Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.
Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.
Shechter
I
I
Eleanor Roosevelt Institute, Denver, Colorado 80206.
Klinger
E
E
Rucker
M L
ML
Engstrom
R G
RG
Spirito
J A
JA
Islam
M A
MA
Boettcher
B R
BR
Weinstein
D B
DB
D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000378:metabolism
D011480:Protease Inhibitors; D013186:Farnesyl-Diphosphate Farnesyltransferase; D014357:Trypsin; D004229:Dithiothreitol