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M H Gelb

Publications and source records attributed to M H Gelb.

At least 91 records · Page 5Linked to original sources
PubMed1997

Zero-order interfacial enzymatic degradation of phospholipid tubules.

The first study of enzymatic hydrolysis of phospholipid tubules is reported. Phosphatidylcholines with acyl chains containing diacetylene groups are known to form tubular microstructures in which the lipids are tightly packed and crystalline. These tubules can be used to probe the role of microstructural form in the mechanics of interfacial enzymatic degradation by such enzymes as phospholipase A2 (PLA2). Hydrolysis by PLA2 may occur most rapidly in regions having the greatest number of bilayer packing defects, such as those that must be found at tubule ends. A microstructure that degrades primarily from its ends should exhibit zero-order kinetics, because the area of the degrading tubule and remains constant as the length of the microstructure decreases. Free fatty acid concentration was measured to follow the generation of PLA2 hydrolysis products in suspensions of diacetylenic phospholipid tubules. The kinetics of tubule hydrolysis were essentially zero-order until conversion was complete, as predicted. However, microscopy of partially hydrolyzed tubules revealed the formation of multiple discrete anionic product domains along the length of degrading tubules as well as in insoluble reaction product microstructures. Furthermore, the rate of tubule hydrolysis was only moderately enhanced by increasing the number of tubule ends, which is consistent with the conclusion that tubule ends are not the only sites of hydrolysis. A model that reconciles the overall kinetics with the morphological evidence is proposed.

Calorimetry, Differential Scanning↗
PubMed1997

Prenylation of proteins in Trypanosoma brucei.

Prenyl modification of proteins by farnesyl and geranylgeranyl isoprenoids occurs in a variety of eukaryotic cells. Culturing of Trypanosoma brucei in the presence of [3H]mevalonolactone (which is hydrolyzed in cells to give mevalonic acid, the precursor of protein prenyl groups) and an inhibitor of mevalonic acid biosynthesis leads to the radiolabeling of a specific set of proteins when analyzed by gel electrophoresis. T. brucei proteins were also labeled when cells were cultured in the presence of [3H]farnesol or [3H]geranylgeraniol, and each prenol labels a distinct set of proteins. Unlike mammalian cells, only a few T. brucei proteins of molecular weights similar to those of the mammalian Ras superfamily of GTPase (20-30 kDa) were labeled with [3H]farnesol or [3H]geranylgeraniol. When the 0-55% ammonium sulfate fraction of T. brucei cytosol was fractionated on anion exchange chromatography, protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PGGT-I) activities were detected and elute as two distinct peaks. Partially purified T. brucei PFT and PGGT-I display partly different specificities toward prenyl acceptor substrates from those of mammalian protein prenyltransferases. As shown previously, rat PFT utilizes proteins ending in CVLS and CVIM as efficient prenyl acceptors and rat PGGT-I utilizes proteins ending in CVLL and CVIM in vitro. On the contrary, T. brucei PFT farnesylates a protein ending in CVIM but not CVLS or CVLL, and T. brucei PGGT-I preferentially geranylgeranylates a protein ending in CVLL.

Alkyl and Aryl Transferases↗
PubMed1997

Pharmacological control of the mevalonate pathway: effect on arterial smooth muscle cell proliferation.

The mevalonate (MVA) pathway is involved in cell proliferation. We investigated drugs acting at different enzymatic steps on rat aorta smooth muscle cell (SMC) proliferation. Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (0.1-10 microM) dose-dependently decreased (up to 90%) SMC proliferation. This effect was prevented by 100 microM MVA, 10 microM all-trans farnesol (F-OH) and 5 microM all-trans geranylgeraniol (GG-OH), precursors of protein prenyl groups, but not by 2-cis GG-OH, precursor of dolichols, squalene and ubiquinone. The same inhibitory effect was obtained with 6-fluoromevalonate (1-50 microM), an inhibitor of MVA-pyrophosphate decarboxylase. Partial recovery of cell proliferation was possible by all-trans F-OH and all-trans GG-OH, but not MVA. Squalestatin 1 (1-25 microM), a potent squalene synthase inhibitor, blocked cholesterol synthesis and slightly inhibited (21% decrease) SMC proliferation only at the highest tested concentration. NB-598 (1-10 microM), a potent squalene epoxidase inhibitor, blocked cholesterol synthesis without affecting SMC proliferation. Finally, the benzodiazepine peptidomimetic BZA-5B (10-100 microM), a specific inhibitor of protein farnesyltransferase, time- and dose-dependently decreased SMC proliferation (up to 62%) after 9 days. This effect of BZA-5B was prevented by MVA and all-trans GG-OH, but not by all-trans F-OH. SMC proliferation was not affected by the closely related compound BZA-7B, which does not inhibit protein farnesyltransferase. Altogether, these findings focus the role of the MVA pathway in cell proliferation and call attention to the involvement of specific isoprenoid metabolites, probably through farnesylated and geranylgeranylated proteins, in the control of this cellular event.

