stressed mungbean seedlings

Germination per cent and growth of ML 267 and ML 131 cvs of mungbean (Vigna radiata Wilzec) seedlings was adversely affected by high temperature stress (42°C). Pre-sowing seed soaking in fluridone (flu) significantly increased germination per cent and seedling growth at both normal and high temperatures. Total free sugar accumulation was significantly increased under stress and Flu pre-treatment significantly increased it, both at 30° and 42 °C. Enzyme activities of - and -amylases were found correlated with increased accumulation of sugars under stress. Flu pre-treatment partially ameliorated the effects of heat stress.     

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under P _i limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O₂ limited cultures had similar activities of the carbon catabolic enzymes to P _i-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O₂-limited cells grown of fumarate was 50% lower than in P _i-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.Abbreviations E-D Entner-Doudoroff - EMP Embden-Meyerhof-Parnas - PEPCK phosphoenolpyruvate carboxy kinase - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid]     

The mechanism of ammonia assimilation in nitrogen fixing bacteria

Summary Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium glutamine glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium glutamate glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of -ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N₂-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH₃-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K _m for ammonium predominates in the N₂-grown cell.Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.Abbreviation DON 6-diazo-5-oxo-l-norleucine     

Activities of some oxidative and hydrolytic enzymes in musculus biceps brachii of rats after tonic stress

Summary Employing histochemical and biochemical techniques the authors studied the activities of some oxidative and hydrolytic enzymes in musculus biceps brachii of rats which from day 15 of life (at this time the muscle is differentiated) were stressed by tonic exercise on ladders. After six months of stress significant changes in the proportions of basic fiber types were observed (an increase of intermediate and decrease of white fibers). Significant changes were found in the activities of succinate dehydrogenase, -glucan phosphorylase, and nonspecific esterase (increase of activities), whereas no such differences were found in the activities of lactate dehydrogenase and mitochondrial -glycerol phosphate dehydrogenase. The difficulties encountered in the classification of basic fiber types are pointed out. The findings furnished evidence that tonic stress influences the metabolism and typology of the striated muscle of the growing rat.     

Kinetic properties of hexose-monophosphate dehydrogenases. II. Isolation and partial purification of 6-phosphogluconate dehydrogenase from rat liver and kidney cortex

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60–75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 M and 258 M respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37°C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (K_i) of 21 M. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 M and 56 M respectively. The specific activity at pH 8.0 and 37°C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a K_i of 41 M. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.Publication No 170 from Drugs, Environmental Toxics and Cell Metabolism research group. Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain     

Quantitative aspects of enzyme histochemistry on sections of freeze-substituted glycol methacrylate-embedded rat liver

Freeze-substituted rat liver embedded in glycol methacrylate (GMA) has been used to demonstrate the activities of several enzymes. The following enzymes could be detected in GMA-sections by the indicated histochemical procedure(s): 5-nucleotidase (lead salt, cerium-diaminobenzidine), alkaline phosphatase (indoxyl-tetrazolium salt), catalase (diaminobenzidine), acid phosphatase (diazonium salt), lactate dehydrogenase (tetrazolium salt) and glutamate dehydrogenase (tetrazolium salt). The activities of all these enzymes were dramatically decreased compared with the activities demonstrated in unfixed cryostat sections, with the exception of catalase. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt), xanthine oxidoreductase (tetrazolium salt), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide) and glucose-6-phosphatase (cerium-diaminobenzidine). The possible role of restricted penetration of reagents into the resin was studied by measuring cytophotometrically the enzyme activities in GMA-sections of 3 and 6 m in thickness. For all the enzymes that could be detected, the 6 m : 3 m ratio varied from 1.4 to 2.7. An eventual retarded penetration of reagents into the resin was investigated by measuring cytophotometrically the amount of final reaction product during incubation for acid phosphatase and glutamate dehydrogenase activities. In both cases linear relationships without a lag phase were found for the specific enzyme activities with incubation time. Chemical denaturation of proteins or masking of active sites in proteins due to embedding in the resin monomer may be considered to be the main cause of decreased enzyme activities.     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).