Secretory hydrolases of Entamoeba histolytica

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Analysis of glycerol dehydrogenase activities present in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> YR-1

Two inducible NADP⁺-dependent glycerol dehydrogenase (GlcDH) activities were identified in Mucor circinelloides strain YR-1. One of these, denoted iGlcDH2, was specifically induced by n-decanol when it was used as sole carbon source in the culture medium, and the second, denoted iGlcDH1, was induced by alcohols and aliphatic or aromatic hydrocarbons when glycerol was used as the only substrate. iGlcDH2 was found to have a much broader substrate specificity than iGlcDH1, with a low activity as an ethanol dehydrogenase with NAD⁺ or NADP⁺ as cofactor. Both isozymes showed an optimum pH for activity of 9.0 in Tris-HCl buffer and are subject to carbon catabolite repression. In contrast, the constitutive NADP⁺-dependent glycerol dehydrogenases (GlcDHI, II, and III) were only present in cell extracts when the fungus was grown in glycolytic carbon sources or glycerol under oxygenation, and their optimum pH was 7.0 in Tris-HCl buffer. In addition to these five NADP⁺-dependent glycerol dehydrogenases, a NAD⁺-dependent alcohol dehydrogenase is also present in glycerol or n-decanol medium; this enzyme was found to have weak activity as a glycerol dehydrogenase.     

Studies on the metabolism of galactose-6-phosphate in rat heart and brain.

The subcellular distribution of NADP⁺ and NAD⁺-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP⁺ or NAD⁺ as cofactor. While all of the tissues examined had NADP⁺-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP⁺-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (K_m's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP⁺ or NAD⁺ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (K_i = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase.     

Cholesterol esters and hydrolytic cholesterol esterase during Wallerian degeneration

To study the involvement of cholesterol esters in myelination and demyelination, we determined the concentration of free cholesterol and cholesterol esters and the activity of hydrolytic cholesterol esterase (sterol ester hydrolase; EC 3.1.1.13) in hen sciatic nerve during Wallerian degeneration. A progressive increase in the ratio of cholesterol ester to free cholesterol was observed in the degenerating nerve at 8, 16 and 32 days after nerve section. Hydrolytic cholesterol esterase activity decreased progressively in the degenerating nerves at the same time. In addition we measured the ratio of RNA to DNA, and the activity of the NADP⁺-dependent isocitrate dehydrogenase [L₈-isocitrate: NADP oxidoreductase (decarboxylating); EC 1.1.1.42] at 8, 16 and 32 days after nerve section. The RNA to DNA ratios decreased progressively in the degenerating nerves. NADP⁺-dependent isocitrate dehydrogenase increased in activity after nerve section, reaching a peak at 16 days.     

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

Summary 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 -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, 5nucleotidase. glucose-6-phosphatase, -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.This study was made possible by grants from the Jan Dekker Foundation for Biomedical Research and the Niels Stensen Foundation, The Netherlands, to the first author     

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.     

Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps (Hemiptera: Scutelleridae): Enzyme markers of microvillar and perimicrovillar membranes

The subcellular distributions of six digestive and non-digestive enzymes (α-glucosidase, β-glucosidase, alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster integriceps have been studied. The subcellular distributions of acid phosphatase and α-glucosidase are similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially membrane-bound enzymes, alkaline phosphatase and β-glucosidase have similar distributions in differential centrifugation fractions, which are different from that of α-glucosidase. Sucrose gradient ultracentrifugation of membranes from luminal contents showed that β-glucosidase carrying membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile of proteins extracted from β-glucosidase carrying membranes is different from that of α-glucosidase carrying membranes. We conclude that β-glucosidase and aminopeptidase are markers of microvillar membrane (MM) and perimicrovillar space, respectively, while α-glucosidase and acid phosphatase are perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is used to resolve these membranes.     

Purification of Alcohol Dehydrogenase fromEntamoeba histolyticaandSaccharomyces cerevisiaeUsing Zinc-Affinity Chromatography

We have developed a single-step method for the purification of NADP⁺-dependent alcohol dehydrogenase fromEntamoeba histolyticaand NAD⁺-dependent alcohol dehydrogenase fromSaccharomyces cerevisiae.It is based on the affinity for zinc of both enzymes. The amebic enzyme was purified almost 800 times with a recovery of 54% and the yeast enzyme was purified 30 times with a recovery of 100%. The kinetic constants of the purified enzymes were similar to those reported for other purification methods. With mammalian alcohol dehydrogenase, we obtained a 40-kDa band suggestive of purified alcohol dehydrogenase, but we failed to retain enzymatic activity in this preparation. Our results suggest that the described method is more applicable to the purification of tetrameric alcohol dehydrogenases.     

Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation

Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP⁺-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD⁺-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.Non-standard abbreviations AADC acetoacetate decarboxylase - AcaDH acetaldehyde dehydrogenase - BuaDH butyraldehyde dehydrogenase - CoA-TF acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase - NAD-ADH, NAD⁺ dependent alcohol dehydrogenase - NADP-ADH, NADP⁺ dependent alcohol dehydrogenase     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP⁺-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol l^-1 of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h^-1, and then sharply dropped. In the N-sufficient cultures both NAD⁺- and NADP⁺-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3-hours delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.     

