Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A*

Abstract

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.

Chymotryptic inhibitor I from potatoes: The amino acid sequences of subunits B, C and D

“…'Ulster Prince'), and the subunits Reduced and Scarboxymethylated samples of the subunits were cleaved with cyanogen bromide and Nbromosuccinimide, and by dilute acid hydrolysis as described previously [2]. In addition, some samples were also cleaved by treatment with 2 N-hydroxylamine [3].…”

“…In addition, some samples were also cleaved by treatment with 2 N-hydroxylamine [3]. Peptides were obtained from the intact subunits and fragments by digestion with trypsin, thermolysin, pepsin and papain, as described previously [2]. Certain fragments and peptides were also further digested with chymotrypsin (Worthington Biochemical Corp; enzyme/substrate ratio 1:50, in 0.1 M ammonium carbonate pH 8.0, at 37°C for 2 hr).…”

“…Mixtures of fragments and peptides were fractionated by gel-filtration on Sephadex G-SO and G-25, by high-voltage paper electrophoresis at pH 6.5,4.7, and 1.9, and by paper chromatography using butan-lol-acetic acid-water-pyridine (15:3: 12: 10, by volume) and isoamyl alcohol-pyridine-water (70: 70:60, by volume) as the solvents [2].…”

“…The amino acid sequences of peptides were determined by the dansyl-Edman procedure and by digestion with carboxypeptidase A, as described previously [2] . Amide positions were assigned where possible by using the electrophoretic mobilities of the peptides at pH 6.5, by the release of certain residues with carboxypeptidase A and by direct dansylation without subsequent acid hydrolysis at suitable stages of the Edman degradation.…”

“…Investigation in this laboratory has shown that the subunit A isola$ed by this method gives a single band when examined by polyacrylamide gel disc electrophoresis and a unique amino acid sequence exhibiting only a very low level of micro-heterogeneity [2]. On the other hand, the subunits B, C and D did not give single bands on electrophoresis, even after their peaks were separately re-chromatographed on SE-cellulose or SP-Sephadex in 8 M urea (M. Richardson, unpub!ished results).…”