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. 1974 Mar;53(3):402–410. doi: 10.1104/pp.53.3.402

An Apparent Oligomer of Malate Dehydrogenase from Bean Leaves 1

William Habig a,2, David Racusen a
PMCID: PMC543231  PMID: 16658714

Abstract

Two forms of malate dehydrogenase of widely differing molecular weight have been examined from primary leaves of Phaseolus vulgaris. In addition to the normal 69,000 molecular weight enzyme, an unusual form of 280,000 molecular weight may be detected by sucrose density gradient centrifugation or gel filtration with Sephadex G-200. Isopycnic density gradient centrifugation showed that both forms of malate dehydrogenase differed markedly from the bulk of the leaf protein by their low bouyant density of 1.261 g/cm3.

High molecular weight (280,000) malate dehydrogenase could be converted to active low molecular weight (69,000) malate dehydrogenase by treatment with 2.5 m CsCl, 1.0 m NaCl, 6 m urea, pH 6.5 or below, or one freeze-thaw cycle. Simple removal of salt or raising the pH were not effective in reforming the high molecular weight malate dehydrogenase after dissociation. The high molecular weight enzyme was not dissociated during prolonged dialysis against 0.1 m NaCl or 0.05 m phosphate, pH 7.0. Calcium at concentrations up to 0.1 m produced no activation or differential response in the two MDH forms.

High and low molecular weight malate dehydrogenase were nearly identical in susceptibility to inhibition by various unreactive substrate analogs. However, there was a marked difference in the ability of the two forms of malate dehydrogenase to reduce 3-acetylpyridine-deamino-NAD. This difference in activity was the basis of a convenient assay for determining the ratio of high to low molecular weight malate dehydrogenase in crude extracts. The pH activity profiles and Michaelis constant for malate were nearly identical for the two molecular weight forms.

