Search PubMed⌕ Search

Biomedical subjects

J A Glomset

Publications and source records attributed to J A Glomset.

At least 19 recordsLinked to original sources
PubMed1992

Substrate specificity of a CoA-dependent stearoyl transacylase from bovine testis membranes.

We identified a CoA-dependent stearoyl transacylase activity in bovine testis membranes, then examined the enzyme's specificity in mixed micelle systems containing the neutral detergent Triton X-100. The enzyme transferred stearoyl groups from a variety of phospholipids to sn-2-arachidonoyl lysophosphatidic acid (lysoPA), but showed very little palmitoyl transacylase activity. Its ability to transfer stearoyl groups was both donor- and acceptor-dependent. For example, it used weakly acidic phospholipids, such as sn-1-stearoyl-2-acyl species of phosphatidylinositol (PI), as donors, but did not use phosphatidylinositol-4,5-bisphosphate or sn-1-stearoyl-2-arachidonoyl phosphatidylcholine. Moreover, it used sn-2-acyl species of lysoPA and sn-2-arachidonoyl lysoPI as acceptors but did not use sn-2-arachidonoyl species of lysophosphatidylserine, lysophosphatidylethanolamine, or lysophosphatidylcholine. When taken together, our results raise the possibility that sn-1-stearoyl-2-acyl species of PI may be the primary acyl donors in the transacylase reaction in vivo, while sn-2-acyl species of lysoPA may be the primary acyl acceptors. Available evidence suggests that the PA that is formed may subsequently be converted into PI, but the metabolic fate of the other reaction product, sn-2-acyl lysoPI, remains to be determined.

Acyl Coenzyme A↗
PubMed1992

Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.

Arachidonic Acids↗
PubMed1991

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

Acyltransferases↗
PubMed1991

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.

Adrenal Gland Neoplasms↗
PubMed1991

A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity.

A protein geranylgeranyltransferase (PGT) that catalyzes the transfer of a 20-carbon prenyl group from geranylgeranyl pyrophosphate to a cysteine residue in protein and peptide acceptors was detected in bovine brain cytosol and partially purified. The enzyme was shown to be distinct from a previously characterized protein farnesyltransferase (PFT). The PGT selectively geranylgeranylated a synthetic peptide corresponding to the C terminus of the gamma 6 subunit of bovine brain G proteins, which have previously been shown to contain a 20-carbon prenyl modification. Likewise, a peptide corresponding to the C terminus of human lamin B, a known farnesylated protein, selectively served as a substrate for farnesylation by the PFT. However, with high concentrations of peptide acceptors, both prenyl transferases were able to use either peptide as substrates and the PGT was able to catalyze farnesyl transfer. Among the prenyl acceptors tested, peptides and proteins with leucine or phenylalanine at their C termini served as geranylgeranyl acceptors, whereas those with C-terminal serine were preferentially farnesylated. These results suggest that the C-terminal amino acid is an important structural determinant in controlling the specificity of protein prenylation.

Alkyl and Aryl Transferases↗
PubMed1991

Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product.

Protein prenylation occurs by modification of proteins with one of at least two isoprenoids, the farnesyl group and the geranylgeranyl group. Protein farnesyltransferases have been identified, but no such enzyme has been identified for geranylgeranylation. We report the identification of an activity in crude soluble yeast extracts that catalyzes the transfer of a geranylgeranyl moiety from geranylgeranyl pyrophosphate to proteins having the C-terminal sequence Cys-Ile-Ile-Leu or Cys-Val-Leu-Leu but not to a similar protein ending with Cys-Ile-Ile-Ser. This activity is dependent upon the CDC43/CAL1 gene, which is involved in budding and the control of cell polarity, but does not require the DPR1/RAM1 gene, which is known to be required for the farnesylation of Ras proteins. These results indicate that the protein geranylgeranyltransferase activity is distinct from the protein farnesyltransferase activity and that its specificity depends in part on the extreme C-terminal leucine in the protein to be prenylated.

Alkyl and Aryl Transferases↗
PubMed1991

Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus.

We showed previously that a 23-kDa guanine nucleotide-binding protein (G protein) purified from bovine brain membranes is carboxyl methylated and that this modification occurs at or near the membrane-binding domain. In the present study, we identified this small G protein as G25K (formerly termed Gp). We demonstrated that proteolytic digests of 3H-methylated G25K contained radiolabeled material that coeluted with synthetic S-(geranylgeranyl)cysteine methyl ester on reversed-phase HPLC. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the isoprenoid moiety and carboxyl methyl ester are localized on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G25K by Raney nickel treatment positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. These results suggest that geranylgeranyl modification and perhaps methyl esterification function in the membrane localization of this small G protein.

Amino Acid Sequence↗
PubMed1991

Effect of acyl chain unsaturation on the conformation of model diacylglycerols: a computer modeling study.

In a previous modeling study we identified an angle iron-shaped conformation of docosahexaenoic acid and showed that an sn-1-stearoyl diacylglycerol (DG) that contained an sn-2-docosahexaenoyl group in this conformation could adopt a highly regular shape. In the present study we compared the properties of this DG with those of sn-1-stearoyl DGs that contained other unsaturated fatty acyl groups in the sn-2 position. The major findings were that: 1) sn-1-stearoyl DGs that contain polyenoic fatty acids in the sn-2 position can assume regular shapes, and 2) these shapes differ depending on the location of the double bonds. sn-2-Polyenoic fatty acyl groups with a double bond sequence that begins close to the carboxyl ester bond are associated with one type of regular shape, while sn-2-polyenoic fatty acyl groups with a double bond sequence that begins toward the middle of the chain are associated with another. Such shapes would not have been predicted by current ideas relating membrane fluidity to unsaturation. In contrast, another finding of the present study, that sn-1-stearoyl-2-oleoyl DG can adopt, at best, only a highly irregular shape is in good agreement with the results of previous investigators.

