Characterization of (Na+ + K+)-ATPase liposomes. II. Effect of alpha-subunit digestion on intramembrane particle formation and Na+,K+-transport.
The effect of the protein structure of (Na+ + K+)-ATPase on its incorporation into liposome membranes was investigated as follows: the catalytic alpha-subunit of (Na+ + K+)-ATPase was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the (Na+ + K+)-ATPase liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the (Na+ + K+)-ATPase decrease progressively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight alpha-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional (Na+ + K+)-ATPase with intact protein structure and digested, non functional enzyme consisting of fragments of the alpha-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the (Na+ + K+)-ATPase molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of (Na+ + K+)-ATPase.
D000818:Animals; D001693:Biological Transport, Active; D005614:Freeze Fracturing; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008970:Molecular Weight; D010713:Phosphatidylcholines; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D012964:Sodium; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium