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. 1984 Dec 1;224(2):541–547. doi: 10.1042/bj2240541

Quantification of liver and kidney phosphofructokinase by radioimmunoassay in fed, starved and alloxan-diabetic rats.

J C Donofrio, R S Thompson, G D Reinhart, C M Veneziale
PMCID: PMC1144463  PMID: 6240262

Abstract

A newly developed specific radioimmunoassay was used to quantify phosphofructokinase protein directly and independently of assayable activity in liver and kidney cytosol of normal fed, starved and alloxan-diabetic rats. In the fed state, liver phosphofructokinase concentration was 0.096 microM and the kidney enzyme was 0.086 microM (mumol/kg of tissue). In the starved state (24h), liver and kidney phosphofructokinase concentrations decreased by 30%. Prolonged starvation up to 72h did not further decrease enzyme concentration. In liver, total enzyme content during starvation declined by more than 50%, secondary also to a decrease in liver weight. In the alloxan-diabetic rats, there was a 22% decrease in enzyme protein concentration in liver and kidney. Total enzyme content per liver actually decreased much more (46%), because diabetes also resulted in a decrease in liver size. In conjunction with assayable activity measurements, the results of the radioimmunoassay allowed us to calculate the apparent specific activity of the enzyme. The specific activity of the kidney enzyme was 2-3 times that of the liver. Little or no change in specific activity of the liver or kidney enzyme occurred as a result of starvation or chemically induced diabetes. Tissue enzyme concentrations of phosphofructokinase unequivocally reconcile the ultimate results of changing rates of synthesis and degradation and are useful data in the design of spectrophotometric, kinetic, aggregation-disaggregation and other studies.

