Purification of L-3-glycerophosphate dehydrogenase from rat liver mitochondria.
A rapid three-step procedure is presented for the purification of flavin-linked L-3-glycerophosphate dehydrogenase (E.C. 1.1.99.5.) from rat liver mitochondria. Solubilization of the enzyme is achieved selectively by digitonin, at a detergent-to-protein ratio of 0.7 mg/mg (mitochondrial protein concentration 10 mg/ml). The procedure involves chromatography on hydroxymethyl-hexamethylenediamine-succinyl-hexamethylenediamin e Sepharose 4B, followed by anion exchange chromatography using a FPLC technique. Subunit molecular weight of the enzyme was found to be 77,000 when prepared in the presence of the protease inhibitor phenylmethylsulphonyl fluoride. The Kmapp value for glycerophosphate was not influenced by the purification, and the ability of the enzyme to be activated by Ca2+ was preserved as well.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005415:Flavins / Q000302:isolation & purification; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification*; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D012995:Solubility
D005415:Flavins; D002237:Carbohydrate Dehydrogenases; D005993:Glycerolphosphate Dehydrogenase; C056470:3-glycerophosphate dehydrogenase; D002118:Calcium