title: Purification and properties of a protease from developing porcine dental enamel.
abstract: A protease of molecular weight 29,000 was isolated and purified using ammonium sulphate precipitation, lentil lectin-Sepharose affinity chromatography and DEAE-5PW ion-exchange chromatography. The protease had an unusual amino acid composition including 5% serine, 6% proline and 20% tyrosine. It was a glycoprotein containing 12-15% carbohydrate by weight. Activity was optimal at 40-45 degrees C using [3H]-acetyl casein substrate and at 40-55 degrees C using [3H]-acetyl enamel protein substrate. It was irreversibly denatured at 80 degrees C and above. With [3H]-acetyl casein the pH optimum was 8.0-8.5 and with [3H]-acetyl enamel protein it was 6.0-8.0. There was no activity below pH 5.0, and irreversible denaturation occurred at pH 4.0 and below. No autodegradation occurred with storage at 4 degrees C for 30 days at pH 7.0. Phenylmethylsulphonyl fluoride, mercuric chloride, and p-aminobenzoic acid completely inactivated the protease. The enzyme had no requirement for calcium. The sites of cleavage of the oxidized B-chain of insulin were the Cys-Gly and Arg-Gly bonds. The enzyme was therefore an endopeptidase. Cleavage of Na-benzoyl-L-arginine ethyl ester, but not Na-benzoyl-L-tyrosine ethyl ester, suggests that the protease is of the trypsin family. On the basis of its physical and enzymic properties the protease is a serine proteinase and, consistent with existing terminology, has been named proteinase pemB.
authors.affiliation: Department of Oral Biology, School of Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: J
authors.initials: J
authors.lastname: Carter
authors.forename: A C
authors.initials: AC
authors.lastname: Smillie
authors.forename: M G
authors.initials: MG
authors.lastname: Shepherd
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003743:Dental Enamel / Q000201:enzymology*; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
chemical_list: D000596:Amino Acids; D007328:Insulin; D010447:Peptide Hydrolases