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Biomedical subjects

W Harder

Publications and source records attributed to W Harder.

At least 109 records · Page 6Linked to original sources
PubMed1977

Active transport of oxalate by Pseudomonas oxalaticus OX1.

Membrane vesicles isolated from oxalate-grown cells of Pseudomonas oxalaticus accumulated oxalate by an inducible transport system in unmodified form against a concentration gradient. This accumulation was dependent on the presence of a suitable electron donor system such as ascorbate-phenazine-methosulphate. In the presence of this energy source, steady state levels of accumulation of oxalate were 10--20-fold higher than in its absence. The oxalate transport system involved showed a high affinity for oxalate (Km = 11 micron) and was highly specific. Oxalate transport was not affected by the presence of other dicarboxylic acids, such as malate, succinate and fumarate and only partly inhibited by acetate. The energy requirement for oxalate transport is discussed and it is concluded that this requirement is most likely equivalent to 1 mole of ATP per mole of oxalate.

Ascorbic Acid↗
PubMed1976

S-formylgluthathione: the substrate for formate dehydrogenase in methanol-utilizing yeasts.

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The Km values of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathione-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a Km of 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (Km of 40 mM) but a relatively high affinity for S-formylglutathione (Km of 1.1 mM). The Km values of formate dehydrogenase in cell-free extracts of methanol-grown Candida biodinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.

Aldehyde Oxidoreductases↗
PubMed1976

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha.

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes D-aminoacid oxidase and L-alpha-hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.

Alcohol Oxidoreductases↗
PubMed1976

Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism.

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h-1 showed a maximal capacity to oxidize excess methanol (QmaxO2) which was 1.6 times higher than the rate required to sustain the growth rate (QO2). When the dilution rate was decreased to 0.03 h-1, QmaxO2 of cells increased to a value of more than 20 times that of QO2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent Km for methanol of 1.3 mM in air saturated reaction mixtures and the apparent Km of the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM. The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymprpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells X h.

Alcohol Oxidoreductases↗
PubMed1976

The regulation of agarase production by resting cells of Cytophaga flevenis.

The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3 mM calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3 mM calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme

Agar↗
PubMed1976

Characterization of the neoagarotetra-ase and neoagarobiase of Cytophaga flevensis.

The degradation of neoagarotetraose and neoagarobiose by Cytophaga flevensis was investigated. The organism possesses an enzyme that hydrolyzes the tetramer by cleavage of its central beta-galactosidic linkage. The product of this reaction, neoagarobiose, is further hydrolyzed enzymatically to D-galactose and 3,6-anhydro-L-galactose. Both enzyme activities were localized in the cytoplasm. Attempts were made to partially purify the respective enzymes and although a 30-40 fold-purification was achieved, the final preparation contained both neoagarotetra-ase and neoagarobiase activities. Evidence was obtained that these activities were due to different enzymes. Neoagarotetra-ase is highly specific for oligosaccharides containing neoagarobiose units; the rate of hydrolysis is greatest with neoagarotetraose. It cannot hydrolyze pyruvated neoagarotetraose. Optimal conditions for its activity were pH 7.0 and 25 C. Neoagarobiase hydrolyzes only neoagarobiose and neoagarobiitol and optimal conditions for activity were pH 6.75 and 25 C. Both enzymes were inhibited by Ag+, Hg2+ and Zn2+ ions and by p-CMB, which indicates that thiol groups are present in their active centres. Both enzymes were induced by neoagaro-oligosaccharides and melibiose and were repressed when glucose was added to the medium. Neoagarobiase was also induced by D-galacturonic acid. In continuous culture, the rate of enzyme production was maximal at a dilution rate of 0.1 h-1.

Agar↗
PubMed1975

Oxidation of methanol, formaldehyde and formate by catalase purified from methanol-grown Hansenula polymorpha.

Catalase has been partially purified from cell-free extracts of methanol-grown Hansenula polymorpha and its peroxidative properties were studied. It was shown that the enzyme is capable of oxidizing methanol, formaldehyde and formate in the presence of hydrogen peroxide. The physiological significance of these reactions in the transduction of energy from the oxidation of methanol in yeasts is discussed.

Ascomycota↗
PubMed1975

Cytochemical localization of catalase activity in methanol-grown Hansenula polymorpha.

The localization of peroxidase activity in methanol-grown cells of the yeast Hansenula polymorphia has been studied by a method based on cytochemical staining with diaminobenzidine (DAB). The oxidation product of DAB occurred in microbodies, which characteristically develop growth on or methanol, and in the intracristate space of the mitochondria. The staining of microbodies was H2O2 dependent, appeared to be optimal at pH 10.5, diminished below pH 10 and was inhibited by 20 mM 3-amino 1,2,4 triazole (AT). In contrast to these observations, the reaction in the mitochondria was not H2O2 dependent and not notably affected by differences in pH in the range of 8.5 to 10.5. Microbodies and mitochondria were also stained when H2O2 was replaced by methanol. Appropriate control experiments indicated that in this case methanol oxidase generated the H2O2 for the peroxidative conversion of DAB by catalase. These results suggest that catalase is located in the microbodies of methanol-grown yeasts. A model for a possible physiological function of the microbodies during growth on methanol is put forward.

