PMID: 9092511Apr 11, 1997

Monoclonal antibodies against Nalpha-(5'-phosphopyridoxyl)-L-lysine. Screening and spectrum of pyridoxal 5'-phosphate-dependent activities toward amino acids

The Journal of Biological Chemistry
S I Gramatikova, P Christen

Abstract

Cofactors may be expected to expand the range of reactions amenable to antibody-assisted catalysis. The biological importance of pyridoxal 5'-phosphate (PLP) as enzymic cofactor in amino acid metabolism and its catalytic versatility make it an attractive candidate for the generation of cofactor-dependent abzymes. Here we report an efficient procedure to screen antibodies for PLP-dependent catalytic activity and detail the spectrum of catalytic activities found in monoclonal antibodies elicited against Nalpha-(5'-phosphopyridoxyl)-L-lysine. This hapten is a nonplanar analog of the planar, resonance-stabilized coenzyme-substrate adducts formed in the PLP-dependent reactions of amino acids. The hapten-binding antibodies were screened for binding of the planar Schiff base formed from PLP and D- or L-norleucine by competition enzyme-linked immunosorbent assay. The Schiff base (external aldimine) is an obligatory intermediate in all PLP-dependent reactions of amino acids. This simple, yet highly discriminating screening step eliminated most of the total 24 hapten-binding antibodies. Three positive clones bound the Schiff base with L-norleucine, two preferred that with the D-enantiomer. The positive clones were assayed for catalysis o...Continue Reading

References

Apr 1, 1966·Proceedings of the National Academy of Sciences of the United States of America·H C Dunathan
Sep 1, 1995·European Journal of Biochemistry·R GraberP Christen
May 1, 1994·European Journal of Biochemistry·E SandmeierP Christen
Nov 29, 1996·The Journal of Biological Chemistry·S I Gramatikova, P Christen

Related Concepts

Aldimine eliminator
6-N-(5'-phosphopyridoxyl)-2-N-acetyl-lysine methyl ester
Affinity Labels
Monoclonal Antibodies
Digitonin
Lysine Hydrochloride
Pyridoxal Phosphate
Semicarbazides
Fluorescence Spectroscopy
Valine

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