Functional analysis and association state of water channel (AQP-1) isoforms purified from six mammals.
Aquaporin-1 (AQP-1) or CHIP28 occurs in glycosylated (glyCHIP) and non-glycosylated (CHIP) forms and solubilization in octyl-beta-D-glucoside (OG) results in a tight association of glyCHIP and CHIP to form a heterodimer. The tight association did not permit separation of the two forms by affinity chromatography. We examined the mechanism of the tight association by enzymatic removal of sugar moieties, utilized organic solvents for preferential solubilization and purified CHIP28 from six mammals for inspection of glycosylation and association state in OG. Removal of terminal saccharides sustained the dimeric state of human CHIP28, while endo-glycosidases induced the transition into monomers, without leaving an affinity tag for separation purposes. Separation was achieved by preferential solubilization of non-glycosylated CHIP28 in CHCl3/MeOH/H2O mixtures. The two CHIP28 forms were solubilized in SDS, chromatographed in OG, and reconstituted into proteoliposomes; pf values were 1.5 and 1.6 x 10(-14) cm3/s (10 degrees C). Among erythrocytes from cow, pig, sheep, rabbit, dog, and horse CHIP28, one out of two molecules was glycosylated and High Performance Size Exclusion Chromatography (HPSEC) analysis also indicated heterodimers in OG; functional analysis of reconstituted proteoliposomes gave single channel water permeabilities, pf's, ranging from 2.0-3.4 x 10(-14) cm3/s (10 degrees C). The results indicate that CHIP28 structure, function, and association in OG are conserved among mammals and establish procedures to obtain glycosylated and non-glycosylated CHIP28 in functional form.
Schulte
D J
DJ
Department of Medicine, University of California, San Francisco 94143, USA.
van Hoek
A N
AN
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002417:Cattle; D019281:Dimerization; D004285:Dogs; D004912:Erythrocytes / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D010539:Permeability; D011487:Protein Conformation; D011510:Proteolipids; D011817:Rabbits; D012756:Sheep; D013045:Species Specificity; D013552:Swine; D014867:Water / Q000378:metabolism*
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D007473:Ion Channels; D011510:Proteolipids; C020485:proteoliposomes; D014867:Water; D051398:Aquaporin 1