• Journal of Microbiology
• /
• 제34권3호
• /
• pp.270-273
• /
• 1996

Micrococcus luteus purine nucleoside phosphorylase (PNP) has been purified and characterized. The physical and kinetic properties have been described previously. Chemical modification of the enzyme was attempted to gain insight on the active site. The enzyme was inactivated in a time-dependent manner by the arginine- specific modifying reagent phenylglyoxal. There was a linear relationship between the observed rate of inactivation and the phenylglyoxal concentration. At 30 $^{\circ}C$ the bimolecular rate constant for the modification was 0.015 $min^{-1}mM^{-1}$ in 50 mM $NaHCO_3$ buffer, pH 7.5. The plot of logk versus log phenylglyoxal concentration was a strainght line with a slope value of 0.9, indicating that modification of one arginine residue was needed to inactivate the enzyme. Preincubation with saturated solutions of substrates protected the enzyme from inhibition of phenylglyoxal, indicating that reactions with phenylglyoxal were directed at arginyl residues essential for the catalytic functioning of the enzyme.

• 한국식품영양학회지
• /
• 제8권1호
• /
• pp.37-42
• /
• 1995

The amino acid sequence of basic isozyme 55 of Horseradish Peroxidase (HRP E5) was determined by protein sequencing. HRP E5 consisted about 300 residues, and has a molecular weight of approximately 36,000 $\pm$ 500 dalton. The protein was rich In aspartic acid (14%), arginine(13%), and leucine(11%). The primary structure of HRP E5 was established by sequencing its tryptic (T1-T19) and lysylendopeptic (Al-A3) peptides. The sequence homology between HRP E5 and HRP C (neutral isozyme of horseradish peroxidase) is found to be more than 66%. The highest concentration of identical residues are found on residues 29~56, 90~123, and 155~173, but relatively low on 174~271.

• Bulletin of the Korean Chemical Society
• /
• 제24권12호
• /
• pp.1799-1804
• /
• 2003

Acetolatate synthase(ALS) catalyzes the first common step in the biosynthesis of valine, leucine, isoleucine in plants and microorganisms. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. To elucidate the roles of arginine residues in tobacco ALS, chemical modification and site-directed mutagenesis were performed. Recombinant tobacco ALS was expressed in E. coli and purified to homogeneity. The ALS was inactivated by arginine specific reagents, phenylglyoxal and 2,3-butanedione. The rate of inactivation was a function of the concentration of modifier. The inactivation by butanedione was enhanced by borate, and the inactivation was reversible on removal of excess butanedione and borate. The substrate pyruvate and competitive inhibitors fluoropyruvate and phenylpyruvate protected the enzyme against inactivation by both modifiers. The mutation of well-conserved Arg198 of the ALS by Gln abolished the enzymatic activity as well as the binding affinity for cofactor FAD. However, the mutation of R198K did not affect significantly the binding of FAD to the enzyme. Taken together, the results imply that Arg198 is essential for the catalytic activity of the ALS and involved in the binding of FAD, and that the positive charge of the Arg is crucial for the interaction with negatively charged FAD.

• BMB Reports
• /
• 제34권5호
• /
• pp.402-407
• /
• 2001

Based on the currently proposed toxicity model for the different Bacillus thuringiensis Cry $\delta$-endotoxins, their pore-forming activity involves the insertion of the ${\alpha}4-{\alpha}5$ helical hairpin into the membrane of the target midgut epithelial cell. In this study, a number of polar or charged residues in helix 4 within domain I of the 65-kDa dipteranactive Cry11A toxin, Lys-123, Tyr-125, Asn-128, Ser-130, Gln-135, Arg-136, Gln-139 and Glu-141, were initially substituted with alanine by using PCR-based directed mutagenesis. All mutant toxins were expressed as cytoplasmic inclusions in Escherichia coli upon induction with IPTG. Similar to the wild-type protoxin inclusion, the solubility of each mutant inclusion in the carbonate buffer, pH 9.0, was relatively low When E. coli cells, expressing each of the mutant proteins, were tested for toxicity against Aedes aegypti mosquito-larvae, toxicity was completely abolished for the alanine substitution of arginine at position 136. However, mutations at the other positions still retained a high level of larvicidal activity Interestingly, further analysis of this critical arginine residue by specific mutagenesis showed that conversions of arginine-136 to aspartate, glutamine, or even to the most conserved residue lysine, also abolished the wild-type activity The results of this study revealed an important determinant in toxin function for the positively charged side chain of arginine-136 in helix 4 of the Cry11A toxin.

