The biosynthesis of plasmalogens by rat brain: involvement of the microsomal electron transport system.

The biosynthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (ethanolamine plasmalogens) was studied using 1-[1-14C]hexadecyl-sn-glycero-3-phosphoethanolamine as the substrate and EDTA-washed microsomes from brains of 14-day-old rats. It was found that the 1-E11-14C]hexadecyl-sn-glycero-3-phosphoethanolamine was first acylated to form 1-[1-14C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, then was desaturated to form 1-[1-14C]hexadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. The desaturation required O2 and NADH or NADPH and was inhibited by KCN but not by CO. The data indicated that the desaturation is carried out by a mixed-function oxidase system similar to that involved in the desaturation of fatty acids and that the pathway for the biosynthesis of plasmalogens in brain is similar to that previously found in other tissues. The desaturase was not stimulated by ATP and Mg2plus nor inhibited by EDTA. The specific activity of microsomes from brains of rats of different ages was determined; the activity decreased with age until in adults the activity was only 15% that of the 12--14-day-old rats.     

Conversion of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. A novel pathway for the metabolism of ether-linked phosphoglycerides.

Madin Darby canine kidney (MDCK) cells convert 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine [( 3H]alkylacylGPC) to a product tentatively identified as an ethanolamine-containing phosphoglyceride (PE) (Daniel, L. W., Waite, B. M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132). In the present study, analysis of the radiolabeled phosphoglycerides as diradylglycerobenzoate derivatives indicated that [3H] alkylacylGPC was initially converted to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkylacylGPE) which was subsequently desaturated to 1-O-[3H]alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkenylacylGPE). The conversion of [3H]/[32P]alkyl-lysoGPC to [3H]alkenylacylGPE indicated that base exchange enzymes were not involved in this pathway. A phosphono analog of alkyl-lysoGPC, resistant to phospholipase D hydrolysis and radiolabeled in the 1-O-alkyl chain was readily incorporated, acylated, and subsequently metabolized to [3H]alkylacylGPC and [3H]alkenylacylGPE. Therefore, the involvement of phospholipase D in the conversion pathway was ruled out. The conversion of [3H]alkylacylGPC or its phosphono analog to [3H]alkenylacylGPE was significantly enhanced by the addition of 100 microM ethanolamine to the culture media, suggesting that [3H]alkylacylglycerol is an intermediate in the cytidine-dependent pathway of PE synthesis. MDCK cell cytosol and microsomes contained no detectable phospholipase C activity. However, incubation of microsomes with CMP resulted in the degradation of [3H]alkylacylGPC and accumulation of [3H]alkylacylglycerol. Furthermore, the addition of CDP-ethanolamine to microsomes following preincubation with CMP, resulted in a decrease in [3H]alkylacylglycerol with a concomitant increase in [3H]alkenylacylGPE. Overall, these results suggest that the reverse reaction of choline phosphotransferase may be responsible for the conversion of alkylacylGPC to alkylacylGPE.     

Maintenance of respiratory control by beef heart mitochondria incubated at 25 degrees C: response to protective agents and to protective agents and to prior stress.

Lysophospholipase D (EC 3.1.4.-) activity was demonstrated in rat kidneys, intestines, lungs, testes, and liver. The liver enzyme was studied in greatest detail and its labeled products were identified by chemical and Chromatographic techniques. This enzyme hydrolyzes 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphoethanolamine and 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphocholine to yield 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate; the initial product is subsequently dephosphorylated by a phosphohydrolase in microsomes to form 1-[1-¹⁴C]hexadecyl-sn-glycerol. The possibility that phospholipase C and a phosphotransferase were responsible for the formation of 1-[1-¹⁴C]hexadecyl-sn-glycero-3-phosphate was ruled out. Neither 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine nor 1-[1-¹⁴C]hexadecyl-2-acyl-sn-glycero-3-phosphocholine was hydrolyzed. The enzyme requires Mg²⁺, is inhibited by Ca²⁺, and is stimulated by high salt concentrations; it is localized in the microsomal fraction and has a pH optimum between 7.0 and 7.6. Inhibition by sulfhydryl reagents and protection by glutathione and dithiothreitol suggest that a sulfhydryl group is required for activity. The enzyme is inhibited by detergents and by organic solvent extraction. It appears to be tightly bound to the microsomes, since repeated freeze-thawing or sonication did not release the activity, and trypsin digestion (either in the presence or in the absence of 0.04% deoxycholate) did not destroy the activity. Lysophospholipase D was previously known to occur only in brain (R. L. Wykle and J. M. Schremmer, 1974, J. Biol. Chem., 249, 1742–1746).     