Animals↗
PubMed1996

Purification of a protein palmitoyltransferase that acts on H-Ras protein and on a C-terminal N-Ras peptide.

Mammalian H-Ras and N-Ras are GTP-binding proteins that must be post-translationally lipidated to function as molecular switches in signal transduction cascades controlling cell growth and differentiation. These proteins contain a C-terminal farnesyl-cysteine alpha-methyl ester and palmitoyl groups attached to nearby cysteines. Data is presented showing that rat liver microsomes contain an enzyme that transfers the palmitoyl group from palmitoyl-coenzyme A to cysteine residues of H-Ras protein and of a synthetic peptide having the structure of the C terminus of N-Ras. This protein palmitoyltransferase (PPT) was solubilized from membranes and purified 10,500-fold to apparent homogeneity with an overall yield of 10%. On an SDS gel, PPT appears as two proteins of molecular masses of approximately 30 and approximately 33 kDa. If the palmitoylation sites of the N-Ras peptide (the non-farnesylated cysteine) or H-Ras protein (cysteines 181 and 184) are changed to serine, palmitoylation by PPT does not occur. Non-farnesylated H-Ras produced in bacteria as well as in vitro farnesylated bacterial H-Ras are not substrates for PPT nor is the non-farnesylated, methylated N-Ras peptide. These results suggest, but do not prove, that farnesylation and possibly C-terminal methylation are prerequisites for Ras palmitoylation. PPT shows a large preference for palmitoyl-coenzyme A over myristoyl-coenzyme as the acyl donor. Values of Km for palmitoyl-CoA and H-Ras are 4.3 +/- 1.2 and 0.8 +/- 0.3 microM, respectively. PPT is the first protein palmitoyltransferase to be purified, and the availability of pure enzyme should contribute to our understanding of the function and regulation of Ras palmitoylation in cells.

Acyl Coenzyme A↗
PubMed1996

Palmitoylation of Ha-Ras facilitates membrane binding, activation of downstream effectors, and meiotic maturation in Xenopus oocytes.

Ras proteins serve as critical relays in signal transduction pathways that control growth and differentiation and must undergo posttranslational modifications before they become functional. While it is established that farnesylation is necessary for membrane binding and cellular functions of all Ras proteins, the significance of palmitoylation is unclear. We have studied the contribution of Ha-Ras palmitoylation for biological activity in Xenopus oocytes. In contrast to wild-type Ha-Ras, which binds to membranes and induces meiosis when microinjected into oocytes, a nonpalmitoylated but farnesylated and methylated mutant mislocalizes to the cytosol and fails to promote maturation. This lack of responsiveness correlates with the inability of the mutant to induce phosphorylation and activation of mitogen-activated protein kinase and maturation promoting factor, which are both strongly activated by wild-type Ha-Ras. Costimulation of oocytes with insulin increases their responsiveness to Ras and partially rescues the biological activity of the palmitoylation-resistant mutant. However, 25-50 times higher doses of mutant were required to elicit responses equivalent to wild-type Ha-Ras. These results suggest that palmitoylation and membrane association of Ha-Ras is necessary for efficient activation of the mitogen-activated protein kinase cascade in vivo and are consistent with a biochemical function for Ras as a membrane targeting signal for downstream effectors in this pathway.

Animals↗
PubMed1996

Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87.

In bee venom phospholipase A2, histidine-34 probably functions as a Brønsted base to deprotonate the attacking water. Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34. We have prepared mutants at these positions and studied their kinetic properties. The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50-100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity. The interfacial Michaelis constants are changed by less than 10-fold for all mutants. Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis. The pKa of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4'-nitroacetophenone. The pKa is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pKa is unchanged. These pKas are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong. The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis.

Amino Acid Sequence↗
PubMed1996

Identification of phosphorylation sites of human 85-kDa cytosolic phospholipase A2 expressed in insect cells and present in human monocytes.

