Purification, properties, and specificity of rat brain cytosolic fatty acyl coenzyme A hydrolase.
Rat brain cytosolic acyl-CoA hydrolase has been purified 3,500-fold to apparent homogeneity using heat treatment, ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and hydroxyapatite c chromatography. The purified enzyme remains stable only in the presence of a high concentration (30%, vol/vol) of ethylene glycol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme shows a single band of 40.9 kDa. However, on high-performance size-exclusion chromatography the migration rate of the enzyme corresponds with an apparent molecular mass of 148 kDa, indicating that the native enzyme may be a tetramer. The enzyme catalyzes the hydrolysis of fatty acyl-CoAs from six to 18 carbon chains long, having the highest activity for lauroyl (12:0)-CoA. For the purified enzyme the Km for palmitoyl-CoA is 5.8 microM and the Vmax is 1,300 mumol/min/mg of protein. The enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carnitine. Among the sulfhydryl agents, only p-hydroxymercuribenzoate inhibited the enzyme. The enzyme is also inactivated by treatment with a high concentration of diethyl pyrocarbonate, an active center histidine-reacting agent, but not by phenylmethylsulfonyl fluoride (10 mM), a serine esterase inhibitor. The purified enzyme does not appear to possess any O-ester hydrolase, lysophospholipase, transacylase, or acyltransferase activity.
Broustas
C G
CG
Department of Biological Chemistry and Mental Health Research Institute, University of Michigan, Ann Arbor 48104-1687, USA.
Hajra
A K
AK
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D001921:Brain / Q000201:enzymology*; D011232:Chemical Precipitation; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D005590:Fractional Precipitation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010170:Palmitoyl-CoA Hydrolase / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D000596:Amino Acids; D010170:Palmitoyl-CoA Hydrolase; D000645:Ammonium Sulfate