Mechanisms of action of NIP71 on N-myristoyltransferase activity

N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT·II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60^src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (l) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence ofmyristoylatedalanine-richC-kinasesubstrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP₇₁) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291635-639, 1993) potently inhibited highly purified NMT activity (IC₅₀ 23.7 nM). A minor NMT activity (NMT·PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP₇₁ inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP₇₁ having an apparent higher affinity for NMT than the NMT·myristoyl·CoA complex. Inhibition by NIP₇₁ at subsaturating concentrations of myristolyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.Abbreviations NMT N-myristoyl-CoA:protein N-myristoyltransferase - NMT·I mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak I - NMT·II mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak II - NMT·III mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak III - NIP₇₁ 71 kDa heat-stable N-myristoyltransferase inhibitor protein     

The structure of myristoyl-CoA:protein N-myristoyltransferase

Protein N-myristoylation is a covalent modification that occurs co-translationally in eukaryotes. Myristate, a rare 14 carbon saturated fatty acid (C14:0), is attached, via an amide linkage, to the N-terminal glycine of a subset of eukaryotic and viral proteins by myristoyl-CoA:protein N-myristoyltransferase (Nmt). Genetic and biochemical studies have established that Nmt is a target for development of a new class of fungicidal drugs. The enzyme is also a potential target for development of antiviral and antineoplastic agents. The structure of Saccharomyces cerevisiae Nmt1p has been determined recently with bound substrate analogs. The Nmt fold resembles the fold of members of the GCN5-related N-acetyltransferase superfamily. The structure reveals how Nmt's myristoyl-CoA and peptide substrates are recognized and bound, and what elements control the enzyme's ordered kinetic mechanism. Acyl transfer occurs through the nucleophilic addition-elimination reaction: an oxyanion hole formed by main chain atoms polarizes the thioester carbonyl and stabilizes the transition state while deprotonation of the ammonium of the Gly acceptor appears to be mediated by Nmt's C-terminal carboxylate. The use of main chain carboxylate atoms as general base catalyst is a novel feature.     

Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyltransferase deletion mutants and human/yeast Nmt chimeras in Escherichia coli and S. cerevisiae.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, 455-residue, monomeric enzyme. Amino- and carboxyl-terminal deletion mutants of Nmt1p were genetically engineered to determine the minimal domain necessary to maintain catalytic activity. Enzyme activity was assessed by (i) sequentially inducing Nmt1p or its mutant derivatives and one of two eukaryotic substrates for the wild type enzyme (S. cerevisiae Gpa1p and rat Go alpha) in Escherichia coli, a bacterium with no endogenous myristoyltransferase activity, and monitoring Nmt-dependent incorporation of exogenous [3H]myristate into the G protein alpha subunits or (ii) an in vitro enzyme assay using lysates prepared from bacteria producing wild type or mutant Nmts. The data indicate that the minimal catalytic domain of Nmt1p is located between Ile59-->Phe96 and Gly451-->Leu455. Analyses of the ability of mutant nmtps to rescue the lethal phenotype of an nmt1 null allele in a haploid strain of yeast grown on rich media, with or without blockade of cellular fatty acid synthetase, suggest that the amino-terminal 59 residues of Nmt1p may play an important noncatalytic role, functioning as a targeting signal so this cytosolic enzyme can access cellular myristoyl-CoA pools generated from activation of exogenous C14:0 by acyl-CoA synthetase(s). Moreover, there appear to be differences in the location or accessibility of myristoyl-CoA pools derived from fatty acid synthetase and acyl-CoA synthetases. The E. coli co-expression system was used to map structural elements that determine differences in the peptide substrate specificities of Nmt1p and the orthologous human Nmt. Rat Go alpha is a substrate for both enzymes, whereas human Gz alpha is a substrate only for human NMT. Studies of a series of chimeric enzymes composed of elements from the amino- or carboxyl-terminal portions of human and yeast Nmts indicate that (i) recognition/utilization of Gz alpha involves elements distributed from the amino-terminal half through the region defined by Leu352-->Lys410 of the 416 residue human enzyme and (ii) formation of a fully functional peptide binding site and a fully functional myristoyl-CoA binding site in either of these enzymes requires contributions from both their amino-terminal and carboxyl-terminal halves.     

Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl- CoA:protein N-myristoyltransferase

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.     

Assay of N-myristoyl transferase by selective adsorption of myristoyl-coenzyme A on acidic alumina

We have developed a simple and rapid method for detecting the enzyme myristoyl-CoA:protein N-myristoyl transferase. The enzyme catalyzes the transfer of the myristoyl moiety of myristoyl-CoA to the amino-terminal glycine residue of a peptide (protein). Incorporation of the [14C]myristate into the peptide is quantified after separation of the [14C]myristoyl-peptide from unreacted [14C]myristoyl-CoA by selective adsorption of [14C]myristoyl-CoA on acidic alumina. Optimal assay concentrations were 200 microM synthetic peptide, 1 microM [14C]myristoyl-CoA, 10 mM Tris-HCl/1 mM dithiothreitol/0.1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid/aprotinin (10 micrograms/ml) buffer, pH 7.4, and 1-10 micrograms protein.     

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.     

Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyl transferase

Using synthetic octapeptides, we examined the amino-terminal sequence requirements for substrate recognition by myristoyl-CoA:protein N-myristoyl transferase (NMT). NMT is absolutely specific for peptides with amino-terminal Gly residues. Peptides with Asn, Gln, Ser, Val, or Leu penultimate to the amino-terminal Gly were substrates, whereas peptides with Asp, D-Asn, Phe, or Tyr at this position were not myristoylated. Peptides with aromatic residues at this position competitively inhibited myristoylation of substrates, introducing the possibility of developing specific in vivo inhibitors of NMT. Peptides having sequences which correspond to those of known N-myristoyl proteins, including p60src, appear to be recognized by a single enzyme, and yeast and murine NMT have identical substrate specificities. The catalytic selectivity of NMT for myristoyl transfer accounts for the remarkable acyl chain specificity of this enzyme.     

High affinity binding of an N-terminal myristoylated p60src peptide

N-Myristoyl and non-myristoyl peptides corresponding to the N terminus of p60src were used to examine whether N-myristoylation facilitates the binding of p60src to specific protein sites at the plasma membrane. We discovered high affinity protein acceptor sites (Kd = 2.7 nM) to a 15-amino acid N-myristoylated N-terminal p60src peptide in red cell membrane vesicles. Binding was not competed by the non-myristoylated analog of the peptide nor by shorter N-myristoyl src peptides and peptides homologous to the N terminus of other N-myristoylated proteins. Binding was not evident after treatment of vesicles with proteolytic enzymes. Raising the salt concentration of the buffer to 50 mM NaCl caused an apparent inhibition of binding. However, no significant effect of salt was observed on the off-rate of bound ligand under these conditions. The results indicate the existence of N-myristoyl-dependent p60src protein acceptor sites at or near the plasma membrane/skeleton interface of red cells which could be responsible for the localization of p60src to this region and may represent new regulatory components for p60src-mediated tyrosine kinase activity.     

Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 M. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 & mgr;M. The E. coli expressed sNMT was homogenous and showed enzyme activity.     

Amino acid sequence of the diazooxonorleucine binding site of Acinetobacter and Pseudomonas 7A glutaminase--asparaginase enzymes

Acinetobactor glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine, reduced with sodium borohydride, and cleaved with cyanogen bromide. Radioactivity was present only in a 96-residue-N-terminal peptide which eluted as the second peptide peak on Sephadex G-50. Radioactivity was released with the threonine in position 12 during automatic sequencing of this peptide. The amino acid sequence of a 60-residue tn-terminal segment and a 16-residue C-terminal segment of this peptide was determined. Pseudomonas 7 A glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine and reduced with sodium borohydride. Radioactivity was released with the threonine in residue 20 during automatic sequencing of the whole enzyme. Analysis of 26 N-terminal residues showed that an 8-residue segment containing the radioactive threonine was identical with that in Acinetobacter glutaminase-asparaginase and in Escherichia coli asparaginase. Additional identical residues were noted in the N-terminal regions of these enzymes.     

Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C₁₄-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C₁₄-CoA and can also bind the far more abundant palmitoyl-CoA (C₁₆-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C_18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C₁₆-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C₁₆-CoA. These results indicate that ACBD6 can locally sequester C₁₆-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C₁₆-CoA.     

Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major

The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 microM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16-66 microM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.     

Substrate specificity of Saccharomyces cerevisiae myristoyl-CoA: protein N-myristoyltransferase. Analysis of fatty acid analogs containing carbonyl groups, nitrogen heteroatoms, and nitrogen heterocycles in an in vitro enzyme assay and subsequent identification of inhibitors of human immunodeficiency virus I replication.

Covalent attachment of myristic acid (C14:0) to the amino-terminal glycine residue of a variety of eukaryotic cellular and viral proteins can have a profound influence on their biological properties. The enzyme that catalyzes this modification, myristoyl-CoA-protein N-myristoyltransferase (NMT), has been identified as a potential target for antiviral and antifungal therapy. Its reaction mechanism is ordered Bi Bi with myristoyl-CoA binding occurring before binding of peptide and CoA release preceding release of myristoylpeptide. Perturbations in the binding of its acyl-CoA substrate would therefore be expected to have an important influence on catalysis. We have synthesized 56 analogs of myristic acid (C14:0) to further characterize the acyl-CoA binding site of Saccharomyces cerevisiae NMT. The activity of fatty acid analogs was assessed using a coupled in vitro assay system that employed the reportedly nonspecific Pseudomonas acyl-CoA synthetase, purified S. cerevisiae NMT, and octapeptide substrates derived from residues 2-9 of the catalytic subunit of cyclic AMP-dependent protein kinase and the Pr55gag polyprotein precursor of human immunodeficiency virus I (HIV-I). Analysis of ketocarbonyl-, ester-, and amide-containing myristic acid analogs (the latter in two isomeric arrangements, the acylamino acid (-CO-NH-) and the amide (-NH-CO)) indicated that the enzyme's binding site is able to accommodate a dipolar protrusion from C4 through C13. This includes the region of the acyl chain occurring near C5-C6 (numbered from carboxyl) that appears to be bound in a bent conformation of 140-150 degrees. The activities of NMT's acyl-CoA substrates decrease with increasing polarity. This relationship was particularly apparent from an analysis of a series of analogs in which the hydrocarbon chain was terminated by (i) an azido group or (ii) one of three nitrogen heterocycles (imidazole, triazole, and tetrazole) alkylated at either nitrogen or carbon. This inverse relationship between polarity and activity was confirmed after comparison of the activities of the closely related ester- or amide-containing tetradecanoyl-CoA derivatives. Members from all of the analog series were surveyed to determine whether they could inhibit replication of human immunodeficiency virus I (HIV-I), a retrovirus that depends upon N-myristoylation of its Pr55gag for propagation. 12-Azidododecanoic acid was the most active analog tested, producing a 60-90% inhibition of viral production in both acutely and chronically infected T-lymphocyte cell lines at a concentration of 10-50 microM without associated cellular toxicity.     

N-myristoyl transferase assay using phosphocellulose paper binding.

N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.     

In vivo modulation of N-myristoyltransferase activity by orthovanadate

N-Myristoyltransferase (NMT) catalyses the transfer of myristate from myristoyl-CoA to the NH₂-terminal glycine residue of several proteins and are important in signal transduction. STZ-induced diabetes (an animal model for insulin-dependent diabetes mellitus, IDDM) resulted in a 2-fold increase in rat liver NMT activity as compared with control animals. In obese Zucker (fa/fa) rats (an animal model for non-insulin dependent diabetes mellitus, NIDDM) there was a4.7-fold lower liver particulate NMT activity as compared with the control lean rat livers. Administration of sodium orthovanadate to the diabetic rats normalised liver NMT activity. These results would indicate that the rat liver particulate N-myristoyltransferase activity appears to be inversely proportional to the level of plasma insulin, implicating insulin in the control of N-myristoylation.Abbreviations NMT N-myristoyl-CoA:protein N-myristoyltransferase - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - NIP₇₁ 71 kDa N-myristoyltransferase inhibitor protein - NAF₄₅ 45 kDa N-myristoyltransferase activating factor     

