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. 1978 Dec;62(6):968–974. doi: 10.1104/pp.62.6.968

Binding Specificity and Possible Analytical Applications of the Cytokinin-binding Antibody, Anti-N6-Benzyladenosine 1

H A Constantinidou 1,2,3,4, J A Steele 1,2,3,4, T T Kozlowski 1,2,3,4, C D Upper 1,2,3,4
PMCID: PMC1092264  PMID: 16660648

Abstract

Antibodies elicited in rabbits by immunization with an N6-benzyladenosine-bovine serum albumin conjugate bound N6-benzyladenosine specifically. The affinity constants and specific binding site concentrations for a number of cytokinins and related compounds were estimated by nonlinear least squares analysis of direct or competitive ultrafiltration data. The antisera contained 230 to 330 nanomoles of cytokinin binding sites per gram protein. Affinity constants were 8.8 × 108 molar−1 for 6-benzylaminopurine, 8.4 × 107 molar−1 for kinetin, 9.1 × 107 molar−1 for 6-(3-methyl-2-butenylamino)purine, 6.6 × 106 molar−1 for 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine, and 2.0 × 104 molar−1 for 6-methylaminopurine. Affinity constants were below the limit of detectability (<104 molar−1) for benzylamine, adenine, and other adenine derivatives without an N6-side chain. The N6-substituent was thus immunodominant, but the purine moiety was also necessary for binding affinity. The antibodies were immobilized on cyanogen bromide-activated Sepharose with 95% retention of binding capacity and without apparent change in affinity constants. Columns of the immobilized antibody retained 64% of the [3H]6-(3-methyl-2-butenylam-ino)purine from 2 nanomolar solutions and readily trapped [14C]6-benzylaminopurine that had been added to crude extracts of cabbage. Aqueous 10% pyridine adjusted to pH 7.3 with formic acid effectively eluted bound cytokinins from gel columns without loss of binding capacity of the immobilized antibody.

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Selected References

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

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