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 °C using [ 3H]-acetyl casein substrate and at 40–55 °C using [ 3H]-acetyl enamel protein substrate. It was irreversibly denatured at 80 °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 °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.