Purification and sequencing of a 21 kDa and 25 kDa bovine enamel metalloproteinase.
The developing enamel matrix contains metalloproteinases that are presumed to have a role in hydrolysis of enamel matrix proteins. Determination of the identity and function of these proteinases requires further information such as their amino acid composition and sequence. In this study, we purified the 21 kDa and 25 kDa matrix metalloproteinase from secretory stage bovine enamel matrix. After extraction, these proteinases were further purified by sequential separation by ion exchange, Con A affinity chromatography, and reversed phase HPLC. The proteinases were separated by SDS PAGE, transferred to a PVDF membrane and the N-terminus was sequenced. The N-terminal sequences of both proteinases were the same, and showed homology to the porcine enamelysin (MMP 20) cDNA sequence. A cDNA for bovine MMP 20 was isolated from a bovine enamel organ cDNA library using a probe generated by PCR amplification. The coding regions of bovine and porcine MMP 20 cDNAs were highly homologous and contained the same regions of predicted amino acid sequence homology with the proteinase N-terminal sequences. These results suggest that the 21 kDa and 25 kDa enamel matrix metalloproteinases are cleavage products of the initially secreted MMP 20, and that the sequence for MMP 20 is conserved across species.
Den Besten
P K
PK
University of California at San Francisco, 94143-0640, USA. pkdb@itsa.ucsf.edu
Punzi
J S
JS
Li
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D003743:Dental Enamel / Q000201:enzymology* / Q000254:growth & development; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D013552:Swine
D017931:DNA Primers; D018076:DNA, Complementary; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases