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Title: Core proteins of rat liver heterogeneous nuclear ribonucleoprotein (hnRNP) particles
Author: Seraj, Zeba I.
ISNI:       0000 0004 2666 7911
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1986
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To study the nature of the four core proteins A, B, C, and D, of rat liver heterogeneous nuclear ribonucleoprotein (hnRNP) particles, the proteins were isolated and fractionated by preparative sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The structure of the four purified proteins were compared by partial peptide mapping on SDS-polyacrylamide gels, using the Cleveland Method. The enzymes used for the partial peptide mapping were a-Chymotrypsin and S. aureus V8 Protease. Products of the proteolytic treatment were detected on the SDS-polyacrylamide gels either by silver-staining or by autoradiography. In the latter case core proteins iodinated in their tyrosine residues were used and therefore only the tyrosine-containing fragments were observed. Experiments were further conducted to determine whether the four proteins represent glycosylated variants of one protein. As an aid for the structural study of the proteins, polyclonal antibodies were raised to total core proteins and separately to core protein A and B. Immunoblots of the core proteins with the antibody preparation were used to establish the relationship between the four core proteins. Several approaches were made to determine the cause of the observed charge heterogeneity of each of the core proteins when they are fractionated on a pH-gradient. 1) Artefactual formation of the charge isomers was considered. 2) The charge isomers of core proteins A and B were subjectd to partial peptide mapping by the Cleveland Method. One dimensional SDS-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis were used to map the peptides produced after V8 protease treatment. 3) The core proteins were subjected to both in vitro and in vivo phosphorylation. For the in vitro studies, qualitative and quantitative phosphorylation patterns were analysed. The in vivo phosphorylation was performed with core proteins of a Buffalo Rat Liver Cell Line, and 4) Preliminary attempts were made to determine whether the core proteins are poly ADP-ribosylated. Results from the comparison of the partial peptide maps of isolated core proteins A, B, C and D indicated that the four proteins are a family of closely related species. A majority of the peptide products from each protein were identical, and there were relatively few unique bands. The immunological experiments confirmed the close relatedness of the proteins. Since the core proteins were found not to be glycosylated variants of each other, and certain unique peptides were observed, it was concluded that they probably are products of separate genes. The partial peptide maps of the charge isomers of both core protein A and B were shown to be nearly identical. In the A series, charge isomers A1 to A6 are probably post-translational modifications of one protein. The B series of charge isomers B1 to B5 were concluded to be post-translational variants of one or possibly two proteins. It was not possible to identify the nature of the post-synthetic modification responsible for the charge isomers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry