Improved procedures for the synthesis of phosphomevalonate and for the assay and purification of pig liver phosphomevalonate kinase.
An improved procedure for the synthesis of phosphomevalonate using excess free ATP4-, and phenyl agarose to remove contaminating nucleotides, is described. A high-voltage electrophoresis assay, which separates phosphomevalonate from mevalonate 5-diphosphate at pH 3.5, was developed for the assay of phosphomevalonate kinase (ATP:5-phosphomevalonate phosphotransferase, EC 2.7.4.2). High-voltage electrophoresis, at pH 5, could also be used for the separation of mevalonate 5-diphosphate from isopentenyl diphosphate. An alternative method for the purification of phosphomevalonate kinase from pig liver was also developed. The high-voltage electrophoresis assay was used to reassess the metal ion and nucleotide specificity of the pig liver phosphomevalonate kinase. ATP could be partially replaced by ITP and GTP and poorly by CTP and UTP. Apparent activation of the enzyme by free ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224).
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D000818:Animals; D002413:Cations, Divalent; D004586:Electrophoresis; D008099:Liver / Q000201:enzymology*; D008722:Methods; D008798:Mevalonic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D008970:Molecular Weight; D010770:Phosphotransferases / Q000032:analysis* / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor); D013379:Substrate Specificity; D013552:Swine
D002413:Cations, Divalent; C045038:phosphomevalonic acid; D010770:Phosphotransferases; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase; D008798:Mevalonic Acid