Purification of nucleotide-requiring enzymes by immunoaffinity chromatography.
Monospecific (affinity-purified) anti-(yeast glucose-6-phosphate dehydrogenase) IgG inhibits three different NADPH-requiring enzymes, chicken liver dihydrofolate reductase, pigeon liver fatty acid synthetase and chicken liver malic enzyme. The inhibition of all three enzymes was approx. 50% in a 2h incubation with 100 micrograms of IgG. Similarly, with several different NADH-requiring enzymes, an immunocrossreactivity was observed. Monospecific anti-(rabbit muscle glyceraldehyde-3-phosphate dehydrogenase) IgG inhibited yeast alcohol dehydrogenase and pig heart malate dehydrogenase 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 isocitrate dehydrogenase and dihydrofolate reductase from crude rat liver homogenate.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000276:immunology; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D009247:NADH, NADPH Oxidoreductases / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification
D007074:Immunoglobulin G; D007521:Isocitrate Dehydrogenase; D013762:Tetrahydrofolate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases