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     

Expression of human N-myristoyltransferase inEscherichia coli. Comparison with N-myristoyltransferases expressed in different tissues

Myristoyl CoA:protein N-myristoyltransferase catalyzes the addition of myristate to the amino-terminal glycine residue of a number of eukaryotic proteins.Escherichia coli transformed with human NMT expression construct produced high levels of N-myristoyltransferase. Using the combination of ammonium sulfate precipitation, chromatography on SP-Sepharose fast flow and fast protein liquid chromatography on Mono-S, the enzyme was purified more than 100 fold with 40% yield. The hNMT fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-polyacrylamide gel electrophoresis. Upon cleavage by the Enterokinase [(Asp)₄-Lys], the hNMT exhibited an apparent molecular mass of 49 kDa without loss of catalytic activity. The hNMT activity could be greatly activated severalfold with the use of Tris, SDS, ethanol and acetonitrile. The catalytic activity of hNMT was potently inhibited in a concentration dependent manner by NIP₇₁₁ a bovine brain NMT inhibitory protein with a half maximal inhibition of 31.0 nM. TheE. coli expressed hNMT was homogeneous and showed enzyme activity.Abbreviations NMT N-myristoyl CoA:protein N-myristoyltransferase - NIP₇₁ 71 kDa heat stable membrane bound N-myristoyltransferase inhibitor protein - hNMT human NMT - DTNB N-5,5dithiobis (2-nitrobenzoic acid) - FPLC fast protein liquid chromatography - IPTG isopropyl -D-thiogalactopyranoside - cDNA complementarydeoxyribonucleic acid - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride     

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.     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.     

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.     

Coenzyme A dependent myristoylation and demyristoylation in the regulation of bovine spleen N-myristoyltransferase

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the transfer of myristate to the NH₂-terminal glycine residue of a number of important proteins of diverse function. Little is known about the control and regulation of NMT in higher eukaryotes. Bovine spleen N-myristoyltransferase has been purified and characterized [Raju, RVS, Kalra J & Sharma RK (1994) J Biol Chem 269:12080–12083]. The activation of bovine spleen NMT with thiol reducing compounds, and its inhibition by the oxidizing agent sodium iodate, suggest a role for oxidation/reduction in NMT regulation. Available knowledge concerning coenzyme A (CoA), the thiol in the cell, indicated that the agents tested on NMT could also reduce or oxidize CoA. The studies suggested that reduced CoA is the key regulator of NMT activity, while oxidized CoA did not allow NMT to promote myristoylation. Further, the process of myristoylation and demyristoylation may be governed by NMT, depending on the differential concentration of CoA. The process of demyristoylation could be blocked by excess CoA. We therefore hypothesize that the initial event in the regulation of NMT is an increase in cellular CoA concentration which could be coupled to an increase in protein myristoylation. Once the CoA concentration in the cell decreases due to oxidation, the demyristoylation process would be operative.Abbreviations NMT N-myristoyl CoA:protein N-myristoyltransferase - hNMT human NMT - YNMT yeast NMT - DTNB N-55 dithiobis(2-nitrobenzoic acid) - DTT dithiothretol - 2-ME 2-mercaptoethanol     

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.     

A new,robust, and nonradioactive approach for exploring N-myristoylation

Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.     

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.     

Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.     

Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.     

N-myristoylation of p60src. Identification of a myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues.

A 16-residue synthetic peptide corresponding to the N-terminal sequence of p60src was used as the acyl acceptor in an assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues. An additional C-terminal tyrosine amide was added to this peptide to facilitate radioiodination and enhance detectability. Reverse-phase h.p.l.c. enabled the simultaneous detection and quantification of the peptide substrate and its N-myristoylated product. N-Myristoyltransferase activity was highest in the brain with decreasing activities in lung, small intestine, kidney, heart, skeletal muscle and liver. Brain activity was distributed approximately equally between the 100,000 g pellet and supernatant fractions. The soluble enzyme exhibited a Kappm of 20 microM for the src peptide and an optimum between pH 7.0 and 7.5. Maximum N-acylating activity was seen with myristoyl (C14:0)-CoA with lower activities found with the C10:0-CoA and C12:0-CoA homologues. No activity was obtained with palmitoyl (C18:0)-CoA but this derivative inhibited N-myristoyltransferase activity greater than 50% at equimolar concentrations with myristoyl-CoA. With a decapeptide corresponding to the N-terminal sequence of the cyclic AMP-dependent protein kinase catalytic subunit as the acyl acceptor, the brain enzyme displayed a Kapp.m of 117 microM and was about 14-fold less catalytically effective than with the p60src acyl acceptor. Transferase activity was also seen with a 16-residue peptide corresponding to the N-terminal sequence of the HIV p17gag structural protein. Inhibition studies with shorter src peptide analogues indicated an enzyme specificity for the p60src acyl acceptor beyond 9 residues.     

