Characterization and activation of procollagenase from human polymorphonuclear leucocytes. N-terminal sequence determination of the proenzyme and various proteolytically activated forms.
Procollagenase of human polymorphonuclear leucocytes was purified to homogeneity using a rapid and reproducible method. The purification procedure included affinity chromatography on zinc chelate Sepharose, ion exchange chromatography on Q-Sepharose fast flow, followed by affinity chromatography on orange Sepharose and finally a gel-permeation step on Sephacryl S-300. It was shown by SDS/PAGE, under reducing conditions, that the latent collagenase of human polymorphonuclear leucocytes consists of a single polypeptide chain with an apparent relative molecular mass of 85,000. Upon deglycosylation by endoglycosidase F digestion, the apparent relative molecular mass of the procollagenase was reduced to 53,000 which is similar to that of the fibroblast enzyme, and indicates a close relationship between both enzymes. Sequence data were determined by direct automated Edman degradation of the purified polymorphonuclear leucocyte procollagenase. The complete sequence of the propeptide region (residue 1-120) was thereby established. The proteolytic activation of the polymorphonuclear leucocyte procollagenase by various enzymes was investigated by determining the N-terminal sequences of the intermediate and final activated forms. Activation by chymotrypsin and cathepsin G led to the active form (Mr 64,000) by cleaving 79 N-terminal residues from the proenzyme. Trypsin activates in a two-step process. Cleavage of 48 N-terminal residues led to a still latent Mr 70,000 species. The final active form (Mr 65,000) was obtained by splitting off 20 additional N-terminal residues.
Knäuper
V
V
Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Federal Republic of Germany.
Krämer
S
S
Reinke
H
H
Tschesche
H
H
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D017364:Collagenases; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D003012:Microbial Collagenase / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid
D004792:Enzyme Precursors; D010446:Peptide Fragments; D017364:Collagenases; C022440:procollagenase; D003012:Microbial Collagenase; D003488:Cyanogen Bromide