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.     

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.     

N-myristoyl-transferase activity in cancer cells. Solubilization, specificity and enzymatic inhibition of a N-myristoyl transferase from L1210 microsomes

The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.     

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.     

Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.

The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.     

Prediction of N-myristoylation modification of proteins by SVM

Attachment of a myristoyl group to NH(2)-terminus of a nascent protein among protein post-translational modification (PTM) is called myristoylation. The myristate moiety of proteins plays an important role for their biological functions, such as regulation of membrane binding (HIV-1 Gag) and enzyme activity (AMPK). Several predictors based on protein sequences alone are hitherto proposed. However, they produce a great number of false positive and false negative predictions; or they cannot be used for general purpose (i.e., taxon-specific); or threshold values of the decision rule of predictors need to be selected with cautiousness. Here, we present novel and taxon-free predictors based on protein primary structure. To identify myristoylated proteins accurately, we employ a widely used machinelearning algorithm, support vector machine (SVM). A series of SVM predictors are developed in the present study where various scales representing physicochemical and biological properties of amino acids (from the AAindex database) are used for numerical transformation of protein sequences. Of the predictors, the top ten achieve accuracies of >98% (the average value is 98.34%), and also the area under the ROC curve (AUC) values of >0.98. Compared with those of previous studies, the prediction accuracies are improved by about 3 to 4%.     

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acid coenzyme A transferase from rat liver mitochondria.

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acids coenzyme A transferase purified from rat liver mitochondria was assayed. In addition to the previously identified substrates succinyl-CoA, 3-hydroxy-3-methylglutaryl-CoA and malonyl-CoA (Francesconi et al.(1989) Biochim. Biophys. Acta, 999, 163-170) also methylmalonyl-CoA, glutaryl-CoA and adipyl-CoA acted as enzyme substrates, with the latter thioester showing the highest apparent affinity. All corresponding dicarboxylic acids, but not oxaloacetic, citric, alpha-ketoglutaric, malic, fumaric and glutamic acids, acted as coenzyme A acceptor substrates. None of the tested monocarboxylic acids, or the corresponding coenzyme A esters, were enzymatically transformed by the here described coenzyme A transferase.     

Demonstration of multiple forms of bovine brain myristoyl CoA: protein N-myristoyl transferase

Pancreatic -cells are known to respond to hyposmolar stress by releasing insulin. It was evident from perifusion studies using islet cells from oblob-mice mixed with polyacrylamide beads that a similar type of secretory response can be obtained by isosmolar addition of 10–25 mM of the rapidly penetrating urea molecule. There was no effect with hyperosmolar addition of urea. The urea-induced insulin release differed from the ordinary stimulation of secretion in not disappearing but being more pronounced after previous heating to 45°C or removal of extracellular Ca²⁺. Isosmolar urea was exceptional as an insulin secretagogue in being effective also in the presence of the ₂-adrenergic agonist clonidine or when lowering the temperature to 24°C. Further support for the idea that isosmolar addition of rapidly penetrating molecules induces insulin release was obtained by test ing non-metabolizable glucose analogues. Whereas 25 mM 3-O-methyl-D-glucose doubled the secretory rate within 4 min, the non-permeant L-glucose had only a slight initial action. When not compensating for the alterations of the medium osmolarity 3-O-methyl-D-glucose was without effect. Although expansion of -cells cannot explain the existence of a pronounced initial secretory response to D-glucose it may under certain conditions contribute to the stimulatory effects of the sugar.     

链霉菌磷酸泛酰巯基乙胺基转移酶对载体蛋白的底物选择性的研究进展

磷酸泛酰巯基乙胺基转移酶(PPTase)催化脂肪酸合酶(FAS)、聚酮合酶(PKS)和非核糖体肽合成酶(NRPS)中载体蛋白从脱辅基形态转化为全辅基形态,对脂肪酸、PKS产物和NRPS产物的生物合成起着不可或缺的作用。本文介绍并总结了链霉菌PPTase对载体蛋白底物选择性的最新研究进展:Ⅲ型PPTase特异性催化同一个多肽链中ACP的辅基化;Ⅱ型PPTase倾向于催化Ⅰ型PKS中ACP和NRPS中PCP的辅基化;Ⅰ型PPTase倾向于催化Ⅱ型PKS中ACP和Ⅱ型FAS中ACP的辅基化;编码基因位于基因簇内的Ⅰ型/Ⅱ型PPTase倾向于催化编码基因位于同基因簇内的PKS/NRPS中ACP/PCP的辅基化;这些研究结果为阐明并改造链霉菌辅基化网络以提高特定次级代谢产物的产量提供了参考和借鉴。     

Substrate specificity of human alpha-L-fucosidase.