Crystal structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoyl-CoA and inhibitors reveal the functional roles of the N-terminal region

Protein N-myristoylation catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) plays an important role in a variety of critical cellular processes and thus is an attractive target for development of antifungal drugs. We report here three crystal structures of Saccharomyces cerevisiae NMT: in binary complex with myristoyl-CoA (MYA) alone and in two ternary complexes involving MYA and two different non-peptidic inhibitors. In all three structures, the majority of the N-terminal region, absent in all previously reported structures, forms a well defined motif that is located in the vicinity of the peptide substrate-binding site and is involved in the binding of MYA. The Ab loop, which might be involved in substrate recognition, adopts an open conformation, whereas a loop of the N-terminal region (residues 22-24) that covers the top of the substrate-binding site is in the position occupied by the Ab loop when in the closed conformation. Structural comparisons with other NMTs, together with mutagenesis data, suggest that the N-terminal region of NMT plays an important role in the binding of both MYA and peptide substrate, but not in subsequent steps of the catalytic mechanism. The two inhibitors occupy the peptide substrate-binding site and interact with the protein through primarily hydrophobic contacts. Analyses of the inhibitorenzyme interactions provide valuable information for further improvement of antifungal inhibitors targeting NMT.     

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.     

A synthetic peptide corresponding to residues 137 to 157 of p60v-src inhibits tyrosine-specific protein kinases

A 21-residue synthetic peptide corresponding to a part of the noncatalytic domain of p60v-src (residues 137 to 157) was found to inhibit the tyrosine kinase activity of p60v-src. The half inhibition concentration was ca. 7.5 microM. The peptide (peptide A) did not compete with substrate proteins or ATP. Peptide A also inhibited the autophosphorylation of epidermal growth factor receptor/kinase and the tyrosine-specific protein phosphorylation in the acetylcholine receptor-rich membranes isolated from electroplax of Narke japonica. However, serine/threonine-specific protein kinases such as cAMP-dependent and cGMP-dependent protein kinases were not inhibited by peptide A.     

A simplified assay for the enzyme responsible for the attachment of myristic acid to the N-terminal glycine residue of proteins, myristoyl-CoA: glycylpeptide N-myristoyltransferase.

A greatly simplified assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase (NMT) activity is described. The assay is based on the differential solubility of the acyl-peptides produced as a consequence of the NMT activity and yields results comparable with those obtained with the original assay described by Towler & Glaser [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2812-2816], which requires h.p.l.c. to determine the production of the acyl-peptides. The use of the revised assay in the preliminary steps of the purification of rat brain NMT is described, and its use in determining the fatty acid-specificity of the enzyme is illustrated. The results are shown to be comparable with those obtained with the h.p.l.c.-based assay.     

The unique N-terminal sequence of testis angiotensin-converting enzyme is heavily O-glycosylated and unessential for activity or stability.

The testis-specific isozyme of angiotensin-converting enzyme (ACE) is identical, from residue 68 to the C terminus, to the second half or C-terminal domain of somatic ACE. However, the first 67 residues, comprising the signal peptide and a Ser-/Thr-rich 36-residue sequence that constitutes the N terminus of mature testis ACE, are unique. We have expressed a mutant human testis ACE lacking this 36-residue N-terminal sequence and find that compared to the wild-type protein the mutant is 15 kDa smaller due to the loss of greater than 90% of all O-linked sugars, but that it retains full enzymatic activity and is stable in culture. Heavy O-glycosylation is a property of testis ACE that is not shared by the somatic enzyme and is attributable to this unique sequence.