J. Biochem, 1967, Vol. 62, No. 3 364-372
© 1967 Japanese Biochemical Society
research-article |
A Neutral Proteinase from Streptomyces naraensis
II. Its Mode of Action on Baker's Yeast Cytochrome c*
From the Laboratory of Biochemistry, Faculty of Agriculture, Ibaraki University Ami, Ibaraki
To elucidate the specificity of the neutral proteinase from Streptomyces naraensis on protein substrates with known primary structures, its action on baker's yeast (Saccharomyces oviformis) cytochrome c was tested. A hydrolysate of N-ethylsuccinimide-cytochrome c was fractionated by chromatography on a column of Dowex 50x2 into ninteen peaks. Most peaks contained more than two peptides and these were further fractionated until they became homogeneous. In total, twenty-nine peptides and two free amino acids were characterized.
The amino acid and the N-terminal analyses of these purified peptides were carried out to find the exact positions of these peptides in the linear structure of cytochrome c. All the isolated peptides could be located in the known primary structure of cytochrome c.
The neutral proteinase rapidly hydrolyzed the asparaginyl, gluta-minyl and seryl bonds, slowly hydrolyzed the glycyl, alanyl, valyl, leucyl, tyrosyl, phenylalanyl, threonyl, aspartyl, glutamyl, histidyl and lysyl bonds, but did not hydrolyze the isoleucyl, prolyl, methionyl, cystinyl and arginyl bonds in cytochrome c. Therefore this enzyme seems to possess no definite specificity for protein substrates, but its specificity is not so broad as that of the alkaline proteinase from Streptomyces griseus. ATCC 3463 reported previously.
*This work was performed during tenure of a research fellowship at the Institute for Protein Research, Osaka University, Osaka, Japan.