title: Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase.
abstract: Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D008099:Liver / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D012697:Serine Endopeptidases; D013552:Swine
chemical_list: D010732:Phosphofructokinase-1; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase