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Subject: search.filters.subject.Phosphofructokinase

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    The carboxyl terminal sequence of sheep heart phosphofructokinase : a thesis for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1980) Colls, David Selwyn
    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.
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    The amino acid sequence of tryptic peptides of sheep heart phosphofructokinase : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
    (Massey University, 1974) Brennan, Stephen Oliver
    Phosphofructokinase was purified from sheep heart. The sedimentation pattern of the purified enzyme was investigated over a protein concentration range 0.7 to 14.5 mg/ml. Two distinct 7 and 30 S boundaries were observed at all concentrations. A minor amount of 19 S material was also present. The 30 S boundary was asymmetric and its concentration dependence was characteristic of a polymerising system in rapid reversible equilibrium. The dissolved crystalline enzyme usually sedimented as a single trailing 30 S boundary; the molecular weight of this component was estimated at 1.5 x 10 6. This value was consistent with x-ray data, which indicated unit cell dimensions of 600 x 250 x 220 Å, implying a protein weight greater than 106 daltons per asymmetric unit. In one experiment the 30 S component appeared to be undergoing a trimerisation to a 53 S form. Sodium dodecylsulphate gel electrophoresis indicated a protomer molecular weight of 80,000 to 85,000, which was consistent with a corrected sedimentation coefficient of 3.8 S and a molecular weight of 90,000 for maleyl-phosphofructokinase, and with a corrected sedimentation coefficient of 3.9 S for the urea-dissociated enzyme. When maleylation was carried out on carboxymethyl-phosphofructokinase in 7.5 M urea, the enzyme was further dissociated to a 40,000 molecular weight subunit. Peptide mapping of tryptic peptides; in which arginine-, histidine-, tryptophan- and tyrosine-containing peptides were located; was consistent with an 85,000 form composed of two identical subunits. The enzyme was digested with trypsin. Forty-three different peptides were isolated using a combination of: gel filtration, ion exchange chromatography (on Dowex 50 and DE 32 cellulose), paper electrophoresis, and paper chromatography. The complete amino acid sequence was established for 38 of these peptides. The amino acid compositions (and partial sequences) were established for the other five tryptic peptides. A summary of the amino acid sequence data obtained for the tryptic peptides is shown in Table V. Seven carboxymethylcysteine-containing peptides were isolated in this investigation, while eight have been isolated from rabbit muscle (Coffee et al. 1973). Six of these peptides had very similar compositions between the two species. The rabbit enzyme contained two carboxymethylcysteine-containing peptides which were not found in sheep heart and the sheep enzyme contained a 20 residue peptide not found in rabbit muscle. This probably reflects genetic variation between the two species.