The phosphorylation sites on the human, 85-kDa cytosolic phospholipase A2 (cPLA2) were identified using recombinant cPLA2 expressed in Spodoptera frugiperda (Sf9) cells. Analysis by high performance liquid chromatography of tryptic digests of 32P-labeled recombinant cPLA2 showed four major peaks of radiolabeled phosphopeptides. The phosphorylated residues were identified as Ser-437, Ser-454, Ser-505, and Ser-727 using mass spectrometry and automated Edman sequencing. Sf9 cells infected with recombinant virus expressing cPLA2 exhibited a time-dependent release of arachidonic acid in response to the calcium ionophore A23187 or the protein phosphatase inhibitor okadaic acid, which was not observed in Sf9 cells infected with wild-type virus. Stimulation of Sf9 cells with A23187 and okadaic acid also increased the level of phosphorylation of cPLA2. Okadaic acid, but not A23187, induced a gel shift of cPLA2 and increased the level of phosphorylation of Ser-727 by 4.5-fold, whereas the level of phosphorylation of the other sites increased by 60% or less in response to both agonists. To determine whether the same sites on cPLA2 were phosphorylated in mammalian cells, human monocytes were studied. Okadaic acid stimulation of monocytes induced a gel shift of cPLA2, increased the release of arachidonic acid, and increased the level of phosphorylation of cPLA2 on serine residues. Comparison of two-dimensional peptide maps of tryptic digests of 32P-labeled recombinant cPLA2 and human monocyte cPLA2 demonstrated that the same peptides on cPLA2 were phosphorylated in mammalian cells as in insect cells. These results show that the Sf9-baculovirus expression system is useful for investigation of the phosphorylation sites on cPLA2. The results also suggest that phosphorylation of the cPLA2 by protein kinases other than mitogen-activated protein kinase may be important for the regulation of arachidonic acid release.

Amino Acid Sequence↗
PubMed1995

Continuous, vesicle-based fluorimetric assays of 14- and 85-kDa phospholipases A2.

This paper describes the synthesis and analysis of new substrates for the 85-kDa, mammalian, cytosolic phospholipase A2 (cPLA2) and the 14-kDa, human nonpancreatic, secreted phospholipase A2 (sPLA2). Phosphatidylcholines containing an arachidonyl chain at the sn-2 position and either a 10-pyrenedecyl or a 10-pyrenedecanoyl chain at the sn-1 position were synthesized and shown to be substrates for cPLA2 in a fluorescence-based assay. Most of the assays make use of small and large unilamellar vesicles of substrate phospholipid, although the assay also works when the substrate is dispersed in Triton X-100 mixed-micelles. The cPLA2 assays can be carried out in a fixed time-point mode in which one of the products, the pyrene-containing lysophospholipid, is detected by rapid HPLC. Alternatively, the assay becomes continuous when bovine serum albumin is present in the aqueous phase; this protein extracts the pyrene-containing lysophospholipid from the vesicle, and this leads to the fluorescence of monomeric pyrene label. These assays are capable of detecting subnanogram amounts of cPLA2. The ester formed between gamma-linolenic acid and 7-hydroxycoumarin is also a substrate for cPLA2, and when incorporated into vesicles of the anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphomethanol, provides an assay in which the enzyme does not leave the vesicle surface (scooting mode). Unlike all of the previously reported, vesicle-based cPLA2 assays, a prolonged linear reaction progress is seen with the DOPM-based assay. An assay of sPLA2 with subnanogram sensitivity was developed which makes use of the substrate 1-palmitoyl-2-(10-pyrenedecanoyl)-sn-glycero-3-phosphomethanol and a lipid sink. The latter is composed of phosphatidylcholine vesicles, in excess of substrate vesicles, which do not bind sPLA2 but provide a trap for enzyme-produced 10-pyrenedecanoic acid. The fluorescence of monomeric pyrene label in sink vesicles is detected. A second sPLA2 assay using a single type of vesicle was developed based on the substrate 1,2-di(10-pyrenedecanoyl)-sn-glycero-3-phosphocholine present at 10 mol% in vesicles of the nonhydrolyzable anionic phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol. The action of sPLA2 on this fluorescent substrate leads to a separation of the pyrene chains resulting in fluorescence emission from monomeric pyrene. These cPLA2 and sPLA2 assays are ideal for inhibitor screening and analysis, and for studying the interfacial kinetics of these enzymes.

Calcium↗
PubMed1995

The farnesyl group of H-Ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase.