Synthesis of Certain 4-Substituted-1-β-D-Ribofuranosyl-3-Hydroxypyrazoles Structurally Related to the Antibiotic Pyrazofurin

Abstract

Several 4-substituted-1-β-D-ribofuranosyl-3-hydroxypyrazoles were prepared as structural analogs of pyrazofurin. Glycosylation of the TMS derivative of ethyl 3(5)-hydroxypyrazole-4-carboxylate (3) with 1-0-acetyl-2,3,5-tri-0-benzoyl-D-ribofuranose in the presence of TMS-triflate gave predominantly ethyl 3-hydroxy-1-(2,3,5-tri-0-benzoyl-β-D-ribofuranosyl)pyrazole-4-carboxylate (4a), which on subsequent ammonolysis furnished 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-carboxamide (5). Benzylation of 4a with benzyl bromide and further ammonolysis gave 3-benzyloxy-1-β-D-ribofuranosylpyrazole-4-carboxamide (8a). Catalytic (Pd/C) hydrogenation of 8a afforded yet another high yield route to 5. Saponification of the ester function of ethyl 3-benzyloxy-1-β-D-ribofuranosylpyrazole-4-carboxylate (7b) gave the corresponding 4-carboxylic acid (6a). Phosphorylation of 8a and subsequent debenzylation of the intermediate 11a gave 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-carboxamide 5′-phosphate (11b). Dehydration of 3-benzyloxy-1-(2,3,5-tri-0-acetyl-β-D-ribofuranosyl)pyrazole-4-carboxamide (8b) with POCl₃ provided the corresponding 4-carbonitrile derivative (10a), which on debenzylation with Cl₃SiI gave 3-hydroxy-1-(2,3,5-tri-0-acetyl-β-D-ribofuranosyl)pyrazole-4-carbonitrile (13). Reaction of 13 with H₂S/pyridine and subsequent deacetylation gave 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-thiocarboxamide (12b). Similarly, treatment of 13 with NH₂OH afforded 3-hydroxy-1-β-D-ribofuranosylpyrazole-4-carboxamidoxime (14a), which on catalytic (Pd/C) hydrogenation gave the corresponding 4-carboxamidine derivative (14b). The structural assignment of these pyrazole ribonucleosides was made by single-crystal X-ray analysis of 6a. None of these compounds exhibited any significant antitumor or antiviral activity in cell culture.     

Potential role of N-myristoyltransferase in cancer

Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. The treatment of colonic cancer remains difficult because of the lack of effective chemotherapeutic agents; therefore it is important to continue to search for cellular functions that can be disrupted by chemotherapeutic drugs resulting in the inhibition of the development and progression of cancer. The current knowledge of the modification of proteins by myristoylation involving myristoyl-CoA: protein N-myristoyltransferase (NMT) is in its infancy. This process is involved in the pathogenesis of cancer. We have reported for the first time that NMT activity and protein expression were higher in human colorectal cancer, gallbladder carcinoma and brain tumors. In addition, an increase in NMT activity appeared at an early stage in colonic carcinogenesis. It is conceivable therefore that NMT can be used as a potential marker for the early detection of cancer. These observations lead to the possibility of developing NMT specific inhibitors, which may be therapeutically useful. We proposed that HSC70 and/or enolase could be used as an anticancer therapeutic target. This review summarized the status of NMT in cancer which has been carried in our laboratory.     

Synthesis and Biological Evaluation of Some Acyclic Pyridine C-Nucleosides. Part Two

Abstract

3-Bromo-5-(2-hydroxyethylthiomethyl)pyridine (7) was synthesized by reaction of 3-bromo-5-chloromethylpyridine hydrochloride (6) with the mono sodium salt of 2-mercaptoethanol. 3-Bromo-5-hydroxymethylpyridine (10) was, after protection as a silyl ether, converted to the 3-carboxy analogue using BuLi and CO₂. After deprotection with NH₄F, the alcohol function was chlorinated using SOCl₂. Finally, attachment of the acyclic chain and ammonolysis gave the acyclic nicotinamide nucleosides. Treatment of the latter compounds with Lawesson's reagent gave the thioamide analogues. All compounds were identified by NMR and DCI-MS. The acyclic pyridine C-nucleosides were evaluated against a series of tumor-cell lines and a variety of viruses. No marked biological activity was found.     

Synthesis and Biological Evaluation of some Acyclic Nucleoside Cyclic Phosphoramidate Derivatives

Abstract

The acyclic nucleosides 2 were treated with 2-chloro-3-methyl-1-oxa-3-aza-2-phosphacyclopentane (3) in the presence of diisopropylethylamine to give the corresponding phosphoramidite derivatives (4). The phosphoramidite intermediates (4) were oxidized with m-chloroperbenzoic acid to the phosphoramidate derivatives (5). Treatment of 5a,b with ZnBr₂ in CH₃NO₂ gave the corresponding acyclic nucleoside cyclic phosphoramidates (6a,b). Attempts to desilylation of 5c by tetrabutylammonium fluoride (TBAF) resulted in opening of the phosphoramidate ring. The newly synthesized compounds were evaluated for antiviral and antitumor cell activity.     

