Myles C. Cabot

Structural and chemical specificity of diradylglycerols for protein kinase C activation

Summary The structural and chemical specificity of diacylglycerols, lipid components of the quaternary complex for protein kinase C activation, have been evaluated. The ether-linked analogs of the diacyl lipids, either dialkyl or alkyl acyl, were not effective activators of protein kinase C and thus had little influence on reducing the Ca ++ requirement of the enzyme. Ester-linked compounds such as 1-palmitoyl- sn -2-butyrylglycerol were as effective as dioleoylglycerol in stimulating protein phosphorylation. Increasing the carbon number at the sn -2 position from two to four resulted in enhanced enzymatic activity, suggesting that the chain length at the secondary hydroxyl is also of importance. These data clearly establish the necessity of the sn -1 carbonyl group of ester-linked glycerolipids in the protein kinase C activation complex.

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

Abstract The metabolism of 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl- sn -glycero-3-phosphocholine.

Manipulation of alkylglycerolipid levels in cultured cells. Fatty alcohol versus alkylglycerol supplements.

Ehrlich ascites cells and monolayers of L-M cell fibroblasts were grown in medium containing either long-chain fatty alcohols or alkylglycerols. The cells were then analyzed to determine the contribution of these lipid precursors to the synthesis of complex lipids for the purpose of defining the most efficient system to elevate the levels of ether phospholipids. Label from high specific activity [1-14C]hexadecanol, [1-14C]octadecanol, and [1-14C]octadecenol was incorporated into alkyl linkages (C-18 : 1 greater than C-16 : 0 greater than C-18 : 0); however, similar labeling of acyl groups occurred. Increasing the amount of hexadecanol in the growth medium resulted in a higher percentage of 14C-labeled acyl groups than alkyl linkages at all concentrations of the alcohol supplement. Supplements of rac-[1-14C]hexadecylglycerol to the growth media were assimilated into phospholipids, which significantly increased as a function of the amounts added. Mass determinations of the alkyl ether phospholipid content in L-M cells incubated for 24 h with an alkylglycerol mixture (10.8 microgram/ml) showed an approximate 70% increase over control levels; supplementation had only a slight effect on the alk-1-enyl content. The systems described will be useful for investigating biophysical and biochemical effects of alkyl ether enrichment in membranes.

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Abstract Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected. Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.

Vertebrate class distribution of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase in serum☆

Serum from numerous mammals and lower vertebrates contains an enzyme activity that is specific for the hydrolysis of the acetate moiety of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF, platelet activating factor). Acetylhydrolase (EC 3.1.1.47, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase) was found in all mammalian sera with activity ranging from 11 (fetal calf) to 178 (rabbit) pmol acetate liberated/microliter serum/min. The enzyme is not present in avian serum but is a constituent of reptiles and bony fishes.

Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56 degrees C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83-86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2-1.0 microM range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the [14C]dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of [14]dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never before documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols.

Serum lipase active in the hydrolysis of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate

Sera from numerous animal sources contain enzymes that degrade the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). The activity was present in rat, mouse, guinea pig, rabbit and goat, and absent in fetal bovine, bovine, porcine, horse, chicken and human sera. The enzyme shows a marked specificity for hydrolysis of the long-chain 12-O acyl group, which results in the formation of phorbol-13-monoacetate as the major product. Enzymatic catalysis displayed two pH optima (5.5 and 8.0) and was completely destroyed by heat inactivation (56 degrees C, 30 min) of the serum. N-Ethylmaleimide and p-chloromercuribenzoate were relatively weak inhibitors, whereas NaF (5.0 mM) produced an approximate 80% reduction of hydrolysis. Esterase-lipase substrates, such as cholesterol oleate, alpha-naphthylacetate, alpha-naphthylpalmitate, and monotetradecanoylglycerol, were not inhibitory when added to the reaction mixture. Porcine pancreatic lipase does not catalyze the removal of the acyl or the acetyl groups from TPA.

Chaulmoogric acid: Assimilation into the complex lipids of mycobacteria

Lipid analysis ofMycobacterium vaccae, grown in the presence of chaulmoogric acid, demonstrates that this cyclopentenyl fatty acid is taken up by the organism and incorporated into cellular phospholipids and triacylglycerols. As cell growth is retarded by the addition of chaulmoorgric acid to the growth medium, it is possible that the antimicrobial properties of this compound result from a perturbation of membrane processes.

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Abstract Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12- O -tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [ 3 H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn -2 position. Palmitic acid was liberated from [1- 14 C]palmitoyl-2-acetyl- sn -glycerol and [1- 14 C]palmitoyl-2-butyryl- sn -glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[ 3 H]acetylglycerol was also used as substrate, but the sn -2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.

Increasing the levels of ether-linked lipids in L-M cells by glyceryl ether supplementation depresses growth and choline utilization☆

To gain insight into the role of alkyl-linked lipids in biological systems, we added hexadecylglycerol (a precursor of complex ether-linked lipids) to medium required for the growth of L-M cells in culture. L-M fibroblasts cultured through several generations in the presence of hexadecylglycerol grow at a reduced rate. Experimental cells at their sixth passage, with 2 microgram supplement/ml, double at 50% the rate of control cell populations. Hexadecylglycerol (10 microgram/ml) added 1 day after cell passage does not retard growth; however, within 1 h it decreases the incorporation of choline into the choline glycerophosphatide fraction. Inhibition is specific for choline; ethanolamine incorporation is not affected. The inhibition of choline utilization by hexadecylglycerol-treated cells is dose-dependent and reaches a maximum 12 h after supplementation. Cellular uptake of choline is reduced (approx. 17%) but not as much as the incorporation of choline into the phospholipids (approx. 60% at 12 h). The assimilation of ether lipid precursor into cellular phospholipids was followed by incubating cells with [1-14C]hexadecylglycerol. Incorporation of radioactivity into cellular phospholipids begins to plateau after 24 h, whereas the interference of hexadecylglycerol with choline metabolism could be detected as early as 1 h. The majority of the radioactivity recovered from cells incubated with labeled hexadecylglycerol is localized in the microsomal fraction (56%), where the label was distributed as free hexadecyglycerol, alkylacyl-phospholipids and alkyldiacylglycerols. These results show that the supplementation of a glyceryl ether to L-M fibroblast growth media selectively inhibits the utilization of choline for choline glycerophospholipid biosynthesis and causes a reduction in cell growth rate when cells are continually passaged in the presence of the glyceryl ether.