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.     

Oxidative enzymes in the development of Fasciola hepatica L. IV. The activity of oxidases and dehydrogenases in redia.

The object of the study was to investigate the occurrence and localization of oxidative enzymes in the redia -- the third larval stage of Fasciola hepatica L. The author detected cytochrome oxidase, peroxidase, NADH and NADPH tetrazolium reductases (diaphorases), as well as succinate, isocitrate, malate, lactate, alpha-glycerophosphate, glyceraldehyde phosphate, glucose-6-phosphate, 6-phosphogluconate, beta-hydroxybutyrate, L-glutamate, and alcohol dehydrogenases. The presence and localization of the enzymes in various periods of development of the redia were detected with histochemical methods. Out of the studied oxidases and dehydrogenases only cytochrome oxidase was found to be absent from the stages of young rediae. It was ascertained that the redia uses all three paths of release of energy i.e. the glycolytic, Krebs, and pentose cycles, glycolysis being presumably the principal mode of energy production.     

Enzymic changes in rabbit and rat mammary gland during the lactation cycle

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.     

Oxidative enzymes in the development of Fasciola hepatica L. III. The activities of oxidases and dehydrogenases in the sporocyst.

The object of the study was the investigation of the occurrence and distribution of some oxidative enzymes in the sporocyst of Fasciola hepatica L. The samples were examined for the presence of cytochrome oxidase, peroxidase, NADH and NADPH tetrazolium reductases, as well as succinate, isocitrate, malate, lactate, alpha-glycerophosphate, glyceraldehyde-3-phosphate, glucose-6-phosphate, beta-hydroxybutyrate, L-glutamate and alcohol dehydrogenases. All of them save cytochrome oxidase were found to occur in the sporocyst. The presence and localization of these enzymes were examined by histochemical methods in various stages of development of the sporocyst. These investigations permitted it to be established that glycolytic processes are the principal way of release of energy for all developmental groups of this larva. Moreover, the functions of the tricarboxyl acid and pentose-phosphate cycles were detected and found to play a less important part in processes of energy production in the sporocyst. In addition, the functioning and metabolism of each larval organ in various stages of its development were discussed in so far as was possible on the basis of the analysis of the above-mentioned oxidative enzymes.     

Cytochemical research on cysts of the sarcosporidian Sarcocystis bovicanis. II. Oxidation-reduction enzymes

By the means of light-microscopic cytological enzymatic methods, the presence of several enzymes (NAD.H and NADP.H-tetrazolium reductases, in addition to alcohol, succinate, isocitrate, glucose-6-phosphate, beta-hydroxybutyrate and glutamate dehydrogenases) has been studied in the tissue cysts of S. bovicanis. A mixed character of oxidative metabolism in the cyst stages is suggested, the involvement of gluconeogenesis being proposed. Neither beta-hydroxybutyrate nor alcohol dehydrogenase activity was demonstrated indicating the absence or a very low rate of lipid metabolism, and suggesting that the process of glycolysis may end with malate formation. From the low activity level of succinate dehydrogenases it is concluded that the citric acid cycle plays presumably a secondary role, if at all, in the energy supply of the cyst stages. Also, a low activity of glucose-6-phosphate dehydrogenases is pointed out. Thus, it is proposed that glycolysis may be primary, if not the only, oxidative pathway in the cyst stages.     

Absence of direct coenzyme transfer in an A-B dehydrogenase system.

Direct transfer of NADPH between two NADP-dependent dehydrogenases, isocitrate dehydrogenase and glutamate dehydrogenase, has been investigated. These enzymes have opposite stereospecificity for hydrogen transfer to the coenzyme. In contrast with the general direct-transfer mechanism postulated for NAD-dependent dehydrogenases [Srivastava & Bernhard (1986) Science 234, 1081-1086], no evidence for direct transfer in either direction was found for these NADP-dependent dehydrogenases.     

Inhibition of Krebs cycle and activation of glyoxylate cycle in the course of chronological aging of Saccharomyces cerevisiae. Compensatory role of succinate oxidation

We investigated oxidative processes in mitochondria of Saccharomyces cerevisiae grown on ethanol in the course of chronological aging. We elaborated a model of chronological aging that avoids the influence of exhaustion of medium, as well as the accumulation of toxic metabolites during aging. A decrease in total respiration of cells and, even more, of the contribution of respiration coupled with ATP-synthesis was observed during aging. Aging is also related with the decrease of the contribution of malonate-insensitive respiration. Activities of citrate-synthase (CS), alpha-ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) were threefold decreased. The activity of NADP-dependent isocitrate dehydrogenase (NADP-ICDH) decreased more significantly, while the activity of NAD-dependent isocitrate dehydrogenase (NAD-ICDH) fell even greater, being completely inactivated on the third week of aging. In contrast, succinate dehydrogenase (SDH), enzymes of glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ACDH)), were activated by 50% or more. The behavior of oxidative enzymes and metabolic pathways are apparently inherent to a more viable, long-lived cells in population, selected in the course of chronological aging. This selection allows cells to reveal the mechanism of their higher viability as caused by shunting of complete Krebs cycle by glyoxylate cycle, with a concomitant increased rate of the most efficient energy source, namely succinate formation and oxidation. Thiobarbituric-reactive species (TAR species) increased during aging. We supposed that to be the immediate cause of damage of a part of yeast population. These data show that a greater succinate contribution to respiration in more active cells is a general property of yeast and animal tissues.     