Acylation↗
PubMed1991

Effect of acyl chain unsaturation on the packing of model diacylglycerols in simulated monolayers.

In a companion study of the effects of acyl chain unsaturation on a series of model sn-1,2-diacylglycerols (DGs) we showed that individual DGs could adopt one of three energy-minimized conformations depending on the number and location of cis double bonds in the sn-2 chain. Here we show that each of these conformations promotes a distinct type of packing arrangement in a simulated DG monolayer. One conformation, shown by sn-1-18:0 DGs containing an sn-2 22:6(n-3)-, 20:4(n-6)-, or 20:3(n-9)- group, determines a regular packing that resembles a known hybrid subcell, HS2, of crystalline hydrocarbon chains. The second conformation, shown by DGs containing an sn-2 18:0-, 18:2(n-6)-, or 18:3(n-3)- group, determines a regular packing that resembles a second known, distinct hydrocarbon subcell, HS1. The third conformation, that of 18:0/18:1(n-9) DG, determines a much looser, less energetically favorable packing. Stable heterogeneous packings are possible for DGs that have similar conformations, but mixed packings of DGs that have dissimilar conformations are less stable. These results raise the possibility that differences in sn-2 acyl chain unsaturation among membrane sn-1,2-diacylglycerophospholipids may promote the formation of different domains.

Acylation↗
PubMed1990

The LDL receptor pathway delivers arachidonic acid for eicosanoid formation in cells stimulated by platelet-derived growth factor.

Animal cells can convert 20-carbon polyunsaturated fatty acids into prostaglandins (PGs) and leukotrienes. These locally produced mediators of inflammatory and immunological reactions act in an autocrine or paracrine fashion. Arachidonic acid (AA), the precursor of most PGs and leukotrienes, is present in the form of lipid esters within plasma lipoproteins and cannot be synthesised de novo by animal cells. Therefore, AA or its plant-derived precursor, linoleic acid, must be provided to cells if PGs or leukotrienes are to be formed. Because several classes of lipoproteins, including low-density lipoproteins (LDL), very-low-density lipoproteins, and chylomicron remnants, are taken up by means of the LDL receptor, and because LDL and very-low-density lipoproteins, but not high-density lipoproteins, stimulate PG synthesis, we have suggested previously that PG formation is directly linked to the LDL pathway. Using fibroblasts with the receptor-negative phenotype of familial hypercholesterolaemia and anti-LDL receptor antibodies, we show here that LDL deliver AA for PG production and that an LDL receptor-dependent feedback mechanism inhibits the activity of PGH synthase, the rate-limiting enzyme of PG synthesis. These results indicate that the LDL pathway has a regulatory role in PG synthesis, in addition to its well-known role in the maintenance of cellular cholesterol homeostasis.

Animals↗
PubMed1990

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.

Animals↗
PubMed1990

Identification of geranylgeranyl-modified proteins in HeLa cells.

Previous studies have shown that animal cells contain isoprenoid-modified proteins and that one of these proteins, lamin B, contains a thioether-linked farnesyl group that is attached to cysteine. In the present study, a novel isoprenoid-modification was identified by labeling HeLa cells with [3H]mevalonic acid and analyzing proteolytic digests of the total cell protein. Radioactive fragments were purified from these digests and treated with Raney nickel. The released, labeled material was analyzed by gas-liquid chromatography (GC) and mass spectrometry (MS). This approach revealed that an all-trans geranylgeranyl group was a major isoprenoid modification.

Chromatography, Gel↗
PubMed1990

Prenyl proteins in eukaryotic cells: a new type of membrane anchor.

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

Animals↗
PubMed1990

Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini.

We have shown previously that guanine nucleotide-binding protein (G protein) beta gamma complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the gamma polypeptide. In the present study, 3H-methylated G beta gamma complexes cleaved to their constituent amino acids by exhaustive proteolysis were shown to contain radiolabeled material that coeluted with geranylgeranylcysteine methyl ester on reversed-phase HPLC and two TLC systems. Further treatment by performic acid oxidation yielded radiolabeled material that coeluted with L-cysteic acid methyl ester, verifying that the prenyl modification occurs on a C-terminal cysteine residue. Analysis by gas chromatography-coupled mass spectrometry of material released from purified G beta gamma by treatment with Raney nickel positively identified the covalently bound lipid as an all-trans-geranylgeranyl (C20) isoprenoid moiety. To delineate the distribution of this modification among gamma subunits, purified G beta gamma complexes were separated into 5-kDa (gamma 5) and 6-kDa (gamma 6) forms of the gamma polypeptide by reversed-phase HPLC. Gas chromatography-coupled mass spectrometry analyses of Raney nickel-treated purified gamma 5 and gamma 6 subunits showed that both polypeptides were modified by geranylgeranylation. These results demonstrate that at least two forms of brain gamma subunit are posttranslationally modified by geranylgeranylation and carboxyl methylation. These modifications may be important for targeting G beta gamma complexes to membranes.

Animals↗
PubMed1990

Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.

smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.

Blood Platelets↗
PubMed1990

Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.

Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast.

Alkyl and Aryl Transferases↗