Solubilization, characterization, and detergent interactions of lymphocyte 5'-nucleotidase.
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 degrees 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.
Loe
D W
DW
Department of Chemistry and Biochemistry, University of Guelph, Ont., Canada.
Glover
J R
JR
Head
S
S
Sharom
F J
FJ
D015720:5'-Nucleotidase; D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D008214:Lymphocytes / Q000201:enzymology*; D008970:Molecular Weight; D009708:Nucleotidases / Q000378:metabolism*; D012995:Solubility; D013501:Surface-Active Agents; D013552:Swine; D013816:Thermodynamics
D003902:Detergents; D013501:Surface-Active Agents; D009708:Nucleotidases; D015720:5'-Nucleotidase