Enzyme histochemical changes in retinal ganglion cells and the optic nerve after goldfish optic nerve lesion. A regenerative and hypertrophic phenomena study

After sectioning of the goldfish optic nerve a number of enzyme histochemical changes are observed in the hypertrophied retinal ganglion cells and in the optic nerve. Between one and eighteen days postoperatively an increase in the amount of acid phosphatase reaction product is noted. The enhanced activity decreased to normal first in the optic nerve, followed by the optic tract and tectum. Four days postoperatively higher levels of activity were noted in the hypertrophic retinal ganglion cells for the enzymes NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase. The same enzymes also showed an activity increase in the lesioned optic nerve after four to ten days postoperatively, beginning at the cut and gradually spreading towards the optic tectum. Between fifteen and eighteen days the activity dropped to normal in the hypertrophic retinal ganglion cells, while in the lesioned nerve raised levels of reaction products could be seen till days thirty-five and/or forty-five. It was concluded that the degeneration of the optic pathway is marked by the increase of acid phosphatase activity, whereas the process of regeneration is characterized by an increase of NADH tetrazolium reductase, cytochrome oxidase, glutamate dehydrogenase and lactate dehydrogenase activities. The possible functional implications of these enzymes in the regenerative phenomena are discussed.     

Cytochemical study of cells of primary and disseminated ascite Yoshida tumor cells

The different distribution of cytochemically demonstrable enzymes: lactate dehydrogenase (LDH, 1.1.1.27), succinate dehydrogenase (SDH, 1.3.99.1), dihydrofolate reductase (DHFR, 1.5.1.3), acid phosphatase (AcP, 3.1.3.2) and alkaline phosphatase (ALP, 3.1.3.1), has been documented in Yoshida ascites hepatoma cells in vivo or stored at 80 degrees C. The dehydrogenase activities (LDH, SDH, DHFR) show a strong reaction in all samples. An increased level of these enzyme activities has been observed in the malignant cells spreading through the organs of tumor bearing rats. On the contrary, in the same samples, acid and alkaline phosphatase activities are very low. The strong dehydrogenase activities observed in Yoshida ascite cells stress the rapid turnover of tumor cells. Our results indicate that the histochemical method may be a useful tool to detect the scattered tumor cells. Furthermore, the cytochemical methods allow the characterization of the metabolic pathways employed by the primary and disseminated tumor cells.     

Histochemical studies on the localization of oxidative and dephosphorylating enzymes and esterases in the peritoneal mesothelial cells

Summary Histochemical localization of various groups of oxidative and dephosphorylating enzymes and esterases has been made in the peritoneal mesothelial cells. These cells were strongly positive for lactic dehydrogenase and succinic dehydrogenase, moderately positive for cytochrome oxidase, simple esterase, acid phosphatase, 5nucleotidase and nonspecific cholinesterase, and negative for monoamine oxidase and specific cholinesterase. They showed moderate positive activity for alkaline phosphatase, localized in the microvilli of these cells. They contain scanty amount of thiamine pyrophosphatase-positive Golgi complex and showed diffuse thiamine pyrophosphatase-positive activity in the cell cytoplasm. Adenosine triphosphatase-positive spaces found in between the adjacent cells and at the bases of the individual cells have been demonstrated. These results are discussed in view of the great absorptive power of the peritoneal mesothelium.     

Histochemical studies of some hepatic enzyme activities in rat during extrahepatic common bile duct obstruction.

Studies on some enzyme activities as measured histochemically in the ligated common bile duct rat liver are reported. They show principally: an increased GGT reaction in the neoductular cells; a diffusion of the LAP reaction to the neoductuli; an immediate increase and diffusion of the alkaline phosphatase reaction; an increase of G-6-PA and AMPA reactions; an increase, during the first week, of the Phyla activity and a contemporary decrease of the UDPG-GS reaction. No significant changes have been observed in other enzyme activities. By means of some dehydrogenase reactions, morphological evidence that the hepatocytes may be involved in neoductular cells formation was obtained.     

