Partial digestion of neurophysins with proteolytic enzymes: unusual interactions between bovine neurophysin II and chymotrypsin

• Published in: Biochemistry (Easton) Vol. 21; no. 5; pp. 817 - 826
• Main Authors: Rabbani, Lillian D, Pagnozzi, Martin, Chang, Paul, Breslow, Esther
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.03.1982
• Subjects: cattle; Chromatography, Affinity - methods; chymotrypsin; Chymotrypsin - antagonists & inhibitors; Chymotrypsin - metabolism; neurophysin II; Neurophysins - metabolism; Oligopeptides - metabolism; Peptide Fragments - pharmacology; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00534a001

Bovine neurophysin II was partially digested by chymotrypsin and by chymotrypsin followed by carboxy-peptidase B to produce large fragments collectively representing deletions of residues 1-5 and 91-95. All such fragments were capable of binding peptides to the principal hormone-binding site of neurophysin with normal or near-normal affinity, indicating that residues 1-5 and 91-95 do not directly participate in binding. In addition, preliminary results with thermolysin-derived fragments suggested that residue 6 does not participate in peptide binding. During the course of chymotrypsin studies, it was demonstrated that bovine neurophysin II behaves as a transient competitive inhibitor of chymotrypsin; for neurophysin-peptide complexes, Ki congruent to 8 x 10(-6) M. This inhibition is dependent on neurophysin conformation and is relieved by the anomalous preferential splitting by chymotrypsin of Arg-Arg and Phe-Pro bonds near the carboxyl terminus of neurophysin II. It is suggested that this phenomenon might reflect the interaction of neurophysin II with a chymotrypsin-related enzyme in the pituitary. One approach used in the study of binding properties of proteolytically modified neurophysin was affinity chromatography; the preparation and properties of a conveniently prepared affinity column for neurophysin are described.