Effect of corticotropin on the dehydrogenase activity of cells and tissues

The authors studied the effect of native ACTH on dehydrogenase activity of isolated strips of rat diaphragm and suspension of E. coli cells, serotype O III:B4, grown on beef extract agar in a medium with different dehydrogenation substrates. ACTH activated dehydrogenase of rat diaphragm in a medium containing pyruvate, alpha-ketoglutarate, malate, beta-hydroxybutyrate, D-aspartic acid and did not alter it in a medium containing succinate. In contradistinction to rat diaphragm, ACTH activated dehydrogenase of E. coli cells whatever the substrates used (oxaloacetate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, pyruvate, lactate, beta-hydroxybutyrate, glucose, D-aspartic acid. Synacthen (ACTH1-24) exerted a similar effect. It is suggested that the effects of ACTH are mediated via its influence on adenylate cyclase in the absence of receptors.     

Histochemical observations on the exoerythrocytic malaria parasite Plasmodium berghei in rat liver

Enzyme histochemical methods were performed on sporozoite infected liver tissue of rats in order to gain insight into the nutrition and metabolism of exoerythrocytic forms of Plasmodium berghei. The following enzymes were demonstrated in the hepatocytic stages of the parasites, obtained 41 and 48 h after inoculation of sporozoites: acid phosphatase, cytochrome oxidase, NADH-tetrazolium reductase, succinate dehydrogenase, NAD+ and NADP+ dependent isocitrate dehydrogenase, NADP+-dependent malate dehydrogenase, lactate dehydrogenases, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenases and alpha-glycerol-phosphate dehydrogenase. The results suggest that a conventional Embden-Meyerhoff pathway, pentose phosphate pathway and Krebs' citric acid cycle may in part be present in these exoerythrocytic parasites. Alkaline phosphatase, nucleoside polyphosphatase, 5' nucleotidase, glucose-6-phosphatase, alpha-glucan phosphorylase, NAD+ dependent malate dehydrogenase, amino-peptidase M and non-specific esterases were not detected by our techniques in the parasite. The enzyme distribution of this intrahepatocytic malaria parasite revealed by histochemistry is compared with the enzyme distribution in the other phases of the parasite's life cycle.     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

1. Superovulated rat ovary was found to contain high activities of NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD-malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP-citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective K(m) values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP-malate dehydrogenase or NADP-isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed.     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.      

Aspects of the development of flight-muscle sarcosomes in the sheep blowfly, Lucilia cuprina, in relation to changes in the distribution of protein and some respiratory enzymes during metamorphosis

1. Changes in the amounts and distribution of protein and respiratory enzymes have been estimated during the life cycle of the fly Lucilia cuprina. 2. The fully fed larva contains about 7mg. of protein, the pupa and newly emerged fly about 4mg., and the mature adult about 3mg. 3. There are two periods of incorporation of protein into particles at the expense of the soluble protein; the first, immediately after pupation, may store protein (0.5mg./insect) for use in adult development; the second, over the period of emergence, was due mainly to the development of the thoracic mitochondria of the adult (0.7mg./insect). 4. In the thorax, cytochrome c oxidase and the dehydrogenases for glycerophosphate, isocitrate (NAD-dependent), succinate and malate appeared initially in small particles (less than 1mu in diameter). 5. In adult development these enzymes were redistributed so that in the mature fly most of the activity was present in larger particles (1-10mu in diameter). 6. During this redistribution the specific activity (mul. of oxygen/hr./mg. of protein) of glycerophosphate dehydrogenase in the small particles was 690 at 1(1/2) days before emergence, 955 at emergence and 980 at 7 days after emergence; the corresponding values for the large particles were 164, 760 and 1220. 7. In the mature fly the highest specific activities (mul. of oxygen/hr./mg. of protein) estimated were: glycerophosphate dehydrogenase 1380, isocitrate dehydrogenase (NAD-dependent and requiring ADP and Mg(2+)) 408, succinate dehydrogenase 122, malate dehydrogenase 190, and cytochrome c oxidase 1360. 8. The results are considered in relation to the development of the flight-muscle sarcosomes.     

A histochemical study of the muscles of Drosophila melanogaster.

