Biosynthesis of phosphatidic acid in rat brain via acyl dihydroxyacetone phosphate.

The enzymes for the biosynthesis of phosphatidic acid from acyl dihydroxyacetone phosphate were shown to be present in rat brain. These enzymes were mainly localized in the microsomal fraction of 12--14 day old rat brains. The brain microsomal acyl C0A:dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42). exhibited a broad pH optimum between pH 5 and 9 with maximum activity at pH 5.4. K, for DHAP at pH 5.4 was 0.1 mM and V,,, was 0.86nmol/min/mg of microsomal protein. The corresponding microsomal enzyme for the glycerophosphate pathway (acyl CoA: sn-gIycerol-3phosphate acyl transferase EC 2.3.1.15) was shown to have a different pH optimum (pH 7.6). On the basis of the differences in pH optima, differential effects of sodium cholate in the enzymes and a common substrate competition study, these acyl transferases were postulated to be two different microsomal enzymes. Acyl DHAP:NADPH oxidoreductase (EC 1.1.1.101) in brain microsomes was found to be quite specific for NADPH as cofactor. being able to utilize NADH only at very high concentrations. This enzyme exhibited a K , of 8 . 6 ~ ~ with NADPH and V,, of 0.81 nmol/min/mg protein. The presence of these two enzymes and the known presence of 1-acyl-sn-glycerol-3-phosphate:acyl CoA acyl transferase in brain (FLEMING & HAJRA, 1977) demonstrated the biosynthesis of phosphatidic acid in brain via acyl dihydroxyacetone phosphate. Phosphatidic acid was shown to form when dihydroxyacetone phosphate, acyl CoA, NADPH and other cofactors were incubated together with brain microsomes Further properties of the enzymes and the probable importance of the presence of this pathway in brain were discussed. PHOSPHAIIDIC acid (1,2-diacyl-sn-glyceroI-3-phosphate) has been shown to be a precursor of all glycerides and phosphoglycerides in different systems (KENNEDY, 1962; VAN DEN BOSCH, 1974). A number of workers have shown that phosphatidate in brain is biosynthesized by the acylation of sn-glycerol-3-phosphate (G-3-P)' (MCMURRAY e t a/., 1957; MARTENSSON & KANFER, 1968; SANCHEZ DE JIMINEZ & CLELAND, 1969; KUWAHARA, 1972; POSMAYER et a / . , 1972). Recently, in liver and other systems, we have shown that phosphatidic acid is also biosynthesized by an alternate pathway via I-acyl dihydroxyacetone-3-phosphate (acyl DHAP) (HAJRA, 1968b, 1977). Acyl DHAP, which is formed by a direct acylation of DHAP with acyl CoA (acyl CoA:DHAP acyl transferase E C 2.3.1.42) (HAJRA, 19686; LABELLE & HAJRA, 1972b) is enzymatically reduced by NADPH (acyl DHAP:NADPH oxidoreductase EC 1.1.1.101) (HAJRA & AGRANOFF. 1968b; LABELLE & HAJRA, 19720) to I-acyl-sn-G-3-P (lysophosphatidic acid). Lysophosphatidate is then converted to phosphatidate by the widely distributed acyl transferase, acyl CoA : I-acyl-G-3-P acyl transferase (FLEMING & HMRA, 1977). Preliminary studies show that the enzymes of the acyl DHAP pathway are present in Abbreuiarions used: G-3-P-sn-glycerol-3-phosphate; DHAP-dihydroxyacetone phosphate. brain (LABELLE & HAJRA, 1972% b ; POLLACK et a/., 1975). However, a detailed study shows that the distribution of the enzymes and their properties are somewhat different than the acyl DHAP pathway enzymes in liver. These results are presented here. A comparison of the acyl CoA:DHAP acyl transferase and the well studied acyl CoA :G-3-P acyl transferase in brain is also made. MATERIALS AND METHODS H332P0, and [1-'4C]palmitic acid were obtained from New England Nuclear (Cambridge, MA.). Coenzyme A, ATP, o-glucose-6-phosphate, , cytochrome e, NADH, NADPH, 2.3'cyclic AMP, 2(-p-iodophenyl)-3(-pnitrophenylt5-phenyl tetrazolium chloride, o~-gIycerol-3-phosphate, 2"-morpholinolethane sullonic acid (MESA N-2-hydroxyethyIpiperazine-N-2-ethane sulfonic acid (HEPES), Tris, and morpholinopropane sulfonic acid (MOPS) were from Sigma Chemical Corp. (St. Louis, MO.). Palmitoyl CoA, stearoyl CoA, oleoyl CoA, and linoleoyl CoA were purchased from P-L Biochemicals (Milwaukee, WI.). Ficoll (MW -400,OOO) came from Pharmacia Fine Chemicals. Fatty acid poor bovine serum albumin (Fr. V) was from Miles Laboratories (Elkhart, IN). Other materials were obtained as described previously (LABELLE & HAJRA, 1972a,b; FLEMING & HAIRA, 1977). Linoleoyl CoA, arachidonoyl CoA. docosatetraenoyl CoA (n-6) and docosahexaenoyl CoA (n-3) were chemically synthesized from CoA and acyi chloride (SEuBERr, 1960;