Changes in NAD(P)+-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation

A sensitive isotope exchange method was developed to assess the requirements for and compartmentation of pyruvate and oxalacetate production from malate in proliferating and nonproliferating human fibroblasts. Malatedependent pyruvate production (malic enzyme activity) in the particulate fraction containing the mitochondria was dependent on either NAD⁺ or NADP⁺. The production of pyruvate from malate in the soluble, cytosolic fraction was strictly dependent on NADP⁺. Oxalacetate production from malate (malate dehydrogenase, EC 1.1.1.37) in both the particulate and soluble fraction was strictly dependent on NAD⁺. Relative to nonproliferating cells, NAD⁺-linked malic enzyme activity was slightly reduced and the NADP⁺-linked activity was unchanged in the particulate fraction of serum-stimulated, exponentially proliferating cells. However, a reduced activity of particulate malate dehydrogenase resulted in a two-fold increase in the ratio of NAD(P)⁺-linked malic enzyme to NAD⁺-linked malate dehydrogenase activity in the particulate fraction of proliferating fibroblasts. An increase in soluble NADP⁺-dependent malic enzyme activity and a decrease in NAD⁺-linked malate dehydrogenase indictated an increase in the ratio of pyruvate-producing to oxalacetate-producing malate oxidase activity in the cytosol of proliterating cells. These coordinate changes may affect the relative amount of malate that is oxidized to oxalacetate and pyruvate in proliferating cells and, therefore, the efficient utilization of glutamine as a respiratory fuel during cell proliferation.     

Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation

The ‘high ammonia pathway’ enzyme glutamate dehydrogenase (NADP⁺) is inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth in reached. Purified glutamate dehydrogenase (NADP⁺) appeared to be a protein composed of six identical subunits with a molecular weight of 54 000. With antibodies raised against purified enzyme it was found that glutamate dehydrogenase (NADP⁺) inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that glutamate dehydrogenase (NADP⁺) inactivation is caused or followed by rapid proteolysis.     

The alpha beta epimerization of reduced nicotinamide adenine dinucleotide.

The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH³ and the nicotinamide ring of αNAD⁺ are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD⁺ is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD⁺.The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10^?3 min^?1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M₄ and H₄ lactate dehydrogenase, are shown to oxidize αNADH to αNAD⁺ at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD⁺in vivo.     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

Purification and properties of the NAD+-xylitol-dehydrogenase from the yeast Pichia stipitis

Cell-free extracts of the xylose fermenting yeast Pichia stipitis exhibited xylitol dehydrogenase activity with NAD⁺ and NADP⁺. During the purification step on DEAE-sephadex A-50 a NAD⁺-dependent xylitol dehydrogenase could be separated from a NADP⁺-dependent. The NAD⁺-xylitol dehydrogenase was further purified to electrophoretic homogeneity via gel and affinity chromatography. The purified enzyme was most active at pH 9 and 35°C. Its molecular weight was determined to be 63,000 dalton by Sephadex G-200 column chromatography, and that of its subunit was 32,000 dalton by sodium dodecyl sulphate polyacrylamide gel electrophoresis. From the results of substrate specificity, the enzyme should be named l-iditol:NAD⁺-5-oxidoreductase (EC 1.1.1.14, sorbitol dehydrogenase).     

Regulation of nitrogen fixation in Rhizobium spp. Isolation of mutants of Rhizobium trifulii which induce nitrogenase activity

This communication describes the isolation and characterization of mutants of Rhizobium trifolii which can induce nitrogenase activity in defined liquid medium. Two procedures were used for the isolation of these mutants from R. trifolii strain DT-6: (1) following chemical mutagenesis, slow growin mutants were selected which were unable to utilize NH₄⁺ as sole source of nitrogen; (2) as spontaneous mutants resistant to the glutamate analogue L-methionine-DL-sulfoximine.Mutants (DT-71, DT-125) isolated by these procedures induced nitrogenase activity in the free-living state, whereas the parent strain lacked this property. Induction of nitrogenase activity in these mutants occurred during the late exponential phase of growth when the rate of protein synthesis was decreasing. The addition of NH₄⁺ to a medium containing glutamate as the nitrogen-source resulted in a 50–70% reduction (repression?) of nitrogenase activity; in contrast, the rate of protein synthesis or the rate of respiration was not influenced by exogenous NH₄⁺.Biochemistry analysis showed that these mutants (strains DT-71 and DT-125) have defects in both nitrogen and carbon metabolism. The levels of glutamate synthase (both NADP⁺-and NAD⁺-dependent activities) and glutamate dehydrogenase (NAD⁺-dependent activity) were markedly lower. In addition, the mutants were found to have no detectable ribitol dehydrogenase or β-galactosidase activity. These findings are discussed in relation to a mechanism of regulation of symbiotic nitrogen fixation.     

NAD+ -dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD⁺-dependent and NADP⁺-dependent malic enzyme activities. The NAD⁺ malic enzyme exhibited more activity with NAD⁺ as cofactor, but also showed some activity with NADP⁺. The NADP⁺ malic enzyme only showed activity when NADP⁺ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NADP⁺ malic enzyme activity (dme) but retained NADP⁺ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N₂. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N₂-fixation would normally begin. These results support the proposal that NAD⁺ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N₂-fixing bacteroids which metabolize C₄-dicarboxylic acids supplied by the plant.     

Detection of 15-hydroxyprostaglandin dehydrogenase in bovine mesenteric blood vessels

Two types of 15-hydroxyprostaglandin dehydrogenase (NAD⁺ and NADP⁺ dependent) were demonstrated in bovine mesentric arteries and veins. The 15-hydroxyprostaglandin dehydrogenase activity was found in the high-speed supernatant, suggesting that these enzymes are associated with the cytoplasmic fraction of the blood vessels. The levels of activities of both NAD⁺- and NADP⁺-dependent dehydrogenases were similar in mesentric blood vessels. Prostaglandin F_2α was preferred to the prostaglandin E₂ as subtrate by both NAD⁺ and NADP⁺ dependent enzymes. The presence of 15-hydroxyprostaglandin dehydrogenase in blood vessels may play a siginificant role in the regulation of intracellular levels of prostaglandins of the E and F series in blood vessels.