	Liposomes in reconstitution of ion-pumps Flemming Cornelius Associate Professor, PhD., DSc. phone: +45 8942 2926, fax:+45 8612 9599 E-mail: fc@biophys.au.dk
PROJECTS In this laboratory we investigate structure, function, and regulation of the sodium-pump and calcium-pump after purification and reconstitution into small lipid vesicles. Since the sidedness of the pump is re-established the asymmetric properties e.g. associated with ion-transport can be studied. Symmetry of pump incorporation and morphological characterization of the proteoliposome population is performed by functional tests and laser diffraction studies (QELS). Protein conformations following phosphorylation is detected by stopped-flow spectro-fluorometry using electrochromic membrane probes. Electrogenic properties with generation of membrane potential associated with ion-transport is studied by potential sensitive fluorescent dyes. The protein-lipid interaction is investigated by biochemical and microcalorimetric measurements (DSC) of pumps after reconstitution into liposomes with defined phospholipid and cholesterol composition. Methods for 2D crystalization in proteoliposomes are investigated. The physiological regulation of the sodium pump by protein kinases (PKA, PKC) is studied in the cell membranes and after purification comparing functioninal and immunochemical studies (SDS-PAGE, 2D-gels, Western, etc.). KEY-REFERENCES Cornelius, F. Rate Determination in Phosphorylation of shark Na,K-ATPase by ATP: Temperature sensitivity and effects of ADP. Biophys. J. 77: 934. Fedosova,N.U., Cornelius,F., and Klodos,I. 1998. E₂P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and P_i by their reactivity towards hydroxylamine and vanadate. Biochemistry 37:13634. Cornelius,F., Fedosova,N.U., and Klodos,I. 1998. E₂P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in P_i phosphorylation of Na,K-ATPase. Biochemistry 37:16686. Gropp,T., Cornelius,F., and Fendler,K. 1998. K⁺-Dependence of electrogenic transport by the Na,K-ATPase. Biochim. Biophys. Acta 1368:184. Cornelius,F. and Logvinenko,N. 1996. Functional regulation of reconstituted Na,K-ATPase by protein kinase A phosphorylation. FEBS lett. 380:277. Cornelius,F. 1995. Cholesterol modulation of molecular activity of reconstituted shark Na,K-ATPase. Biochim. Biophys. Acta 1235:205 Cornelius,F. and Møller,J.V. 1991. Electrogenic pump current of sarcoplasmic reticulum Ca-ATP-ase reconstituted at high lipid/protein ratio. FEBS lett. 284:46. PhD projects: Phosphorylation of Na,K-ATPase by protein kinase A and C. Functional significance in regulation. Yasser A. Mahmmoud Technician: Hanne R. Z. Christensen Front Page Home Department Research Na,K-ATPase E-type-ATPase Teaching Staff ScS TAS Calendar Search Forside Hjem Institut Forskning Na,K-ATPase E-type-ATPase Undervisning Personale VIP TAP Kalender Søgning

Liposomes in reconstitution
of ion-pumps

Flemming Cornelius
Associate Professor, PhD., DSc.
phone: +45 8942 2926, fax:+45 8612 9599
E-mail: fc@biophys.au.dk

PROJECTS

In this laboratory we investigate structure, function, and regulation of the sodium-pump and calcium-pump after purification and reconstitution into small lipid vesicles. Since the sidedness of the pump is re-established the asymmetric properties e.g. associated with ion-transport can be studied.

Symmetry of pump incorporation and morphological characterization of the proteoliposome population is performed by functional tests and laser diffraction studies (QELS).

Protein conformations following phosphorylation is detected by stopped-flow spectro-fluorometry using electrochromic membrane probes.

Electrogenic properties with generation of membrane potential associated with ion-transport is studied by potential sensitive fluorescent dyes.

The protein-lipid interaction is investigated by biochemical and microcalorimetric measurements (DSC) of pumps after reconstitution into liposomes with defined phospholipid and cholesterol composition. Methods for 2D crystalization in proteoliposomes are investigated.

The physiological regulation of the sodium pump by protein kinases (PKA, PKC) is studied in the cell membranes and after purification comparing functioninal and immunochemical studies (SDS-PAGE, 2D-gels, Western, etc.).

KEY-REFERENCES

Cornelius, F. Rate Determination in Phosphorylation of shark Na,K-ATPase by ATP: Temperature sensitivity and effects of ADP. Biophys. J. 77: 934.

Fedosova,N.U., Cornelius,F., and Klodos,I. 1998. E₂P phosphoforms of Na,K-ATPase. I. Comparison of phosphointermediates formed from ATP and P_i by their reactivity towards hydroxylamine and vanadate. Biochemistry 37:13634.

Cornelius,F., Fedosova,N.U., and Klodos,I. 1998. E₂P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in P_i phosphorylation of Na,K-ATPase. Biochemistry 37:16686.

Gropp,T., Cornelius,F., and Fendler,K. 1998. K⁺-Dependence of electrogenic transport by the Na,K-ATPase. Biochim. Biophys. Acta 1368:184.

Cornelius,F. and Logvinenko,N. 1996. Functional regulation of reconstituted Na,K-ATPase by protein kinase A phosphorylation. FEBS lett. 380:277.

Cornelius,F. 1995. Cholesterol modulation of molecular activity of reconstituted shark Na,K-ATPase. Biochim. Biophys. Acta 1235:205

Cornelius,F. and Møller,J.V. 1991. Electrogenic pump current of sarcoplasmic reticulum Ca-ATP-ase reconstituted at high lipid/protein ratio. FEBS lett. 284:46.

PhD projects: Phosphorylation of Na,K-ATPase by protein kinase A and C. Functional significance in regulation. Yasser A. Mahmmoud

Technician: Hanne R. Z. Christensen

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Liposomes in reconstitution of ion-pumps

Liposomes in reconstitution
of ion-pumps