An investigation into the binding of sheep heart phosphofructokinase to an intracellular component : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

Phosphofructokinase (E.C.2.7.1.11) is an ambiquitous enzyme; ie it can be located either in a cytosolic or particulate form, depending on the metabolic status of the cell. Two indepedent methods were used to attempt to determine the particulate location of sheep heart PFK. The first method involved an adaptation of the method of subcellular fractionation of liver, for use with heart tissue. A crude fractionation procedure was first used, in which the PFK was found to be located in a fraction which precipitated at 800g. Specific enzyme assays of acid phosphatase, succinic dehydrogenase and 5'- nucleotidase, were performed to determine the presence of lysosomes, mitochondria and plasma membrane respectively, in the fraction containing PFK. These assays revealed that most of the enzyme activity corresponding to the above organelles, had also precipitated in this fraction. Both mitochondria and myofibrils were purified to determine if PFK copurified with these organelles, but the results obtained were inconclusive. The second method involved immunochemical localisation of PFK. Antibodies against purified sheep heart PFK were raised in rabbits, and an attempt was made to separate the anti-sheep heart PFK antibodies from the other antibodies by affinity chromatography. Both CDI-activated and cyanogen bromide activated sepharose columns were made and used. Successful separation was not achieved. Western blotting followed by staining with a second antibody labelled with alkaline phosphatase, revealed that the antibody preparation was sufficiently specific to proceed to immunochemical localisation. Sections of fresh sheep heart were embedded at low temperature in Lowicryl K4M resin, then incubated with the anti-sheep heart antibodies followed by secondary antibodies labelled with 15nm gold particles. Electron micrographs of the sections revealed the electron dense gold particles, and hence the location of the PFK. By comparing the density of gold particles on the myofibrils with that of the background it was shown that the density on myofibrils was 4 to 4.5 times higher than on the background. Thus it was tentatively shown that the myofibrils was the site of localisation of PFK in sheep heart cells. Investigations into the effect of metabolites on the solubilisation of PFK from the membrane,by including various salts in the extraction buffer, revealed that divalent anions, specifically sulphite, sulphate and thiosulphate ions, were effective in releasing PFK into the cytosol. The effect of fructose-2,6-bisphosphate on the solubilisation and activity of sheep heart PFK was uncertain, but it appeared to inhibit the activity of cytosolic PFK and have no effect on the solubilisation. The possible cross reactivity of the anti-sheep heart antibodies with other sheep tissues and species was studied via polyacrylamide gel electrophoresis of immunoprecipitates, Micro-Ouchterlony plates and alkaline phosphatase staining of Western Blots. Cross reactivity was observed with sheep muscle and to a less extent, rat heart. Only minor cross reactivity was observed against liver PFK (rat or sheep), though this may be due to low levels of PFK in that tissue