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Selected References

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  1. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brand I. A., Söling H. D. Activation and inactivation of rat liver phosphofructokinase by phosphorylation-dephosphorylation. FEBS Lett. 1975 Sep 15;57(2):163–168. doi: 10.1016/0014-5793(75)80707-1. [DOI] [PubMed] [Google Scholar]
  3. Brand I. A., Söling H. D. Rat liver phosphofructokinase. Purification and characterization of its reaction mechanism. J Biol Chem. 1974 Dec 25;249(24):7824–7831. [PubMed] [Google Scholar]
  4. Donofrio J. C., Veneziale C. M., Mazzotta M. Y. Liver fructose bisphosphatase concentration and activity in alloxan induced diabetes. Biochem Pharmacol. 1981 Jan 1;30(1):98–101. doi: 10.1016/0006-2952(81)90291-4. [DOI] [PubMed] [Google Scholar]
  5. Dunaway G. A. A review of animal phosphofructokinase isozymes with an emphasis on their physiological role. Mol Cell Biochem. 1983;52(1):75–91. doi: 10.1007/BF00230589. [DOI] [PubMed] [Google Scholar]
  6. Dunaway G. A., Jr, Weber G. Effects of hormonal and nutritional changes on rates of synthesis and degradation of hepatic phosphofructokinase isozymes. Arch Biochem Biophys. 1974 Jun;162(2):629–637. doi: 10.1016/0003-9861(74)90225-2. [DOI] [PubMed] [Google Scholar]
  7. Dunaway G. A., Jr, Weber G. Rat liver phosphofructokinase isozymes. Arch Biochem Biophys. 1974 Jun;162(2):620–628. doi: 10.1016/0003-9861(74)90224-0. [DOI] [PubMed] [Google Scholar]
  8. Dunaway G. A., Leung G. L., Thrasher J. R., Cooper M. D. Turnover of hepatic phosphofructokinase in normal and diabetic rats. Role of insulin and peptide stabilizing factor. J Biol Chem. 1978 Oct 25;253(20):7460–7463. [PubMed] [Google Scholar]
  9. Etiemble J., Simeon J., Picat C., Boivin P. Phosphofructokinase isozymes from human organs and blood cells. Enzyme. 1979;24(1):61–66. doi: 10.1159/000458629. [DOI] [PubMed] [Google Scholar]
  10. Hansen J. B., Veneziale C. M. Intracellular concentration of hepatic glycerol-3-phosphate dehydrogenase in normal, diabetic, and hormonally manipulated rabbits. Proc Soc Exp Biol Med. 1981 Jan;166(1):44–50. doi: 10.3181/00379727-166-41022. [DOI] [PubMed] [Google Scholar]
  11. Hansen J. B., Veneziale C. M. Intracellular concentration of skeletal and cardiac muscle phosphofructokinase in diabetic and normal animals. J Lab Clin Med. 1980 Jan;95(1):133–143. [PubMed] [Google Scholar]
  12. Johnson M. L., Veneziale C. M. Hormonal regulation of liver pyruvate kinase concentration and activity. Biochemistry. 1980 May 13;19(10):2191–2195. doi: 10.1021/bi00551a030. [DOI] [PubMed] [Google Scholar]
  13. Kasten T. P., Naqui D., Kruep D., Dunaway G. A. Purification of homogeneous rat phosphofructokinase isozymes with high specific activities. Biochem Biophys Res Commun. 1983 Mar 16;111(2):462–469. doi: 10.1016/0006-291x(83)90329-7. [DOI] [PubMed] [Google Scholar]
  14. Massey T. H., Deal W. C., Jr Unusual, metabolite-dependent solubility properties of phosphofructokinase. The basis for a new and rapid purification from liver, kidney, and other tissues. J Biol Chem. 1973 Jan 10;248(1):56–62. [PubMed] [Google Scholar]
  15. Mazzotta M. Y., Veneziale C. M. Concentration of liver and kidney fructose-1,6-diphosphatase determined by specific radioimmunoassay. Biochim Biophys Acta. 1980 Jan 11;611(1):156–167. doi: 10.1016/0005-2744(80)90051-0. [DOI] [PubMed] [Google Scholar]
  16. McLaughlin C. A., Pitot H. C. The effect of various treatments in vitro and in vivo on the binding of 125I-labeled anti-rat serum albumin Fab' to rat tissue polyribosomes. Biochemistry. 1976 Aug 10;15(16):3541–3550. doi: 10.1021/bi00661a023. [DOI] [PubMed] [Google Scholar]
  17. Moore R. E., Hansen J. B., Lardy H. A., Veneziale C. M. The development and application of a radioimmunoassay for rat phosphoenolpyruvate carboxykinase. J Biol Chem. 1982 Nov 10;257(21):12546–12552. [PubMed] [Google Scholar]
  18. Paetkau V., Lardy H. A. Phosphofructokinase. Correlation of physical and enzymatic properties. J Biol Chem. 1967 May 10;242(9):2035–2042. [PubMed] [Google Scholar]
  19. Palacios R., Palmiter R. D., Schimke R. T. Identification and isolation of ovalbumin-synthesizing polysomes. I. Specific binding of 125 I-anti-ovalbumin to polysomes. J Biol Chem. 1972 Apr 25;247(8):2316–2321. [PubMed] [Google Scholar]
  20. Randle P. J., Garland P. B., Hales C. N., Newsholme E. A., Denton R. M., Pogson C. I. Interactions of metabolism and the physiological role of insulin. Recent Prog Horm Res. 1966;22:1–48. doi: 10.1016/b978-1-4831-9825-5.50004-x. [DOI] [PubMed] [Google Scholar]
  21. Reinhart G. D., Lardy H. A. Rat liver phosphofructokinase: kinetic activity under near-physiological conditions. Biochemistry. 1980 Apr 1;19(7):1477–1484. doi: 10.1021/bi00548a034. [DOI] [PubMed] [Google Scholar]
  22. Reinhart G. D., Lardy H. A. Rat liver phosphofructokinase: kinetic and physiological ramifications of the aggregation behavior. Biochemistry. 1980 Apr 1;19(7):1491–1495. doi: 10.1021/bi00548a036. [DOI] [PubMed] [Google Scholar]
  23. Reinhart G. D., Lardy H. A. Rat liver phosphofructokinase: use of fluorescence polarization to study aggregation at low protein concentration. Biochemistry. 1980 Apr 1;19(7):1484–1490. doi: 10.1021/bi00548a035. [DOI] [PubMed] [Google Scholar]
  24. Taketa K. Electrophoretic multiplicity of phospho-fructokinase in rat liver and other tissues and effect of carbon tetrachloride intoxication. Acta Med Okayama. 1973 Dec;27(5):205–209. [PubMed] [Google Scholar]
  25. Tanaka T., An T., Sakaue Y. Studies on multimolecular forms of phosphofructokinase in rat tissues. J Biochem. 1971 Mar;69(3):609–612. [PubMed] [Google Scholar]
  26. Uyeda K. Phosphofructokinase. Adv Enzymol Relat Areas Mol Biol. 1979;48:193–244. doi: 10.1002/9780470122938.ch4. [DOI] [PubMed] [Google Scholar]
  27. Veneziale C. M., Hansen J. B., Johnson M. L. Specific radioimmunoassays for rabbit skeletal and cardiac muscle 6-phosphofructokinase and pyruvate kinase. Methods Enzymol. 1981;74(Pt 100):232–244. doi: 10.1016/0076-6879(81)74016-3. [DOI] [PubMed] [Google Scholar]
  28. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  29. Zalitis J. G., Pitot H. C. The synthesis and degradation of rat liver and kidney fructose bisphosphatase in vivo. Arch Biochem Biophys. 1979 May;194(2):620–631. doi: 10.1016/0003-9861(79)90657-x. [DOI] [PubMed] [Google Scholar]

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