Solubilization and immunochemical identification of mitochondrial glycerol-3-phosphate dehydrogenase.
Lysophosphatidylcholine (contrary to Lubrol WX, Triton X-100, digitonine and deoxycholate) solubilizes hamster brown fat mitochondrial glycerol-3-phosphate dehydrogenase without inactivation. Optimal ratio of lysophosphatidylcholine and membrane protein for solubilization of the enzyme was found to be 0.25 mg of lysophosphatidylcholine per mg protein. The activity of solubilized enzyme, however, was not affected by low concentrations of Lubrol WX, Triton X-100, digitonine, Zwittergent TM 314. Deoxycholate exhibited a pronounced inactivating effect. One-dimensional immunoelectrophoresis of the solubilized membrane proteins revealed 10 protein bands, 3-4 of which exhibited the enzyme activity. Two-dimensional immunoelectrophoresis revealed only a single main band of glycerol-3-phosphate dehydrogenase. This technique thus appears to be the best means for the identification of glycerol-3-phosphate dehydrogenase in the mixture of solubilized membrane proteins and for concentration of the enzyme activity in one major precipitating band.
Rauchová
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H
Haskovec
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Drahota
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D002001:Adipose Tissue, Brown / Q000201:enzymology; D000818:Animals; D006224:Cricetinae; D003902:Detergents; D004586:Electrophoresis; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D007120:Immunochemistry; D008244:Lysophosphatidylcholines; D008647:Mesocricetus; D008722:Methods; D008928:Mitochondria / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D008244:Lysophosphatidylcholines; D005993:Glycerolphosphate Dehydrogenase; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+)