Immunochemical analysis of the soluble hydrogenase from the cyanobacterium Anabaena cylindrica

Abstract

Antibodies were raised in mice against the 42 kDa subunit of the soluble hydrogenase purified from the cyanobacterium Anabaena cylindrica by the method of Ewart, G.D. and Smith, G.D. (Arch. Biochem. Biophys. (1989) 268, 327–337). This protein catalyzes tritium exchange activity. The antibodies did not cross-react with the 50 kDa protein, which appears to be necessary to confer methyl viologen-dependent reductive hydrogenase activity. Reaction of the antibodies with the enzyme was found to enhance its catalytic activity rather than inhibit it. Although nickel ions are required for enzymic activity of the hydrogenase, the protein was synthesized whether or not nickel was included in the growth medium. Qualitatively, the levels of the protein appeared to be the same in nickel-depleted and nickel-replete cells, as judged from an enzyme-linked immunoassay of the protein which had been Western-blotted onto nitrocellulose after polyacrylamide gel electrophoresis in denaturing gels. Using Western blotting procedures we found no evidence for the 42 kDa protein in heterocyst cells. Although the protein was found only in the soluble fraction of vegetative cells from cultures grown in air/CO 2, antibody reactivity was also obtained with the particulate fraction when cultures were grown in nitrogen/4% H 2/0.3% CO 2. Interestingly, whilst such growth conditions are known to enhance hydrogenase activity substantially, qualitative measurements of Western-blotted protein indicated that there was no significant increase in the concentration of the 42 kDa band in the soluble fraction of anaerobically grown cells.