• 한국발생생물학회지:발생과생식
• /
• 제17권1호
• /
• pp.9-16
• /
• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

• Journal of Microbiology and Biotechnology
• /
• 제30권3호
• /
• pp.382-390
• /
• 2020

Periplanetasin-4 is an antimicrobial peptide with 13 amino acids identified in cockroaches. It has been reported to induce fungal cell death by apoptosis and membrane-targeted action. Analogs were designed by substituting arginine residues to modify the electrostatic and hydrophobic interactions accordingly and explore the effect of periplanetasin-4 through the increase of net charge and the decrease of hydrophobicity. The analogs showed lower activity than periplanetasin-4 against gram-positive and gram-negative bacteria. Similar to periplanetasin-4, the analogs exhibited slight hemolytic activity against human erythrocytes. Membrane studies, including determination of changes in membrane potential and permeability, and fluidity assays, revealed that the analogs disrupt less membrane integrity compared to periplanetasin-4. Likewise, when the analogs were treated to the artificial membrane model, the passage of molecules bigger than FD4 was difficult. In conclusion, arginine substitution could not maintain the membrane disruption ability of periplanetasin-4. The results indicated that the attenuation of hydrophobic interactions with the plasma membrane caused a reduction in the accumulation of the analogs on the membrane before the formation of electrostatic interactions. Our findings will assist in the further development of antimicrobial peptides for clinical use.

• BMB Reports
• /
• 제28권2호
• /
• pp.124-128
• /
• 1995

Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136 $M^{-1}min^{-1}$. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157 $M^{-1}min^{-1}$ and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.

• BMB Reports
• /
• 제30권4호
• /
• pp.280-284
• /
• 1997

Phenylglyoxal diethyl pyrocarbonate (DEPC), and 1-cyclohexyl-3-[2-morpholinoethyl]-carbodiimide metho-p-toluenesulfonate (CMC) are modifying reagents specific for arginine, histidine, and aspartate or glutamate, respectively. They were found to inactivate S. cerevisiae farnesyl protein transferase (FPTase). The peptide substrate protected the enzyme against inactivation by CMC and the other substrate farnesyl pyrophosphate showed protection against inactivation by phenylglyoxal. while neither of the two substrates protected the enzyme against DEPC inactivation. These results suggest the presence of aspartate/glutamate, arginine and histidine residues at the active site of this enzyme.

• BMB Reports
• /
• 제31권2호
• /
• pp.139-143
• /
• 1998

The catabolic ${\alpha}-acetolactate$ synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the tryptophane-specific reagent, N -bromosuccinimide, and the arginine-specific reagent, phenylglyoxal. The enzyme was inactivated slowly by the cysteine-specific reagent N-ethylmaleimide. The second-order rate constants for the inactivation by N-bromosuccinimide, phenylglyoxal. and N -ethylmaleimide were $114,749M^{-1}min^{-1}$, $304.3M^{-1}min^{-1}$, and $5.1M^{-1}min^{-1}$, respectively. The reaction order with respect to N-bromosuccinimide, phenylglyoxal, and N-ethylmaleimide were 1.5,0.71, and 0.86, respectively. The inactivation of the catabolic aacetolactate synthase by these modifying reagents was protected by pyruvate. These results suggest that essential tryptophane, arginine, and cysteine residues are located at or near the active site of the catabolic ${\alpha}-acetolactate$ synthase.

• Journal of Microbiology and Biotechnology
• /
• 제16권9호
• /
• pp.1441-1447
• /
• 2006

The light perception and phototransformation of phytochromes are the first process of the phytochrome-mediated light signal transduction. The chromophore ligation and its photochromism of various site-specific and deletion mutants of pea phytochrome A and bacterial phytochrome-like protein (Cph1) were analyzed in vitro. Serial truncation mutants from the N-terminus and C-terminus indicated that the minimal N-terminal domain for the chromophore ligation spans from the residue 78 to 399 of pea phytochrome A. Site-specific mutants indicated that several residues are critical for the chromophore ligation and/or photochromism. Histidine-324 appears to serve as an anchimeric residue for photochromism through its H-bonding function. Isoleucine-80 and arginine-383 playa critical role for the chromophore ligation and photochromism. Arginine-383 is presumably involved in the stabilization of the Pfr form of pea phytochrome A. Apparently, the amphiphilic ${\alpha}$-helix centered around the residue-391 is in the chromophore pocket and critical for the chromophore ligation.