Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1985 Feb;47(2):489–495. doi: 10.1128/iai.47.2.489-495.1985

Lactate dehydrogenase from Streptococcus mutans: purification, characterization, and crossed antigenicity with lactate dehydrogenases from Lactobacillus casei, Actinomyces viscosus, and Streptococcus sanguis.

P Sommer, J P Klein, M Schöller, R M Frank
PMCID: PMC263197  PMID: 3917978

Abstract

A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.

Full text

PDF
489

Images in this article

492Image
on p.492
493Image
on p.493

Selected References

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

  1. Brown A. T., Christian C. P., Eifert R. L. Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate dehydrogenase from Actinomyces viscosus. J Bacteriol. 1975 Jun;122(3):1126–1135. doi: 10.1128/jb.122.3.1126-1135.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown A. T., Wittenberger C. L. Fructose-1,6-diphosphate-dependent lactate dehydrogenase from a cariogenic streptococcus: purification and regulatory properties. J Bacteriol. 1972 May;110(2):604–615. doi: 10.1128/jb.110.2.604-615.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. Garvie E. I., Bramley A. J. Streptococcus bovis--an approach to its classification and its importance as a cause of bovine mastitis. J Appl Bacteriol. 1979 Jun;46(3):557–566. doi: 10.1111/j.1365-2672.1979.tb00855.x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gasser F., Gasser C. Immunological relationships among lactic dehydrogenases in the genera Lactobacillus and Leuconostoc. J Bacteriol. 1971 Apr;106(1):113–125. doi: 10.1128/jb.106.1.113-125.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gordon G. L., Doelle H. W. Purification, properties and immunological relationship of L (+)-lactate dehydrogenase from Lactobacillus casei. Eur J Biochem. 1976 Aug 16;67(2):543–555. doi: 10.1111/j.1432-1033.1976.tb10720.x. [DOI] [PubMed] [Google Scholar]
  11. HIGUCHI W. I., GRAY J. A., HEFFERREN J. J., PATEL P. R. MECHANISMS OF ENAMEL DISSOLUTION IN ACID BUFFERS. J Dent Res. 1965 Mar-Apr;44:330–341. doi: 10.1177/00220345650440020501. [DOI] [PubMed] [Google Scholar]
  12. Hensel R., Mayr U., Yang C. Y. The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei. Eur J Biochem. 1983 Aug 15;134(3):503–511. doi: 10.1111/j.1432-1033.1983.tb07595.x. [DOI] [PubMed] [Google Scholar]
  13. Jonas H. A., Anders R. F., Jago G. R. Factors affecting the activity of the lactate dehydrognease of Streptococcus cremoris. J Bacteriol. 1972 Aug;111(2):397–403. doi: 10.1128/jb.111.2.397-403.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lehner T., Russell M. W., Caldwell J., Smith R. Immunization with purified protein antigens from Streptococcus mutans against dental caries in rhesus monkeys. Infect Immun. 1981 Nov;34(2):407–415. doi: 10.1128/iai.34.2.407-415.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McGhee J. R., Michalek S. M. Immunobiology of dental caries: microbial aspects and local immunity. Annu Rev Microbiol. 1981;35:595–638. doi: 10.1146/annurev.mi.35.100181.003115. [DOI] [PubMed] [Google Scholar]
  18. Perch B., Kjems E., Ravn T. Biochemical and serological properties of Streptococcus mutans from various human and animal sources. Acta Pathol Microbiol Scand B Microbiol Immunol. 1974 Jun;82(3):357–370. doi: 10.1111/j.1699-0463.1974.tb02338.x. [DOI] [PubMed] [Google Scholar]
  19. Russell R. R., Beighton D., Cohen B. Immunisation of monkeys (Macaca fascicularis) with antigens purified from Streptococcus mutans. Br Dent J. 1982 Feb 2;152(3):81–84. doi: 10.1038/sj.bdj.4804751. [DOI] [PubMed] [Google Scholar]
  20. Schöller M., Klein J. P., Frank R. M. Common antigens of streptococcal and non-streptococcal oral bacteria: immunochemical studies of extracellular and cell-wall-associated antigens from Streptococcus sanguis, Streptococcus mutans, Lactobacillus salivarius, and Actinomyces viscosus. Infect Immun. 1981 Jan;31(1):52–60. doi: 10.1128/iai.31.1.52-60.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schöller M., Klein J. P., Sommer P., Frank R. Protoplast and cytoplasmic membrane preparations from Streptococcus sanguis and Streptococcus mutans. J Gen Microbiol. 1983 Oct;129(10):3271–3279. doi: 10.1099/00221287-129-10-3271. [DOI] [PubMed] [Google Scholar]
  22. Smith D. J., Taubman M. A., Ebersole J. L. Effect of oral administration of glucosyltransferase antigens on experimental dental caries. Infect Immun. 1979 Oct;26(1):82–89. doi: 10.1128/iai.26.1.82-89.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Weeke B. Crossed immunoelectrophoresis. Scand J Immunol Suppl. 1973;1:47–56. doi: 10.1111/j.1365-3083.1973.tb03778.x. [DOI] [PubMed] [Google Scholar]
  27. Wittenberger C. L., Angelo N. Purificationa and properties of a fructose-1,6-diphosphate-activated lactate dehydrogenase from Streptococcus faecalis. J Bacteriol. 1970 Mar;101(3):717–724. doi: 10.1128/jb.101.3.717-724.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Yamada T., Endo K., Araya S. A fructose 1,6-diphosphate-independent L-lactate dehydrogenase in a strain of Streptococcus mutans. Arch Oral Biol. 1976;21(4):233–236. doi: 10.1016/0003-9969(76)90040-6. [DOI] [PubMed] [Google Scholar]
  30. Zakin M. M., Hirth C., Garel J. R., Cohen G. N. Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens. Application to the glyceraldehyde 3-phosphate dehydrogenases from different species. Mol Immunol. 1980 Nov;17(11):1373–1379. doi: 10.1016/0161-5890(80)90006-1. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES