Use of an Isotope-Coded Mass Tag (ICMT) Method To Determine the Orientation of Cholesterol Oxidase on Model Membranes

Abstract

Although the interfacial membrane protein cholesterol oxidase is structurally and kinetically well-characterized, its orientation in and mode of interaction with cholesterol-containing membranes have not been established. Cholesterol oxidase can alter the structure of the cell membrane in pathogenic bacteria and is thus a potential antimicrobial drug target. We recently developed a mass spectrometry-based isotope-coded mass tag (ICMT) labeling method to monitor the real-time solvent-accessible surface of peripheral membrane proteins, such as cholesterol oxidase. The ICMT strategy utilizes maleimide-based isotope tags that covalently react with cysteine residues. In this study, by comparing the ICMT labeling rates of cysteine variants of cholesterol oxidase, we determined which residues of the protein were engaged with the protein–lipid interface. We found that upon addition of cholesterol-containing lipid vesicles, four cysteine residues in a cluster near the substrate entrance channel are labeled more slowly with ICMT probes than in the absence of vesicles, indicating that these four residues were in contact with the membrane surface. From these data, we generated a model of how cholesterol oxidase is oriented when bound to the membrane. In conclusion, this straightforward method, which requires only microgram quantities of protein, offers several advantages over existing methods for the investigation of interfacial membrane proteins and can be applied to a number of different systems.

The corrective action of original cholesterol oxidase preparation: in vitro and in vivo use

“…Modern biotechnological use of preparations of the enzyme ChO, obtained from various sources, its chemical and physiological properties are of great interest to researchers abroad. 3,4 In Russia, the ChO enzyme itself is used in laboratory diagnostics to determine the level of total Ch and HDL-Ch in serum. Created original means based on ChO-active agent-PChO from Streptomiceslavendulae strain VKMA-5921 of soil origin is studied for the first time.…”

“…One of the authors of the article, Senior Reseacher IV Okunevich took part in the development of experimental nutritional models of DLP. 4 In rats, alimentary DLP was createdfor 20 and 30 days by feeding an atherogenic diet in the form of a hypercholesterinemic diet (HCh diet) enriched with dietary Ch. The HCh diet contained a very heated mixture of 5% cholesterol in sunflower oil and 0.12% methyl thiouracil (MTU), an essential thyroid suppressor agent.…”

Lipid-lowering and anti-atherosclerotic activity of the natural original enzyme preparation in the experiment

“…В научной литературе отмечен значительный интерес к проблемам, связанным с изучением модификаций ХСО. Современное биотехнологическое применение ХСО, получаемой из различных источников, ее новые физико-химические, биологические и физиологические свойства активно изучаются во многих зарубежных лабораториях [2,4,6,8].…”

Effect of lipid-lowering activity of the natural original enzyme preparation in the experiment

“…ХО применяют в лабораторной диагностике для определения в сыворотке крови уровня общего холестерина (ХС) и ХС-липопротеинов высокой плотности (ХС-ЛПВП) энзиматическим методом в наборах фирмы "Vital Diagnostics" (Россия) [3]. Современное биотехнологическое использование ХО, получаемой из различных источников, её новые физико-химические и физиологические свойства активно изучаются во многих зарубежных лабораториях [4,5].…”