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Biochemical and immunological studies on gap junctions

Rahman, Salman; (1991) Biochemical and immunological studies on gap junctions. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

A panel of polyclonal anti-peptide antibodies were generated towards selected sequences of the major rat liver gap junction polypeptide, connexin 32. The amino acid sequences were derived from different putative intra- and extracellular domains based on a low resolution, two-dimensional topographical model in which connexin 32 traverses the junctional membrane four times. Six antipeptide antibodies that recognized the parent polypeptide were generated from the use of eleven peptide immunogens. Within this panel of antibodies were reagents capable of recognizing both denatured and native forms of connexin 32. In topographical studies of connexin 32 these antibodies directly demonstrated the cytoplasmic disposition of the amino and carboxyl termini of connexin 32, by immune-localization to 'intact' and 'split' gap junctions. Similarly, the cytoplasmic disposition of a protease hypersensitive loop peptide, predicted to connect the second and third transmembrane domains, was also demonstrated. By characterizing the peptide products of connexin 32 generated by the controlled proteolysis of gap junctions, these site-directed reagents further supported the immunolocalization studies, and in addition, identified a disulphide bridge(s) between the two predicted extracellular domains of connexin 32. Taken together these studies endorse and extend previous topographical models of the arrangement of connexin 32 in the junctional membrane. In separate studies, the subcellular distribution of connexin 32 in liver was analysed. These investigations implied that after synthesis on ribosomes, connexin 32 is transferred to the Golgi complex, where it accumulates without detectable modification by glycosylation, en route to the cell surface. In addition, biochemical and immunological approaches have identified several putative novel connexins in liver and brain tissues.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Biochemical and immunological studies on gap junctions
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences; Polyclonal anti-peptide antibodies
URI: https://discovery.ucl.ac.uk/id/eprint/10113804
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