Heterogeneous long chain acyl-CoA synthetases control distribution of individual fatty acids in newly-formed glycerolipids of neuronal cells undergoing neurite outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growt h factor (NGF) were labeled with [H-3]glycerol, [H-3]oleic acid (OA) or [H- 3]arachidonic acid (AA) in the presence of other unlabeled fatty acids of e ndogenous or exogenous origin. Triacsin C (4.8 mu M), an inhibitor of acyl- CoA synthetase, decreased the incorporation of exogenous [H-3]OA into glyce rolipids by 30-90%, and increased by about 60% the accumulation of free [H- 3]OA in the cells. However it did not affect the incorporation of endogenou s fatty acids nor of exogenous [H-3]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is spe cific for AA (ACS), or of the non-specific LCAS which activates OA and othe r fatty acids (OCS), were much higher in microsomal and cytoplasmic fractio ns than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 mu M, respect ively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 mu M, respectively. Triacsin C (2-33 mu M) did not affect ACS activity in micros omal or cytoplasmal fractions, but inhibited OCS activities dose-dependentl y and competitively: IC50 and apparent Ki values were 13.5 mu M and 14 mu M in microsomes, and 3.8 mu M and 4 mu M in cytoplasm. NGF stimulated the ac tivities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heteroge neous with respect to their intracellular locations, substrate specificitie s, kinetic characteristics and sensitivities to triacsin C; and that this h eterogeneity affects the extents to which individual fatty acids are utiliz ed to form glycerolipids.