1887

Abstract

Glycerol : NAD 2-oxidoreductase (EC 1.1.1.6) was purified to homogeneity from the non-methylotrophic yeast H 122. Results of electrophoresis in polyacrylamide gels, gel filtration and ultracentrifugation were compatible with the enzyme’s consisting of two subunits with a molecular weight 38000. No heterogeneity was observed by isoelectric focusing. The pH optima were10·0 for glycerol oxidation and 7·5 for dihydroxyacetone reduction. The values for glycerol, dihydroxyacetone, NAD and NADH were 5·8 10 , 7·7 10 , 1·4 10 and 4·8 10 , respectively. 1,2-Propanediol also served as substrate in the forward and -glyceraldehyde in the reverse reaction. The oxidation product of glycerol was identified as dihydroxyacetone. An ordered bi-bi mechanism was deduced from product-inhibition studies. Of several anions and cations tested, pyrophosphate and ammonium ions stimulated the dehydrogenating activity the most. 2-Mercaptoethanol, ethylene glycol and Tris inhibited activity; an inhibition was also observed by phosphorylated coenzymes and substrates. The purified enzyme, which is labile at low concentrations, was stabilized by the addition of neutralized filtrate from the culture liquid.

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1984-12-01
2024-04-19
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References

  1. Andrews P. 1964; Estimation of the molecular weights of proteins by Sephadex gel filtration. Biochemical Journal 91:222–223
    [Google Scholar]
  2. Babel W., Hofmann K.H. 1981; The conversion of triosephosphate via methylglyoxal, a bypass to the glycolytic sequence in methylotrophic yeasts?. FEMS Microbiology Letters 10:133–136
    [Google Scholar]
  3. Babel W., Hofmann K.H. 1982; The relation between the assimilation of methanol and glycerol in yeasts. Archives of Microbiology 132:179–184
    [Google Scholar]
  4. Böhme H.-J., Kopperschläger G., Schulz J., Hofmann E. 1972; Affinity chromatography of phosphofructokinase using Cibacron blue F3G-A. Journal of Chromatography 69:209–214
    [Google Scholar]
  5. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  6. Burton R.M. 1955; Glycerol dehydrogenase from Aerobacter aerogenes. Methods in Enzymology 1:397–400
    [Google Scholar]
  7. Cleland W.W. 1963; The kinetics of enzyme-catalyzed reactions with two or more substrates or products. Biochimica et biophysica acta 67:104–137
    [Google Scholar]
  8. Dunker A.K., Kenyon A.J. 1976; Mobility of sodium dodecyl sulphate-protein complexes. Biochemical Journal 153:191–197
    [Google Scholar]
  9. Eisenthal R., Cornish-Bowden A. 1974; The direct linear plot.A new graphical procedure for estimating enzyme kinetic parameters. Biochemical Journal 139:715–720
    [Google Scholar]
  10. Flynn T.G., Cromlish J.A. 1982; Glycerol dehydrogenase from rabbit muscle. Methods in Enzymology 89:237–242
    [Google Scholar]
  11. Gancedo C., Gancedo J.M., Sols A. 1968; Glycerol metabolism in yeasts: pathways of utilization and production. European Journal of Biochemistry 5:165–172
    [Google Scholar]
  12. Hanes C.S. 1932; Studies on plant amylases. I. The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley. Biochemical Journal 26:1406–1421
    [Google Scholar]
  13. Hochuli E., Taylor K.E., Dutler H. 1977; Dihydroxyacetone reductase from Mucor javanicus. 2.Identification of the physiological substrate and reactivity towards related compounds. European Journal of Biochemistry 75:433–439
    [Google Scholar]
  14. Hofmann K.H., Babel W. 1979; Synthese und Akkumulation der Glycerin-Dehydrogenase in Candida validaH 122 in Abhängigkeit von der C-Quelle. VII. Reinhardsbrunner Symposium der SektionMikrobiologie der BiologischenGesellschaft der DDR, vom 6. bis 12. Mai. In Abhandlungen der Akademie der Wissenschaften der DDR N3 pp. 229–234 Berlin: Akademie-Verlag 1981;
    [Google Scholar]
  15. Hofmann K.H., Babel W. 1983; Methylglyoxalein toxisches Fermentationsprodukt. Acta Bio technologica 3:21–25
    [Google Scholar]
  16. Itoh N. 1982; Purification and characterization of glycerol dehydrogenase isoenzymes from Geotrichum candidum. Agricultural and Biological Chemistry 46:3029–3039
    [Google Scholar]
  17. Jervis L., Schmidt C.N.G. 1977; Affinity chromatography of potato lactate dehydrogenase. Biochemical Society Transactions 5:1767–1770
    [Google Scholar]
