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.
Highlights
In our preliminary experiments, we have observed that the against the enzyme was induced in a rabbit, and the inactivation of purified phosphofructokinase by tryptic digesIgG fraction was obtainedbychromatographyon a tion is inhibited by addition of fructose 2,6-bisphosphate.l Protein A-Sepharose CL-4B column
Since Schimke [18]reported that the change in arginase activity in rat liver under a dietary restriction was due to the change in degradation rate, the physiological importance of enzyme catabolism has been discussed by many groups
B abc were further purified by two-dimensional electrophoresis in an ISOTACHO/DALT system
Summary
Dithiothreitol, ATP, AMP, fructose 6-phosphate, fructose 2,6-bisphosphate, antipain, leu- Dunaway and his co-workers [1,2,3,4] have concluded that the reduction in liver PFK-L2 A small portion of IgG was further purified by affinity chromatography ona phosphofructokinase-conjugated Affi-Gel 10 column. The immunoaffinity-purified IgG was used as a firstantibody for enzyme-linked immunostaining of phosphofructokinase and its intermediate catabolic products on a nitrocellulose membrane. Radioiodinated polypeptides were purified again by SDS-polyacrylamide gel electrophoresis, and electrophoretically eluted in buffer L (25 mM Tris, 0.192 M glycine, 0.1% (w/w) SDS). The digestion products were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography
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