Biochemical evidence against protein-mediated uptake of myristic acid in the bioluminescent marine bacterium Vibrio harveyi

The bioluminescent marine bacterium, Vibrio harveyi, can utilize exogenous myristic acid (14:0) for beta-oxidation, phospholipid and lipid A synthesis, and as an source of myristyl aldehyde for light emission in the V. harveyi dark mutant M17. A variety of genetic and biochemical strategies were employed in an attempt to isolate V. harveyi mutants defective in myristate uptake and to characterize proteins involved in this process. Although [3H]myristate uptake in a tritium suicide experiment decreased the survival of nitrosoguanidine-treated M17 cells by a factor of 10(5), none of the surviving cells characterized were defective in either incorporation of exogenous myristate into phospholipid or stimulation of light emission. These parameters were also unaffected when intact M17 cells were treated with proteases. Moreover, M17 double mutants selected on the basis of diminished luminescence response to myristate all incorporated [3H]myristate into lipids normally. Finally, no resistant colonies were obtained using the bacteriocidal fatty acid analogue, 11-bromoundecanoate, and experiments with decanoate (10:0) indicated that the V. harveyi cell envelope is very sensitive to physical disruption by fatty acids. Taken together, these results support an unfacilitated uptake of myristic acid in V. harveyi, in contrast with the regulated vectorial transport and activation of long chain fatty acids in Escherichia coli.     

Exogenous myristic acid can be partially degraded prior to activation to form acyl-acyl carrier protein intermediates and lipid A in Vibrio harveyi.

To study the involvement of acyl carrier protein (ACP) in the metabolism of exogenous fatty acids in Vibrio harveyi, cultures were incubated in minimal medium with [9,10-3H]myristic acid, and labeled proteins were analyzed by gel electrophoresis. Labeled acyl-ACP was positively identified by immunoprecipitation with anti-V. harveyi ACP serum and comigration with acyl-ACP standards and [3H]beta-alanine-labeled bands on both sodium dodecyl sulfate- and urea-polyacrylamide gels. Surprisingly, most of the acyl-ACP label corresponded to fatty acid chain lengths of less than 14 carbons: C14, C12, C10, and C8 represented 33, 40, 14, and 8% of total [3H]14:0-derived acyl-ACPs, respectively, in a dark mutant (M17) of V. harveyi which lacks myristoyl-ACP esterase activity; however, labeled 14:0-ACP was absent in the wild-type strain. 14:0- and 12:0-ACP were also the predominant species labeled in complex medium. In contrast, short-chain acyl-ACPs (< or = C6) were the major labeled derivatives when V. harveyi was incubated with [3H]acetate, indicating that acyl-ACP labeling with [3H]14:0 in vivo is not due to the total degradation of [3H]14:0 to [3H]acetyl coenzyme A followed by resynthesis. Cerulenin increased the mass of medium- to long-chain acyl-ACPs (> or = C8) labeled with [3H]beta-alanine fivefold, while total incorporation of [3H]14:0 was not affected, although a shift to shorter chain lengths was noted. Additional bands which comigrated with acyl-ACP on sodium dodecyl sulfate gels were identified as lipopolysaccharide by acid hydrolysis and thin-layer chromatography. The levels of incorporation of [3H] 14:0 into acyl-ACP and lipopolysaccharide were 2 and 15%, respectively, of that into phospholipid by 10 min. Our results indicate that in contrast to the situation in Escherichia coli, exogenous fatty acids can be activated to acyl-ACP intermediates after partial degradation in V. harveyi and can effectively label products (i.e., lipid A) that require ACP as an acyl donor.     

Inhibition of Vibrio harveyi bioluminescence by cerulenin: in vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis.

Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with [3H]myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. In contrast, in vitro acylation of both the synthetase and transferase subunits, as well as the activities of luciferase, transferase, and aldehyde dehydrogenase, were not adversely affected by cerulenin. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10 micrograms/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from [14C]acetate, whereas uptake and incorporation of exogenous [14C]myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with [3H]tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with [3H]tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which [3H]myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction.     

A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Elo1p-dependent carboxy-terminal elongation of C14:1Delta(9) to C16:1Delta(11) fatty acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation). In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.     

Control of aldehyde synthesis in the luminous bacterium Beneckea harveyi.

Some of the Beneckea harveyi dim aldehyde mutants, all of which emit light upon addition of exogenous long-chain aldehyde, also emit light when myristic acid is added. Analysis of these myristic acid-responsive mutants indicates that they are blocked before fatty acid formation, whereas another class of mutants, which respond only to aldehyde, appear to be defective in the enzyme(s) involved in the conversion of acid to aldehyde. Evidence is presented that this activity, designated myristic acid reductase, is coinduced with luciferase and is involved in the recycling of acid produced in the luciferase reaction, with specificity for the C14 compounds.     

