G. Hübscher

Metabolism of phospholipids IX. Phosphatidate phosphohydrolase in rat liver

Abstract 1. 1. Rat-liver homogenates were fractionated into mitochondrial, lysosomal, microsomal, and particle-free supernatant fractions which were characterised by specific marker enzymes, and the intracellular distribution of phosphatidate phosphohydrolase ( l -α-phosphatidate phosphohydrolase, EC 3.1.3.4) was studied. 2. 2. The lysosomal fraction had the highest specific activity with respect to phosphatidate phosphohydrolase activity but the mitochondrial and microsomal fractions contained the relatively highest proportions of this enzyme. It was concluded that phosphatidate phosphohydrolase is a true constituent of all three particulate sub-cellular fractions, with some activity occurring in the supernatant. 3. 3. Repeated extraction of the three particulate subcellular fractions by freezing and thawing in 0.3 M sucrose solubilised 60, 25 and 16% of the mitochondrial, lysosomal, and microsomal phosphatidate phosphohydrolase activity, respectively. 4. 4. Investigation of Michaelis constants, pH optima, and the effect of detergents and cations revealed significant differences between the mitochondrial enzyme which had been extracted by freezing and thawing and that which remained insoluble. 5. 5. Examination of an extract obtained from a mitochondrial preparation by repeated freezing and thawing showed that of a number of important enzymes in glyceride and phospholipid biosynthesis only phosphatidate phosphohydrolase was extracted.

The intracellular distribution of the enzymes catalysing the biosynthesis of glycerides in the intestinal mucosa

Abstract 1. 1. The biosynthesis of glycerides in subcellular fractions from the mucosa of cat small intestine was studied with respect to its intracellular localisation. The purity of the subcellular fractions was estimated with the aid of marker enzymes. 2. 2. The enzymes catalysing the biosynthesis of glycerides via the glycerophosphate pathway as well as those of the monoglyceride pathway were localised mainly in the microsomal fraction. 3. 3. Subfractionation of the microsomal proteins into a fraction containing most of the RNA and glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) activity, and a fraction almost completely void of these constituents showed that the former was responsible for the synthesis of glycerides. 4. 4. Further subfractionation of the microsomal fraction into one rough-surface vesicle fraction and two smooth-surface vesicle fractions revealed that the enzymes of the glycerophosphate and monoglyceride pathways were mainly in the roughsurface vesicle fraction. Lesser but significant amounts were in one of the smoothsurface vesicle fractions, while the second smooth-surface vesicle fraction contained biosynthetic activities which might have been due to contamination with the other two fractions. 5. 5. The results are discussed in relation to the intracellular distribution of CDP-choline: 1,2-diglyceride cholinephosphotransferase (EC 2.7.8.2) and to chylomicron formation.

Metabolism of phospholipids II. Isolation and propeties of phosphatidic acid from mammalian liver

Abstract 1. 1. A procedure is described for the isolation of phosphatidic acid from mammalian liver (ox, pig and rat). 2. 2. Analytical data are presented identifying this compound as phosphatidic acid. 3. 3. Between 80 and 90% of the fatty acids of phosphatidic acid are unsaturated, the majority being linoleic acid. 4. 4. The biochemical significance of phosphatidic acid is discussed.

The effect of chain length on the activation and subsequent incorporation of fatty acids into glycerides by the small intestinal mucosa

Summary 1. A modification of the hydroxamic acid method for determining acyl-CoA synthetase is described. 2. Using cat intestinal mucosa, maximum reaction rates for acyl-CoA synthetase were obtained with palmitate and stearate, unsaturated fatty acids giving much lower reaction rates than their saturated analogues. Comparison of activities of the homogenate with the microsomal fraction indicated that acetyl-CoA synthetase and one of the acyl-CoA synthetases (EC 6.2.1.2) was not of microsomal origin. 3. The microsomal fraction of guinea-pig intestinal mucosa had maximum reaction rates for acyl-CoA synthetases with myristate, unsaturated acids having rates similar to their saturated analogues. 4. The incorporation of fatty acids into glycerides by the total homogenate of cat intestinal mucosa was studied. Using l -3-glycerophosphate and single fatty acids, maximum incorporations were obtained with myristate, palmitate and stearate. A different pattern of incorporation was found with 2-monopalmitin, i -monopalmitin and i -monoolein, but again the preference was for long-chain acids. If equimolar mixtures of acids were substituted for the single fatty acids, then irrespective of glyceride-glycerol precursor, myristate was incorporated to the greatest extent. 5. It is suggested that the specificity of the enzymes studied contributes to the partition of fatty acids between portal blood and chyle during fat absorption.

Metabolism of phospholipids VIII. Biosynthesis of phosphatidylcholine in the intestinal mucosa

Abstract 1. 1. The occurrence of the enzyme cholinephosphotransferase (CDPcholine: 1,2-diglyceride cholinephosphotransferase, EC 2.7.8.2) has been demonstrated in the intestinal mucosa of the cat, guinea pig and rabbit. 2. 2. The characteristics of the enzyme with respect to substrate concentrations, pH optimum, Mg2+ requirement, inhibition by Tween 20 and stability to freezing and thawing were studied, using a mixed particulate subcellular fraction of cat intestinal mucosa as source of the enzyme and 1,2-diglyceride and [32P]CMPcholine as substrates. 3. 3. A fractionation of homogenates of the small intestinal mucosa of the cat and guinea pig showed that the enzyme is largely concentrated in the microsomal fraction, though comparison using enzyme markers indicated that it is also a true mitochondrial constituent. 4. 4. Further subfractionation of the microsomal fraction from cat small intestinal mucosa into two smooth- vesicle and one rough-vesicle fractions revealed that the cholinephosphotransferase was ahnost exactly divided between the rough-vesicle fraction and one of the smooth-vesicle fractions, whereas the second smooth-vesicle fraction contained negligible amounts of this enzyme. 5. 5. The enzyme cholinephosphate cytidyltransferase (CTP: phosphorylcholine cytidyltransferase, EC 2.7.7.15) also occurs in the intestinal mucosa of the cat indicating that the CDPcholine-dependent biosynthetic pathway for phosphatidylcholine is operating in the intestinal mucosa. 6. 6. The possibility of alternative pathways for the biosynthesis of phosphatidylcholine in the small intestinal mucosa were examined in studies in vivo using cats injected with inorganic 32P. From activity-time curves of the individual phospholipids it was concluded that neither the methylation of phosphatidylethanolamine nor the Ca2+-activated exchange reaction of free choline with preformed phospholipid could be a major pathway for the formation of phosphatidylcholine. 7. 7. The results are discussed in relation to fat absorption and formation of chylomicrons.

Monoglyceride transacylase of rat-intestinal mucosa

Abstract 1. 1. Monoglyceride transacylase occurs in the small intestinal mucosa of the rat in two forms; one is a soluble protein in the cell sap, while the other is tightly associated with subcellular structures such as mitochondria and microsomes. 2. 2. A partially purified particulate monoglyceride transacylase of rat small-intestinal mitochondria is studied with respect to cofactor requirement, pH optimum and substrate specificity using various monoglycerides. Palmitic acid, ATP and CoA can be replaced by palmityl CoA. 3. 3. The reaction product is identified as diglyceride.

Metabolism of phospholipids. VII. On the lipid requirement for phosphatidic acid phosphatase activity.

Abstract 1. 1. The treatment of phosphatidic acid phosphatase ( l -α-phosphatidate phosphohydrolase, EC 3.1.3.4) with a variety of organic solvents causes a reduction of enzymic activity. 2. 2. Under specified conditions, the addition of lipid preparations brings about an almost complete reactivation of phosphatidic acid phosphatase preparations which have been solvent-deactivated. 3. 3. The results are discussed in relation to the function of membrane-bound enzymes.

The effect of unsaturated fatty acids and the particle-free supernatant on the incorporation of palmitate into glycerides

Abstract 1. 1. Biosynthesis of glycerides was measured using rat-liver mitochondria or the microsomal fraction of cat intestinal mucosa. With glycerolphosphate as precursor, the incorporation of radioactive palmitate into glycerides was stimulated up to 2.6-fold by the addition of 20 to 100 μM. unsaturated long-chain fatty acids which were themselves incorporated. At the same time, the amount of labelled phosphatidate present showed a corresponding decrease. 2. 2. Out of 5 unsaturated long-chain fatty acids tested, linoleate and linolenate gave the largest stimulations, while saturated fatty acids were ineffective. Under optimum conditions, the molar ratio of palmitate to linoleate incorporated into glycerides was approx. 2.5. 3. 3. The addition of particle-free supernatant to either the mitochondrial or microsomal system brought about a much larger stimulation of palmitate incorporation than did unsaturated fatty acids. Evidence is presented that the stimulating effect of the particle-free supernatant is partially due to the presence of unsaturated fatty acids in this fraction. 4. 4. The formation of higher glycerides from 1-monopalmitin or from 1,2-diglyceride prepared from egg lecithin was not or only slightly stimulated by the addition of unsaturated fatty acids or the particle-free supernatant.