Biomedical Chemistry: Research and Methods最新文献_第6页

The Study of Sensitivity to Proteolysis of Full-length and Truncated Forms of Recombinant Human Renalase Expressed in the Prokaryotic System 原核系统中表达的全长和截短重组人Renalase对蛋白水解敏感性的研究

Biomedical Chemistry: Research and Methods Pub Date : 1900-01-01 DOI: 10.18097/bmcrm00164

V. Fedchenko, A. Kaloshin, S. Kaloshina, A. Kopylov, A. E. Medvedev

{"title":"The Study of Sensitivity to Proteolysis of Full-length and Truncated Forms of Recombinant Human Renalase Expressed in the Prokaryotic System","authors":"V. Fedchenko, A. Kaloshin, S. Kaloshina, A. Kopylov, A. E. Medvedev","doi":"10.18097/bmcrm00164","DOIUrl":"https://doi.org/10.18097/bmcrm00164","url":null,"abstract":"Renalase (RNLS) is a flavoprotein; its N-terminal peptide (amino acid residues 1-17) performs various important functions. Inside cells, it is involved in the Rossmann fold formation (residues 2-35), which is necessary for the binding of the FAD cofactor and the manifestation of the enzymatic activity of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). When RNLS is secreted into the extracellular space, this peptide is cleaved off, and the resulting truncated extracellular RNLS can no longer bind FAD and, therefore, numerous effects described in the literature are carried out by non-catalytic mechanisms. In this work, we have investigated the sensitivity to trypsinolysis of two recombinant forms of human RNLS expressed in prokaryotic cells: (a) full-length RNLS containing the FAD cofactor; (b) a truncated RNLS lacking the 1-17 N-terminal peptide (truncatedRNLS, tRNLS) unable to bind the FAD cofactor. Trypsin (1 unit/20 μL of medium) effectively cleaved both forms of renalase (RNLS and tRNLS). When exposed to a lower concentration of trypsin (0.1 U/20 μL of medium), full length RNLS was more trypsin resistant than tRNLS. We suggest that the different sensitivity of RNLS and tRNLS is apparently determined by the presence of the FAD cofactor in the full-length recombinant protein, which contributes to the formation of a spatial structure that is more resistant to the action of certain proteases.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"32 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"132211044","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generalized predictive model of estimation of inhibition of muscarinic receptors M1-M5 毒蕈碱受体M1-M5抑制的广义预测模型

Biomedical Chemistry: Research and Methods Pub Date : 1900-01-01 DOI: 10.18097/bmcrm00129

A. Mikurova, Vladlen S. Skvortsov, V. Grigoryev

{"title":"Generalized predictive model of estimation of inhibition of muscarinic receptors M1-M5","authors":"A. Mikurova, Vladlen S. Skvortsov, V. Grigoryev","doi":"10.18097/bmcrm00129","DOIUrl":"https://doi.org/10.18097/bmcrm00129","url":null,"abstract":"A general predictive model for assessing the inhibition constant (Ki) value of human acetylcholine muscarinic receptors M1-M5 by potential ligands has been constructed. We used information on the three-dimensional structure of human M1, M2, M4, and M5 receptors, as well as a model of the M3 receptor constructed according to homology based on the structure of the rat M3 receptor. A set of complexes of known inhibitors with the target receptor constructed by means of molecular docking, was selected using an additional option: the coincidence of the spatial position of 4 pharmacophore points of a tested inhibitor and tiotropium, for which the position in the crystal structure was known. For five types of M receptors 199 complexes with known Ki values were selected. Based on the data obtained during molecular dynamics simulation of these complexes by means of the MM-PBSA/MM-GBSA methods, their energy characteristics were calculated. They were used as independent variables in linear regression equations for pKi value prediction. The R2 prediction for the generalized equation was 0.7, and the mean prediction error was 0.55 logarithmic units with a range for pKi=4.7.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"185 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"115380245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Electrochemical Analysis of Metabolites as a Method for Cytochromes P450 Activity Determination 电化学分析代谢物测定细胞色素P450活性的方法

