Molecular Dynamic Simulations of the Fatty Acid Bilayer Containing Very Long Chain Transmembrane Dicarboxylic Acids

Choi,Yong-Hoon,Yang,Chul-Hak,Kim,Hyun-Won,Jung,Seun-Ho The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.1

Recent research results regarding the very long chain transmembrane α,ω-dicarboxylic components in the membrane of extremophilic eubacteria, such as Sarcina ventriculi, Thermotoga maritima, and Thermoanaerobacter ethanolicus have raised interesting questions concerning the physical and biochemical function on these components in the membrane. In order to understand the dynamic characteristics of these acids which reside in the bilayer membrane, 580 ps molecular dynamic simulations at 300 K were performed for two model systems. These systems were the bilayer with regular chain (C16:0 or C18:1) fatty acid methyl and the fatty acid bilayer containing very long chain transmembrane dicarboxylic acid methyl esters (α,ω-15,16-dimethyltriacotane-dioate dimethyl ester; C32:0). Our analyses indicate that very long chain transmembrane dicarboxylic acids have a noticeable influence on the bilayer dynamics at a sub-nanosecond time scale. The center-of-mass mean-squared-displacement (MSD) of regular chain fatty acids adjacent to the very long chain transmembrane dicarboxylic acids decreased, the long-axis order parameter increased, and the reorientational motions of methylene groups were slowed along the hydrocarbon chains. These results indicate that the very long chain transmembrane dicarboxylic acids reduce the molecular order of the whole bilayer membrane.

Mechanism of Antibiotic Action and Biosynthesis of Centipedin Purified from Scolopendra subspinipes multilans L. Koch (Centipede)

Kim,Ki-Tae,Cho,Key-Seung,Lee,Jong-Ho,Hong,Sa-Weon,Park,Kyung-Bae The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

The 8-hydroxyisocoumarin, named Centipedin, which has a significant antibiotic activity, was separated and solubilized with organic solvents, such as diethyl ether from centipede Scolopendra subspinipes multilans L. Koch. The Centipedin was purified by silicic acid column and high S cation exchange chromatography followed by reverse-phase HPLC. It was confirmed that Centipedin has a potent antibiotic effectiveness against Gram-negative Klebsiella pneumoniae ATCC 8308. The results showed that Centipedin blocks both DNA replication and RNA transcription during the growth of this pathogen in vivo. The biosynthesis of antibiotic 8-hydroxyisocoumarin was studied in vivo by feeding [¹⁴C]-labelled compound as a precursor to live centipede, in which [¹⁴C] acetate was the most efficiently incorporated into the Centipedin within 30h after injection. Also, in vitro study on the biosynthesis of Centipedin showed that efficient incorporation of [¹⁴C] acetate occurred at pH range 5.0-7.0 for 10h incubation and decreased significantly after then. It is suggested that 8-hydroxyisocoumarin is one of the defense compounds acting on bacterial infection in Scolopendra subspinipes.

Existence of "25 kDa Thiol Peroxidase" in Retina : Evidence for An Antioxidative Role

Kim,Il-Han,Cha,Mee-Kyung The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

We isolated and sequenced a human retina cDNA fragment that encodes 25 kDa thiol peroxidase. A search of a databank showed that the 25 kDa thiol peroxidase from retina is the same type of thiol peroxidase which exists in human brain and red blood cells. This type of thiol peroxidase was distributed in all of the tested tissues including retina. This result suggests a physiological role for the 25 kDa thiol peroxidase as an important antioxidant.

Active-Site Mutants of Human Glutathione S-Transferase P1-1 : Effects of the Mutations on Substrate Specificity and Inhibition Characteristics

Kong,Kwang-Hoon,Park,Hee-Joong,Yoon,Suck-Young The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with theronine resulted in a drastic decrease in the specific activities to <10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the l50 values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be inthe vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.

In Vitro Enhancement of Microsomal Cytochrome P450-Dependent Monooxygenases by Organic Solvents in Rat Liver

Lee,Dong Wook,Moon,Ja Young,Lim,Heung Bin,Park,Ki Hyun The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

In vitro effects of acetone, methanol, and dimethylsulfoxide (DMSO) on liver microsomal cytochrome P450(P450) content, and P450-dependent arylhydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were studied in rats. Acetone at 1%(v/v) enhanced the content of P450, assayed spectrally in 3-methylcholanethrene (MC)- AND β-naphthoflavone(BNF)-inducible micorsomes by 18 and 7%, respectively. Methanol, up to 5% (v/v) applied, also showed enhancement effects on P450 content in liver microsomes from rats treated with phenobarbital (PB), MC, and BNF, as well as uninduced microsomes with similar but low strength. DMSO, however, did not show such enhancing effects at the ranges of the concentrations applied. AHH and ECOD activities in MC-inducible microsomes were also enhanced by acetone at 1%, which was in proportion to the increase in P450 content by the same concentration. However, the P450 content, and AHH and ECOD activities, were decreased by increasing the concentration of acetone. Methanol at the same concentration with acetone also enhanced ECOD activity but not AHH activity in MC-inducible microsomes. The enhancing effect of acetone on the enzymes was negligible when the micorsomes were pretreated with a specific monoclonal antibody of MC-inducible isozyme. The difference in the effects of these solvents on P450 system might be due to their different properties that cause the P450 active site to be exposed in milieu.

