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Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures

1981; Elsevier BV; Volume: 110; Issue: 1 Linguagem: Inglês

10.1016/0003-2697(81)90138-x

1096-0309

Anthony T. Yeung, Katherine J. Turner, Newell F. Bascomb, Robert R. Schmidt,

Diatoms and Algae Research

A modified five-step purification procedure was developed which gave an 80–87% yield of pure NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella. The enzyme was shown to be composed of six identical subunits with alanine as the C-terminal amino acid. The purified enzyme was covalently coupled to CNBr-activated Sepharose-4B, and then the subunits were linked together with dimethyl suberimidate to make a stable antigen affinity column for purification of anti-NADP-GDH IgG from rabbit antiserum. When the subunits of the column-bound holoenzyme were not linked together, elution of the anti-NADP-GDH IgG resulted in a 50% loss of enzyme subunits from the column. This loss of subunits inactivated the column. The monospecific, affinity-purified anti-NADP-GDH IgG was used in an indirect immunoprecipitation procedure with purified sheep anti-rabbit IgG or in an indirect procedure with Staphylococcus Protein A Sepharose 4B to obtain a 95–98% recovery (by either procedure) of 35S-labeled NADP-GDH from radioactive Chlorella cell homogenates. As shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the [35S]NADP-GDH recovered by these procedures was free of contaminating radioactive cellular proteins. When direct precipitation was used with the purified antibody, only an 85–90% recovery of the radioactive enzyme was obtained. Thus, the indirect procedures would be the ones of choice for measurements of the in vivo rates of synthesis and degradation of the NADP-GDH which comprises approximately 0.2% of the total soluble protein of Chlorella.