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H Holzer

Publications and source records attributed to H Holzer.

At least 109 records · Page 6Linked to original sources
PubMed1985

Effect of sulfite on the energy metabolism of mammalian tissues in correlation to sulfite oxidase activity.

Mammalian tissues show significant differences in the activity of sulfite oxidase (EC 1.8.3.1) which detoxifies sulfite by oxidation to sulfate. Lung tissue and phagocytic cells such as alveolar macrophages, peritoneal macrophages, Kupffer cells and granulocytes show very low activities of sulfite oxidase. Liver tissue and hepatocytes, however, exhibit high activities of sulfite oxidase. Lung tissue and macrophages show an almost 100% decrease of the intracellular ATP levels when incubated with 1 mM sulfite at pH 6 for 30 min. In addition, the O2 consumption of lung tissue is inhibited by 1 mM sulfite at pH 6 by more than 80%. This sulfite-induced decrease of the ATP level and of the O2 consumption of lung tissue is enhanced between pH 6.0 and pH 7.4 with decreasing pH value of the incubation medium. In contrast, the ATP levels in liver tissue and hepatocytes are not affected by 1 mM sulfite at pH 6. The O2 consumption of liver tissue and hepatocytes is significantly increased by sulfite due to the high activities of sulfite oxidase. Therefore, the activity of the 'sulfite-detoxifying enzyme' sulfite oxidase and the sensitivity of the energy metabolism to sulfite show a reciprocal relationship in the tissues and cells studied.

Adenosine Triphosphate↗
PubMed1985

Effects of m-Cl-peroxy benzoic acid on glycolysis in Saccharomyces cerevisiae.

Concentrations of m-Cl-peroxy benzoic acid (CPBA) higher than 0.1 mM decrease the ATP-content of Saccharomyces cerevisiae in the presence of glucose in 1 min to less than 10% of the initial value. In the absence of glucose, 1.0 mM CPBA is necessary for a similar effect. After the rapid loss of ATP in the first min in the presence of glucose caused by 0.2 mM CPBA, the ATP-content recovers to nearly the initial value after 10 min. Aerobic glucose consumption and ethanol formation from glucose are both completely inhibited by 1.0 mM CPBA. Assays of the activities of nine different enzymes of the glycolytic pathway as well as analysis of steady state concentrations of metabolites suggest that glyceraldehyde-3-phosphate dehydrogenase is the most sensitive enzyme of glucose fermentation. Phosphofructokinase and alcohol dehydrogenase are slightly less sensitive. Incubation for 1 or 10 min with concentrations of 0.05 to 0.5 mM CPBA causes a) inhibition of glyceraldehyde-3-phosphate dehydrogenase, b) decrease of the ATP-content and c) a decrease of the colony forming capacity. From these findings it is concluded that the disturbance of the ATP-producing glycolytic metabolism by inactivation of glyceraldehyde-3-phosphate dehydrogenase may be an explanation for cell death caused by CPBA.

Adenosine Triphosphate↗
PubMed1985

Effect of sulfite or nitrite on the ATP content and the carbohydrate metabolism in yeast.

Low concentrations of sulfite or nitrite (about 0.5 mmol) when applied at pH 3.6, caused a rapid and drastic decrease of the concentration of ATP in yeast cells. Under these conditions, alcoholic fermentation was inhibited by sulfite and to a lesser extent by nitrite. Ethanol consumption under aerobic conditions was shown to be more sensitive to nitrite than to sulfite. This indicates a higher sensitivity of respiratory processes to nitrite than to sulfite. Among 15 enzyme activities assayed in extracts from yeast cells after incubation with sulfite or nitrite, glyceraldehyde-3-phosphate dehydrogenase was shown to be the most sensitive. Analysis of the steady-state concentrations of intermediates of alcoholic fermentation in intact yeast cells also implies inhibition by sulfite or nitrite of the glyceraldehyde-3-phosphate dehydrogenase step of fermentation. In contrast to nitrite, sulfite had an additional effect by accumulating the intracellular steady state concentration of glyceraldehyde-3-phosphate 10 to 100-fold over the concentration in the absence of sulfite. In vitro studies on the equilibrium catalyzed by triosephosphate isomerase or aldolase confirmed the postulated shift of equilibrium concentrations by a formation of complex of glyceraldehyde-3-phosphate with sulfite.

Adenosine Triphosphate↗
PubMed1985

Accumulation of nitrite and sulfite in yeast cells and synergistic depletion of the intracellular ATP content.

When nitrite or sulfite are applied to yeast cells below pH 5.0, an enormous intracellular accumulation occurs. It is assumed that nitrite and sulfite penetrate the cell membrane in their undissociated forms as nitrous acid (pK = 3.3) or sulfurous acid (pK = 1.8), respectively. Due to the neutral intracellular pH they are trapped inside the cell in their anionic forms, which are impermeable to the cell membrane. It has previously been shown that sulfite causes a rapid depletion of the ATP content of yeast cells [Schimz, K.L. and Holzer, H. (1979) resp. Hinze et al. as above]. Similarly, millimolar concentrations of nitrite decrease the ATP level to less than 10% of the initial value. Nitrite and sulfite in combination deplete the ATP content of yeast cells much stronger than expected for the sum of the separate effects of these compounds ("synergistic effect").