  18. Kawagishi T., Nishio N., Matsuno R., Kamikubo T. 1980; Purification of NAD-dependent 1,2-propanediol dehydrogenating enzyme from Glycerol dehydrogenase from Candida valida Microcycluseburneus. Agricultural and Biological Chemistry 44:949–950
    [Google Scholar]
  19. Koike M., Hamada M. 1971; Preparation of calcium phosphate gel deposited on cellulose. Methods in Enzymology 22:339–342
    [Google Scholar]
  20. Kopperschläger G., Storch H., Birkenmeier G. 1977; Einsatz der Porengradienten-Polyacryl-amidgelelektrophorese zur Diagnostik von Paraproteinamien. Zeitschrift für medizinische Laboratoriumsdiagnostik 18:300–308
    [Google Scholar]
  21. Laas T., Fast-Johansson A. 1979; Isoelectric focusing with Pharmalyte in gel rods. In Protides of Biological Fluids. 27th Colloquium pp. 693–697 Edited by Peeters H. Oxford: Pergamon Press;
    [Google Scholar]
  22. Lin E.C.C. 1976; Glycerol dissimilation and its regulation in bacteria. Annual Review of Microbiology 30:535–578
    [Google Scholar]
  23. Lin E.C.C., Magasanik B. 1960; The activation of glycerol dehydrogenase from Aerobacteraerogenes by monovalent cations. Journal of Biological Chemistry 235:1820–1823
    [Google Scholar]
  24. May J.W., Sloan J. 1981; Glycerol utilization by Schizosaccharomycespombe: dehydrogenation as the initial step. Journal of General Microbiology 123:183–185
    [Google Scholar]
  25. May J.W., Marshall J.H., Sloan J. 1982; Glycerol utilization by Schizosaccharomyces pombe: phosphorylation of dihydroxyacetone by a specific kinase as the second step. Journal of General Microbiology 128:1763–1766
    [Google Scholar]
  26. Mcgregor W.G., Phillips J., Suelter C.H. 1974; Purification and kinetic characterization of a monovalent cation-activated glycerol dehydrogenase from Aerobacter aerogenes. Journal of Biological Chemistry 249:3132–3139
    [Google Scholar]
  27. Nishio N., Kawagishi T., Matsuno R., Kamikubo T. 1978; Metabolism of 1,2-propanediol by methanol-utilizing bacteria and some properties of 1,2-propanediol dehydrogenating enzyme. Agricultural and Biological Chemistry 42:1095–1100
    [Google Scholar]
  28. Reader V. 1927; The relation of the growth of certain micro-organisms to the composition of the medium. I. The synthetic culture medium. Biochemical Journal 21:901–907
    [Google Scholar]
  29. Ruch F.E., Lin E.C.C., Kowit J.D., Goldberg A.L. 1980; In-vivo inactivation of glycerol dehydrogenase in Klebsiellaaerogenes : properties of active and inactivated proteins. Journal of Bacteriology 141:1077–1085
    [Google Scholar]
  30. Scharschmidt M., Pfleiderer G., Metz H., Brümmer W. 1983; Isolierung und Charakterisierung von Glycerin-Dehydrogenase aus Bacillus megaterium. Zeitschrift für physiologischeChemie 364:911–921
    [Google Scholar]
  31. Scrimgeour K.G. 1977; Molecular weight. In Chemistry and Control of Enzyme Reactions pp. 14–l6 London: Academic Press;
    [Google Scholar]
  32. Selwyn M.J. 1965; A simple test for inactivation of an enzyme during assay. Biochimica et biophysica acta 105:193–195
    [Google Scholar]
  33. Sheys G.H., Doughty C.C. 1971a; Subunits of aldose reductase from Rhodotorula. Biochimica et biophysica acta 235:414–417
    [Google Scholar]
  34. Sheys G.H., Doughty C.C. 1971b; The reaction mechanism of aldose reductase from Rhodotorula. Biochimica et biophysica acta 242:523–531
    [Google Scholar]
  35. Sprague G.F., Cronan J.E. 1977; Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism. Journal of Bacteriology 129:1335–1342
    [Google Scholar]
  36. Strickland J.E., Miller O.N. 1968; Inhibition of glycerol dehydrogenase from Aerobacteraerogenes by dihydroxyacetone, high ionic strength, and monovalent cations. Biochimica et biophysica acta 159:221–226
    [Google Scholar]
  37. Tang C.-T., Ruch F.E., Lin E.C.C. 1979; Purification and properties of a nicotinamide adenine dinucleotide-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism. Journal of Bacteriology 140:182–187
    [Google Scholar]
  38. Viswanath-Reddy M., Pyle J.E., Howe H.B. 1978; Purification and properties of NADP+-linked glycerol dehydrogenase from Neurospora crassa. Journal of General Microbiology 107:289–296
    [Google Scholar]
  39. Weber K., Osborn M. 1969; The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244:4406–4412
    [Google Scholar]
  40. Yamada H., Nagao A., Nishise H., Tani Y. 1982; Glycerol dehydrogenase from Cellulomonassp. NT3060: purification and characterization. Agricultural and Biological Chemistry 46:2333–2339
    [Google Scholar]
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