Polysulfide reductase and formate dehydrogenase from Wolinella succinogenes contain molybdopterin guanine dinucleotide

Pterin derivatives were extracted from formate dehydrogenase and from polysulfide reductase of Wolinella succinogenes and converted to 6-carboxypterin. The amounts of 6-carboxypterin were consisted with the molybdenum content of the enzymes. The bis(carboxamidomethyl) derivatives of the cofactors showed absorption spectra that were identical with that of the corresponding molybdopterin guanine dinucleotide derivative (cam MGD). After hydrolysis of the derivatives with nucleotide pyrophosphatase in the presence of alkaline phosphatase, guanosine was formed together with a compound showing the properties of dephospho-bis(carboxamidomethyl)-molybdopterin. It is conluded that both formate dehydrogenase and polysulfide reductase of W. succinogenes contain molybdopterin guanine dinucleotide.Abbreviations MPT molybdopterin - MGD molybdopterin guanine dinucleotide - cam MPT bis(carboxyamidomethyl)-molybdopterin - cam MGD bis(carboxyamidomethyl)-molybdopterin guanine dinucleotide     

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.     

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves: Cellular Distribution of Enzymes and Energy Sources for Nitrate Reduction

The localization of enzymes responsible for nitrate assimilation and the generation of NADH for nitrate reduction were studied in corn (Zea mays L.) leaf blades. The techniques used effectively separated mesophyll and bundle sheath cells as judged by microscopic observations, enzymic assays, chlorophyll a/b ratios and photochemical activities. Nitrate reductase, nitrite reductase, and the nitrate content of leaf blades were localized primarily in the mesophyll cells, although some nitrite reductase was found in the bundle sheath cells. Glutamine synthetase, NAD-malate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glutamate dehydrogenase were found in both types of cells, however, more NADP-glutamate dehydrogenase was found in the bundle sheath cells than in the mesophyll cells. These data indicate that the mesophyll cells are the major site for nitrate assimilation in the leaf blade because they contained an ample supply of nitrate and the enzymes considered essential for the assimilation of nitrate into amino acids. Because the specific activity of nitrate reductase was severalfold lower than the other enzymes involved in nitrate assimilation, nitrate reduction is indicated as the rate-limiting step in situ. A sequence of reactions is proposed for nitrate assimilation in the mesophyll cells of corn leaves as related to the C-4 pathway of photosynthesis.     

In vitro dephosphorylation inhibits the activity of soybean lysine-ketoglutarate reductase in a lysine-regulated manner

In plant seeds, the essential amino acid lysine auto-regulates its own level by modulating the activity of its catabolic enzyme lysine-ketoglutarate reductase via an intracellular signaling cascade, mediated by Ca²⁺ and protein phosphorylation/dephosphorylation. In the present report, it has been further tested whether the activity of soybean lysine-ketoglutarate reductase, as well as that of saccharopine dehydrogenase, the second enzyme in the pathway of lysine catabolism, are modulated by direct phosphorylation of the bifunctional polypeptide containing both of these linked activities. Incubation of purified lysine-ketoglutarate reductase/ saccharopine dehydrogenase with casein kinase II resulted in a significant phosphorylation of the bifunctional enzyme. Moreover, in vitro dephosphorylation of the bifunctional polypeptide with alkaline phosphatase significantly inhibited the activity of lysine-ketoglutarate reductase, but not of its linked enzyme saccharopine dehydrogenase. The inhibitory effect of alkaline phosphatase on lysine-ketoglutarate reductase activity was dramatically stimulated by binding of lysine to the enzyme. Our results suggest that in plant seeds, active lysine-ketoglutarate reductase is a phospho-protein, and that its activity is modulated by opposing actions of protein kinases and phosphatases. Moreover, this modulation is subject to a compound regulation by lysine.     