Adenosine Triphosphate↗
PubMed1985

Control of yeast neutral trehalase by distinct polyphosphates and ribonucleic acid.

The activity of yeast trehalase when assayed at pH 7 in a crude extract was found to increase 2- to 3-fold upon incubation with 0.1% (v/v) polyethyleneimine or other polycations such as polylysine (0.075-mMol) and calf thymus histones (0.08 mMol). Incubation with 3 mM-Mn2+ and 5 mM-Ca2+ also led to 3- and 1.6-fold increases in trehalase activity, respectively. The activities of 11 other enzymes assayed in the crude yeast extract did not increase after addition of polyethylene imine. At concentrations of polyethyleneimine that maximally stimulated trehalase activity, 97% of the total RNA present in the crude extract, 40% of total protein, and 60% of the polyphosphate (assayed as inorganic phosphate liberated during 7 min incubation at 95 degrees C and pH O) were found to be precipitated. A similar finding was made with trehalase-stimulating concentrations of Mn2+. Activation of trehalase by polyethylene imine rendered this enzyme susceptible to inhibition by a preparation of total yeast RNA, inorganic polyphosphates, and related polyanions. We present further evidence that the removal of a distinct RNA and/or polyphosphate is the basic principle of polyethyleneimine-induced activation of trehalase. A more pronounced stimulation of trehalase activity (4-fold) could be obtained by enzymatic phosphorylation with ATP in the presence of cyclic AMP and Mg2+ as described by van Solingen and van der Plaat (1975) [9].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1985

Oxidative inactivation of yeast fructose-1,6-bisphosphatase.

Active, non-phosphorylated fructose-1,6-bisphosphatase from yeast is partially inactivated by two different mixed-function oxidation systems: the ascorbate-FeC13-02 system described by Levine (Levine, R. (1983) J. Biol. Chem. 258, 11823-11827) and the NADH oxidase-NADH-FeC13-O2 system described by Fucci et al. (Fucci, L., Oliver, C.N., Coon, M.J., and Stadtman, E.R. (1983) Proc. Natl. Acad. Sci. 80, 1521-1525). Fructose-2,6-bisphosphate (1 microM) or histidine (10 mM) partially protect from oxidative inactivation. The inactivation is characterized by the following changes in the kinetic properties of fructose-1,6-bisphosphatase: decrease of the ratio of activity at pH 7 to that at pH 9 and decrease of the ratio of activity with 10 mM Mg2+ to that with 2 mM Mn2+. These changes of the kinetic properties are very similar to the ones previously observed following phosphorylation (Holzer, H. (1984) in "Enzyme regulation by reversible phosphorylation - further advances" (P. Cohen, ed.) pp. 143-154, Elsevier Science publisher) and limited proteolysis of fructose-1,6-bisphosphatase with yeast proteinase B (Pohlig, G., Schäfer, W., v. Herrath, M. and Holzer, H. (1984) in "Current topics in cellular regulation" (S. Shaltiel and P. Boon Chock, eds.) in press).

Ascorbic Acid↗
PubMed1985

Phosphorus kinetics during haemodialysis and haemofiltration.

Phosphorus excretion during haemodialysis positively correlates with the plasma inorganic phosphorus (Pi) concentration and the dialyser Pi clearance. Therefore, by using highly efficient dialysers or haemofilters, disciplined patients may achieve a well regulated P-balance. The plasma Pi concentration time curve during haemodialysis or haemofiltration must be seen as the result of passive diffusion combined with an active mobilisation of Pi from a rapidly exchangeable P-pool. The plasma Pi concentration hardly falls below the normal range in dialysis patients, regardless of the quantity of Pi removed by haemodialysis or haemofiltration. The stability of plasma Pi demonstrates the existence of a mechanism for phosphate regulation in body fluids, independent of the calcium homeostasis.

Blood↗
PubMed1984

Characterization of fructose 1,6-bisphosphatase from bakers' yeast.

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.

Adenosine Monophosphate↗
PubMed1983

Cyclic AMP and fructose-2,6-bisphosphate stimulated in vitro phosphorylation of yeast fructose-1,6-bisphosphatase.

Phosphorylation of purified yeast fructose-1,6-bisphosphatase was studied using purified preparations from yeast of two different cyclic AMP-independent protein kinases and a cyclic AMP-dependent protein kinase. Incorporation of 32P into fructose-1,6-bisphosphatase could be demonstrated only with the cyclic AMP-dependent protein kinase. Phosphorylation of fructose-1,6-bisphosphatase was stimulated by 3 microM fructose-2,6-bisphosphate and inhibited by 1 mM 5'-AMP.

Cyclic AMP↗
PubMed1982

Characterization of the proteolytic activity firmly attached to yeast phoshoenolpyruvate carboxykinase.

