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. 1981 Apr;78(4):2478–2482. doi: 10.1073/pnas.78.4.2478

Antibody-mediated activation of genetically defective Escherichia coli beta-galactosidases by monoclonal antibodies produced by somatic cell hybrids.

R S Accolla, R Cina, E Montesoro, F Celada
PMCID: PMC319370  PMID: 6787606

Abstract

Six hybridomas producing monoclonal antibodies against Escherichia coli beta-galactosidase (beta-D-galactoside galoctohydrolase, EC 3.2.1.23) have been derived from two separate somatic cell fusions. Three of these antibodies can activate defective enzymes produced by strains of E. coli carrying Z-gene point mutations. In antigen excess, one monoclonal antibody shows similar enzyme binding and mutant-activating capacity. Characteristically, the former reaction has a 200-fold higher equilibrium constant. These data provide direct evidence that the enzyme-activation reaction is a single-hit event in which one antibody site favors the correct conformation of one active center of the enzyme. Because each "activating" hybridoma is able to activate several but not all point mutant enzymes tested, it appears that the correction of the genetic defect is produced by binding key sites of the protein three-dimensional structure rather than the sites affected by the mutation.

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Selected References

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

  1. Accolla R. S., Carrel S., Mach J. P. Monoclonal antibodies specific for carcinoembryonic antigen and produced by two hybrid cell lines. Proc Natl Acad Sci U S A. 1980 Jan;77(1):563–566. doi: 10.1073/pnas.77.1.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Accolla R. S., Celada F. Antibody-mediated activation of a deletion-mutant beta-galactosidase defective in the alpha region. FEBS Lett. 1976 Sep 1;67(3):299–302. doi: 10.1016/0014-5793(76)80551-0. [DOI] [PubMed] [Google Scholar]
  3. Accolla R. S., Celada F. Immune response against the beta-galactosidase enzyme of E. coli at precursor cell level. I. Analysis of the secondary repertoire in BALB/c mice. Eur J Immunol. 1978 Oct;8(10):688–692. doi: 10.1002/eji.1830081004. [DOI] [PubMed] [Google Scholar]
  4. CINADER B., LAFFERTY K. J. MECHANISM OF ENZYME INHIBITION BY ANTIBODY. A STUDY OF THE NEUTRALIZATION OF RIBONUCLEASE. Immunology. 1964 Jul;7:342–362. [PMC free article] [PubMed] [Google Scholar]
  5. Celada F., Ellis J., Bodlund K., Rotman B. Antibody-mediated activation of a defective beta-D-galactosidase. II. Immunological relationship between the normal and the defective enzyme. J Exp Med. 1971 Sep 1;134(3 Pt 1):751–764. doi: 10.1084/jem.134.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Celada F., Macario A. J., Conway de Macario E. Enzyme activation by antibodies: a method to determine the binding constant of the activating antibody towards one determinant of E. coli beta-D-galactosidase. Immunochemistry. 1973 Dec;10(12):797–804. doi: 10.1016/0019-2791(73)90183-3. [DOI] [PubMed] [Google Scholar]
  7. Celada F., Strom R. Antibody-induced conformational changes in proteins. Q Rev Biophys. 1972 Aug;5(3):395–425. doi: 10.1017/s0033583500000998. [DOI] [PubMed] [Google Scholar]
  8. Celada F., Ullmann A., Monod J. An immunological study of complementary fragments of beta-galactosidase. Biochemistry. 1974 Dec 31;13(27):5543–5547. doi: 10.1021/bi00724a014. [DOI] [PubMed] [Google Scholar]
  9. Conway de Macario E., Ellis J., Guzman R., Rotman B. Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme. Proc Natl Acad Sci U S A. 1978 Feb;75(2):720–724. doi: 10.1073/pnas.75.2.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crumpton M. J. Conformational changes in sperm-whale metmyoglobin due to combination with antibodies to apomyoglobin. Biochem J. 1966 Jul;100(1):223–232. doi: 10.1042/bj1000223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fowler A. V., Zabin I. The amino acid sequence of beta-galactosidase of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1507–1510. doi: 10.1073/pnas.74.4.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gefter M. L., Margulies D. H., Scharff M. D. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 1977 Mar;3(2):231–236. doi: 10.1007/BF01551818. [DOI] [PubMed] [Google Scholar]
  13. Horibata K., Harris A. W. Mouse myelomas and lymphomas in culture. Exp Cell Res. 1970 Apr;60(1):61–77. doi: 10.1016/0014-4827(70)90489-1. [DOI] [PubMed] [Google Scholar]
  14. Köhler G. Frequency of precursor cells against the enzyme beta-galactosidase: an estimate of the BALB/c strain antibody repertoire. Eur J Immunol. 1976 May;6(5):340–347. doi: 10.1002/eji.1830060507. [DOI] [PubMed] [Google Scholar]
  15. Köhler G., Howe S. C., Milstein C. Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. Eur J Immunol. 1976 Apr;6(4):292–295. doi: 10.1002/eji.1830060411. [DOI] [PubMed] [Google Scholar]
  16. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  17. LITTLEFIELD J. W. SELECTION OF HYBRIDS FROM MATINGS OF FIBROBLASTS IN VITRO AND THEIR PRESUMED RECOMBINANTS. Science. 1964 Aug 14;145(3633):709–710. doi: 10.1126/science.145.3633.709. [DOI] [PubMed] [Google Scholar]
  18. Melchers F., Messer W. Enhanced stability against heat denaturation of E. coli wild type and mutant beta-galactosidase in the presence of specific antibodies. Biochem Biophys Res Commun. 1970 Aug 11;40(3):570–575. doi: 10.1016/0006-291x(70)90940-x. [DOI] [PubMed] [Google Scholar]
  19. Mäkelä O., Karjalainen K. Inherited immunoglobulin idiotypes of the mouse. Immunol Rev. 1977;34:119–138. doi: 10.1111/j.1600-065x.1977.tb00370.x. [DOI] [PubMed] [Google Scholar]
  20. Roth R. A., Rotman B. Inactivation of normal beta-D-galactosidase by antibodies to defective forms of the enzyme. J Biol Chem. 1975 Oct 10;250(19):7759–7765. [PubMed] [Google Scholar]
  21. Rotman M. B., Celada F. Antibody-mediated activation of a defective beta-D-galactosidase extracted from an Escherichia coli mutant. Proc Natl Acad Sci U S A. 1968 Jun;60(2):660–667. doi: 10.1073/pnas.60.2.660. [DOI] [PMC free article] [PubMed] [Google Scholar]

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