Isolation of type IV procollagen-like polypeptides from glomerular basement membrane. Characterization of pro-alpha 1(IV).
Type IV procollagen-like constituents of glomerular basement membrane were solubilized by reduction and alkylation of disulfide bonds under denaturing conditions. Four polypeptides were observed with apparent Mr = 185,000, 175,000, 164,000, and 152,000. The two largest chains correspond to pro-alpha 1(IV) and pro-alpha 2(IV), described in model systems which secrete a basement membrane-like matrix, while the smaller chains appear to be shortened forms of these polypeptides. Fractionation of the four polypeptides into two groups was achieved by ion exchange chromatography. Pro-alpha 1(IV) and 164,000 polypeptide are relatively acidic with respect to pro-alpha 2(IV) and 152,000 polypeptide, which is due in part to a relatively high content of arginine in the latter. Based on amino acid analysis of the collagenase-sensitive regions of these polypeptides, pro-alpha 1(IV) is the parent molecule from which alpha 1(IV) is derived on pepsin digestion of basement membranes and pro-alpha 2(IV) is the parent molecule of alpha 2(IV). Pro-alpha 1(IV) was isolated by gel filtration and ion exchange chromatography and characterized. It has a molecular weight of 194,000 as determined by sedimentation equilibrium. The polypeptide contains 14% carbohydrate in the form of both disaccharide, glucosylgalactosylhydroxylysine, and heteropolysaccharide units. The polypeptide backbone mass is calculated to be 167,000 daltons. Digestion of pro-alpha 1(IV) with bacterial collagenase resulted in two resistant segments of mass = 31,000 and 33,000 dalton, which make up approximately 30% of the polypeptide.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001485:Basement Membrane / Q000032:analysis*; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D007678:Kidney Glomerulus / Q000032:analysis*; D003012:Microbial Collagenase; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification*; D011347:Procollagen / Q000302:isolation & purification*
D000596:Amino Acids; D002241:Carbohydrates; D010455:Peptides; D011347:Procollagen; D003012:Microbial Collagenase