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity.

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0-8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 microM) or propanolol (IC50 = 180 microM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.     

Studies on the hydrolysis of 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine by microsomes from myelinating rat brain.

Microsomal fractions of 14-day-old rat brain were incubated at pH 7.1 with 1-[1'-14C]-alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen). 1-[1'-14C]alkenylglycerol was produced by hydrolyzing enzyme activities, which were stimulated by Mg2 and inhibited by SH-group reagents. Hydrolysis of 1-[1'-14C]alkyl-sn-glycero-3-phosphoethanolamine is very similar in this respect, but the Km value is higher in the former case. The 1-alkyl compound acts as a non-competitive inhibitor of the hydrolyzing enzyme activity described, whereas the hydrolysis of the 1-alkyl derivative is not inhibited by the 1-alkenyl compound.     

Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes.

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PtdIns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 micrograms/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.     

5'-Iodothyronine deiodinase of rat liver: activity in microsomes prepared by various methods, solubilization by detergents and partial purification

Iodothyronine deiodinase activities of rat liver microsomes prepared by (1) differential centrifugation, (2) column chromatography, (3) precipitation with Ca2+, (4) precipitation at low pH, or combinations of these were compared. Method 2 or 2 followed by 4 provided microsomes with specific activities 4.6- and 7.4-times higher than method 1, respectively. Both Triton X-100 at 0.1% (w/v) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) at 4-6 mM efficiently solubilized deiodinase and were not inhibitory at low concentrations. The Chaps-soluble enzyme could be moderately purified by fractionation with ammonium sulfate but more effectively with poly(ethylene glycol).     

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.     

Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings.

Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein.h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein.h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds.     

Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes

Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 mumol/min per mg from rat liver microsomes with 51% of the activity recovered. Purification was accomplished by solubilization of the membrane-associated enzyme with octylglucoside and chromatographic resolution on sequential DEAE cellulose and hydroxylapatite (HPLC) columns in the presence of octylglucoside. The partially purified enzyme, specific for the 2-deacylated plasmalogen, lysoplasmalogen (1-alk-1'-enyl-sn-glycero-3-phosphocholine or -ethanolamine), had no hydrolytic activity with intact plasmalogens or 1-acyl-sn-glycero-3-phosphoethanolamine. Kinetic analyses of enzymic activity demonstrated apparent Km values of 5.5 and 42 microM for 1-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine, respectively. The Vmax values were 11.7 and 13.6 mumol/min per mg with the choline and ethanolamine substrates, respectively. The optimal pH range was between 6.6 and 7.1 with both substrates; the energy of activation for the purified enzyme was 15,200 cal. The enzyme required no cofactors and was unaffected by low millimolar concentrations of Ca2+, Mg2+, Mn2+ or EDTA. It was inhibited by the sulfhydryl-reacting reagent, p-chloromercuribenzoate. Mono- or diradylglycerophospholipids or sphingomyelin did not affect the enzymic activity at 37 degrees C. Activity of the purified enzyme, destroyed by freezing at -20 degrees C, was preserved if stored at this temperature in the presence of 300-600 microM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, facilitated this purification. This is the first reported purification of alkenylhydrolase.     

Phospholipase C from human sperm specific for phosphoinositides

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.     

Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.     

Plasmalogen biosynthesis in Madin-Darby canine kidney cells: selectivity in the acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphoethanolamine and the subsequent desaturation step

Acyl group specificity in the acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphoethanolamine (1-alkyl-2-lyso-GroPEtn) to form 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (1-alkyl-2-acyl-GroPEtn) and the subsequent desaturation of 1-alkyl-2-acyl-GroPEtn to form plasmalogens (1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine, i.e., 1-alk-1'-enyl-2-acyl-GroPEtn) was investigated in intact Madin-Darby canine kidney (MDCK) cells and cell-free membrane preparations. We found 1-[3H]alkyl-2-lyso-GroPEtn was selectively acylated with polyunsaturated fatty acids in the order 20:4 greater than 20:5 greater than 20:3 (n-9) greater than 22:6 by cell-free membrane preparations of MDCK cells. The same pattern of acyl specificity was seen in intact MDCK cells, although the intact cells produced significantly larger amounts of 1-[3H]alkyl-2-acyl-GroPEtn containing oleic acid. There was an increased desaturation of the 1-[3H]alkyl-2-acyl-GroPEtn species containing docosahexaenoic acid to plasmalogens (1-[3H]alk-1'-enyl-2-acyl-GroPEtn) by both intact MDCK cells and the cell-free membrane preparations. The relatively rapid disappearance of the 1-[3H]alk-1'-enyl-2-docosahexaenoyl-GroPEtn species during a 20-h incubation of prelabeled intact MDCK cells suggests a more rapid turnover of this molecular species. Our results indicate there is a high selectivity in the final acylation and desaturation steps of the biosynthetic pathway for plasmalogens.     

Phospholipase B-like activity in rabbit brain membranes.

1. Phospholipase B-like activity was found in rabbit brain membranes. 2. The activity was greatly enhanced by 0.025% (w/v) Triton X-100 and was inhibited by both Ca2+ and Mg2+. 3. With increasing pH of the reaction mixture, the activity was augmented. 4. The characteristics of the enzyme activity possibly suggest that phospholipase B in rabbit brain may be distinct from those previously reported.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.     

Studies on phospholipases from Streptomyces. III. Purification and properties of Streptomyces hachijoensis phospholipase C.

1. Phospholipase C [EC 3.1.4.3] found in the growth medium of Streptomyces hachijoensis was purified about sixty-fold by dialysis and column chromatography on Sephadex G-50. 2. The active fraction was separated by isoelectric focusing into two fractions, phospholipase C-I (pI 6.0) and phospholipase C-II (pI 5.6). 3. Both purified phospholipases C were homogeneous by immunodiffusion and were not differentiated as regards antigencity. 4. Phospholipase C-I had maximal activity at pH 8.0 and the optimal temperature was 50degree. Phospholipase C-I was stable at 50degrees for 30 min and was stable at neutral pH. 5. The activity of phospholipase C-I was inhibited by high concentrations of various detergents such as Triton X-100, sodium, cholate, SDS and was also inhibited by Ca2+, Ba2+, Al3+, and EDTA, but was stimulated by Mg2+, and ethyl ether. 6. The Km value of phospholipase C-I was 0.9 mM, using phosphatidylcholine as a substrate. 7. By the gel filtration procedure, the molecular weights of phospholipase C-I and -II were both determined to be 18,000. 8. Phosphatidylcholine, phosphatidylinositol, cardiolipin, sphingomyelin, and lysophosphatidylcholine were hydrolyzed by phospholipase C-I, but phosphatidylethanolamine and phosphatidylserine were hydrolyzed with difficulty under the same conditions, Phospholipase C-I also hydrolyzed phosphatidic acid.     

Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.

Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidylserine into phosphatidylethanolamine in 7777 hepatoma mitochondria is inhibited by KCN. This study provides further evidence of abnormal mitochondrial biogenesis in the 7777 hepatoma. Our earlier study indicated a greatly increased mitochondrial activity of CTP:phosphatidate cytidylyltransferase in the 7777 hepatoma (Hostetler, Zenner, and Morris. 1978. J. Lipid Res. 19: 553-560). The presence in mitochondria of these two enzymes, which are primarily microsomal in normal liver, does not appear to be due to rapid growth alone, since their intracellular distribution was not altered in fetal or regenerating rat liver.-Hostetler, K. Y., B. D. Zenner, and H. P. Morris. Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma.     

