Comparison of c.d. spectra of Zn-insulin with Zn2+-free insulin demonstrated significant differences. It has been proposed that these differences are due to either changes in the structure of insulin monomers within aggregated insulins or the results of insulin aggregation. The effect of Zn2+ on the immunological activity of insulin indicated that the antigenic determinants of insulin were also altered. The apparent loss of immunological activity of monoiodotyrosylinsulin was demonstrated to be due to the loss of Zn2+ rather than the substitution of iodine. The immunological activity of Zn-insulin and Zn2+-free insulin was compared in both the radioimmune and immune haemolysis-inhibition assays by using an identical population of antibodies and concentrations of inhibitor. Relative to Zn-insulin, Zn2+-free insulin had a markedly attenuated immunological activity in the immune haemolysis-inhibition assay, whereas in the radioimmune assay slightly greater immunological activity was observed with the Zn2+-free insulin. These observations are submitted as evidence that the removal of Zn2+ perturbs the conformation of determinants that react with antibodies operative in the immune haemolysis-inhibition assay (immune haemolysis determinants) and has a minimal effect on the conformation of determinants that react with antibodies operative in the radioimmune assay (radioimmune determinants).
We investigated the ability of a calcium channel blocker nifedipine, given as an aerosol, to attenuate bronchoconstriction induced by citric acid, Ascaris antigen, and methacholine in Basenji-Greyhound dogs. citric acid 10% increased pulmonary resistance (RL) 4.2-fold +/- 1.0 (mean +/- SEM) and 1.7-fold +/- 0.7 in untreated and nifedipine-pretreated dogs (P less than 0.05). Dynamic compliance (Cdyn) fell to 0.58 +/- 0.07 and 0.66 +/- 0.07 of control values in untreated and nifedipine-treated dogs, respectively (p greater than 0.05). Ascaris antigen increased RL 5.1-fold +/- 0.83 and 3-fold +/- 0.48 in untreated and nifedipine-treated dogs, respectively (p less than 0.05); Cdyn decreased to 0.33 +/- 0.04 and 0.48 +/- 0.03 in untreated and nifedipine-treated dogs, respectively (p less than 0.05). In 5 dogs challenged with 0.3 mg/ml methacholine, RL increased 6.1-fold +/- 0.7 and 4.4-fold +/- 1.0 in untreated and nifedipine-treated dogs, respectively (p less than 0.05); Cdyn fell to 0.41 +/- 0.-3 and 0.46 +/- 0.07 of control values in untreated and nifedipine-treated dogs (P greater than 0.05). Neither 40% ethanol nor nifedipine-ethanol altered resting RL and Cdyn. We conclude that nifedipine effectively attenuates bronchoconstriction induced by citric acid, Ascaris antigen, and methacholine in dogs with hyperreactive airways.
These studies suggest that the immunologic indicator in the radioimmune assay, 125I-iodoinsulin, selects antibody populations from within the antiserum that interact with determinants distant from the solvent surface on the insulin molecule to which iodine is substituted. Evidence is presented that the connecting peptide of proinsulin is in close proximity to regions on the solvent surface of the A-chain of insulin that include the tyrosyl residues at A-14 and A-19. A marked immunologic cross-reaction between derivatives of insulin with perturbations in the regions of tyrosyl A-14 and A-19 was noted in the radioimmune assay employing desalanine-(B-30)-desasparagine-(A-21)-insulin antiserum. This observation is consistent with the presence of a restricted population of antibodies in such antisera that is directed toward immunologic determinants in or near the insulin dimer site. The apparent immunologic activity of insulin derivatives depends on which antibody populations from the antiserum pool can react with the immunologic indicatory employed on the one hand and on the composition of antibodies in that antiserum on the other. These studies indicate that the specificity of antibody populations in a given antiserum can be identified and their levels quantitated with several assay systems, each employing one of a variety of indicators.
Desoctapeptide-and desalanine-desasparagine insulins have similarly altered circular dichroic spectra compared to insulin. In contrast, the results of immunological studies clearly demonstrate significant conformational differences between these two types of structurally modified insulin molecules. Removal of alanine B-30 alone from insulin yields a derivative indistinguishable from insulin. It is concluded, therefore, that changes in structure of desalaninedesasparagine-insulin are due to the removal of Asn A-21 and not Ala B-30. The presence of a one-way cross-reaction between insulin and antibodies against desalanine-desas-T A he structural and functional integrity of the insulin molecule is dependent on the interaction of the COOH-terminal B chain with the core of the molecule (Carpenter, 1966; Arquilla and Bromer, 1967; Arquilla et al., 1972;Hodgkin and Mercola, 1972). This interaction results in a hydrophobic center rich in aromatic amino acids (Arquilla et al., 1969;Hodgkin and Mercola, 1972). Specific molecular alterations which affect the interaction of the COOH-terminal B chain with the core of the molecule are, therefore, of interest. The two derivatives most widely studied in this regard have been desalanine (B-30)-desasparagine-(A-21)-insulin and desoctapeptide (B-23 to B-30)-insulin, both of which have attenuated biologic activity (Carpenter, 1966;Arquilla et al., 1969), as well as similarly altered circular dichroic (CD) spectra (Morris et al., 1970), immunologic properties (Arquilla et al., 1969), and ability to aggregrate (Goldman, 1967).We previously proposed that the structural role of Asn A-21 may involve stabilization of the COOH-terminal B chain via electrostatic interaction with the guanidinium residue of Arg B-22, suggesting that deletion of Asn A-21, or the octapeptide of the COOH-terminal B chain may induce similar conformational changes.In order to investigate possible structural similarities between insulin, desoctapeptide-insulin, and des-Ala-des-Aspinsulin, antisera prepared against each of them were tested for cross-reactivity. These experiments have shown that desoctapeptide-and des-Ala-des-Asp-insulins differ immunologically and hence, structurally in spite of their similar circular dichroic spectra. In addition, we demonstrate an antigenic determinant which is expressed in des-Ala-des-Asp-insulin but not in native insulin.
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