Abstract
The complete amino acid sequence of rhizopuspepsin, an aspartic proteinase from the fungus Rhizopus chinensis was determined by conventional protein sequencing, using peptide fragments obtained mainly by several enzymatic cleavages of the reduced and carboxymethylated (RCm-) protein. The RCm-protein was first cleaved by trypsin, and the resulting peptides were purified and their amino acid sequences determined extensively. These tryptic peptides were aligned by the aid of overlapping peptides isolated from a tryptic and a chymotryptic digest of the citraconylated RCm-protein and the RCm-protein, respectively. The amino acid sequence thus deduced was further confirmed by isolation and sequence determination of peptides obtained by digestion of the RCm-protein with Staphylococcus aureus V8 protease. The location of the disulfide bonds was determined by isolation and analysis of cystine-containing peptides from a chymotryptic digest of intact rhizopuspepsin. These results showed that the protein is composed of a single polypeptide chain of 325 amino acid residues cross-linked by two disulfide bonds, and shows overall homology with other aspartic proteinases, including 36% identity with penicillopepsin and 38% identity with porcine pepsin.
Highlights
In the present studies we have determined the complete boxymethylated (RCm-) protein
Staphylococcus aureus V8 protease.Thelocationof the disulfide bonds was determined by isolation and analysis of cystine-containingpeptides froma chymotryptic digest of intact rhizopuspepsin
These results showed that the protein is composed of a single polypeptide chain of326 amino acid residuescross-linked by two disulfide bonds, and shows overall homology digested with trypsin and the resulting peptides were isolated by chromatography on a Sephadex G-50 column followed by high performance liquid chromatography
Summary
Aminoacidsequencethusdeduced was furthercon- In thepresent studies, the enzyme was first reduced and carboxyfirmedby isolation and sequence determinatoifonpep- methylated, and the NH*-terminal 32-residue sequence was estabtides obtained by digestion of the RCm-protein with lished by direct Edman degradation. Staphylococcus aureus V8 protease.Thelocationof the disulfide bonds was determined by isolation and analysis of cystine-containingpeptides froma chymotryptic digest of intact rhizopuspepsin These results showed that the protein is composed of a single polypeptide chain of326 amino acid residuescross-linked by two disulfide bonds, and shows overall homology digested with trypsin and the resulting peptides were isolated by chromatography on a Sephadex G-50 column followed by high performance liquid chromatography. Two kinds of residues were identified at positions 15 (Val and Ile), 61 (Lys and Asn), 116 (Asn and Ser), 162 tigated the three-dimensional structure of this protein at 3-A resolution These results indicateadclose structural homology of rhizopuspepsin with other aspartic proteinaseIsn. order to ' Portions of this paper (including "Experimental Procedures," part of "Results,))Tables I-VI, and Figs.
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