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)