Purification of β-hydroxy-β-methylglutaryl-coenzyme A reductase from yeast

Abstract

Beta-Hydroxy-beta-methylglutaryl-coenzyme A reductase of yeast has been solubilized by two different methods and then purified approximately 5000-fold. The purified enzyme shows a single precipitin band on immunodiffusion, and it moves as a single band of protein and enzyme activity on gel filtration and diethylaminoethylcellulose column chromatography. It also shows one major band on polyacrylamide gel electrophoresis. The specific activity of the pure enzyme is 18 000 to 22 000 nmol of reduced nicotinamide adenine dinucleotide phosphate oxidized per min per mg of protein. The molecular weights of the enzyme, estimated by gel filtration, and the subunits, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 2.6 X 10(5) and 6.0 X 10(4), respectively.

Genetic and Structural Analysis of Hmg2p-induced Endoplasmic Reticulum Remodeling inSaccharomyces cerevisiae

“…Next we tested whether the Hmg2p cytoplasmic hN-domain fused to GFP, as in the ER altering Hmg2p s -TM-hN-GFP mentioned above, could stimulate membrane reorganization (Ole1p i -2hN-GFP). We again did not observe any structures, even when TDH3pr-driven The CD of mammalian HMGR forms homodimers and homotetramers, and biochemical evidence indicates that the same holds true for the highly conserved yeast CD (Qureshi et al, 1976). To determine whether the CDs of yeast HMGR have an intrinsic ability to reorganize membranes, potentially via multimerization, we generated a fusion between Ole1p i and the entire 425 amino acid CD of Hmg2p (Ole1p i -Hmg2p CD) and expressed it from the TDH3pr.…”

Purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver.

“…In a previous study (12) we succeeded in purifying yeast HMG-CoA reductase to homogeneity. This enzyme had a specific activity of approximately 10,000 nmol of mevalonate formed per min/mg of protein.…”

“…In addition, workers in three laboratories have reported the preparation of enzyme that yields only one band on immunodiffusion or sodium dodecyl sulfate (NaDodSO4) disc gel electrophoresis (9-11). However, the enzyme activities of these preparations were very low (10-516 nmol of mevalonate formed per min/mg of protein).In a previous study (12) we succeeded in purifying yeast HMG-CoA reductase to homogeneity. This enzyme had a specific activity of approximately 10,000 nmol of mevalonate formed per min/mg of protein.…”

Purification and characterization of 3‐hydroxy‐3‐methylglutaryl coenzyme a reductase from chicken liver

“…The isolated enzyme has a specific activity of approximately 1700 nmol/min/mg, which is significantly greater than that generally reported for the rat enzyme. A molecular weight by SDS electrophoresis 128 of 18 000 for avian reductase is lower than the molecular weight of 50 000-65 000 reported for the rat [10,16,19] and yeast [20] enzyme. In our laboratory, rat HMG-CoA reductase, purified by sequential chromatography on agarose-blue dextran and agarosehexane-coenzyme A also had an apparent monomer molecular weight of approx.…”

“…Affinity chromatography employing agarosehexane-coenzyme A has been utilized to partially purify rat-liver HMG-CoA reductase* and has been recently incorporated into the purification procedures for the isolation of reductase from rat microsomes [19] and yeast [20] . In the present studies, we have employed dextran-blue affinity chromatography for the final step in purification of chicken HMG-CoA reductase.…”