[70] Lactate Dehydrogenase-X from Mouse Testes and Spermatozoa

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This chapter describes the assay method, purification procedure, and properties of lactate dehydrogenase-X from mouse testes and spermatozoa. LDH-X from mouse testes has a high affinity for other α-keto and α-hydroxy acids. To estimate LDH-X in a mixture of LDH isozymes, pyruvate is replaced in the reaction mixture by 60 nmoles of α-ketovalerate. The ratio of α-ketovalerate/pyruvate activity is a close approximation of the percentage of LDH-X in a testes extract. The purification procedure includes steps, such as crude extraction, heat step, first ammonium sulfate step, second ammonium sulfate step, third ammonium sulfate step, chromatography on DEAE-sephadex (A-50), final purification with ammonium sulfate, and crystallization of the enzyme. The birefringent crystals of LDH-X from mouse testes are thin laths about 0.15 mm thick, 0.1 mm wide and over 0.4 mm long. The crystalline preparation is homogeneous by ultracentrifugal analysis, polyacrylamide gel electrophoresis, and immunological analysis. The crystalline suspension is stable for several months at 4°. One of the more striking characteristics of LDH-X is its rather broad substrate specificity. The enzyme catalyzes reversibly, the reduction of α-ketobntyrate and α-ketovalerate as well as α-ketoglutarate, in the presence of NADH. LDH-X is generally less thermolabile than the other isozymes. In the mouse, the half-life of each isozyme at 65° is 9 min for LDH-5, 10 min for LDH-1, and 43 min for LDH-X. Oxamate and oxalate are competitive inhibitors of pyruvate reduction and lactate oxidation respectively catalyzed by LDH-X. The turnover number of LDH-X with pyruvate is considerably lower than the other isozymes. The chapter also discusses the purification of LDH-X from spermatozoa by affinity chromatography.

Original languageEnglish (US)
Pages (from-to)318-323
Number of pages6
JournalMethods in Enzymology
Issue numberC
StatePublished - Jan 1 1975

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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