Biosynthesis of Phosphatidic Acid in Mitochondria and Microsomes Via the Acylation of sn-Glycero-3-phosphate

Abstract

Nuclei-free homogenate, prepared from guinea pig livers, was fractionated into subcellular particles which were then examined for the activities of two microsomal marker enzymes, glucose-6-phosphatase and NADPH: cytochrome c reductase. In an incubation system containing sn-glycero-3-phosphate, fatty acid, and various cofactors the intracellular distribution of acyl-CoA: sn-glycero-3-phosphate acyltransferase(s) was studied and compared with the distribution of the two microsomal marker enzymes.Results obtained showed that the highest specific activity for the acylation of sn-glycero-3-phosphate was associated with the microsomal fraction and the activity in each subcellular fraction paralleled activities of the two microsomal marker enzymes. Furthermore, the amount of acyl-CoA: sn-glycero-3-phosphate acyltransferase activity observed in the mitochondrial and submitochondrial fractions could be accounted for by the content of endoplasmic reticulum as determined by the marker enzymes. This observation was also true for brain, heart, and kidney, as well as for rat liver.These results are interpreted as evidence that isolated mitochondria are unable to synthesize phosphatidic acid by direct acylation of sn-glycero-3-phosphate.

Evidence for the Biosynthetic Difference between Isolated Mitochondria and Microsomes from Guinea-Pig and Rat Liver Regarding Lysophospharidic Acid, Phosphatidic Acid, CDP-Diglyceride, Phosphatidylglycerol, and Cardiolipin

“…in previous communications from this laboratory (2,4). Results presented in this communication were obtained from four independent enzyme preparations and repre-…”

“…sn-Glycero-3-phosphate-2-3H and all other chemicals and reagents were of the origin and purity previously deseribed (2).…”

“…Mitochondria and microsomes were prepared from guinea-pig and rat liver homogenates as described (2).…”

“…Assays for determination of NADPH -cytochrome c reductase and glucose-6-phosphatase were described elsewhere (2). The assay of acyl-CoA:sn-glycero-3-phosphate acyltransferase (2) was modified as follows: the incubation volume was 0.25 ml with the amounts of all components reduced by one half, 0.5 rng of bovine serum albumin was added, the incubation time was 3 min, and labelled lipids were extracted according to the acidic Bligh and Dyer procedure (3).…”

Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes

“…Guinea pig liver mitochondria and microsornes were prepared as described (5). NADPH -cytochrome c redarctase activity was determined as described (5).…”