Human α-l-fucosidase is a soluble lysosomal enzyme which hydrolyzes α-l-fucose residues linked to the 2 position of galactose or the 3, 4, or 6 position ofN-acetylglucosamine. Demonstration of activity towards natural oligosaccharide or glycosphingolipid substrates was achieved by measuring liberated l-fucose by coupling to fucose dehydrogenase and NAD and measuring NADH production spectrophotometrically. Activity of purified human spleen, brain, and cultured skin fibroblast or crude cell extracts towards 4-methylumbelliferyl-α-l-fucoside had a pH optimum of 4.5 to 5.5 and was unaffected by the presence of neutral detergents such as Triton X-100. However, the addition of sodium taurocholate or other bile salts to the incubation mixture caused a marked inhibition at pH 5 and a shift in pH optimum to the pH 6–7 region. Sodium taurocholate effected a threefold reduction in the apparent K_m for α-l-fucosidase at pH 6.0, but studies on fucosidosis tissue (α-fucosidase deficiency) or subcellular fractions derived from rat liver failed to indicate the existence of a membrane-bound α-l-fucosidase. The response of other lysosomal hydrolases to the presence of bile salts was investigated and was found to be variable, perhaps depending upon the hydrophilic or hydrophobic nature of the natural substrate and/or the state of association of the active enzyme.     

Substrate specificity of ascorbate oxidase.

Ascorbate oxidase oxidizes leuco 2, 6-dichloroindophenol to the blue quinoid dye and produces spectral changes in the UV spectra of certain substituted polyhydric and amino phenols at pH 5.7. The new peaks produced by the addition of enzyme to the dichlorohydroquinones (2,5 and 2,6) and hydroxyhydroquinone correspond to the respective p-quinones of these substrates. At pH 5.7, the enzyme does not oxidize hydroquinone, barely oxidizes chlorohydroquinone, but oxidizes 2,6- and 2,5-dichlorohydroquinone and hydroxyhydroquinone at a rate about $\text{1}\text{12}$ that of ascorbic acid, with the uptake of one gram atom of oxygen per mole of substrate. A correlation has been found between the concentration of anion present in solution at pH 5.7 and the rate of oxidation of compounds of the hydroquinone series by the enzyme. The results indicate that an anionic form of the substrate is an important requirement of the enzyme specificity.     

Substrate specificity of vetebrate collagenase.

Substrate specificity of purified tadpole collagenase (EC 3.4.24.3) has been studied using eleven synthetic peptides. A pentapeptide, t-butyloxycarbonylprolylalanylglycylisoleucylalanine amide, was susceptible to the action of the enzyme and an octapeptide, acetylprolylglutaminylglycylisoleucylalanylglycylglutaminylarginine ethyl ester, was proposed to be the best substrate for vertebrate collagenase among the peptides tested.     

Down-regulation of N-myristoyl transferase expression in human T-cell line CEM by human immunodeficiency virus type-1 infection

The present study focuses on the expression level of N-myristoyl transferase (NMT) in the course of human immunodeficiency virus type-1 (HIV-1) infection. HIV-1 structural proteins were gradually expressed during the process of infection of the human T-cell line CEM, whereas the expression levels of NMT subsequently decreased under the same conditions. In addition, the chronically HIV-1-infected T-cell line CEM/LAV-1 exhibited low expression levels of NMT. We hypothesize that the decrease in the expression level of NMT due to HIV-1 infection may be related to the virus' strategy that leads to its persistent replication.     

Amino-terminal processing of actins mutagenized at the Cys-1 residue.

Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.     

Substrate specificity of rat brain ceramidase.

In this study, the substrate specificity of a newly identified rat brain ceramidase (CDase) was investigated. To this end, the major functional groups and stereochemistry of ceramide (Cer) were evaluated for their influence on the hydrolysis of substrate by this CDase. The results showed that, of the four possible stereoisomers of Cer, only the natural d-e-C(18)-Cer isomer was used as substrate (K(m) of 1.1 mol% and V(max) of 5 micromol/min/mg). Removal of the 4-5 trans double bond to generate dihydroceramide decreased the affinity of the enzyme toward its substrate by around 90%, whereas changing the configuration of the double bond from the natural trans configuration into cis or introduction of a hydroxyl group (phytoceramide) resulted in loss of hydrolysis. Shortening the chain length of the sphingosine backbone resulted in decreased affinity. Methylation of either the primary or the secondary hydroxyl groups resulted in loss of activity. Results also indicated that Cer species that harbor long saturated or monounsaturated fatty acyl chains are preferred substrates of the enzyme. alpha-Hydroxylated Cer demonstrated considerably higher affinity, indicating a preference of the enzyme to those Cer molecular species. These results disclose a very high specificity of nonlysosomal CDase for its substrate, Cer.     

Substrate specificity of carboxypeptidase from Watermelon.

The substrate specificity of carboxypeptidase (F-II) purified from watermelon for various synthetic peptides and esters was examined kinetically. The enzyme showed a broad substrate specificity against various carbobenzoxy- and benzyl-dipeptides. Peptides containing glycine or proline were hydrolyzed slowly by the enzyme. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal resulted in an increase in the rate of hydrolysis. Inhibition studies with diisopropyl flurophosphate and diastereomers of carbobenzoxy-Phe-Ala demonstrated that the peptidase and esterase activities of the enzyme are both catalyzed by the same site of the enzyme molecule, but the binding sites for peptides and esters seem not to be the same. The enzyme also had amidase activity, which was optimal at pH 7.0.