The antioxidants of legume nodule mitochondria

The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation. They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites. We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L. cv. Contender x Rhizobium leguminosarum bv. phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix. This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase. Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids. Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts. We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane. The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol. In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase.     

Development and differentiation of fetal rat sensory ganglia and spinal cord segments in vitro

Summary Fetal spinal ganglia and spinal cord segments with adhering spinal ganglia were explanted on collagen-coated coverslips. They were investigated with enzyme histochemical methods for the existence of hydrolases and dehydrogenases up to 54 days of cultivation.Alkaline phosphatase was located in arachnoid cells and in mantle cells (satellite cells). Butyrylcholinesterase and alpha-glycerophosphate-menadione-tetrazolium reductase were found in mantle cells. Acetylcholinesterase and indoxylesterase were active in the whole neuron; acid phosphatase and alpha-naphthylacetate esterase were restricted to the perikarya.During the process of cell differentiation in vitro alkaline phosphatase decreased in mantle cells. Acid phosphatase became diminished distinctly in spinal ganglion cells. The other neuronal enzymatic activities remained unaltered during the whole period of cultivation. Proliferated Schwann cells were conspicuous by their activity for butyrylcholinesterase. In newly formed myelin sheaths arylsulphatase was active. Lactate dehydrogenase was contained in the perineurium which had developed. Cultures of long duration could contain cytological formations which were reminiscent of sensory end-organs with respect to their enzyme patterns.The enzyme activities of nervous tissues in vitro in their approximation to the situation in situ are discussed.A preliminary report was presented at the 17th Meeting of the Association of German Neuropathologists and Neuroanatomists, in Freiburg, September 1972.The skilled technical assistance of Miss Johanna Sixel and Miss Charlotte Beyer is gratefully acknowledged.     

Demonstrability of some oxidative enzymes in early rodent embryos with and without fixation

Several oxidative enzymes [NADH-TR (reduced nicotinamide-adenine dinucleotide-tetrazolium reductase), NADPH-TR (reduced nicotinamide-adenine dinucleotide phosphate-tetrazolium reductase), SDH (succinic dehydrogenase) and LDH (lactate dehydrogenase)] were studied by histochemical means during early development of rat and mouse. All investigated enzymes could be easily demonstrated in zygote and also to some extent in somitic stages without any pretreatment. However, in cleavage and early postimplantation stages enzyme activity could be revealed only after the embryos were pretreated in some way. This pretreatment can be fixation with formalin or acetone, freezing and thawing, slight mechanical damage or very prolonged incubation time. The formazan granules as a sign of enzymatic activity were present in all stages of embryonic development and were more abundant in reactions for NADH-TR and LDH than in reactions for NADPH-TR and SDH. Our results suggest that the investigated enzymes are present in all embryonic cells during early development. It seems that the permeability of embryonic cells for histochemical media must be increased otherwise the histochemical reactions cannot be accomplished.     

Modulation of rat liver hydroxymethylglutaryl-CoA reductase by protein phosphatases: purification of nonspecific hydroxymethylglutaryl-CoA reductase phosphatases

Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (Mr 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I1 (Mr 180,000) and I3 (Mr 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I2 (Mr 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the Mr of all phosphatases changed; protein phosphatases I2, I3, and II were brought to an Mr of 35,000, while phosphatase I1 was reduced to an Mr of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I2, I3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low-Mr forms. A comparison has been made with other protein phosphatases previously reported in the literature.     

The purification and characterization of Dictyostelium discoideum plasma membranes.

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5'-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome c reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.     

Expression of 11 beta-hydroxysteroid dehydrogenase using recombinant vaccinia virus

Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11 beta-hydroxysteroid dehydrogenase. To determine the kinetic properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed both 11 beta-dehydrogenation of corticosterone to 11-dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kcat/Km for dehydrogenase activity exceeded that for reductase (63 vs. 38 min-1 x 10(-4], whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min-1 x 10(-4] at pH 7.0. These results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP+ produced by the reductase reaction significantly increased reductase activity. Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase reaction at slightly higher concentrations (50% inhibitory concentration, less than 5 nM for dehydrogenase, 10-20 nM for reductase). Partial inhibition of glycosylation with A1-tunicamycin decreased dehydrogenase activity 50% without affecting reductase activity. The data demonstrate that a single polypeptide catalyzes both dehydrogenation and reduction, although the presence of additional enzyme forms catalyzing one or the other activity has not been ruled out.     

Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.     

Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis.

In plant and mammalian cells, excess lysine is catabolized by a pathway that is initiated by two enzymes, namely, lysine-ketoglutarate reductase and saccharopine dehydrogenase. In this study, we report the cloning of an Arabidopsis cDNA encoding a bifunctional polypeptide that contains both of these enzyme activities linked to each other. RNA gel blot analysis identified two mRNA bands-a large mRNA containing both lysine-ketoglutarate reductase and saccharopine dehydrogenase sequences and a smaller mRNA containing only the saccharopine dehydrogenase sequence. However, DNA gel blot hybridization using either the lysine-ketoglutarate reductase or the saccharopine dehydrogenase cDNA sequence as a probe suggested that the two mRNA populations apparently are encoded by the same gene. To test whether these two mRNAs are functional, protein extracts from Arabidopsis cells were fractionated by anion exchange chromatography. This fractionation revealed two separate peaks-one containing both coeluted lysine-ketoglutarate reductase and saccharopine dehydrogenase activities and the second containing only saccharopine dehydrogenase activity. RNA gel blot analysis and in situ hybridization showed that the gene encoding lysine-ketoglutarate reductase and saccharopine dehydrogenase is significantly upregulated in floral organs and in embryonic tissues of developing seeds. Our results suggest that lysine catabolism is subject to complex developmental and physiological regulation, which may operate at gene expression as well as post-translational levels.     

Subcellular localization of gamma-glutamyltransferase in calf thymocytes.

The subcellular localization of gamma-glutamyltransferase in calf thymocytes was investigated and compared with that of alkaline phosphodiesterase I, alkaline nitrophenyl phosphatase, succinate-tetrazolium oxidoreductase (succinate-INT reductase) and lactate dehydrogenase after two different methods of cell disruption and differential centrifugation. Most of the activity was recovered in the crude membrane fractions (43.0%), but significant amounts co-pelleted with the large-granule (mitochondria) fractions (31%). The specific activity of the gamma-glutamyltransferase in the purified plasma membrane was 30-50 times that of the enzyme in the cell homogenate and had a similar subcellular distribution to the plasma-membrane markers, alkaline phosphodiesterase I and alkaline nitrophenyl phosphatase. It was concluded that gamma-glutamyltransferase was primary a plasma-membrane-bound enzyme, and that its location in other subcellular fractions was probably due to their contamination with plasma-membrane vesicles.     

The stereospecificity of sequential nicotinamide-adenine dinucleotide-dependent oxidoreductases in relation to the evolution of metabolic sequences.

The generalization that 'when a metabolic sequence involves consecutive nicotinamide-adenine dinucleotide-dependent reactions, the dehydrogenases have the same stereospecificity' was tested and confirmed for three metabolic sequences. (1) NAD+-xylitol (D-xylulose) dehydrogenase and NADP+-xylitol (L-xylulose) dehydrogenase are both B-specific. (2) D-Mannitol 1-phosphate dehydrogenase and D-sorbitol 6-phosphate dehydrogenase are both B-specific. (3) meso Tartrate dehydrogenase and oxaloglycollate reductive decarboxylase are both A-specific. Other dehydrogenases associated with the metabolism of meso-tartrate in Pseudomonas putida, such as hydroxypyruvate reductase and tartronate semialdehyde reductase, were also shown to be A-specific. Malate dehydrogenase from Pseudomonas putida was A-specific, and the proposition is discussed that the common A-stereospecificity among the dehydrogenases involved in meso-tartrate metabolism reflects their origin from malate dehydrogenase.