Immunochemistry of creatine-kinase

Cherif, Safi (1979) Immunochemistry of creatine-kinase. PhD thesis, University of Glasgow.

Abstract

Rabbit muscle creatine kinase has been found to be weakly immunogenic in sheep. Of twelve sheep immunized with the enzyme only one animal gave an immune response. Of six chickens tested with the same enzyme, antiserum could be detected in four but the solubility properties of the antibodies isolated rendered them less suitable for experimental work. Further work was pursued using antiserum from the single responsive sheep. The animal was killed in order to obtain large amounts of antisera. This work therefore in part represents an attempt to investigate the immunological properties of a weak immunogen. Creatine kinase was covalently attached to sepharose 4B gel by the cyanogen bromide technique. This immobilized enzyme was used to investigate the stoicheiometry of binding of the antibodies. Two bleedings of the secondary response of the responsive sheep were made at an interval of 3.5 days. It was observed that the antibodies of the first bleeding precipitated CK more readily either in gel media or liquid media than did the antibodies of the second bleeding. The immobilized dimeric enzyme was found to bind 3.5 antibody molecules of the first bleeding and 2.1- 2.9 antibody molecules of the second bleeding. Work with other and, in all cases, stronger immunogens suggests that values of 5 or 6 might be expected for a protein of this size. Most of the antibody protein bound could be eluted by a buffer of pH 2.2 from the immobilized immune complex using antibodies of the second bleeding; with the first bleeding elution was only partial. Most of the antigen dimer remained associated under these conditions. The antibody protein eluted was found to have lost most of its antigen-binding capacity. Immobilized monomeric creatine kinase was prepared; using the second bleeding a valency of 1.2 was found. The two essential thiol groups of the creatine kinase dimer were alkylated by reaction with iodoacetamide. Single radial immunodiffusion studies using the first bleeding were carried out to compare the antigenicity of the native and modified enzyme; there was an unusual cross-reaction pattern which could be interpreted as a higher affinity for the antibody population by the modified enzyme. The alkylated enzyme was found to bind 3.8 antibody molecules; none of them could be eluted with pH 2.2 buffer. With the second bleeding the modified enzyme behaved like the native enzyme. Both antibody populations were found to inhibit the enzyme activity of soluble creatine kinase. Those of the second bleeding appeared to inhibit completely in excess but those of the first bleeding did not. With the immobilized enzyme inhibition by an excess of either of the antibody populations, especially with those of the second bleeding, was much less. The unretarded antibodies resulting from immunoabsorption of antibodies of the second bleeding inhibited creatine kiniase almost as effectively as the unabsorbed antibodies; those of the first bleeding showed much less inhibition. Some differences of the antigenicity of various liganded forms of creatine kinase were found using antibodies of the first bleeding; with those of the second bleeding the antigenicity was mostly the same as for the native enzyme. When the enzyme was present as the tightly-bound creatine-NO-3 - MgADP complex, which is believed to be an analogue of the transition state of the enzyme catalysed reaction, it showed a significant decrease of immune precipitate and bound only 1.6 antibody molecules of the first bleeding; these could not be eluted using a pH 2.2 buffer. The unretarded antibodies resulting from this immunoabsorption were found to inhibit the enzyme activity of soluble creatine kinase. In the presence of Mg ADP and creatine but without NO3- ions the antigenic valency was the same as for the native enzyme but none of the antibodies were found to be eluted at pH 2.2 The experiments described indicate that in immunoabsorption only high affinity antibodies are detected whereas in solution both high and low affinity antibodies are likely to be detected due to their higher avidity in the immuno-complex. These results have been interpreted in terms of a model system where each subunit of the antigen has two determinants, one with high affinity for homologous antibodies, the other with a lower affinity. Some or all of the low affinity antibodies have the property of inhibiting enzymic activity. The affinities of all the corresponding antibodies of the first bleeding appear to be higher than those of the second bleeding.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: A R Williamson
• Keywords: Immunology, Biochemistry
• Date of Award: 1979
• Depositing User: Enlighten Team
• Unique ID: glathesis:1979-72257
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 May 2019 15:12
• Last Modified: 24 May 2019 15:12
• URI: https://theses.gla.ac.uk/id/eprint/72257