SPECIFIC CLEAVAGE OF A RECOMBINANT MURINE AMELOGENIN AT THE CARBOXY-TERMINAL REGION BY A PROTEINASE FRACTION ISOLATED FROM DEVELOPING BOVINE TOOTH ENAMEL

A proteinase fraction of 48-70-kDa was isolated from developing bovine tooth enamel by size exclusion and reversed-phase high-pressure liqui d chromatography (HPLC) techniques. Proteolytic activity in the HPLC f raction was visualized by enzymography using gelatin as substrate. A r ecombinant murine amelogenin (M179) composed of 179 amino acid residue s (20 kDa) was used as a substrate to examine the specificity of the e nzymes in the isolated fractions. Incubation of M179 with the proteina se fraction at 37 degrees C generated a major proteolytic product elut ing at about 42% acetonitrile from the reversed-phase column. This pro duct had an amino-terminal sequence Pro-Leu-Pro-Pro-His-Pro- in confor mity with that of the M179 parent protein. These data indicated that t he product resulted from the cleavage of the M179 recombinant protein in the carboxy-terminal region. Mass spectroscopic analysis of the pro duct isolated by reversed-phase HPLC gave a molecular mass of 18.89 kD a. Given an intact amino-terminal sequence, this mass figure suggests that this product terminates at pro(168) Of the M179 residue sequence. The presence of EDTA in proteolysis experiments when M179 was used as substrate inhibited production of the 18.89-kDa product. Antipain, ap rotinin, leupeptin and 4,(amidino-phenyl)methanesulphonyl fluoride, wh ich are serine proteinase inhibitors, did not affect the proteolytic a ctivity. In addition, replacement of Ca2+ with Zn2+, Mn2+ or Co2+ in t he proteolysis buffer inhibited the enzymatic activity. It is conclude d that the 'high molecular-weight' proteinase cleaving M179 at pro(168 )-Ala(169) is a specific 'calcium-dependent metalloproteinase'.