Analysis of membrane proteome of Paracoccus denitrificans by two-dimensional electrophoresis

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Publikace nespadá pod Fakultu sportovních studií, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.
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BOUCHAL Pavel KUČERA Igor

Rok publikování 2002
Druh Článek ve sborníku
Konference XVIII. biochemický zjazd. Zborník.
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Obor Biochemie
Klíčová slova Paracoccus denitrificans; membrane proteins; proteome; 2-D electrophoresis
Popis In facultative anaerobic bacterium P. denitrificans, a broad range of substrates can serve as carbon sources and terminal electrone acceptors. Successfull adaptation of this bacterium to altered conditions depends on expression of metabolically active proteins. For example, nitrate induces expression of some denitrification enzymes. Many important redox proteins are membrane-associated. For comprehensive study on changes of proteins level in this bacterium, we have optimized a method using two-dimensional electrophoresis (2-DE). Our work represents the first analysis of P. denitrificans proteome using 2-DE. Membrane suspension of anaerobically grown cells of Paracoccus denitrificans was prepared by combination of enzymatic and osmotic lysis, centrifugation and ulgtracentrifugation. We have tested various detergents and procedures for membrane proteins presolubilization (e.g., SDS, CHAPS, dodecyl-â-D-maltoside with 6-aminocaproic acid). Sample buffer and IEF gel composition effects on 2-D gel patterns were observed. Other parametres of 2-DE analysis were also optimized: conditions of final centrifugation, dilution ratio (presolubilized sample:sample buffer) and focusation conditions of carrier-ampholyte isoelectric focusation. 12% homogenous SDS-PAGE gel (16x20 cm) was used in the second-dimension. Using the optimized protocol, we have analyzed protein composition of membrane of P. denitrificans cells grown under aerobic and anaerobic conditions. 2-D gel patterns were compared using PDQUEST software. We have found 15 proteins significantly more expressed under anaerobic conditions a 5 proteins significantly more expressed under aerobic conditions. We have also sucessfully analyzed periplasmic proteins using this method.
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