Many Kinds Of Enzymes Research Articles

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.

Spatiotemporal expression of Prdm genes during Xenopus development.

Epigenetic regulation is known to be important in embryonic development, cell differentiation and regulation of cancer cells. Molecular mechanisms of epigenetic modification have DNA methylation and histone tail modification such as acetylation, phosphorylation and ubiquitination. Until now, many kinds of enzymes that modify histone tail with various functional groups have been reported and regulate the epigenetic state of genes. Among them, Prdm genes were identified as histone methyltransferase. Prdm genes are characterized by an N-terminal PR/SET domain and C-terminal some zinc finger domains and therefore they are considered to have both DNA-binding ability and methylation activity. Among vertebrate, fifteen members are estimated to belong to Prdm genes family. Even though Prdm genes are thought to play important roles for cell fate determination and cell differentiation, there is an incomplete understanding of their expression and functions in early development. Here, we report that Prdm genes exhibit dynamic expression pattern in Xenopus embryogenesis. By whole mount in situ hybridization analysis, we show that Prdm genes are expressed in spatially localized manners in embryo and all of Prdm genes are expressed in neural cells in developing central nervous systems. Our study suggests that Prdm genes may be new candidates to function in neural cell differentiation.

Effects of Intracellular Process on the Therapeutic Activation of Nanomedicine

In this article, we review the endocytosis ways of nanoscale materials and how they work. Surrounded by FBS in culture medium, the special nanomedicines, entering into lysosome via endosome or autophagosome, are degraded by many kinds of enzymes, and are finally snagged. During this process, they are interacting with lysosome membrane, hence the lysosome membrane changes in permeation. With a weak damage extent of lysosome, the nanoparticles escape the damage effect of lysosome, and interact with other organelles (for example mitochondria, proteins), resulting in their defeature and being swallowed by autophagosomes. Now that lysosomes are damaged, the autophagosomes won’t be digested by lysosomes. With accumulation of endosomes, the cancer cells accelerate the aging process or apoptosis. If lysosomes are strongly destabilized, the cathepsin B/D will interact with caspase family, causing necrosis or apoptosis of cancer cells. So we recognise that the lysosome is key for cancer cell death.

亚热带3种树种凋落叶厚度对其分解速率及酶活性的影响

对中国亚热带树种杉木(<em>Cunninghamia lanceolata</em>)、香樟(<em>Cinnamomum camphora</em>)、银杏(<em>Ginkgo biloba</em>)3个树种在不同凋落物厚度下凋落物分解速率和分解酶活性进行了探究。利用分解网袋法,根据浙江省的平均酸雨水平,在酸雨(pH4.0)条件下设置了凋落物40g、凋落物20g、凋落物10g 3个梯度。结果表明:凋落物分解速率随厚度的增加呈加快的趋势,杉木凋落物10、20、40g的年分解系数<em>K</em>分别为0.24、0.27、0.34,香樟凋落物10、20、40g的年分解系数<em>K</em>分别为0.25、0.3、0.32,银杏凋落物10、20、40g的年分解系数<em>K</em>分别为0.42、0.5、0.58;脲酶活性表现为:凋落叶40g>凋落叶20g>凋落叶10g,纤维素酶活性表现为:凋落叶40g、凋落叶20g>凋落叶10g,蔗糖酶活性表现为:后期凋落叶40g>凋落叶20g>凋落叶10g,凋落物分解过程是多种酶共同作用的结果。

Regulation of metabolism and transport of sphingosine-1-phosphate in mammalian cells

Sphingosine-1-phosphate (S1P), which is generated from the sphingosine kinase-catalyzed phosphorylation of sphingosine, is now recognized as a critical regulator of many kinds of physiological and pathological processes, including cancer, cardiovascular function, and diabetes. It can also trigger a wide variety of biological effect, such as cell movement, differentiation, survival, inflammation, immunity, calcium homeostasis, and angiogenesis. As we know, a number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and the secretion of S1P are regulated by various endogenetic or ectogenous stimuli and involve many kinds of enzymes and transporters. In this review, we discuss the regulation of S1P synthesis by many kinds of enzymes and mainly introduce the process of ceramide to S1P. Moreover, S1P deterioration is important balance in physiologic adjustment. We also describe the role of verified or potential transporters in S1P release in detail.

Design, Synthesis, and Evaluation of [188Re]Organorhenium-Labeled Antibody Fragments with Renal Enzyme-Cleavable Linkage for Low Renal Radioactivity Levels

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl Nepsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated with Nepsilon-tert-butoxycarbonyl-glycyl-lysine or Nepsilon-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippuryl Nepsilon-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While [188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acid at a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higher than those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.

キャピラリー／マイクロチップ電気泳動による糖分析ならびに糖鎖‐タンパク質結合解析

Carbohydrates are widely distributed in nature and have multilateral functions. Recently glycobiology has evoked from the special interest in diverse functions of carbohydrates covalently bound to proteins and lipids. It has been revealed that the carbohydrate chains in such glycoconjugatges on cell surface are deeply associated with various cell events including division, differentiation, morphogenosis, fertilization, inflammation and metastasis. Although they are not formed by direct translation as in genomes and proteomes, they are important because many kinds of enzymes relating to their biosynthesis are under the control of genomes. Therefore, the carbohydrates in glycoconjugates are attractive as postscriptomes, and the method development for carbohydrate analysis is an urgent theme in glycobiology. Since a number of structurally resembling species of carbohdrates coexist in biological samples, their analysis requires the most efficient methods capable of high resolution and sensitive detection. For this reason capillary electrophoresis (CE) has become the most important tool for carbohydrate analysis. CE is, however, basically a method for ions, hence carbohydrates, which are generally neutral, are not good objects of CE. In addition, carbohydrates are normally difficult to sensitively detect, because they lack in chromophore/fluorophore. Chemical/enzymatic derivatization is the most appropriate strategy to solve these problems simultaneously. In this paper a number of methods for derivatization of carbohydrates for CE are compared and criticized, and the 1-phenyl-3-methyl-5-pyrazolone (PMP) method and the methylglycamine-4-nitro-2, 1, 3-benzodiazole tagging method (MG-NBD method) are selected as the most suitable methods for routine and ultramicro analyses of carbohydrates, respectively. A number of examples for their application are presented. A technique of automation of such analysis by CE with in-capillary derivatization is also presented. Miniaturization of the PMP method to microchip electrophoresis (ME) has realized rapid analysis of carbohydrates in few ten seconds. The versatile functions of carbohydrates stem from their binding with particular proteins, and therefore the study of carbohydrate-protein binding is also important. CE can be performed in free solution, not necessitating addition of any supporting materials. Owing to this single-phase property of CE one can observe interaction occurring in a capillary as the migration time change of either of these reactants in running buffer containing the counterpart. By mathematical treatment of such migration time change, we could develop a series of methods for micro-ultramicro estimation of association constant. On the other hand we could succeed in finding out a number of proteins in biological samples, which can specifically bind to glycans and glycoconjugates.

Use of a Microtiter Plate Screening Method for Obtaining Leuconostoc mesenteroides Mutants Constitutive for Glucansucrase

A new method using microtiter plates for screening mutants for the production of glucansucrases of Leuconostoc mesenteroides was developped. The method significantly increased the efficiency of the screening process for enzyme production of mutants. Several high-producing constitutive mutants for glucansucrase were selected from L. mesenteroides B-512FMC, B-742CB, B-1299CB, and B-1355C. Enzyme production of the mutants were stable for at least 6 months with culture transfer every two weeks and as lyophilized cultures. The method has wide-ranging capabilities beyond the screening for glucansucrase-producing mutants and it could be used for the screening of many kinds of enzymes or compounds produced by microbial mutants.