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

D 2002

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

BOUCHAL, Pavel and Igor KUČERA

Basic information

Original name

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

Authors

BOUCHAL, Pavel and Igor KUČERA

Edition

Stará Lesná, XVIII. biochemický zjazd. Zborník. p. 160-160, 2002

Publisher

Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV

Other information

Language

English

Type of outcome

Proceedings paper

Field of Study

10600 1.6 Biological sciences

Country of publisher

Slovakia

Confidentiality degree

is not subject to a state or trade secret

RIV identification code

RIV/00216224:14310/02:00006413

Organization unit

Faculty of Science

Keywords in English

Paracoccus denitrificans; membrane proteins; proteome; 2-D electrophoresis

Abstract

In the original language

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.

Links

GA203/01/1589, research and development project

Name: Mechanizmy regulace respiračních systémů denitrifikačních bakterií faktory prostředí

Investor: Czech Science Foundation, Mechanisms involved in regulation of respiratory systems in denitrification bacteria by environmental factors

Cite

BOUCHAL, Pavel and Igor KUČERA. Analysis of membrane proteome of Paracoccus denitrificans by two-dimensional electrophoresis. In XVIII. biochemický zjazd. Zborník. Stará Lesná: Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV, 2002, p. 160.

@inproceedings{405321,
   author = {Bouchal, Pavel and Kučera, Igor},
   address = {Stará Lesná},
   booktitle = {XVIII. biochemický zjazd. Zborník.},
   keywords = {Paracoccus denitrificans; membrane proteins; proteome; 2-D electrophoresis},
   language = {eng},
   location = {Stará Lesná},
   pages = {160-160},
   publisher = {Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV},
   title = {Analysis of membrane proteome of Paracoccus denitrificans by two-dimensional electrophoresis},
   year = {2002}
}

TY  - CONF
ID  - 405321
AU  - Bouchal, Pavel - Kučera, Igor
PY  - 2002
TI  - Analysis of membrane proteome of Paracoccus denitrificans by two-dimensional electrophoresis
PB  - Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV
CY  - Stará Lesná
KW  - Paracoccus denitrificans
KW  - membrane proteins
KW  - proteome
KW  - 2-D electrophoresis
N2  - 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.
ER  -

BOUCHAL, Pavel and Igor KUČERA. Analysis of membrane proteome of Paracoccus denitrificans by two-dimensional electrophoresis. In \textit{XVIII. biochemický zjazd. Zborník.}. Stará Lesná: Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV, 2002, p.~160.

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