Serine and ethanolamine incorporation into different plasmalogen pools: Subcellular analyses of phosphoglyceride synthesis in cultured glioma cells

In cultured glioma cells, plasma membrane (PM) is enriched in phosphatidylserine (PtdSer) and plasmalogens (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine). Serine can be a precursor of headgroups of both ptdSer and ethanolamine phosphoglycerides (PE) including plasmalogens and non-plasmalogen PE (NP-PE). Synthesis of phospholipids was investigated at the subcellular level using established fractionation procedures and incorporation of [³H(G)]L-serine and [1,2-¹⁴C]ethanolamine. Specific radioactivity of PtdSer from [³H]serine was 2-fold greater in PM than in microsomes, reaching maximum by 2–4 h. Labeled plasmalogen from [³H]serine appeared in PM by 4 h and increased to 48 h, whereas almost no plasmalogen accumulated in microsomes within 12 h. In contrast, labeled plasmalogen from [1,2-¹⁴C]ethanolamine appeared in both PM and microsomes at early incubation times and became enriched in PM beyond 12 h. Thus, in glioma cells: (1) greater and faster accumulation of labeled PtdSer in PM may reflect direct synthesis from serine within PM; (2) PM is a major source of PtdSer for decarboxylation and PE synthesis; (3) NP-PE in both PM and microsome provides headgroup for synthesis of plasmalogen; and, (4) plasmalogen synthesis may involve different intracellular pools depending on headgroup origin.Abbreviations NP-PE nonplasmenylethanolamine phosphoglycerides including both diacyl and alkylacyl species - PE total ethanolamine phosphoglycerides: plasmalogen-plasmenylethanolamine or alkenylacyl ethanolamine phosphoglyceride (1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) - PL phospholipid - PM plasma membrane - PtdCho phosphatidylcholine - PtdSer phosphatidylserine     

The metabolism of lyso-platelet-activating factor (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by a calcium-dependent lysophospholipase D in rabbit kidney medulla

A Ca2+-dependent lysophospholipase D activity in microsomal preparations from the rabbit kidney medulla hydrolyzes the choline moiety from 1-O-[9,10-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) to form 1-O-[9,10-3H]hexadecyl-2-lyso-sn-glycero-3-P; the latter is subsequently dephosphorylated by a phosphohydrolase to 1-O-[9,10-3H]hexadecyl-sn-glycerol. Sodium vanadate, which is known to inhibit phosphohydrolases, reduces the proportion of hexadecylglycerol and increases the formation of hexadecyl-lysoglycerophosphate. Essentially no hydrolysis occurs when the sn-2 position of the hexadecyllysoGPC substrate contains an acyl moiety. The lysophospholipase D in rabbit kidney is of microsomal origin and has a broad pH optimum between 8.0 and 8.8, with the activity decreasing sharply from pH 7.6 to 7.2. Wykle et al. (Biochim. Biophys. Acta 619 (1980) 58-67) have previously demonstrated the existence of a microsomal lysophospholipase D (specific for ether lipid substrates) in rat tissues that requires Mg2+ and exhibits a pH optimum of 7.2; high activities of the Mg2+-dependent lysophospholipase D were found in liver and brain, but not in kidney. In contrast to the Mg2+-dependent lysophospholipase D in rat tissues, the renal enzyme from rabbits requires Ca2+ (5 mM), whereas Mg2+ (5 mM) exhibits little stimulatory action. Under optimal assay conditions (0.1 M Tris-HCl (pH 8.4)/5 mM CaCl2), lysophospholipase D in the rabbit kidney medulla has an activity of 2.7 nmol/min per mg protein compared to 0.9 nmol/min per mg protein for the lysophospholipase D in the rat kidney medulla (0.1 M Tris-HCl (pH 7.2)/5 mM MgCl2). The Ca2+-dependent lysophospholipase D is highest in the liver and kidney medulla from rabbits, but is very low in rat tissues; similar activities were found in male and female rabbits. Our data indicate that the divalent metal ion requirements for expression of maximum lysophospholipase D activities can differ markedly among animal species and also suggest the microsomal Ca2+-dependent lysophospholipase D is an important catabolic route for lyso-PAF metabolism in rabbit renomedullary tissue.