Establishment of a Binding Assay System for Screening of the Inhibitors of p56lck SH2 Domain

Yun,Yungdae,Hur,Eun Mi,Kim,Jynho The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

Src-Homology 2(SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SH2 domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the p56lck SH2 domain. In our assay system, SH2 domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the botinylated phosphotyrosine (pY)-containing synthetic peptides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated steptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction wit reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of p56lck SH2 function.

Characterization of dnaK Mutants in Streptococcus pneumoniae

Pyo,Suhk-Neung,Kim,Seung-Whan,Rhee,Dong-Kwon The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.1

DnaK is a major heat shock protein and known to be highly conserved in all species. Previously, the dnaK in Streptococcus pneumoniae was cloned and the immunogenic nature characterized. In this study, dnaK mutants were generated by insertion of duplication mutagenesis and their characteristics examined. They had defective growths at all temperatures (20℃-42℃) and cell divisions, and formed filaments after a temperature shift from 30 to 42. A unique feature of the dnaK mutants of S. pneumoniae, unlike those of E. coli and B. subtilis, was the growth capability at high temperature (42℃) without producing the putative GroES. Our results suggest that DnaK may serve as a regulator and/or modifier in GroEL gene expression.

Purification and Characterization of a 25 kDa Cathepsin L-like Protease from the Hemocyte of Coleopteran Insect, Tenebrio molitor Larvae

Lee,Bok Luel,Lee,Kang Moon,Kim,Mi Hee,Choi,Hye Won,Cho,Mi Young,Kurata, Shoichiro,Natori, Shunji,Jang,Kyung Suk,Lee,Young Un The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

Insect plasma protein is abundant in the hemolymph of holometabolous insect larvae and is used as a source of amino acids and energy for construction of adult structures during metamorphosis. In order to understand the mechanism of decomposition of larval plasma proteins by hemocyte protease, we tried to purify a cysteine protease from the hemocyte lysate by using Carbobenzoxy-L-Phenylalanyl-L-Arginine-4-Methyl-Coumaryl-7-Amide (Z-Phe-Arg-MCA) as substrate and to identify plasma proteins that are selectively susceptible to the purified protease. Here, we describe the purification and characterization of a cysteine protease that specifically hydrolyzes the plasma protein of the coleopteran insect, Tenebrio molitor, larvae. The molecular mass of this enzyme was 25 kDa, as determined by SDS-PAGE under reducing conditions. The amino acids sequence of its NH₂-terminus was determined to be Leu-Pro-Gly-Gln-Ile-Asp-Trp-Arg-Asp-Lys-Gly. This sequence contained Pro, Asp, and Arg residues, conserved in many papain superfamily enzymes. The specific cysteine protease inhibitors, such as E-64 and leupetin, inhibited its hydrolytic activity. One plasma protein with a molecular mass of 48 kDa was selectively hydrolyzed within 3 h when the purified enzyme and plasma proteins were incubated in vitro. However, the 48 kDa protein was not hydrolyzed by the purified 25 kDa protease in the presence of E-64. Western blotting analysis at various developmental stages showed that the purified enzyme was detected at larvae, pupae, and adult stages, but not the embryo stage.

Rat Duodenal Mucosa Inositol Monophosphatase; Novel Enzyme of Which Properties are Distinct from Brain Enzyme

Kwon, Hyeok Yil,Lim, Bong Hee,Park, Hyung Seo,Lee, Yun Lyul,Lee, Eun Hee,Choi,Soo Young,Park, Hyoung Jin The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.3

An inositol monophosphatase (IMPase) was purified to homogeneity from rat duodenal mucosa for the first time and its enzymatic properties were investigated. Rat duodenal mucosa peculiarly exhibited the highest IMPase activity among various rat tissues examined. By means of ammonium sulfate precipitation, followed by Q-Sepharose, polylysine agarose, reactive-red agarose column chromatography, Uno-Q FPLC, and Bio-Silect FPLC, duodenal IMPase was purified 223-fold to a specific activity of 13.6U/mg protein. The molecular mass of the native enzyme was estimated to be 48,000 Da on gel filtration. The subunit molecular mass was determined by SDS-PAGE to be 24,000 Da. These results indicate that duodenal IMPase is a dimeric protein made up of identical subunits. Rat duodenal IMPase has distinct properties from brain IMPase. It has a broad spectrum of substrate specificity and is insensitive to Li+. Duodenal IMPase does not absolutely require Mg²+ for its catalytic activity. Furthermore, duodenal IMPase is less stable to heat than brain enzyme. It is suggested that the rat duodenal mucosa needs a large amount of IMPase whose properties are quite different from that of the brain enzyme.

Purification and Properties of Phenylalanine Ammonia-lyase from Chinese Cabbage

Lim, Chang-Jin,Lim, Hye-Won,Sa, Jae-Hoon,Kim, Tae-Soo,Park, Eun-Hee,Park, Soo-Sun The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.1

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme in the phenylpropanoid biosynthesis, catalyzes the elimination reaction of ammonium ion from L-phenylalanine. PAL was purified from the cytosolic fraction of Chinese cabbage (Brassica campestris ssp. napus var. pekinensis) through ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-200 chromatography, and Q-Sepharose chromatography. It consists of four identical subunits, the molecular mass of which was estimated to be about 38,000 daltons on SDS-PAGE. The optimal pH and temperature of the purified enzyme are 8∼9 and 45℃, respectively. Its activity is greatly inhibited by Zn²?? ion, and strongly activated by caffeic acid. The purified PAL has some different characteristics compared to those obtained with other PALs.