Incubation of partially purified yeast phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) with 5% mercaptoethanol and 0.01% sodium dodecyl sulfate at 37 degrees C results in degradation of the enzyme. The degradation can be partially prevented by addition of proteinase B inhibitor 2 or phenylmethylsulfonyl fluoride, an inhibitor of proteinase B and carboxypeptidase Y. The degradation can be completely inhibited by addition of proteinase B inhibitor 2 together with pepstatin, and inhibitor of proteinase A. Thus it appears that proteolytic activities are firmly attached to phosphoenolpyruvate carboxykinase and are identical with the yeast proteinases A and B. The latter conclusion was supported by experiments using the pure yeast proteinases.

Aspartic Acid Endopeptidases↗
PubMed1982

Neurophysiological findings and serum aluminium in dialysis encephalopathy.

64 patients on hemodialysis were investigated. The mean duration of dialysis was 43 months. In all the patients, serum aluminium levels, systolic blood pressure (averaged over a period of 6 weeks) and the EEG were investigated. Psychological testing to assess the level of intelligence (IQ) was also performed. The serum aluminium levels have been assayed by flameless atomic absorption (Perkin-Elmer atomic absorption spectrophotometer model 420). In 6 patients, the diagnosis of dementia was made on the basis of psychological testing and clinical observations. The demented patients showed significantly (p less than 0.01) higher aluminium levels (mean 409 microgram/l, SD 235) than the 58 non-demented patients (mean 189 microgram/l, SD 152), whereas the age of the patients, duration of dialysis and blood pressure were the same in both groups. The EEG was abnormal in all 6 demented patients. Only in 23 of the 58 non-demented patients was the EEG pathological (p less than 0.05). A significant correlation was found between serum aluminium levels and the EEG data with regard to bilateral slow waves, focal slow waves and epileptic potentials (p less than 0.05). The EEG, age of the patients, duration of dialysis and blood pressure showed no correlation. Electromyography, nerve conduction velocity and latency showed no correlation with aluminium levels or dementia. It can be concluded from our findings that in dialyzed patients there is a correlation between serum aluminium levels and the appearance of dementia and EEG changes.

Aluminum↗
PubMed1981

Purification and properties of proteinase B from yeast.

Proteinase B (EC 3.4.22.9) was purified from commercial baker's yeast and from wild type strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis. For large scale purification a procedure was developed involving hydrophobic chromatography on octyl-Sepharose 4B and gel filtration on Sephadex G-100. A rapid purification of small amounts of proteinase B was achieved by affinity chromatography on the nitrated proteinase B inhibitor, immobilized on CH-Sepharose according to Bünning and Holzer (Bünning, P. and Holzer, H. (1977). J. Biol. Chem. 252, 5316-5323). The enzyme prepared from all three sources appeared to be homogeneous and exhibited a molecular weight of 33 000 in SDS-polyacrylamide gel electrophoresis. Homogeneity and molecular weight were confirmed for the enzyme from baker's yeast by ultracentrifugation studies. Polyacrylamide gel electrophoresis without SDS and electrofocusing however, indicated microheterogeneity of the proteinase B activity. The aminoterminal residue of the enzyme was found to be glycine. Proteinase B turned out to be a glycoprotein, containing 8-9% neutral sugars and 1.5% amino sugars. The enzyme is blocked by p-hydroxymercuribenzoate and by the serine proteinase inhibitors DFP and PMSF. Among the proteinase inhibitors from microbial origin, chymostatin and antipain were the most powerful inhibitors of proteinase B.

Amino Acids↗
PubMed1981

The substrate specificity of proteinase B from baker's yeast.

The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied. Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity. The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA. The Km value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only 1/20 that of chymotrypsin A. Km and kcat for Bz-Arg-OEt are 1/50 and 1/7 as high as the corresponding values determined for trypsin. Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25). Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26). These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin. When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.

Binding Sites↗
PubMed1981

Immunochemical studies on catabolite inactivation of phosphoenolpyruvate carboxykinase in Saccharomyces cerevisiae.

Phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was purified to homogeneity. The enzyme is composed of four subunits of Mr = 64,000. Specific antibodies against phosphoenolpyruvate carboxykinase were raised in rabbits and purified by affinity chromatography. Phosphoenolpyruvate carboxykinase is rapidly inactivated when glucose is added to cells starved for carbon (Haarasilta, S., and Oura, E. (1975) Eur. J. Biochem. 52, 1-7; Gancedo, C., and Schwerzmann, K. (1976)( ARch. Microbiol. 109, 221-225). In the present study this inactivation has been analyzed by immunochemical techniques. It was found that the loss of catalytic activity is paralleled by a decrease in cross-reacting material which suggests degradation of the enzyme. In the absence of glucose the enzyme is degraded very slowly, which indicates that glucose-induced inactivation cannot simply be due to repression of enzyme synthesis in the presence of a rapid rate of degradation. Experiments with a proteinase-deficient mutant showed that proteinase B, carboxypeptidase Y, and carboxypeptidase S are not involved in the inactivation system.

Antibodies↗