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 liquid chromatography (HPLC) techniques. Proteolytic activity in the HPLC fraction was visualized by enzymography using gelatin as substrate. A recombinant murine amelogenin (M179) composed of 179 amino acid residues (20 kDa) was used as a substrate to examine the specificity of the enzymes in the isolated fractions. Incubation of M179 with the proteinase fraction at 37 degrees C generated a major proteolytic product eluting at about 42% acetonitrile from the reversed-phase column. This product had an amino-terminal sequence Pro-Leu-Pro-Pro-His-Pro- in conformity with that of the M179 parent protein. These data indicated that the product resulted from the cleavage of the M179 recombinant protein in the carboxy-terminal region. Mass spectroscopic analysis of the product isolated by reversed-phase HPLC gave a molecular mass of 18.89 kDa. Given an intact amino-terminal sequence, this mass figure suggests that this product terminates at Pro168 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, aprotinin, leupeptin and 4,(amidinophenyl)methanesulphonyl fluoride, which are serine proteinase inhibitors, did not affect the proteolytic activity. In addition, replacement of Ca2+ with Zn2+, Mn2+ or Co2+ in the proteolysis buffer inhibited the enzymatic activity. It is concluded that the 'high molecular-weight' proteinase cleaving M179 at Pro168-Ala169 is a specific 'calcium-dependent metalloproteinase'.
Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles 90033.
J
J
Moradian-Oldak
J P
JP
Simmer
P E
PE
Sarte
M
M
Zeichner-David
A G
AG
Fincham
D000566:Amelogenesis; D053523:Amelogenin; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003743:Dental Enamel / Q000201:enzymology*; D003746:Dental Enamel Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008963:Molar / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D053523:Amelogenin; C507200:Amelx protein, mouse; D003746:Dental Enamel Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D002118:Calcium