Solubilization, purification, and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei.
Succinate dehydrogenase (SDH) was solubilized from membranes of Mycobacterium phlei by Triton X-100 with a recovery of about 90%. The solubilized SDH was purified about 90-fold by Sephacryl S-300, DEAE-cellulose, hydroxylapatite, and isoelectric focusing in the presence of Triton X-100 with a 20% recovery. SDH was homogeneous, as determined by polyacrylamide gel electrophoresis in nondenaturing gels containing Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme revealed two subunits with molecular weights of 62,000 and 26,000. SDH is a flavoprotein containing 1 mol of flavin adenine dinucleotide, 7 to 8 mol of nonheme iron, and 7 to 8 mol of acid-labile sulfide per mol of protein. Using phenazine methosulfate and 2,6-dichloroindophenol as electron acceptors, the enzyme had an apparent Km of 0.12 mM succinate. SDH exhibited a sigmoidal relationship of rate to succinate concentration, indicating cooperativity. The enzyme was competitively inhibited by fumarate with a Ki of 0.15 mM. In the absence of Triton X-100, the enzyme aggregated, retained 50% of the activity, and could be resolubilized with Triton X-100 with full restoration of activity. Cardiolipin had no effect on the enzyme activity in the absence of Triton X-100, but it stimulated the activity by about 30% in the presence of 0.1% Triton X-100 in the assay mixture. Menaquinone-9(2H), isolated from M. phlei, had no effect on the enzyme activity either in the presence or absence of Triton X-100.
D002308:Cardiolipins / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009161:Mycobacterium / Q000201:enzymology*; D009168:Mycobacterium phlei / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012995:Solubility; D013385:Succinate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D002308:Cardiolipins; D013386:Succinates; D011092:Polyethylene Glycols; D017830:Octoxynol; D019802:Succinic Acid; D013385:Succinate Dehydrogenase