3,3'-Diaminobenzidine↗
PubMed1975

Microbodies in methanol-assimilating yeasts.

Cells of 3 yeast species capable of assimilating methanol have been examined by electron microscopy. When grown on methanol as the sole source of carbon and energy they contained many microbodies. Cells grown on glucose or ethanol either did not contain such bodies at all, or only to a limited extent.

Ascomycota↗
PubMed1975

Substrate inhibition in Pseudomonas oxalaticus OX1: a kinetic study of growth inhibition by oxalate and formate using extended cultures.

Pseudomonas oxalaticus OX1 has been grown in a mineral salts medium with oxalate or formate as the sole source of carbon and energy. At concentrations of these substrates above 50 mM inhibition of growth was indicated by a long and variable lag phase in batch culture. This inhibition was further studied by estimating maximum specific growth rates at different substrate concentrations using the extended culture technique for control of the substrate concentration. With formate, inhibition became apparent at substrate concentrations above 20 mM, whereas oxalate inhibited growth at concentrations above 15 mM. Complete inhibition was not observed even at concentrations of 100 mM. A number of inhibition functions were fitted with the experimental data using computer analysis. The results indicated that the Haldane equation was the simplest function to describe quantitatively the kinetics of the observed substrate inhibition. Studies on the rate of oxygen uptake at different concentrations of oxalate indicated that respiration was much more sensitive to inhibition than growth. However with formate, inhibition of respiration was not observed up to concentrations of 50 mM, indicating that different mechanisms may underlie the observed growth inhibition by the two substrates.

Computers↗
PubMed1975

Oxidation of organic C1 compounds by Hyphomicrobium spp.

Washed cell suspensions of Hyphomicrobium spp. were able to oxidize methanol, formaldehyde and formate. This suggested that enzymes for the oxidation of these compounds were present. The pathway of the oxidation of methanol to carbon dioxide and water has been investigated using cell-free extracts. An ammonium-ion-activated, phenazine methosulphate-linked methanol dehydrogenase was detected. This enzyme has a dual substrate specificity for normal primary alcohols and formaldehyde. It has a high pH optimum for activity of 9.5. The pathway is completed by an NAD-linked formate dehydrogenase. This enzyme is inhibited by low concentrations of potassium cyanide, copper sulphate and hypophosphite.

Alcohol Oxidoreductases↗
PubMed1975

Production and characterization of the agarase of Cytoplaga flevensis.

Cytophaga flevensis produced an inducible agarase which was extracellular under most conditions tested. The effect of cultural conditions on the production of enzyme was studied in batch and continuous culture. In batch culture, production was optimal when Cytophaga flevensis was incubated at 20 C in a mineral medium with agar as the sole carbon source and ammonium nitrate as the nitrogen source at an initial pH of 6.6-7.0. The enzyme appeared to be subject to catabolite repression, since its synthesis was repressed when glucose was added to the medium in batch culture. Furthermore, in continuous culture, enzyme production decreased with increasing growth rate. Extracellular agarase was partially purified and the enzyme preparation obtained was very stable. The enzyme has a molecular weight of 26 000 daltons. It is a beta-agarase which is highly specific for polysaccharides containing neoagarobiose units. The final products of hydrolysis of agarose by the endo-acting enzyme were neoagarotetraose and neoagarobiose. Optimal conditions for its activity were pH 6.3 and 30C. When agarose was used as a substrate, an apparent temperature optimum of 35C was found, due to gelling of the substrate during the assay procedure.

Carbohydrates↗
PubMed1975

Studies on the physiological significance of the lack of a pyruvate dehydrogenase complex in Hyphomicrobium sp.

Hyphomicrobium X was grown in media containing either methanol or ethanol as a carbon and energy source, with or without additional organic carbon sources. The organism transported pyruvate, malate and succinate into the cells, and incorporated their carbon skeletons into cellular material, but when each of these compounds was added as sole carbon and energy source none supported growth of the organism. Enzymic analysis of crude cell-free extracts failed to detect either a complete pyruvate dehydrogenase complex or an active E1 component. Furthermore, oxygen uptake experiments with whole cell suspensions did not show any oxidation of pyruvate, succinate or malate. The distribution of radioactivity amongst the amino acids in hydrolysates of cell protein obtained from organisms grown in the presence of [14C]pyruvate, [14C]acetate or [14C]succinate indicated that the organism is limited in its ability to metabolize pyruvate. Growth in the presence of [14C]pyruvate resulted in 93% of the total radioactivity recovered being associated with amino acids derived directly from pyruvate. In contrast, growth in the presence of [14C]acetate or [14C]succinate resulted in more-or-less uniform labelling of all biogenic classes of amino acids. These results are consistent with the lack of an active pyruvate dehydrogenase complex which would make it impossible for Hyphomicrobium X to convert pyruvate into acetyl-CoA and to generate energy from carbon compounds for which the energy metabolism relies on oxidation through tricarboxylic acid (TCA) cycle intermediates.

Acetates↗