Studies on the Native and Reduced Alkylated Renal Glomerular Basement Membrane

Abstract

Abstract The solubility of the bovine glomerular basement membrane was assessed in a variety of reagents, including salt, urea, and detergent solutions, and found to be negligible except in urea and in sodium dodecyl sulfate (SDS), where a limited solubilization of about 20% was effected. The material so solubilized differed in composition from the whole membrane, containing fewer of the amino acids characteristic of collagens, and was primarily of very high molecular weight, failing to penetrate 5% polyacrylamide gels on electrophoresis. Extraction of the basement membrane with urea or SDS in the presence of 2-mercaptoethanol resulted in a rapid solubilization of the membrane. Moreover, the S-carboxymethylated basement membrane formed by the alkylation of the reduced membrane demonstrated a markedly enhanced solubility over that of the native membrane in all of the reagents. About 80% of the reduced alkylated membrane could be extracted into 5% SDS, and polyacrylamide electrophoresis in SDS revealed the presence of at least 11 components ranging in molecular weight from 30,000 to 220,000, as well as some material which failed to penetrate the 5% gel. No free sulfhydryl groups could be detected in the basement membrane, indicating that all of the cysteine residues occur in disulfide linkage. A number of different amino acids were found in NH2-terminal and COOH-terminal positions in this membrane. On the basis of the total NH2-terminal residues, an average molecular weight of 134,000 was calculated for the peptide chains. Cyanogen bromide treatment of the basement membrane resulted in a maximum of 70% conversion of methionine to homoserine. The incomplete action of the cyanogen bromide was shown to be due to the presence in the membrane of 30% of the methionine residues in the sulfoxide form. Reduction of the methionine sulfoxide permitted almost complete reaction with the cyanogen bromide to take place. Gel filtration on Sephadex G-75 of these cyanogen bromide products revealed the presence of peptide components which spanned a wide range of molecular weights. Examination of a number of other proteins indicated that most contain only insignificant amounts of methionine sulfoxide. However, the anterior lens capsule of the calf, another basement membrane, was found to have about 30% of its methionine in this oxidized form, which led in the case of this protein also to an incomplete reaction with cyanogen bromide.