No abstract

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.

1. The metabolism of phosphatidylinositol in pig thyroid has been investigated as a basis for understanding the specific stimulation of the synthesis of this phospholipid in the gland by thyrotropin. 2. The gland contained an active Ca(2+)-dependent phosphatidylinositol-splitting enzyme with an optimum pH of 5.3-5.5. 3. The major water-soluble product (65%) formed by this catabolic enzyme was not phosphorylinositol but a related compound, which may be a cyclic phosphorylinositol. Both this and phosphorylinositol (35%) were released simultaneously from the phosphatidylinositol substrate. 4. The phosphatidylinositol-splitting enzyme was found almost exclusively in the supernatant fraction obtained by homogenization of the gland. It was not present in the acid-phosphatase-containing particulate fraction. 5. The incorporation of [2-(3)H(1)]inositol into phosphatidylinositol in the presence of either CDP-diglyceride or CTP+ATP was most active in the microsomal fraction. 6. When thyroidal microsomes were labelled with [(3)H]inositol and (32)P, and then incubated with unlabelled inositol, there was a dramatic loss of (3)H labelling from the phosphatidylinositol, which was not accompanied by an equivalent loss of (32)P from the phosphate moiety. This turnover of the inositol moiety required nucleotide coenzymes. It is postulated that the phosphatidylinositol is split into inositol and a phosphorus-containing lipid precursor of the phospholipid that remains on the microsomal membrane and is recycled. 7. Isolated thyroidal mitochondria synthesized phosphatidylinositol from [2-(3)H(1)]inositol only because of their contaminating microsomal component. 8. Some evidence has been obtained of a rapid transfer of phosphatidylinositol molecules from thyroidal microsomes to mitochondria when these were incubated together in the presence of a supernatant fraction. 9. Both phosphatidylinositol breakdown by the supernatant fraction of the gland and synthesis by the microsomes were totally inhibited by 1mm-chlorpromazine. This drug is known to suppress thyrotrophin-induced stimulation of activity in thyroid slices.

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.

The incorporation of choline into the phosphatidylcholine of subcellular fractions of rat liver has been examined. The mitochondrial fraction shows a minimal phosphatidylcholine synthesis compared with that produced by adding microsomes providing an incubation medium which allows adequate phospholipid synthesis is used. The small mitochondrial incorporation can be partly if not wholly explained by microsomal contamination or a non-energy dependent exchange process. It is concluded that the appreciable turnover of mitochondrial phosphatidylcholine observed in vivo can be explained most satisfactorily by assuming a transfer of newly synthesized phospholipid molecules from the endoplasmic reticulum.Although phospholipids play an essential role in many reactions of the mitochondrial electron transport system [l] their site of synthesis within the cell is a subject of some controversy. The phospholipid present in highest concentration in liver mitochondria is phosphatidylcholine and from experiments in vivo this appears to be turned over a t a rate which is only slightly slower than the microsomal phosphatidylcholine [2]. Early experiments suggested that liver mitochondria are devoid of the phosphatidylcholine synthesizing enzyme CDP-choline : 1,2 diglyceride cholinephosphotransferase which was found mainly in the microsomes [3,4]. Later studies of Stoffel and Schiefer [5] indicated that some of the enzyme was present in the outer mitochondrial membranes of rat liver, but these studies have been criticised on the grounds that no recovery of total activity or degree of microsomal contamination was provided [2,6,7 ]. Recently experiments were performed in this laboratory which indicated that the limited phosphatidylcholine synthesis from [32P]phosphate or CDP-[W]choline carried out by isolated liver mitochondria was largely due to microsomal contamination of these organelles [2]. An exchange of phosphatidylcholine between isolated microsomes and mitochondria was demonstrated and it was suggested that this was the process by which the dynamic turnover of phosphatidylcholine in the mitochondria is maintained. Contrasting markedly with these conclusions, Bygrave and Kaiser [8] chondria by an energy-dependent process with little or no contribution from contaminating or added microsomes. The experiments reported in this paper were performed in an attempt to resolve these differences. EXPERIMENTAL PROCEDUREIsolation of Mitochondria, Microsomes and Cell Supernatant The procedure followed was essentially the same as described by McMurray and Dawson [2]. Female rats of the Wistar albino strain, 200-250 gin weight were decapitated and the livers rapidly excised and chilled. All subsequent operations were performed a t 0"-4". The livers, usually pooled from two animals, were minced with scissors and homogenized in a motor-driven homogenizer with 0.008 in clearance [ l l ] for 1 min in 10 volumes of cold 0.25 M sucrose-0.1 mM EDTA, pH 7.4. The homogenate was centrifuged a t 2500 rev./min (800 xg) for 10 min in a Servall RC-2 centr...