흰쥐 간조직 5'-Nucleotidase와 그 Isoenzyme에 관한 연구

Abstract

51-Nucleotidase is an intrisic membrane glycoprotein which has been shown in a variety of cell types to be an ectoenzyme_ It is widely used as a plasma membrane marker in subcellular fractionation studies and used in monitoring cell membrane changes during neoplastic transformation. It has been reported as that 5ιnucleotidase has broad specificities for nucleoside 5' -monophosphate Attempts have been made for such a reason to resolve this enzyme into isoenzymes, but in most cases evidence for isoenzymes were not obtained. The present investigation was carried out to prepare lipid free 5ιnucleotidase from rat liver and to study its properties and isoenzymes with the following results. 1. Lipid-free 5ιnucleotidase obtained by Sephadex G-200 gel filtration or DEAE-cellulose chromatography from rat liver was resolved into two isoenzyme bands by polyacrylamide gel disk electrophoresis. 2. 5'-Nucleotidase was purified about 45 folds from rat liver homogenate with 42% yield by nbutanol extraction and gel filtration, but the enzyme isoenzymepurified by the treatment of n-butanol extraction and DFAE'callulose chromatography showed the 11 folds of purification with 5% yield. 3. The pH optimum and KM values of the two differently purified enzymes were identical; pH 7.5, O. 33mM, respectively. 4. The enzymes showed different pattern in the effect of EDTA; 5ιnucleotidase purified by gel filtration method revealed strongly inhibited activity, in contrast with that by DEAE-cellulose chromatography. 5. Effects of metal ions(Ca",Mg & Mn") on the activity of 5ιnucleotidase by two procedures used were similar to that by microsomal 5'-nucleotidase. 6. The effects of detergents (Triton X-IOO, sodium deoxycholate and sodium dodecylsulfate) on the activity of the enzymes were prominently different; the enzyme by gel filtration showed increased activity, while that by DEAE'cellulose chromatography, de' creased activity. The KM value , however was not altered, while Vmax, changed