Abstract
The endopeptidase, post-proline cleaving enzyme, has been purified 10,500-fold in an overall yield of 18% from lamb kidney. The enzyme possesses a specific activity of 45 mumol/mg/min as tested with the substrate Z-Gly-Pro-Leu-Gly (Km = 6.0 X 10(-5)), has a molecular weight of 115,000, is comprised of two subunits with a molecular weight of 57,000, and exhibits maximal activity at pH 7.5 to 8.0. With the exception of the -Pro-Pro linkage, the -Pro-X-peptide bond (X equals L- and D-amino acid residues) located internally in the peptide sequence can be hydrolyzed (cleavage occurs faster when X = lipophilic side chain as compared to X = acidic side chain). The appropriate -Pro-X- bonds in zinc-free porcine insulin, oxytocin, arginine vasopressin, angiotensin II, bradykinin-potentiating factor were cleaved. Human gastrin, adrenocorticotropic hormone, denatured guinea pig skin collagen, and ascaris cuticle collagen were not degraded. Dipeptides with the structure Z-Pro-LD-X competitively inhibit post-proline cleaving enzyme.
Highlights
The purification scheme found to give the best results to date yields a post-proline cleaving enzyme of a 10,500-fold purification on an average (Table IV)
It was confirmed that peptides without proline residues were not degraded, while all types of -Pro-Xbonds, with the exception of a -Pro-Pro- bond, can be hydrolyzed there are considerable differences in rates
In order to obtain detectable cleavage the proline residue cannot occupy the NH,terminal position of an unprotected peptide
Summary
Human svnthetic adrenocorticotropic hormone I-& [15], based on the structure of Lee et al [16], was kindlv suunlied bv Dr R. L. Colescott; human aastrin [17,18] and its NHz-terminal fragment,
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