Differential regulation of enzyme activities involved in aldehyde metabolism in the luminescent bacterium Vibrio harveyi.

The effects of catabolite repression and nutrient abundance on the activities of Vibrio harveyi enzymes known to be related to aldehyde metabolism were investigated. The growth of cells in complex medium containing glucose, which decreases in vivo luminescence and luciferase synthesis, also resulted in decreases in the specific activities of V. harveyi aldehyde dehydrogenase and acyl carrier protein acyltransferase as well as in the degree of fatty acylation of three bioluminescence-specific polypeptides (32, 42, and 57 kilodaltons), as monitored by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This repression was partially alleviated in glucose medium containing cyclic AMP. The acylation of the above-mentioned proteins, in addition to light emission and luciferase and acyltransferase activities, was also repressed when cells were grown in minimal medium, with partial recovery of these functions upon the addition of arginine. In contrast, aldehyde dehydrogenase activity was increased in minimal medium. These results suggest that the 42-, 57-, and 32-kilodalton proteins, which are responsible for the supply and reduction of fatty acids to form aldehydes for the luciferase reaction, are regulated in the same way as luciferase under the above-described conditions. However, aldehyde dehydrogenase, whose role in V. harveyi aldehyde metabolism is not yet known, is regulated in a different way with respect to nutrient composition.     

Myristic acid utilization in Chinese hamster ovary cells and peroxisome-deficient mutants.

Chinese hamster ovary (CHO) cells convert [9,10-3H]myristic acid ([3H]14:0) to several lipid-soluble, radioactive metabolites that are released into the medium. The main products are lauric (12:0) and decanoic (10:0) acids. Some of the 12:0 formed also is retained in cell lipids. Similar metabolites are not synthesized from palmitic (16:0), oleic (18:1), or arachidonic (20:4) acids, and the addition of these fatty acids does not reduce the conversion of [3H]14:0 to 12:0. Two peroxisome-deficient CHO cell lines do not convert [3H] 14:0 to any polar metabolites, but, they elongate, desaturate, and incorporate [3H]14:0 into intracellular lipids and proteins normally. While BC3H1 muscle cells convert some [3H]14:0 to 12:0, they also produce at least nine lipid-soluble polar products from [3H]12:0. These findings suggest that a previously unrecognized function of myristic acid is to serve as a substrate for the synthesis of 12:0, which can be either secreted into the medium or converted to other oxidized metabolites. The absence of this peroxisomal oxidation pathway, however, does not interfere with other aspects of myristic acid metabolism, including protein myristoylation.     

Both the anaerobic pathway and aerobic desaturation are involved in the synthesis of unsaturated fatty acids in Vibrio sp. strain ABE-1.

Vibrio sp. strain ABE-1 is a unique marine bacterium in terms of its ability to synthesize delta 9-trans-hexadecenoic acid and delta 7-cis-tetradecenoic acid (14:1(7c); Okuyama, H., Sasaki, S., Higashi, S. and Murata, N. (1990) J. Bacteriol. 172, 3515-3518). The present study, involving labeling with [1-14C]acetate, demonstrated that 14:1 is synthesized by the anaerobic pathway. When cells of this bacterium were grown in the presence of [1-14C]myristic acid (14:0), this compound was converted to palmitic (16:0) and hexadecenoic (16:1) acids but not to 14:1, under aerobic conditions. These results suggest that the incorporated 14:0 was elongated to 16:0 and then converted to 16:1 by the aerobic desaturation of 16:0. It appears that the anaerobic pathway and aerobic desaturation are both involved in the synthesis of unsaturated fatty acids during aerobic growth of Vibrio sp. strain ABE-1.     

Heart fatty acid uptake is decreased in heart fatty acid-binding protein gene-ablated mice

Cell culture systems have demonstrated a role for cytoplasmic fatty acid-binding proteins (FABP) in lipid metabolism, although a similar function in intact animals is unknown. We addressed this issue using heart fatty acid-binding protein (H-FABP) gene-ablated mice. H-FABP gene ablation reduced total heart fatty acid uptake 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6 compared with controls, respectively. Similarly, the amount of fatty acid found in the aqueous fraction was reduced 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6, respectively. Less [1-(14)C]16:0 entered the triacylglycerol pool, with significant redistribution of fatty acid between the triacylglycerol pool and the total phospholipid pool. Less [1-(14)C]20:4n-6 entered each lipid pool measured, but these changes did not alter the distribution of tracer among these pools. In gene-ablated mice, significantly more [1-(14)C]16:0 was targeted to choline and ethanolamine glycerophospholipids, whereas more [1-(14)C]20:4n-6 was targeted to the phosphatidylinositol (PtdIns) pool. H-FABP gene ablation significantly increased PtdIns mass 1.4-fold but reduced PtdIns 20:4n-6 mass 30%. Consistent with a reported effect of FABP on plasmalogen mass, ethanolamine plasmalogen mass was reduced 30% in gene-ablated mice. Further, 20:4n-6 mass was reduced in each of the three other major phospholipid classes, suggesting H-FABP has a role in maintaining steady-state 20:4n-6 mass in heart. In summary, H-FABP was important for heart fatty acid uptake and targeting of fatty acids to specific heart lipid pools as well as for maintenance of phospholipid pool mass and acyl chain composition.     

Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts

Human skin fibroblasts actively elongate a portion of incorporated C20 polyunsaturated fatty acids to their respective C22 derivatives. As much as 40% of incorporated [14C]eicosapentaenoate is elongated within 8 h and 85% by 48 h. Elongation of [14C]arachidonate is initially less than half that of [14C]eicosapentaenoate and plateaus at 20-30% of incorporated 14C-labeled fatty acid. The elongation of 5,8,11-[14C]eicosatrienoate is intermediate between that of 20:4(n-6) and 20:5(n-3). Docosatetraenoate is not an effective inhibitor of the elongation of arachidonate, thus suggesting that the observed plateau is not due to product inhibition. When concentrations of exogenous fatty acids are increased, these cells elongate substantial quantities of C20 polyunsaturated fatty acids; elongation of eicosapentaenoate is consistently more extensive than that of arachidonate. Eicosapentaenoate is also an effective inhibitor of the elongation of [14C]arachidonate. Increases in exogenous arachidonate up to 10 microM result in an increase in elongation of [14C]arachidonate both in absolute quantities and as a percentage of that incorporated; the arachidonate thus acts as a positive modulator of its own elongation. Increased eicosapentaenoate also enhances the elongation of [14C]eicosapentaenoate, but only at lower concentrations (0.02-0.15 microM). The factors which regulate the elongation of C20 polyunsaturated fatty acids in human skin fibroblasts serve to permit extensive elongation of eicosapentaenoate while retaining incorporated arachidonate primarily in its C20 form.     

Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum

The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [35S]cysteine, [35S]methionine, [14C]myristic acid (14C-14:0), [14C]palmitic acid (14C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.     

Mechanism of the apparent regulation of Escherichia coli unsaturated fatty acid synthesis by exogenous oleic acid.

Starvation of strains of Escherichia coli which are glycerol auxotrophs and are also defective in beta oxidation results in the accumulation of large amounts of free fatty acid (Cronan, J. E., Jr., Weisberg, L. W., and Allen, R. G. (1975) J. Biol. Chem. 250, 5835-5840). We now report that addition of exogenous oleic acid to these cultures results in no decrease in the synthesis of the unsaturated acids of the free fatty acid fraction although a 40 to 60% decrease of [14C]acetate incorporation into phospholipid unsaturated acyl moieties occurs under these conditions. This result indicates that the decreased synthesis of phospholipid unsaturated acyl moieties observed by others during oleic acid supplementation can be attributed to competition between exogenous and endogenously synthesized unsaturated fatty acids rather than a curtailment of unsaturated fatty acid synthesis per se.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Acylation of steryl glucosides by phospholipids. Solubilization and properties of the acyl transferase.

Particulate enzyme preparations of cotton fibers catalyze the acylation of exogenous steryl glucoside to form acylated steryl glucoside. The acyl transferase involved in this reaction was solubilized by treatment of the membrane fractions with Triton X-100 and was partially purified by chromatography on DEAE-cellulose and gel filtration. This solubilized enzyme had an absolute requirement for Triton X-100 and phospholipid in order to catalyze the acylation of the steryl glucoside. The best phospholipid substrate was phosphatidylethanolamine but egg and soybean phosphatidylcholine were also active. The phospholipid was shown to function as an acyl donor by demonstrating that [¹⁴C]fatty acid from ¹⁴C-labeled phospholipid could be transferred to steryl-[³H]glucoside to form [¹⁴C,³H]acylated steryl glucoside. Saponification of this compound yielded [¹⁴C]fatty acid and steryl-[⁷H]glucoside.