AR(:HLv,Gs OF IJIOCHICMISTItY AND BIOPHYSICS 128, 124-128 (1968) Optimal Conditions for Palmitate Oxidation by Rat Heart Homogenates’ SUSANA PASSERON; MICHAEL A. SAVAGEAU,3 AND ISAAC HARARY Department of Biophysics, Laboratory of Nuclear Medicine and Radiation Biology, and Department of Biological Chemistry, UCLA School of Medicine, and Institute of Molecular Biology, University of California, Los Angeles, California 90024 Received March 28, 1968, accepted July 9, 1968 A study of the oxidation of palmitate-1-C I4 to CY402 by heart homogenates was undertaken and the opt.imal conditions were determined. The syst.em has an absolute requirement for carnitine, CoA, ATP, and Mg*+. The levels of ATP and Mg2+ for the optimal rate of palmitate oxidation are interdependent. Excess of ATP or Mg2+ in- hibits the production of COP. The amount of fatty acid oxidized is dependent on the molar ratio of fatty acid to albumin in the incubation medium: an optimal ratio of approximately 5 was found for all t,he concentrations of fatt,y acid and albumin tested. A tentative mechanism for the albumin effect is presented. The heart readily utilizes long-chain free fatty acids (FFA) as oxidative substrates (l-3). However, most studies on fatty acid oxidation have been concerned with short- chain fatty acids and their derivatives (4-6) presumably because of solubility problems with long-chain fatty acids (6, 7). FFA are present in the blood plasma as a complex with albumin (8). It thus appears that albu- min serves as a transport vehicle for FFA and that the albumin-FFA complex must be thought of as an entity with considerable biological significance. The present work was undertaken to investigate the optimal con- 1 These studies were supported in part by Con- tract AT(04-l)GEN-12 between the Atomic Energy Commission and the University of Cali- fornia and by Research Grant SROlHE-11216-09 from the National Institutes of Health, U. S Public Health Service. 2 Present address: Inatituto de Investigaciones Bioquimicas, “Fundaci6n Campomar” and Facul- tad de Ciencias Exact,as y Naturales, Obligado 2490, Buenos Aires (28), Republica Argentina. 3 Supported by postdoctoral fellowship l-F2- HE-29, 840-01 from the National Heart Institute. Present address: Fleischmann Laboratories, Stan- ford University School of Medicine, Palo Alto, California 94304. ditions for palmitate oxidation in rat heart homogenates. The data presented here are consistent with the hypothesis that in cell- free preparations t’he rate of FFA oxidation is determined in part by the ratio FFA: albu- min and also by t’he ratio ATP: Mg”+. MATERIALS AND METHODS Chemicals. Palmitate labeled in the carboxyl group was purchased from New England Nuclear Corporation (Boston, Massachusetts). Unlabeled palmitate and oL-carnitine were obtained from California Corporation of Biochemical Research (Los Angeles, California), NAD and ATP from Sigma Chemical Co. (St. Louis, Missouri), and CoA from Boehringer Mannheim, Germany. Bovine plasma albumin (fraction X) was obtained from Pentex and extracted by the met,hod of Chen (9) so that the endogenous fatty acid was reduced to less than 0.02 eq/mole protein. Sephadex G-25 was purchased from Pharmacia A. G. (Uppsala, Sweden). All other reagents used were of analyti- cal grade. Preparation of heart eztracts. Male and female albino Wist.ar rats weighing 100-300 g were used. They were maintained on a standard laboratory diet and water ad l&turn. The animals were sacri- ficed by a sharp blow on t.he head, and the heart (0.7-1.0 g) was quickly removed and finely diced with scissors. The cut pieces were then homoge- 124