Purification of nucleotide-requiring enzymes by immunoaffinity chromatography

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Abstract
Monospecific (affinity-purified) anti-(yeast glucose-6-phosphate dehydrogenase [EC 1.1.1.49]) IgG inhibits 3 different NADPH-requiring enzymes, chicken liver dihydrofolate reductase [EC 1.5.1.3], pigeon liver fatty acid synthetase and chicken liver malic enzyme [EC 1.1.1.40]. The inhibition of all 3 enzymes was .apprx. 50% in a 2 h incubation with 100 .mu.g of IgG. Similarly, with several different NADH-requiring enzymes, an immunocross reactivity was observed. Monospecific anti-(rabbit muscle glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12]) IgG inhibited yeast alcohol dehydrogenase [EC 1.1.1.1] and pig heart malate dehydrogenase [EC 1.1.1.37] by 39% and 55% respectively. The cross-reactivity observed was tested by affinity chromatography. Immunoaffinity columns made with each monospecific IgG were able to bind each of the enzymes it immunotitrated. Enzymes were eluted with a nondenaturing solvent with little loss of activity. The immunoaffinity column with monospecific anti-(glucose-6-phosphate dehydrogenase) IgG as the bound ligand was also used to purify partially (over 150-fold) both isocritrate dehydrogenase and dihydrofolate reductase from crude rat liver homogenate.