The monoclonal antispermine antibody Spm8-2 was obtained by immunizing mice with a thyroglobulin-spermine conjugate. The molecular requirements for polyamines binding to this antibody were investigated by ELISA binding and inhibition tests, using a variety of natural polyamines and synthetic polyamine analogs. Four major structural determinants are important for the binding of polyamines by the antibody: (1) terminal amino groups: N-alkylation of both terminal amino groups of the polyamines leads to an important drop in the affinity for the antibody; (2) number of methylene groups spacing the amino groups: the four carbon chains appear to present the optimum length since the antibody binds polyamines with repeats of the aminobutyl moiety more actively than their homologues with shorter or longer carbon chains; (3) number of amino groups: the affinity of Spm8-2 for free homologous polyamines varied in the following order: pentamines > tetramines > triamines > diamines, showing the importance of the number of positive charges of the polyamines in the antibody-antigen reaction; the importance of charges is further emphasized by the dependence of antibody binding on the ionic strength of the medium; (4) N-acylation of one terminal amino group: the antibody binds more actively N1-acetylspermidine than spermidine or spermine. The binding properties of Spm8-2 suggest the presence of two recognition sequences, one selective for N-acylaminopropyl moieties, the second for the aminobutyl moiety.
Polyamines are ubiquitous low molecular weight organic cations which are essential for cell proliferation and differentiation [l].Their functions are still not fully understood. Cellular localisation of these species should enable a more precise study of their molecular functions. For this purpose antipolyamines antibodies may be useful tools [2,3].The molecular structure of the binding site of antipolyamine antibodies has not been defined yet. The monoclonal antibody SPM8-2 raised against spermine has been characterized in our laboratory [4,5]. Its amino acid sequence was deduced from the nucleotide sequence of the cDNA and molecular modeling studies using the programmes AbM (Oxford Molecular) and Modeller and Insight I1 97 (MSI) were performed in an attempt to identify residues involved in the antibody spermine interaction. Computer generated models of the antibody binding site show a positively charged cleft containing acidic and aromatic residues, predominantly tyrosine and aspartic acid residues from the heavy chain CDR,s. These amino acids probably interact with the terminal amino groups of spermine by electrostatic interactions andor hydrogen bonding. It has been shown experimentally that the strength of the antibody spermine interaction, as measured by Elisa, is strongly dependent on ionic strength and pH. This supports the hypothesis that negatively charged acidic residues in the antibody binding site combine with the positively charged amine groups of the polyamine. The affinity of the antibody for spermine decreased dramatically when the NaCl concentration in the ELISA buffer was increased from 0.14 to 0.75M as well as when the pH was decreased below 6 or increased over 8 (Table 1). Table 1 : Effect of ionic strength and pH on the relative affinity (Kax) of Spm8-2 for spermine NaCl (M) 0.15 0.25 0.50 0.75 1
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