Analysis by polyacrylamide gel electrophoresis revealed one high molecular weight and two low molecular weight malate dehydrogenase zones. Dissociation of high molecular weight malate dehydrogenase resulted in formation of low molecular weight enzyme whose electrophoretic properties differed from the normal low molecular weight forms.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BEERS R. F., Jr, SIZER I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952 Mar;195(1):133–140. [PubMed] [Google Scholar]
  3. Breidenbach R. W. Characterization of some glyoxysomal proteins. Ann N Y Acad Sci. 1969 Dec 19;168(2):342–347. doi: 10.1111/j.1749-6632.1969.tb43120.x. [DOI] [PubMed] [Google Scholar]
  4. Bronovitskaia Z. S., Kretovich V. L. Molekuliarnyi ves malatdegidrogenazy semiadolei soi. Dokl Akad Nauk SSSR. 1970 Jan;190(2):461–463. [PubMed] [Google Scholar]
  5. CIOTTI M. M., KAPLAN N. O., STOLZENBACH F. E. Reaction of pyridine nucleotide analogues with dehydrogenases. J Biol Chem. 1956 Aug;221(2):833–844. [PubMed] [Google Scholar]
  6. Chilson O. P., Kitto G. B., Pudles J., Kaplan N. O. Reversible inactivation of dehydrogenases. J Biol Chem. 1966 May 25;241(10):2431–2445. [PubMed] [Google Scholar]
  7. Consiglio E., Varrone S., Covelli I. Characterization of the heavy form (9S) of mitochondrial malate dehydrogenase. Eur J Biochem. 1970 Dec;17(3):408–414. doi: 10.1111/j.1432-1033.1970.tb01180.x. [DOI] [PubMed] [Google Scholar]
  8. DAVIES D. D., KUN E. Isolation and properties of malic dehydrogenase from ox-heart mitochondria. Biochem J. 1957 Jun;66(2):307–316. doi: 10.1042/bj0660307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DAVIES D. D. The specificity of malic dehydrogenase from higher plants. Biochem J. 1961 Jul;80:93–99. doi: 10.1042/bj0800093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  11. GRIMM F. C., DOHERTY D. G. Properties of the two forms of malic dehydrogenase from beef heart. J Biol Chem. 1961 Jul;236:1980–1985. [PubMed] [Google Scholar]
  12. Greenway H. Salt responses of enzymes from species differing in salt tolerance. Plant Physiol. 1972 Feb;49(2):256–259. doi: 10.1104/pp.49.2.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hiatt A. J., Evans H. J. Influence of Salts on Activity of Malic Dehydrogenase from Spinach Leaves. Plant Physiol. 1960 Sep;35(5):662–672. doi: 10.1104/pp.35.5.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. JOYCE B. K., GRISOLIA S. Variations in malic dehydrogenase activity, with lyophilization, dialysis, and conditions of incubation. J Biol Chem. 1961 Mar;236:725–729. [PubMed] [Google Scholar]
  15. KAPLAN N. O., CIOTTI M. M. Evolution and differentiation of dehvdrogenases. Ann N Y Acad Sci. 1961 Nov 2;94:701–722. doi: 10.1111/j.1749-6632.1961.tb35567.x. [DOI] [PubMed] [Google Scholar]
  16. Koen A. L., Shaw C. R. Multiple substrate specificities of some dehydrogenase molecules. Biochem Biophys Res Commun. 1964 Feb 18;15(1):92–99. doi: 10.1016/0006-291x(64)90109-3. [DOI] [PubMed] [Google Scholar]
  17. Kuramitsu H. K. The effects of adenine nucleotides on pig heart malate dehydrogenase. Biochem Biophys Res Commun. 1966 May 3;23(3):329–334. doi: 10.1016/0006-291x(66)90550-x. [DOI] [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Levitzki A., Koshland D. E., Jr Ligand-induced association-dissociation as a means for enzyme purification. Biochim Biophys Acta. 1970 Jun 10;206(3):473–475. doi: 10.1016/0005-2744(70)90162-2. [DOI] [PubMed] [Google Scholar]
  20. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  21. Mukerji S. K., Ting I. P. Malic dehydrogenase isoenzymes in green stem tissue of Opuntia: isolation and characterization. Arch Biochem Biophys. 1969 May;131(2):336–351. doi: 10.1016/0003-9861(69)90406-8. [DOI] [PubMed] [Google Scholar]
  22. Murphey W. H., Barnaby C., Lin F. J., Kaplan N. O. Malate dehydrogenases. II. Purification and properties of Bacillus subtilis, Bacillus stearothermophilus, and Escherichia coli malate dehydrogenases. J Biol Chem. 1967 Apr 10;242(7):1548–1559. [PubMed] [Google Scholar]
  23. Murphey W. H., Kitto G. B., Everse J., Kaplan N. Malate dehydrogenases. I. A survey of molecular size measured by gel filtration. Biochemistry. 1967 Feb;6(2):603–610. doi: 10.1021/bi00854a031. [DOI] [PubMed] [Google Scholar]
  24. O'sullivan S. A., Wedding R. T. Malate dehydrogenase isoenzymes from cotton leaves: molecular weights. Plant Physiol. 1972 Feb;49(2):117–123. doi: 10.1104/pp.49.2.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Racusen D. Double-disc electrophoresis of proteins. Nature. 1967 Mar 4;213(5079):922–922. doi: 10.1038/213922a0. [DOI] [PubMed] [Google Scholar]
  26. Rocha V., Ting I. P. Malate dehydrogenases of leaf tissue from Spinacia oleracea: properties of three isoenzymes. Arch Biochem Biophys. 1971 Nov;147(1):114–122. doi: 10.1016/0003-9861(71)90316-x. [DOI] [PubMed] [Google Scholar]
  27. SIEGEL L., ENGLARD S. Beef-heart malic dehydrogenases. I. Properties of the enzyme purified from extracts of acetone-dried powders. Biochim Biophys Acta. 1961 Nov 25;54:67–76. doi: 10.1016/0006-3002(61)90938-6. [DOI] [PubMed] [Google Scholar]
  28. THORNE C. J. Properties of mitochondrial malate dehydrogenases. Biochim Biophys Acta. 1962 Jun 4;59:624–633. doi: 10.1016/0006-3002(62)90642-x. [DOI] [PubMed] [Google Scholar]
  29. Ting I. P. Malic dehydrogenases in corn root tips. Arch Biochem Biophys. 1968 Jul;126(1):1–7. doi: 10.1016/0003-9861(68)90552-3. [DOI] [PubMed] [Google Scholar]
  30. WOLFE R. G., NEILANDS J. B. Some molecular and kinetic properties of heart malic dehydrogenase. J Biol Chem. 1956 Jul;221(1):61–69. [PubMed] [Google Scholar]
  31. Wedding R. T., Hansch C., Fukuto T. R. Inhibition of malate dehydrogenase by phenols and the influence of ring substituents on their inhibitory effectiveness. Arch Biochem Biophys. 1967 Jul;121(1):9–21. doi: 10.1016/0003-9861(67)90004-5. [DOI] [PubMed] [Google Scholar]
  32. Weimberg R. Effect of sodium chloride on the activity of a soluble malate dehydrogenase from pea seeds. J Biol Chem. 1967 Jun 25;242(12):3000–3006. [PubMed] [Google Scholar]
  33. YOSHIDA A. ENZYMIC PROPERTIES OF MALATE DEHYDROGENASE OF BACILLUS SUBTILIS. J Biol Chem. 1965 Mar;240:1118–1124. [PubMed] [Google Scholar]
  34. YOSHIDA A. PURIFICATION AND CHEMICAL CHARACTERIZATION OF MALATE DEHYDROGENASE OF BACILLUS SUBTILIS. J Biol Chem. 1965 Mar;240:1113–1117. [PubMed] [Google Scholar]

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