Analysis of the Molecular Structure of StrePtomyces chromofuscus-derived Phospholipase D

Abstract

When we separated the purified specimen of Streptomyces chromofuscus phospholipase D (PLase D) by SDS-PAGE, two protein bands were observed. Molecular weights of these proteins were determined to be approximately 57 and 40 kDa, respectively. As a result of N-terminal amino acid sequence analysis of these two proteins, up to 20 residues from the N-terminal amino acid were completely identical. Therefore, to evaluate the intermolecular structure of the 57 kDa protein, we performed limited proteolysis of this protein with Staphylococcus aureus V8 protease. Furthermore, regarding peptide fragments obtained as main products of limited proteolysis of the 57 kDa protein, N-terminal amino acid sequence analysis was performed. As a result, the amino acid sequence of all eight peptide fragments with molecular weights of 10, 14, 16, 18, 20, 30, 42 and 46 kDa, which were obtained by limited proteolysis of the 57 kDa protein, were found in the amino acid sequence of Streptomyces chromofuscus PLase D obtained from DNA analysis. Based on these results, it was suggested that the amino acid sequence of the 57 kDa protein was identical to that of Streptomyces chromofuscus PLase D obtained from DNA analysis.