TAILIEUCHUNG - Báo cáo khoa học: Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit

ThemaltoseATP-binding cassette transporter ofSalmonella typhimurium is composed of a membrane-associated com-plex (MalFGK2) and a periplasmic receptor (MalE). In addition to its role in transport, the complex acts as a repressor ofmaltose-regulatedgene expressionand is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively. . | Eur. J. Biochem. 269 4074-4085 2002 FEBS 2002 doi Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit Anke Stein1 Martina Seifert2 Rudolf Volkmer-Engert2 Jorg Siepelmeyer3 Knut Jahreis3 and Erwin Schneider1 1 Humboldt Universitat zu Berlin Institut fur Biologie Berlin Germany 2Humboldt Universitat zu Berlin Institut fur Medizinische Immunologie Berlin 3 Universitat Osnabruck Fachbereich Biologie Chemie Germany The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of a membrane-associated complex MalFGK2 and a periplasmic receptor MalE . In addition to its role in transport the complex acts as a repressor of maltose-regulated gene expression and is subject to inhibition in the process of inducer exclusion. These activities are thought to be mediated by interactions of the ATPase subunit MalK with the transcriptional activator MalT and nonphosphorylated enzyme IIA of the glucose phosphotransferase system respectively. To gain further insight in protein regions that are critical for these functions we have generated nine MalK-specific monoclonal antibodies. These bind to four nonoverlapping linear epitopes 60-LFig-63 5B5 113-RVNQVAEVLQL-123 represented by 4H12 309-GHETQI-314 2F9 and 352-LFREDG SACR-361 represented by 4B3 . All mAbs recognize their epitopes in soluble MalK and in the MalFGK2 complex with Kd values ranging from 10 6 to 10 8 M. ATP reduced the affinity of the mAbs for soluble MalK indicating a conformational change that renders the epitopes less accessible. 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE maltose-stimulated ATPase activity of proteolipo-somes while their Fab fragments displayed no significant effect. The results suggest a similar solvent-exposed position of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in

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