Solubilization, characterization, and detergent interactions of lymphocyte 5′-nucleotidase

Publisher Website
Google Scholar
Abstract
5′-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5′-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5′-nucleotidase showed "break points" for all detergents in the temperature range 30–37 °C. SDS-PAGE of pure 5′-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200–630 kDa, suggesting that lymphocyte 5′-nucleotidase may be present in high molecular mass aggregates in its native state.Key words: 5′-nucleotidase, plasma membrane, detergents, solubilization, stability, activation energy.