Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.

Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.     

Stimulation of acyl-CoA:cholesterol acyltransferase activity in rat liver microsomes by phosphatidylcholine

Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent.Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification.     

Extraction and partial purification of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase from rat liver microsomes

Acyl-CoA:l-acyl-sn-glycero-3-phosphochoh'ne acyltransferase (EC 2.3.1.23) was extracted from rat liver microsomes with an aqueous dispersion of 1-acylsn-glycero-3-phosphocholine, a substrate of the enzyme, and purified up to 30-fold. The procedure includes removal of unrelevant proteins and lipids by washings of microsomes with a buffer of high ionic strength and with buffers containing detergents, extraction of the enzyme with an aqueous dispersion of 1-acyl-sn-glycero-3-phosphocholine, and chromatography by gel filtration. The acyltransferase was eluted from a Ultrogel AcA 34 column at a position with a K_av of 0.122; an elution position of a protein with a molecular weight of 225 000.The partially purified enzyme was active over a wide range of pH with an optimum at around pH 8. Depending on the acyl donors, different rates of the reaction were obtained by the preparation. The order was: arachidonoyl-CoA > linoleoyl-CoA = oleoyl-CoA > palmitoyl-CoA. The enzyme preparation acylated 1-acyl-sn-glycero-3-phosphocholine, 1-acyl-sn-glycero-3-phosphoethanolamine and 1-acyl-sn-glycero-3-phosphoinositol but not acylated 2-acyl-sn-glycero-3-phosphocholine, 1-acyl-sn-glycerol 3-phosphate or diacylglycerol. Some sulfhydryl-binding reagents inactivated the enzyme.     

Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.     

Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.     

Localization and characterization of the sn-glycerol-3-phosphate acyltransferase in Rhodopseudomonas sphaeroides

The membrane localization and properties of the Rhodopseudomonas sphaeroides sn-glycerol-3-phosphate acyltransferase have been examined utilizing enzymatically prepared acyl-acyl carrier protein (acyl-ACP) substrates as acyl donors for sn-glycerol-3-phosphate acylation. Studies conducted with membranes prepared from chemotrophically and phototrophically grown cells show that sn-glycerol-3-phosphate acyltransferase activity is predominantly (greater than 80%) associated with the cell's cytoplasmic membrane. Enzyme activity associated with the intracytoplasmic membranes present in phototrophically grown R. sphaeroides was within the range attributable to cytoplasmic membrane contamination of this membrane fraction. Enzyme activity was optimal at 40 degrees C and pH 7.0 to 7.5, and required the presence of magnesium. No enzyme activity was observed with any of the long-chain acyl-CoA substrates examined. Vaccenoyl-ACP was the preferred acyl-ACP substrate and vaccenoyl-ACP and palmitoyl-ACP were independently utilized to produce lysophosphatidic and phosphatidic acids. With either vaccenoyl-ACP or palmitoyl-ACP as sole acyl donor substrate, the lysophosphatidic acid formed was primarily 1-acylglycerol-3-phosphate and the Km(app) for sn-glycerol-3-phosphate utilization was 96 microM. The implications of these results to the mode and regulation of phospholipid synthesis in R. sphaeroides are discussed.     

Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli.

Overproduction of the sn-glycerol-3-phosphate acyltransferase in Escherichia coli leads to incorporation of this integral membrane protein into ordered tubular arrays within the cell. Freeze-fracture-etch shadowing was performed on suspensions of partially purified tubules and whole bacteria. This procedure revealed the presence of ridges and grooves defining a set of long-pitch left-handed helical ridges. The long-pitch helices represented chains of acyltransferase dimers. Tubules observed within the cell were often closely packed, with an apparent alignment of grooves and ridges in adjacent tubules. Fracture planes passing through the tubules indicated the presence of a bilayer structure, with some portion of the enzyme being associated with the membrane. The major portion of the enzyme extended from the hydrophilic surface, forming a large globular structure that, in favorable views, displayed a central cavity facing the cytoplasm. Computer analysis of shadowed tubules revealed that the left-handed helices were six stranded, with a pitch of 1,050 A (105.0 nm) and a spacing of 75 A (7.5 nm) between acyltransferase dimers along the chains. Analysis of the predicted secondary structure failed to reveal obvious transmembrane segments, suggesting that very little of the protein was inserted into the bilayer.     

Solubilization and modulation of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity in rat liver microsomes

The acylation of 1-acyl-glycerophosphocholine is an important mechanism for the maintenance of the asymmetrical distribution of acyl groups in phosphatidylcholine. The majority of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase is located in the microsomal fraction. In this study, the rat liver microsomes were incubated with various detergents, and the solubilized enzyme was separated from the remainder by centrifugation. Sodium cholate, sodium deoxycholate and octylglucopyranoside caused the Solubilization of 14–25% of the enzyme activity. The acyl specificity of the solubilized enzyme was similar to the insoluble enzyme, indicating that there was no selective solubilization of any acyl specific acyltransferase. The solubilized enzyme did not display any lipid requirement, and its activity was inhibited by phosphatidylcholine, phosphatidylethanolamine and 1,2-diacylglycerol. Kinetic studies with varying concentrations of acyl-CoAs revealed that the inhibition by 1,2-diacylglycerol was essentially uncompetitive. The modulation of acyltransferase activity by 1,2-diacylglycerol may be an important mechanism for controlling the acylation of lysophosphatidylcholine.     

