Creatinine hydrolase and creatine amidinohydrolase: II. Partial purification and properties

Summary

The optimal conditions for growing A. ureafaciens for producing maximum amounts of creatinine hydrolase and creatine amidinohydrolase relative to total protein are described. This required a medium in which either creatinine or creatine was the sole source of carbon.

The specific activity of a crude, cell-free sonicate of creatinine hydrolase was increased 110- to 140-fold by a series of purification steps including heat treatment which inactivated creatine amidinohydrolase, precipitation of nucleic acids with streptomycin, ammonium sulfate precipitation of inert proteins, Sephadex Gel-200 filtration and DEAE-cellulose column chromatography. The most highly purified preparation of creatinine hydrolase still contained a number of inert proteins as demonstrated by acrylamide gel electrophoresis but was completely devoid of creatine amidinohydrolase activity.

The pH optimum of creatinine hydrolase was 8.3 and the K_m was 0.125 M. The molecular weight of creatinine hydrolase as determined by filtration on a Sephadex Gel-200 column was approximately 240,000 and appeared to be composed of eight subunits. The molecular weight of creatine amidinohydrolase was in the vicinity of 100,000.

The inhibitory effects of heavy metals and sulfhydryl compounds on the activities of the enzymes were studied.