Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1982 Aug 1;205(2):313–320. doi: 10.1042/bj2050313

Purification and radioimmunoassay of rat lactate dehydrogenase A and B subunits.

T J Beebee, D S Carty
PMCID: PMC1158483  PMID: 7138505

Abstract

We have developed procedures for purifying lactate dehydrogenase isoenzymes from rat tissues that involve two affinity-chromatography steps and that facilitate the isolation of milligram quantities of highly purified proteins within 2--3 days. Antibodies raised against pure A and B subunits in rabbits and hens were used in radioimmunoassays and showed negligible cross-reactivity with heterologous subunits. The radioimmunoassays provide a sensitive method for measuring nanogram amounts of A-subunit and B-subunit polypeptides in tissue homogenates and were employed to characterize the enzyme purification procedures.

Full text

PDF
316

Images in this article

316Fig. 1.
on p.316
316Fig. 2.
on p.316

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brodelius P., Mosbach K. Separation of the isoenzymes of lactate dehydrogenase by affinity chromatography using an immobilized AMP-analogue. FEBS Lett. 1973 Sep 15;35(2):223–226. doi: 10.1016/0014-5793(73)80290-x. [DOI] [PubMed] [Google Scholar]
  2. Carlotti R. J., Garnett G. F., Hsieh W. T., Smucker A. A., Vestling C. S., Morris H. P. Comparison of purified lactate dehydrogenases from normal rat liver and Morris hepatomas in rats and in culture. Biochim Biophys Acta. 1974 Apr 25;341(2):357–365. doi: 10.1016/0005-2744(74)90228-9. [DOI] [PubMed] [Google Scholar]
  3. Chang S. M., Lee C. Y., Li S. S. Structural relatedness of mouse lactate dehydrogenase isozymes, A4 (muscle), B4 (heart), and C4 (testis). Biochem Genet. 1979 Aug;17(7-8):715–729. doi: 10.1007/BF00502130. [DOI] [PubMed] [Google Scholar]
  4. Dietz A. A., Lubrano T. Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Anal Biochem. 1967 Aug;20(2):246–257. doi: 10.1016/0003-2697(67)90030-9. [DOI] [PubMed] [Google Scholar]
  5. Eventoff W., Rossmann M. G., Taylor S. S., Torff H. J., Meyer H., Keil W., Kiltz H. H. Structural adaptations of lactate dehydrogenase isozymes. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2677–2681. doi: 10.1073/pnas.74.7.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  7. Fritz P. J., Morrison W. J., White E. L., Vesell E. S. Comparative study of methods for quantitation of lactate dehydrogenase isozymes. Anal Biochem. 1970 Aug;36(2):443–453. doi: 10.1016/0003-2697(70)90381-7. [DOI] [PubMed] [Google Scholar]
  8. Nadal-Ginard B. Regulation of lactate dehydrogenase levels in the mouse. J Biol Chem. 1978 Jan 10;253(1):170–177. [PubMed] [Google Scholar]
  9. Nickols G. A., Brooker G. Potentiation of cholera toxin-stimulated cyclic AMP production in cultured cells by inhibitors of RNA and protein synthesis. J Biol Chem. 1980 Jan 10;255(1):23–26. [PubMed] [Google Scholar]
  10. O'Carra P., Barry S. Affinity chromatography of lactate dehydrogenase Model studies demonstrating the potential of the technique in the mechanistic investigation as well as in the purification of multi-substrate enzymes. FEBS Lett. 1972 Apr 1;21(3):281–285. doi: 10.1016/0014-5793(72)80183-2. [DOI] [PubMed] [Google Scholar]
  11. Peterson G. L. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem. 1979 Dec;100(2):201–220. doi: 10.1016/0003-2697(79)90222-7. [DOI] [PubMed] [Google Scholar]
  12. Rajewsky K. Kreuzreagierende antigene Determinanten auf Lactatdehydrogenasen I und V durch Acetylierung. Biochim Biophys Acta. 1966 May 26;121(1):51–68. [PubMed] [Google Scholar]
  13. Ryan L. D., Vestling C. S. Rapid purification of lactate dehydrogenase from rat liver and hepatoma: a new approach. Arch Biochem Biophys. 1974 Jan;160(1):279–284. doi: 10.1016/s0003-9861(74)80035-4. [DOI] [PubMed] [Google Scholar]
  14. Spielman H., Erickson R. P., Epstein C. J. The production of antibodies against mammalian LDH-1. Anal Biochem. 1974 Jun;59(2):462–467. doi: 10.1016/0003-2697(74)90299-1. [DOI] [PubMed] [Google Scholar]
  15. Spielmann H., Erickson R. P., Epstein C. J. The separation of lactate dehydrogenase X from other lactate dehydrogenase isozymes of mouse testes by affinity chromatography. FEBS Lett. 1973 Sep 1;35(1):19–23. doi: 10.1016/0014-5793(73)80568-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES