Isolation and immunological characterization of the four non-identical subunits of the soluble NAD-linked hydrogenase from Alcaligenes eutrophus H16.
The soluble NAD-linked hydrogenase of Alcaligenes eutrophus H16 is a tetramer consisting of 4 non-identical subunits with molecular weights of 63,000, 56,000, 30,000 and 26,000. Conditions have been elaborated to separate and isolate each of these subunits as a single polypeptide by a preparative scale of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS). Against each of the 4 subunits, polyclonal antibodies were produced. From the crude sera isolated from rabbits, the antibodies (IgG fractions) were purified by Protein A-Sepharose chromatography. By the double immunodiffusion method, comparison of the 4 types of subunits revealed that they are in fact different polypeptides. Subunit 1 (Mr = 63,000) and subunit 2 (Mr = 56,000) only reacted with their own specific antibodies and showed no cross-reaction whatsoever with the antibodies raised against the other subunits. The only immunological relationship among the different subunits was observed with subunit 3 (Mr = 30,000) and subunit 4 (Mr = 26,000); the type of cross-reaction indicated that they are partially identical. A. eutrophus H16 contains, in addition to the soluble hydrogenase, a membrane-bound hydrogenase which is a dimer composed of 2 subunits with Mr of 61,000 and 30,000. Whereas the 2 native enzymes did not show any immunological cross-reaction with the respective antibodies, it was demonstrated by double immunofluorescence labeling on nitrocellulose filters that the larger subunit of the membrane-bound hydrogenase cross-reacted significantly with the antibodies raised against subunit 2 of the soluble hydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000421:Alcaligenes / Q000201:enzymology*; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002462:Cell Membrane / Q000201:enzymology; D006864:Hydrogenase / Q000276:immunology / Q000302:isolation & purification*; D005779:Immunodiffusion; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009615:Nocardia / Q000201:enzymology
D000906:Antibodies; D000941:Antigens; D046911:Macromolecular Substances; D009243:NAD; D006864:Hydrogenase