흰쥐 간세포 원형질막 5'-Nucleotidase의 특성에 관한 연구

Abstract

5'-Nucleotidase bas been shown to be localized in the plasma membranes of various cell types. It is widely used as a marker enzyme for plasma membrane in subcellular fractionation. It has been reported as well that 5' -nucleotidase has broad specificities for nucleotide 5'-monophosphates. Attempts have been made for such a reason to resolve this enzyme into isoenzymes, but in most cases evidences for isoenzymes were not obtained. 5'-Nucleotidase has been reported recently to Increase in its activity upon aging the plasma membrane fraction in vitro at low temperature. A mechanism involved for the increase has been put foward in effect that a regulatory substance is bound to the enzyme, forming a latent 5f-nucleotidase. The present investigation was carried out to prepare membrane-free 5'- nucleotidase from rat liver plasma membrane and to study the kinetic properties of the enzyme. Attempts also were made to resolve 5'nucleotidase into isoenzymes and to confirm and characterize the proposed regulatory substance. The results obtained are summarized as follows: I. Membrane-free 5' -nucl eotidase obtained by sonification from rat liver plasma membrane was resolved into two isoenzyme bands by disk electrophoresis on the polyacrylamide gel. The slow-moving isoenzyme was major component whereas the fast-moving Isoenzyme, a minor component. 2. Membrane-free 5f -nuclcotidase compnzmg the major isoenzyme was purified about 12-fold from rat liver homogenate by differential centrifugation and gel filtration on Sephadex G-200 column equilibrated with 20mM sodium deoxycholate-20mM Tris, pH 9.2. 3. Activation of membrane·free 5f-nucleotidase was brought about to the same extent(5%) by the much lower concentration of MnH (0. 01-0. ImM) and by much higher concentration of MgH(IOmM). 4. The enzyme was strongly inhibited by EDTA and recovered completely by the addition of about 5 time as much MgH in molar concentration as EDTA. 5. The enzyme was strongly inhibited by ADP and to a much less extent by GDP and GTP. Purine nucleosides did not affect the enzyme activity. 6. The pH optimum and KM value of the enzyme were 8. a and O. 15mM respectively. 7. Phospholipid was detected in the membrane-free 5f-nucleotidase and the enzyme was activated by widely used detergents such as sodium dodecyl sulfate (SDS) , sodium deoxycholate(SDC) and Triton x-roo. Vma x of the enzyme was increased about 2-fold by addition of 5mg of SDC and 0.2 I' moles of MnH to 2ml of the enzyme assay system, but KM value of the enzyme was not altered. And the slope of the decrease in enzyme activity with time due to thermal denaturation at 30°C was greater in the SDC and Mnt t-containing assay system than in the control assay system. Based on the above results, it was discussed that the phospholipid bound to 5' -nucleotidase could be regulatory substance which could be released by the detergents, most effectively by SDC combined with MnH , and by incubation itself, causing the enzyme to increase in activity.