A New Synthesis of 7-Methyl-8-Oxoguanosine

Abstract

A new, facile synthesis of 7-methyl-8-oxoguanosine is reported. 2-Chloro-7-methylpurine-6, 8-dione (5) was silylated with hexamethyldi-silazane and the silylated intermediate, 6, glycosylated with 1-0-acetyl-2, 3, 5-tri-0-benzoyl-D-ribofuranose to yield 2-chloro-7-methyl-9-(2′, 3′,-5′-tri-0-benzoyl-β-D-ribofuranosyl) purin-6, 8-dione (8). Deprotection of 8 with sodium hydroxide in aqueous methanol gave 2-chloro-7-methyl-9-(β-D-ribofuranosyl) purine-6,8-dione (9), which was aminated with liquid ammonia or methanolic ammonia to yield 7-methyl-8-oxoguanosine (3).     

Synthesis of Some Novel Acyclolumazine N-1 Nucleosides

Abstract

Reactjon of (2-acetoxyethoxy)methyl bromide with the silylated lumazine bases (1-6) in the presence of n-Bu₄NI leads to the formation of the nucleosides 8, 10, 12, 14, 16 and 18 respectively. Deacetylation with methanolic ammonia afforded the free nucleosides 9, 11, 13, 15, 17 and 19, respectively, in good yields. Structural proofs of the newly synthesized compounds are based on elemental analyses, UV and ¹H-NMR spactra. None of the acyclic nucleosides exhibited antiviral activity against HSV-1 in vitro.     

Synthesis and Biological Properties of 9-(2, 4-Dihydroxybutyl) Adenine and Guanine: New Analogues Of 9-(2, 3-Dihydroxyprqpyl)adenine (Dhpa) and 9-(2-Hydroxyethoxymethyl)guanine (Acyclovir)

Abstract

New analogues of antiviral agents 9-(2, 3-dihy-droxyproply) adenine (DHPA, 1a.) and 9-(2-hydroxyethoxymethyl) guanine (acyclovir, Ib) - compounds Ic and Id were prepared and their biological activity was investigated. Racemic 1, 2, 4-butanetriol (2) was converted to the corresponding benzylidene derivative (3a) by acetalation with benzalde-hyde and triethyl orthoformate. Acetal 3a and p-toluene- sul-fonyl chloride in pyridine gave the corresponding p-toluenes fonate 3b. Alkylation of adenine 5a via sodium salt of 5a with 3b in dimethylformamide or in the presence of tetra-n-butylammonium fluoride in tetrahydrofuran gave intermediate 6a. Reaction of 2-amino-6-chloropurine (5b) with 3b effected by K₂CO₃ in dimethylsulfoxide gave compound 6b and a smaller amount of 7-alkylated proauct 7. A similar transformation catalyzed by tetra-n-butylammonium fluoride afforded only intermediate 5b. Acid-catalyzed de-protection (hydrolysis) of 6b and 6a gave the title compounds Ic and Id. The S-enantiomer of Ic was deaminated with adenosine deaminase. Our results argue against the presence of a methyl group-binding site of adenosine deaminase. Compounds Ic and Id exhibited little or no activity in antiviral assays with several DNA and RNA viruses.     

人Src蛋白N端区段的表达、纯化和体外豆蔻酰化底物活性

利用RT PCR技术 ,从来源于人结肠癌Caco 2细胞总RNA中 ,扩增得到编码人Src蛋白N端 147氨基酸的DNA序列片段。进而构建T7启动子控制下的C端His tag融合的表达质粒pMF SrcHT ,并转化大肠杆菌BL2 1(DE3)。通过SDS PAGE等分析结果显示 ,在 37°C培养条件下经IPTG诱导 ,C端His tag融合的人Src蛋白N端区段 (命名为SrcHT ,2 1kD)得到高效表达 ,并且主要以可溶性形式存在。进一步利用Ni IDA亲和层析分离 ,从表达菌裂解上清液中一步纯化获得重组蛋白SrcHT ,SDS PAGE分析纯度达 95 %以上。在此基础上 ,以 [3H]豆蔻酰 CoA为同位素标记底物进行SrcHT的体外NMT豆蔻酰化反应测定。SDS PAGE分离和放射自显影分析结果表明 ,SrcHT蛋白可被NMT有效豆蔻酰化而具有NMT的底物活性。这些为深入详细研究Src蛋白豆蔻酰化作用和构建以Src蛋白豆蔻酰化为靶标的分子筛药体系等打下了重要基础。