Microsomal palmitoyl-coenzyme A synthetase from rat liver. Partial and exchange reactions

Abstract

The partial and exchange reactions of long-chain fatty acid activation were determined by using purified microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3). No significant ATP formation from palmitoyl-AMP and PP(i), nor palmitoyl-AMP-dependent CoA disappearance could be demonstrated. Similarly, no palmitate-dependent [(32)P(2)]PP(i)-ATP exchange was catalysed by the pure enzyme. The above partial and exchange reactions were, however, catalysed by the parent microsomal fraction at a rate similar to that of the overall reaction. The implications of these results are discussed.

Palmitoyl-coenzyme A synthetase. Mechanism of reaction

“…Whereas enzyme-bound palmitate is the intermediate ofthe Bi Bi Uni Uni Ping Pong mechanism, the ternary complex consisting of AMP, palmitate and enzyme is the intermediate of the Bi Uni Uni Bi Ping Pong alternative. As pointed out by Bar-Tana et al (1972b), no partial reactions with exogenous palmitoyl-AMP as substrate could be shown with the pure enzyme fraction, indicating perhaps that the enzyme-bound palmitate alternative is a better description of the intermediate than is enzyme-bound palmitoyl-AMP.…”

“…In support of this hypothesis, rat liver microsomal fractions were shown to catalyse the formation of ATP or palmitoyl-CoA from palmitoyl-AMP in the presence of PP, or CoA respectively, as well as a [32P2]PPI-ATP exchange reaction. However, by using a purified long-chain fatty acyl-CoA synthetase (Bar-Tana et al, 1971) the activity of these partial reactions and the palmitatedependent [32P2]PP1-ATP exchange reaction were found to be nearly negligible (Bar-Tana et al, 1972b).…”

“…Vol. 131 Experimental Materials These were as described by Bar-Tana et al (1972b) except for inorganic pyrophosphatase, obtained from Sigma Chemical Co., St. Louis, Mo., U.S.A., bis-(trimethylsilyl)acetamide, obtained from Aldrich Chemical Co. Inc., Milwaukee, Wis., U.S.A., and Dowex 1 (X2; Cl-form; 200-40 mesh) from Dow Chemicals Co. (through Fluka A.G., Buchs SG, Switzerland). [9,10-3H]Palmiticacid, [1-14C]palmitic acid and [U-14C]ATP were obtained from The Radiochemical Centre, Amersham, Bucks., U.K. Palmitoyl-CoA and [y-32P]ATP were prepared as described previously (Bar-Tana et al, 1971).…”

Palmitoyl-coenzyme A synthetase. Isolation of an enzyme-bound intermediate

“…The distinction between the alternatives presented seemed to be of interest, since no partial reactions using synthetic palmitoyl-AMP as substrate, to yield ATP or palmitoyl-CoA in the presence of PPi or CoA respectively, were found to be catalysed by the purified enzyme (Bar-Tana et al, 1972). In the present paper the isolation of the enzyme-bound intermediate is described and its nature is discussed.…”

Effect of Dexamethasone on the Synthesis of Dipalmitoyl Phosphatidylcholine

“…The assay for this enzyme was modified from Bar-Tana et al [3]. Because of the presence of very active ATP-ases in lung microsomes, an ATPgenerating system was used.…”