The carboxyl terminal sequence of sheep heart phosphofructokinase : a thesis for the degree of Master of Science in Biochemistry at Massey University

Abstract

The aim of this project was to investigate the carboxyl terminal sequence of sheep heart phosphofructokinase. Existing methods for the preparation of the enzyme from sheep heart proved to be unsuitable because of the insoluble nature of the purified enzyme. Consequently it was necessary to develop a suitable purification scheme before sequencing work could be commenced. Purification strategies involving magnesium ion precipitation of phosphofructokinase, DEAE-cellulose chromatography and agarose chromatography were tried before a suitable method was found. The carboxyl terminal sequence of phosphofructokinase was investigated by the tritium labelling method of Matsuo, by carboxypeptidase Y digestion and by isolation of the carboxyl terminal peptide generated by tryptic digestion. Digestion of phosphofructokinase by carboxypeptidase Y resulted in the release of leucine, isoleucine and phenylalanine. However attempts to characterise the carboxyl terminal residue by tritium laberling, and to isolate the carboxyl terminal peptide were unsuccessful. The carboxyl terminal sequence suggested by these results and the possible amidation of the carboxyl terminal residue are discussed.