PMID: 39613Sep 12, 1979

Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans. Isolation of the hydrogenase

Biochimica Et Biophysica Acta
E Sim, P M Vignais

Abstract

The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break'. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.

References

Dec 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·D L ErbesW H Orme-Johnson
Jun 17, 1975·Biochemistry·P H Gitlitz, A I Krasna
Mar 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·T KakunoM D Kamen
Jul 25, 1977·Biochemical and Biophysical Research Communications·M W Adams, D O Hall
Jan 1, 1978·Biochimie·B Schink, H G Schlegel
Oct 16, 1978·Biochemical and Biophysical Research Communications·K Schneider, H G Schlegel
Apr 1, 1977·Archives of Microbiology·K Schneider, H G Schlegel
Jun 22, 1971·Biochemistry·G Nakos, L E Mortenson
Jan 1, 1970·Archiv für Mikrobiologie·J PfitznerH G Schlegel
Sep 16, 1970·Biochimica Et Biophysica Acta·D Kleiner, R H Burris

Citations

Oct 10, 2007·Biochemistry. Biokhimii︠a︡·A A TsygankovA A Karyakin
Nov 6, 1989·European Journal of Biochemistry·V M FernandezE C Hatchikian
Sep 14, 1981·FEBS Letters·E SimR B Sim
Oct 23, 2019·International Journal of Molecular Sciences·Pierre AlbinaBenjamin Erable

Related Concepts

Plasma Membrane
Hydrogen
Hydrogen-Ion Concentration
Oxidase
Paracoccus denitrificans
Vigilon

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