The isolation of phosphorylated polypeptide components of the organic matrix of embryonic bovine enamel

Abstract

1. (1)The organic matrix of developing embryonic enamel was obtained from bovine fetuses of various ages. This material was extracted sequentially using three neutral and two acid pH extractions at 4° and the amount of protein in each extract measured. 2. (2)Fractionation of the proteins which were extracted at neutral pH was achieved by three procedures: (a) Free flow, horizontal electrophoresis in 6 M urea at pH 8.3; (b) Preparative polyacrylamide gel electrophoresis at pH 8.8 in 6 M urea; and (c) gel filtration using columns of Bio-gel P-10 and P-20 in 6 M urea at pH 8.3. 3. (3)Four highly purified organic phosphorus containing polypeptides were obtained by preparative polyacrylamide gel electrophoresis. The four polypeptides (E 1, E 2, E 3, E 4) contained 0.39; 0.45; 1.60 and 2.10% phosphorus, respectively. Two of the fractions (E 1, E 2) constitute only approx. 0.2% of the enamel proteins which are soluble at neutral pH, while the other two fractions (E 3, E 4) represent 15–20% of the neutral pH-soluble proteins. 4. (4)The E 3 fraction was characterized by its high content of leucine while the E 4 fraction contained relatively large amounts of tyrosine. The results of molecular sieving indicate that these two components had similar mol. wt. of approx. 6000.