IMP dehydrogenase. I. Studies on regulatory properties of crude tissue extracts based on an improved assay method.
Inhibition of conversion from IMP to uric acid, which interferes with both spectrophotometric and radioisotopic assays of IMP dehydrogenase, by addition of allopurinol (0.1 mM), an inhibitor of xanthine oxidase, to the incubation system made it possible to determine the enzyme activity in crude liver extracts. With this improved assay method, the regulatory properties of the enzyme in crude extracts of liver and Yoshida sarcoma ascites cells were examined. In both tissues IMP dehydrogenase was found in the postmicrosomal supernatant. However, further centrifugation resulted in precipitation of the enzyme, the enzyme from Yoshida sarcoma ascites cells being precipitated more easily than that from rat liver. It was also found that IMP dehydrogenase activity increased during liver regeneration and that this increase was associated with the precipitate from the postmicrosomal fraction. These findings suggest that such a large sedimentable complex including IMP dehydrogenase might be formed in relation to cell growth. Most of the enzyme activity in rat liver and Yoshida sarcoma ascites cells was extracted in the supernatant obtained by centrifugation at 105,000 X g for 4 h after treatment of tissue homogenates with 1 M KCl, 0.75 M (NH4)2SO4, 2 M dimethylsulfoxide, 2 M KSCN, 25% glycerol, or 0.8 M guanidine-HCl. Treatment with 2% deoxycholate, 2% Triton X-100 or 2 M urea gave limited extraction. The enzyme was retained on a phenyl-Sepharose CL-6B or octyl-Sepharose CL-6B column and eluted with 0.8 M guanidine-HCl. These results suggested that the enzyme molecule has not only ionic but also hydrophobic domains, through which it interacts with other molecules of the enzyme itself and/or postmicrosomal cellular components.(ABSTRACT TRUNCATED AT 250 WORDS)
D000493:Allopurinol / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007168:IMP Dehydrogenase / Q000378:metabolism*; D066298:In Vitro Techniques; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008115:Liver Regeneration; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012517:Sarcoma, Yoshida / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology
D000493:Allopurinol; D007168:IMP Dehydrogenase; D007658:Ketone Oxidoreductases