Biomedical Chemistry: Research and Methods Pub Date : 1900-01-01 DOI: 10.18097/bmcrm00176

A. Kuzikov, R. Masamrekh, T. Filippova, V. Shumyantseva

{"title":"Electrochemical Analysis of Metabolites as a Method for Cytochromes P450 Activity Determination","authors":"A. Kuzikov, R. Masamrekh, T. Filippova, V. Shumyantseva","doi":"10.18097/bmcrm00176","DOIUrl":"https://doi.org/10.18097/bmcrm00176","url":null,"abstract":"The review deals with the electrochemical methods for determination of metabolites of cytochromes P450 catalyzed reactions. We have focused on the electrochemical determination of metabolites of drugs and some endogenous compounds. We have reviewed bielectrode systems for determination of cytochrome P450 activity, where one electrode serves as a matrix for enzyme immobilization and a source of electrons for heme iron ion reduction and initialization of the catalytic reaction towards a substrate and the second one is being used for quantification of the products formed by their electrochemical oxidation. Such systems allow one to elude additional steps of separation of reaction substrates and products. The review also includes discussion of the ways to increase the analytical sensitivity and decrease the limit of detection of the investigated metabolites by chemical modification of electrodes. We demonstrate the possibilities of these systems for cytochrome P450 kinetics analysis and the perspectives of their further improvement, such as increasing the sensitivity of metabolite electrochemical determination by modern electrode modificators, including carbon-based, and construction of devices for automatic monitoring of the products.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"95 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"122479710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

The Study of Type I Collagen by Immunoblotting in Samples of Bone-Plastic Biomaterials 骨塑生物材料中I型胶原蛋白的免疫印迹研究

Biomedical Chemistry: Research and Methods Pub Date : 1900-01-01 DOI: 10.18097/bmcrm00189

T. Medvedeva, L. Volova, L. N. Kulagina

{"title":"The Study of Type I Collagen by Immunoblotting in Samples of Bone-Plastic Biomaterials","authors":"T. Medvedeva, L. Volova, L. N. Kulagina","doi":"10.18097/bmcrm00189","DOIUrl":"https://doi.org/10.18097/bmcrm00189","url":null,"abstract":"The type I collagen was studied in samples of two types of osteoplastic materials produced in the Biotech Research Institute of the Samara State Medical University using immunoblotting. The demineralized samples used in the work were compact bone powder and crushed material of human cancellous bone tissue. Collagen and its polypeptides were separated in a 5% polyacrylamide gel with 3.6 M urea according to the method of Hayashi and Nagai (1979). The advantage of the method is the separation under these conditions of type I and III collagen, as well as the α1(I) and α2(I) chains of type I collagen. Immunoblotting was carried out by diffusion method according to the method of Towbin et al. (1979) using nitrocellulose membranes (Santa Cruz, USA). Primary goat polyclonal antibodies to denatured collagen, 1:500 dilution (Millipore) were used. Peroxidase-conjugated secondary antibodies (mouse vs. goat), 1:80000 dilution (Sigma) were used also. It has been established that the bulk of the compact bone protein is localized between the α1- and α2-fractions of collagen. In samples of cancellous bone tissue, a molecular reduction of the protein is noted. Protein macromolecules with a gradually decreasing molecular weight and low molecular weight polypeptides migrating in the gel with a wide front up to the indicator line are detected. Due to the low specificity of osteoblast integrins in regenerating bone tissue, collagen polypeptides, as well as protein molecules retained in implants, can act as inducers of synthetic processes occurring in osteoblast nuclei. Protein fragmentation products in the implant can act as signaling molecules that trigger cascades of enzymatic reactions and intracellular signaling pathways.","PeriodicalId":286037,"journal":{"name":"Biomedical Chemistry: Research and Methods","volume":"1 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"1900-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130203936","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0