The Primary Structure of Porcine Pancreatic Ribonuclease

作者：Richard L. Jackson, C.H.W. Hirs · 发表于：Journal of Biological Chemistry · 年份：1970 · DOI：10.1016/s0021-9258(18)63377-0 · 被引用次数：115 · 研究领域：Glycosylation and Glycoproteins Research、Enzyme Structure and Function、Biochemical and Molecular Research

Abstract Reduced, S-aminoethylated porcine ribonuclease was subjected to tryptic hydrolysis. A sequence of fractionation steps that included gel filtration and chromatography over SE-Sephadex C-50, Dowex 50-X2, and Dowex 1-X2 afforded a series of glycopeptide fractions which were recognized to derive from three separate regions of the molecule. Heterogeneity of the attached polysaccharide chains precluded the isolation of glycopeptides homogeneous with respect to polysaccharide and hampered the preparation of fractions suitable for amino acid sequence determination. However, fractions representative of individual segments of the primary structure of the protein moiety were obtained. Their amino acid sequences were determined by conventional procedures. These fractions represent 35 of the 124 amino acid residues in the molecule. By reference to the amino acid sequence of porcine ribonuclease, determined in concurrent experiments, the three sites of polysaccharide attachment were recognized to be at positions 21, 34, and 76, designated as Sites I, II, and III, respectively. Aspartic acid occupies each of these positions and it is presumed that the carbohydrate-peptide attachment in each is represented by a β-N-aspartamido-2-acetamido-1,2-dideoxyglucopyranoside unit. By reference to the structure of bovine ribonuclease, the three attachment sites are recognized to be at residues which have external side chains in regions of the molecule remote from the active site. The polysacch...