Rat sn-glycerol-3-phosphate acyltransferase: molecular cloning and characterization of the cDNA and expressed protein.

Rat mitochondrial glycerol-3-phosphate acyltransferase (GPAT) cDNA was cloned and characterized. We identified a cDNA containing an open reading frame of 828 amino acids that had an 89% homology with the coding region of the previously characterized mouse mitochondrial GPAT and a predicted amino acid sequence that was 96% identical. The rat 5' UTR was only 159 nucleotides, in contrast to the 926 nucleotide 5' UTR of the mouse cDNA and had an internal deletion of 167 nucleotides. GPAT was expressed in Sf21 insect cells, and specific inhibitors strongly suggest that, like the Escherichia coli GPAT, the recombinant mitochondrial GPAT and the mitochondrial GPAT isoform in rat liver contain critical serine, histidine, and arginine residues.     

Inactivation of acyl-CoA:cholesterol acyltransferase by ATP+Mg2+ in rat liver microsomes

The molecular modulation of acyl-CoA:cholesterol acyltransferase (EC 2.3.2.26) was studied in the microsomes of rat liver. Acyl-CoA: cholesterol acyltransferase was specifically inactivated by ATP and ADP, requiring Mg2+ as a cofactor. The inactivation was not due to substrate diminution nor to inhibition by the activity of acyl-CoA hydrolase, which was not affected by Mg2+ or ATP+Mg2+. Enhancement of inactivation of acyl-CoA: cholesterol acyltransferase by ATP+Mg2+, NaF and a heat-labile cytosolic factor (or factors) is consistent with a protein-kinase catalyzed phosphorylation being involved in the short term regulation of this enzyme.     

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma.

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.     

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.     

The triose-phosphate site of homogeneous reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli

The sn-glycerol-3-phosphate (glycerol-phosphate) acyltransferase of Escherichia coli was purified to near homogeneity and its activity reconstituted with phospholipids (Green, P.R., Merrill, A.M., Jr. and Bell, R.M. (1981) J. Biol. Chem. 256, 11151-11159). The competency of glycerol-P analogues to serve as inhibitors and as substrates was investigated. Dihydroxyacetone-P, ethyleneglycol-P, 1,3-propanediol-P, 3,4-dihydroxybutylphosphonate and DL-glyceraldehyde-3-P were inhibitors of the reconstituted purified glycerol-phosphate acyltransferase. The kinetics of inhibition, while formally of the mixed type, most closely resembled that of a simple competitive inhibition with respect to glycerol-3-P. Inorganic phosphate was also found to be a competitive inhibitor. All of the glycerol-3-P analogues except DL-glyceraldehyde-3-P were substrates. Of these, dihydroxyacetone-P proved to be the best substrate. The secondary hydroxyl was not necessary for activity. Glycerol-phosphate acyltransferase catalyzed the hydrolysis of palmitoyl-CoA in the presence of DL-, but not D-glyceraldehyde-3-P. This suggests that the gem diol of L-glyceraldehyde-3-P may be a substrate, and that the acylated adduct may be unstable. The enzyme was inactivated by phenylglyoxal and butanedione, suggesting that arginine may be at or near the active site.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.     

Asymmetric reconstitution of homogeneous Escherichia coli sn-glycerol-3-phosphate acyltransferase into phospholipid vesicles

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside. Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50. Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000. These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules. Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle. The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1. The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid). Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles. Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles. Disruption of vesicles with Triton X-100 did not reveal significant latent activity. These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.     

Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction

Alignment of amino acid sequences from various acyltransferases [sn-glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I-IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I-IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases. Using Escherichia coli GPAT (PlsB) as a model acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I-IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine and aspartate in block I, the glycine in block III, and the proline in block IV all play a role in E. coli GPAT catalysis. The phenylalanine and arginine in block II and the glutamate and serine in block III appear to be important in binding the glycerol 3-phosphate substrate. Since blocks I-IV are also found in LPAAT, DHAPAT, and LPEAT, we believe that these conserved amino acid motifs are diagnostic for the acyltransferase reaction involving glycerol 3-phosphate, 1-acylglycerol 3-phosphate, and dihydroxyacetone phosphate substrates.     

Phospholipid dependence of homogeneous, reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli

A novel mixed micelle assay for the sn-glycerol-3-phosphate acyltransferase of Escherichia coli was developed using the nonionic detergent octaethylenegly-coldodecyl ether. The assay permitted investigation of the phospholipid dependence of enzyme activity at phospholipid/detergent ratios of 5:1 (w/w) to 2:1 depending on the phospholipid employed. The higher ratio yielded maximal activity when E. coli phospholipids were used; the lower ratio was observed with cardiolipin(E. coli). Phosphatidylglycerol(E. coli) and phosphatidylethanolamine(E. coli) also restored enzyme activity. Activation by phosphatidylethanolamine(E. coli) was pH-dependent and relatively inefficient. The synthetic, disaturated (1,2-palmitoyl)phosphatidylglycerol reconstituted only 25% of the total enzyme activity as that observed with the monounsaturated (1-palmitoyl, 2-oleoyl) species. Full activation of enzyme was achieved with (1,2-dioleoyl)phosphatidylglycerol. Phosphatidylcholine and phosphatidic acid were unable to reconstitute enzyme activity. Chromatographic sizing of the sn-glycerol-3-phosphate acyltransferase, following reconstitution in cardiolipin(E. coli)/octaethyleneglycoldodecyl ether mixed micelles, suggested that the monomeric form of the enzyme was active.