To study the function of the farnesyl modification of Ras, the farnesyl group and a variety of its structural analogs, which lack one or more double bonds and/or the methyl groups, were enzymatically incorporated into recombinant H-Ras in vitro. These proteins were used in a cell- and membrane-free, Ras-dependent mitogen-activated protein kinase (MAP kinase) activation system derived from Xenopus laevis eggs to examine the contribution of the farnesyl group toward the activation of the kinase. Whereas non-farnesylated H-Ras is unable to activate MAP kinase, farnesylation of H-Ras alone, in the absence of further processing, is sufficient to cause the activation of MAP kinase in this system. All of the analogs of the farnesyl group, when incorporated into H-Ras, support the activation of the kinase to variable extents. These results suggest a direct but fairly nonspecific interaction of the farnesyl moiety of H-Ras with a soluble upstream activator of MAP kinase.

Animals↗
PubMed1995

Binding of prenylated and polybasic peptides to membranes: affinities and intervesicle exchange.

The small GTP-binding protein G25K and the protein K-Ras 4B contain prenyl groups (geranylgeranyl and farnesyl, respectively) that are thioether linked to a C-terminal cysteine which is methylated on its alpha-carboxyl group. These proteins, like many other prenyl proteins, also have a string of basic residues near their C-termini. A series of prenylated peptides based on the C-terminal sequences of human brain G25K and human K-Ras 4B were synthesized and analyzed for their membrane binding affinities. G25K peptides containing an N-terminal N-acetyltryptophan group were studied because their binding to membranes containing a trace of dansylated phospholipid could be detected by fluorescence resonance energy transfer. The G25K peptide lacking a prenyl group and a C-terminal methyl ester did not detectably bind to vesicles, and binding was enhanced by more than 500-fold if the peptide was geranylgeranylated. For the farnesylated peptide, methylation of the C-terminus increased membrane affinity by at least 60-fold if the vesicles contained phosphatidylserine and by 3-fold if they lacked this acidic lipid. The geranylgeranylated and methylated G25K peptide remains irreversibly attached to vesicles over several minutes only if the vesicles contain phosphatidylserine, whereas the corresponding nonmethylated or farnesylated and methylated peptides dissociate rapidly (less than a few seconds) from neutral or anionic vesicles. Farnesylation of the nonmethylated K-Ras 4B peptide enhances its affinity to vesicles containing acidic phospholipids (phosphatidylglycerol or phosphatidylserine) by 70-fold, and methylation leads to an additional dramatic (150-fold) increase in membrane affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2.

The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses.

Allergens↗
PubMed1995

Translocation of the 85-kDa phospholipase A2 from cytosol to the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen.

The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.

Animals↗
PubMed1995

Multiple enzymatic activities of the human cytosolic 85-kDa phospholipase A2: hydrolytic reactions and acyl transfer to glycerol.

The recombinant human 85-kDa cytosolic phospholipase A2 (cPLA2), when assayed in the presence of glycerol, catalyzes the transfer of acyl chains of radiolabeled phosphatidylcholine and para-substituted phenyl esters of fatty acids to glycerol, in addition to hydrolyzing these substrates. The product of the transacylation reaction is monoacylglycerol (MAG), and the acyl chain is predominantly esterified (> or = 95%) to a primary hydroxyl group of glycerol (sn-1/3); the stereochemistry is not known. Increasing concentrations of glycerol accelerate enzyme turnover both by providing an additional mechanistic pathway for the enzyme-substrate complex to form products and by increasing the intrinsic hydrolytic and transacylation activities of the enzyme. Significant enzymatic hydrolysis of sn-1/3-arachidonylmonoacylglycerol was measured, while sn-1/3-alpha-linolenoyl- and sn-2-arachidonylmonoacylglycerols were not detectably hydrolyzed. 1,3-Propanediol also serves as an acyl acceptor for the enzyme. cPLA2 hydrolyzes analog of lysophosphatidylcholine that lacks the sn-2 hydroxyl group. The enzyme will hydrolyze sn-1-acyl chains of rac-1-(arachidonyl, alpha-linolenoyl, palmitoyl)-2-O-hexadecyl-glycero-3-phosphocholine lipids and transfer the acyl chain to glycerol. Thus, cPLA2 has phospholipase A1 activity but only if an ether linkage rather than an ester linkage is present at the sn-2 position, and it is shown that the sn-1 acyl chains of both enantiomers of phosphatidylcholine are hydrolyzed. Phenyl [14C]-alpha-linolenate and five para-substituted phenyl esters of [3H]-alpha-linolenic acid with pKa values ranging from 7.2 to 10.2 for the phenol leaving groups were incorporated into 1,2-ditetradecyl-sn-glycero-3-phosphomethanol/Triton X-100 mixed micelles as substrates for the transacylation/hydrolysis reactions of the enzyme. Average product ratios, which are defined as the amount of monoacylglycerol formed to phenyl ester hydrolyzed, were 2.1 +/- 0.1 (n = 5) for the para-substituted phenyl esters and 2.0 +/- 0.3 (n = 7) for phenyl alpha-linolenate. The similarity of the ratios, despite the range of pKa values for the leaving groups, is consistent with the formation of a common enzyme intermediate that partitions to give either fatty acid or MAG. That intermediate may be a covalent acyl enzyme. Finally, the acyl chain specificity of cPLA2 was investigated to better understand the preference of the enzyme for phospholipids with sn-2-arachidonyl chains.

