Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning

Abstract

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.

Biosynthesis of Pyruvate Kinase Isozymes in Rat Liver

“…After this the rate was restored slightly just before weaning to about 75 of that at birth. Weaning dramatically boosted the synthesis of the L isozyme, which was 44 higher than that at birth and 11 times of that on day 3 which was in accordance with that observed by Hopkirk and Bloxham [20].…”

“…Our anti-(L-pyruvate kinase) antibodies also demonstrated at least two distinct parallel arcs upon diffusion against the crude liver extract which was in agreement with the finding of Kohl and Cottam [23]. In the present study this problem was bypassed by subjecting the immunoprecipitate to sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by slicing and counting the radioactivity resided in the region corresponding to the subunit of the L-type pyruvate kinase according to the procedure of Hopkirk and Bloxham [20]. In all cases the sufficiency of antibody was established by an absence of a precipitin line upon double diffusion of the supernatant after immunoprecipitation against fresh antibodies.…”

“…However, the change in activity at the weaning phase has not been intensively studied in the present report as our major attention was paid to the general development pattern and also it has been demonstrated previously by Hopkirk and Bloxham [20] that induction began between 6 h and 24 h after weaning and reached a maximum 120 h after weaning, which in their case took place on the 20th day. Therefore, it was possible in the present study that the rats had already entered the early phase of weaning since the rats might already gain access to the laboratory chow even in the presence of the mother.…”

“…1 h after the injection the animals were sacrificed and their body weights recorded. The L and M, pyruvate kinases were precipitated by their respective antibodies and washed in the manner adopted by Hopkirk and Bloxham [20]. The washed precipitate was then subjected to dodecyl sulphate electrophoresis in 5.6 % polyacrylamide gel, sliced and counted for radioactivity as described previously [20].…”

“…The L and M, pyruvate kinases were precipitated by their respective antibodies and washed in the manner adopted by Hopkirk and Bloxham [20]. The washed precipitate was then subjected to dodecyl sulphate electrophoresis in 5.6 % polyacrylamide gel, sliced and counted for radioactivity as described previously [20]. The incorporation of ~-[4,5-~H]leucine into total protein was determined by the filter-paper-disc method [21].…”