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. 1986 Jun 15;236(3):721–727. doi: 10.1042/bj2360721

Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms.

R A Williams, P Andrews
PMCID: PMC1146904  PMID: 3790089

Abstract

The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.

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Selected References

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  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Babul J., Stellwagen E. Measurement of protein concentration with interferences optics. Anal Biochem. 1969 Apr 4;28(1):216–221. doi: 10.1016/0003-2697(69)90172-9. [DOI] [PubMed] [Google Scholar]
  4. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
  5. Crossley L. G., Jago G. R., Davidson B. E. Partial sequence data for the L-(+)-lactate dehydrogenase from Streptococcus cremoris US3 including the amino acid sequences around the single cysteine residue and at the N-terminus. Biochim Biophys Acta. 1979 Dec 14;581(2):342–355. doi: 10.1016/0005-2795(79)90254-x. [DOI] [PubMed] [Google Scholar]
  6. Crow V. L., Pritchard G. G. Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation. J Bacteriol. 1977 Jul;131(1):82–91. doi: 10.1128/jb.131.1.82-91.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dynon M. K., Jago G. R., Davidson B. E. The subunit structure of lactate dehydrogenase from Streptococcus cremoris US3. Eur J Biochem. 1972 Oct;30(2):348–353. doi: 10.1111/j.1432-1033.1972.tb02104.x. [DOI] [PubMed] [Google Scholar]
  8. Edelstein S. J., Schachman H. K. The simultaneous determination of partial specific volumes and molecular weights with microgram quantities. J Biol Chem. 1967 Jan 25;242(2):306–311. [PubMed] [Google Scholar]
  9. Garvie E. I. Bacterial lactate dehydrogenases. Microbiol Rev. 1980 Mar;44(1):106–139. doi: 10.1128/mr.44.1.106-139.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garvie E. I. Lactate dehydrogenases of Streptococcus thermophilus. J Dairy Res. 1978 Oct;45(3):515–518. doi: 10.1017/s0022029900016745. [DOI] [PubMed] [Google Scholar]
  11. Hardman M. J., Crow V. L., Cruickshank D. S., Pritchard G. G. Kinetics of activation of L-lactate dehydrogenase from Streptococcus lactis by fructose 1,6-bisphosphate. Eur J Biochem. 1985 Jan 2;146(1):179–183. doi: 10.1111/j.1432-1033.1985.tb08636.x. [DOI] [PubMed] [Google Scholar]
  12. Harold F. M., Spitz E. Accumulation of arsenate, phosphate, and aspartate by Sreptococcus faecalis. J Bacteriol. 1975 Apr;122(1):266–277. doi: 10.1128/jb.122.1.266-277.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hensel R., Mayr U., Stetter K. O., Kandler O. Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. I. Purification and kinetics of the allosteric L-lactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus. Arch Microbiol. 1977 Feb 4;112(1):81–93. doi: 10.1007/BF00446658. [DOI] [PubMed] [Google Scholar]
  14. Hoagland V. D., Jr, Teller D. C. Influence of substrates on the dissociation of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase. Biochemistry. 1969 Feb;8(2):594–602. doi: 10.1021/bi00830a020. [DOI] [PubMed] [Google Scholar]
  15. Jago G. R., Nichol L. W., O'Dea K., Sawyer W. H. Physicochemical studies on the lactate dehydrogenase of Streptococcus cremoris US3: the effects of modifiers. Biochim Biophys Acta. 1971 Nov 13;250(2):271–285. doi: 10.1016/0005-2744(71)90184-7. [DOI] [PubMed] [Google Scholar]
  16. Kelly N., Delaney M., O'Carra P. Affinity chromatography of bacterial lactate dehydrogenases. Biochem J. 1978 Jun 1;171(3):543–547. doi: 10.1042/bj1710543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee J. C., Timasheff S. N. The calculation of partial specific volumes of proteins in guanidine hydrochloride. Arch Biochem Biophys. 1974 Nov;165(1):268–273. doi: 10.1016/0003-9861(74)90164-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. O'Carra P., Barry S., Griffin T. Spacer arms in affinity chromatography: use of hydrophilic arms to control or eliminate nonbiospecific adsorption effects. FEBS Lett. 1974 Jul 15;43(2):169–175. doi: 10.1016/0014-5793(74)80993-2. [DOI] [PubMed] [Google Scholar]
  20. Penke B., Ferenczi R., Kovács K. A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal Biochem. 1974 Jul;60(1):45–50. doi: 10.1016/0003-2697(74)90129-8. [DOI] [PubMed] [Google Scholar]
  21. Pesce A., Fondy T. P., Stolzenbach F., Castillo F., Kaplan N. O. The comparative enzymology of lactic dehydrogenases. 3. Properties of the H4 and M4 enzymes from a number of vertebrates. J Biol Chem. 1967 May 10;242(9):2151–2167. [PubMed] [Google Scholar]
  22. Teller D. C. Characterization of proteins by sedimentation equilibrium in the analytical ultracentrifuge. Methods Enzymol. 1973;27:346–441. doi: 10.1016/s0076-6879(73)27017-9. [DOI] [PubMed] [Google Scholar]
  23. Thomas T. D., Ellwood D. C., Longyear V. M. Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. J Bacteriol. 1979 Apr;138(1):109–117. doi: 10.1128/jb.138.1.109-117.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thompson S. T., Cass K. H., Stellwagen E. Blue dextran-sepharose: an affinity column for the dinucleotide fold in proteins. Proc Natl Acad Sci U S A. 1975 Feb;72(2):669–672. doi: 10.1073/pnas.72.2.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. WOLIN M. J. FRUCTOSE-1,6-DIPHOSPHATE REQUIREMENT OF STREPTOCOCCAL LACTIC DEHYDROGENASES. Science. 1964 Nov 6;146(3645):775–777. doi: 10.1126/science.146.3645.775. [DOI] [PubMed] [Google Scholar]
  26. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]
  27. Yamada T., Carlsson J. Regulation of lactate dehydrogenase and change of fermentation products in streptococci. J Bacteriol. 1975 Oct;124(1):55–61. doi: 10.1128/jb.124.1.55-61.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

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