The thoracic muscles of Drosophila melanogaster can be classified into two classes, the fibrillar and the tubular muscles, on morphological grounds. Histochemical techniques were used to characterize these two classes of muscle according to their content of various enzymes (alpha-glycerophosphate, NAD-dependent isocitrate, malate and succinate dehydrogenases, fumarase, acid phosphatase, adenosine triphosphatase and acetylcholinesterase) and of glycogen. These investigations showed that the two muslces types are histochemically very different and, further, that the morphologically similar tubular muscles are heterogeneous with respect to their enzyme content. In particular, the tergal depressor of the trochanter of the second leg, the largest of the tubular muslces, has considerably less of all the enzymes studied, with the exception of acetylcholinesterase, than all the other tubular muscles examined. The histochemical techniqes were also used to follow the changes in enzyme levels that occur during development of the indirect flight muscle fibres. All the enzymes that are present in adult flight muslces showed an increase in staining intensity throughout muscle development. Some minor differences were observed in the time of appearance and rate of increase of intensity of the different enzymes.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

Comparative Intermediary Metabolism of Vegetative Cells and Microcysts of Myxococcus xanthus

Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate.     

Ovarian dehydrogenase activities: suggested adrenal involvement in luteolysis.

Immature rat ovarian dehydrogenase activity was studied during corpus luteum regression following withdrawal of prior pregnant mare serum gonadotrophin. Glucose-6-phosphate dehydrogenase activity declined to nontreatment levels whereas 6-phosphogluconate, malate, and isocitrate dehydrogenase dehydrogenases exhibited a partial return to normal. Adrenalectomy prior to PMS withdrawal enhanced the decline in MAD while sharply elevating G6PD and 20alpha-hydroxysteroid dehydrogenase. Corticosterone and progesterone prevented the G6PD changes induced by adrenalectomy and moderated the rise in 20alpha-OHSD. Adrenalectomy appears to enhance the process of luteolysis.     

Selectivity in the binding of NAD(P)+ analogues to NAD- and NADP-dependent pig heart isocitrate dehydrogenases. A nuclear magnetic resonance study.

The coenzyme selectivity of pig heart NAD-dependent and NADP-dependent isocitrate dehydrogenase has been investigated by nuclear magnetic resonance through the use of coenzyme analogues. For both isocitrate dehydrogenases, more than 10-fold lower maximal activity is observed with thionicotinamide adenine dinucleotide [sNAD(P)+] than with NAD(P)+ or acetylpyridine adenine dinucleotide [acNAD-(P)+] as coenzyme. Nuclear Overhauser effect measurements failed to reveal any differences in the adenine-ribose conformations among the enzyme-bound analogues. The 2'-phosphate resonance of the enzyme-bound NADP+ analogues showed the same change in chemical shift observed for the natural coenzyme and revealed the same lack of pH dependence in the range from pH 5.4 to 8.2. NADP-dependent isocitrate dehydrogenase exhibits only small differences in Michaelis constants for the coenzymes with various nicotinamide substituents, reflecting a predominant role for the adenosine moiety in binding. The conformation of the bound nicotinamide-ribose of the natural coenzymes was appreciably different from that of the coenzyme, sNAD(P)+, which shows low catalytic activity. For both isocitrate dehydrogenases, sNAD(P)+ bound to the enzymes exhibits a mixture of syn and anti conformations while only the anti conformation can be detected for NAD(P)+. Chemical shifts of NAD(P)+ enriched with 13C in the carboxamide indicate that interaction of this group with the enzymes may play a role in positioning the nicotinamide ring to participate in catalysis. Our results suggest that, although interaction of the nicotinamide moiety with the enzymes contributes relatively little to the energy of interaction in the binary complex, the enzymes must correctly position this group for the catalytic event.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors influencing the histochemical demonstration of coenzyme-dependent dehydrogenases and diaphorases

Procedures for the histochemical demonstration of DPN and TPN diaphorases have been presented by other workers. These techniques rely on the coenzyme-dependent dehydrogenases present in the tissue slice to generate the substrate required by the diaphorases. In vitro studies were carried out on kidney and adrenal tissue of the rat, using NT (neotetrazolium) and INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) with various substrates of DPN-dependent dehydrogenases. The solutions used for study contained alcohol and alcohol dehydrogenase, glutamate and malate, malate, glutamate, beta-hydroxybutyrate, or DPNH. It has been possible to demonstrate (1) that histological distribution of dehydrogenases may differ from that of the flavoprotein oxidizing reduced coenzyme I; (2) characteristic patterns of distribution of particular dehydrogenases in the tissue proper; (3) different levels of dehydrogenase in kidney and adrenal; and (4) differences in dehydrogenase distribution in the kidneys of man and rat. The evidence presented clearly indicates the limitations inherent in the accepted procedures for the demonstration of DPN and TPN diaphorases. The possible application of the tetrazolium salts to the study of particular coenzyme-dependent dehydrogenases and the pitfalls which might occur are also discussed.