생체막 구조의 변화에 따른 5'-Nucleotidase 활성의 변화

Abstract

Assay of 5'-nucleotidase is a biochemical tool to identify the plasma membrane in subcellular fractlonationc fy-Nucleotidase was reported to be activated by Mg" ion but the authors observed that the degree of activation depended on the structural state of the plasma membrane. This experiment was performed to study further in detail the change in 5'-nucleotidase activity relating to the chan· ge of the structural state of the plasma membrane. The results obtained are summarized as follows: 1. Rat livers were homogenized in O. 25M sucrose and the homogenate was fractionated into 2000 X g fraction, mitochondrial fraction and microsomal fraction according to the particle size of the plasma membrane. The activation effect of Mg" ions on 5'-nucleotidase was tested on each fraction. The 'effect was most pronounced in the microsomal frac- lion and the most effective concentration of Mg" 'ions was 10mM. 2. Microsomal vesicles increased in size in the presence of lOmM Mg" ions in the assay system of '5'-nucleotidase, which was confirmed by Tyndall effect and electron microscopy. 3. Microsomal vesicles were coagulated in the presence of 10mM Ca* ions in the assay system of '5'-nucleotidase, which was confirmed by Tyndall . effect and electron microscopy and Ca" ions inhibited 5'-nucleotidase about 30% at the concentration 'of 10mM. 4. From Tyndall effect of the microsomal vesicles it was shown that the vesicles were slowly enlarged in the hypotonic assay system at 30'C, but the vesicles did not increase in size at 16'C. 5. When the activities of 5'-nucleotidase were 'measured by starting the enzyme reaction with the .substrate, the activities of the enzyme were increased with increasing incubation period of the en' zyme preparation in the enzyme assay buffer at 30'C 'before addition of the substrate. From this result it is concluded that the activities of 5' -nucleotidase 'should be measured by starting the enzyme reaction with the enzyme preparation. 6. The activity of 5'-nucleotidase in the micro' somal fraction was increased with aging the mi- crosomal fraction in the refrigerator at 4 'C, which was accompanyed by the increase in lipid peroxide. Thus the increase in the enzyme activity of the microsomal fraction was about 40% after 4 days' aging. The activation effect of Mg" ions on 5'-nu. cleotidase was decreased with aging.