Acylation↗
PubMed1995

The inhibition of protein prenyltransferases by oxygenated metabolites of limonene and perillyl alcohol.

The monoterpenes limonene and perillyl alcohol are effective therapeutic agents against advanced rat mammary cancer. Limonene is currently undergoing clinical testing in cancer patients. These monoterpenes and their oxygenated metabolites have been previously shown to inhibit protein prenylation in cultured cells. Since farnesylation of ras protein is critical for its ability to cause oncogenic transformation, inhibition of protein prenylation may be the basis of the anti-tumor effects of limonene and perillyl alcohol. In this study we test the ability of limonene and its oxygenated analogs to inhibit protein prenylation enzymes in vitro. Limonene and perillyl alcohol and their major in vivo metabolite, perillic acid, are weak inhibitors of both mammalian and yeast protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT). In contrast, a minor metabolite of both limonene and perillyl alcohol, perillic acid methyl ester, is a potent inhibitor of both enzymes. Perillic acid methyl ester is a competitive inhibitor of yeast PFT with respect to farnesyl pyrophosphate. These studies suggest that if the inhibition of protein prenylation is a mechanism for limonene's and perillyl alcohol's anti-cancer activities, these monoterpenes may be prodrugs that are converted into pharmacologically-active substances by metabolic modification.

Animals↗
PubMed1995

Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus.

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Amino Acid Sequence↗
PubMed1995

Mammalian protein geranylgeranyltransferase-I: substrate specificity, kinetic mechanism, metal requirements, and affinity labeling.

Protein geranylgeranyltransferase-I (PGGT-I) catalyzes the transfer of the 20-carbon prenyl group from geranylgeranyl pyrophosphate to the cysteine residue near the C-termini of a variety of eukaryotic proteins. Kinetic analysis of homogenous PGGT-I from bovine brain reveals that the reaction follows a sequential pathway in which either prenyl donor or acceptor can bind first to the enzyme and that the reaction operates at steady-state rather than at rapid equilibrium. Substrate inhibition by prenyl acceptor but not by prenyl donor suggests that geranylgeranyl pyrophosphate binding first to free enzyme is the kinetically preferred pathway. This is supported by isotope trapping experiments which show that the ternary complex goes on to products faster than the release of geranylgeranyl pyrophosphate from the complex. The KM for the interaction of geranylgeranyl pyrophosphate with PGGT-I is markedly affected by the structure of the prenyl acceptor bound to the enzyme. A detailed analysis of the substrate specificity of PGGT-I reveals that peptides which contain a C-terminal leucine are preferred (kcat/KM = 1-5 x 10(5) M-1 s-1) to those that end in serine (kcat/KM = 2-4 x 10(3) M-1 s-1) or phenylalanine (kcat/KM = 0.5 x 10(3) M-1 s-1). PGGT-I also catalyzes the farnesylation of peptides that have a C-terminal leucine; kcat for farnesylation and KM for farnesyl pyrophosphate are similar to those for geranylgeranylation, but the KM for the peptide is 30-fold higher. Geranyl pyrophosphate is utilized by PGGT-I but is a poor substrate. Optimal activity of PGGT-I is obtained in the presence of micromolar amounts of Zn2+ and mM amounts of Mg2+. Mn2+ or Cd2+ but not Co2+ can substitute for Zn2+ and for Mg2+. Metals are not required for tight-binding of geranylgeranyl pyrophosphate to PGGT-I, and the measured dissociation equilibrium constant for this binary complex is 16 nM. Photoaffinity analogues of geranylgeranyl pyrophosphate and farnesyl pyrophosphate were prepared and shown to exclusively label the beta-subunit. The implication of the results for the substrate specificity of protein prenylation in cells is briefly discussed.

Affinity Labels↗