Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain.
Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10mum-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl(2), and 5,5'-dithiobis-(2-nitrobenzoic acid), and exhibit the same K(m) (1.8mum) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0 degrees C), but there is no change in the K(m) with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10mum-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D004355:Drug Stability; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010170:Palmitoyl-CoA Hydrolase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013869:Thiolester Hydrolases / Q000378:metabolism*
D013439:Sulfhydryl Reagents; D010171:Palmitoyl Coenzyme A; D013869:Thiolester Hydrolases; D010170:Palmitoyl-CoA Hydrolase