Studies on the proteins of rat liver heterogeneous nuclear ribonucleoprotein particles

Wilks, Andrew F (1980) Studies on the proteins of rat liver heterogeneous nuclear ribonucleoprotein particles. PhD thesis, University of Glasgow.

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Abstract

1. HnRNP particles were prepared by either low salt (pH8.0) extraction, or sonic lysis of purified nuclei. These complexes exhibited a characteristic sedimentation coefficient (4OS) and buoyant density (1.39g.cm-3). Possible artefactual protein adsorption to the particles during their preparation was looked for by the addition of exogenous labelled proteins. Under normal circumstances, no adventitious binding could be observed. Approximately 22 polypeptides, dominated by a group of 4 major and 5 minor "core polypeptides" of molecular weight 30,000-45,000, were detected when the particle proteins were fractionated by electrophoresis on one dimensional SDS- polyacrylamide gels. No ribosomal proteins or histones were present. Two-dimensional analysis, employing isoelectric focussing (IEF) in the charge separation dimension, revealed the neutral and acid polypeptides as a complex mixture, but failed to resolve the major core species, in optimised non-equilibrated pH gradient electrophoresis technique (NEPHGE) was employed to circumvent the short-comings of the IEF-based system. Under these conditions the core polypeptides could be resolved into three chains of spots. In total over 90 species could be resolved by two-dimensional protein fractionation. An examination of the tissue and species specificity of these proteins by two-dimensional electrophoresis revealed a general conservation of the core species, but a number of minor differences were detected in the neutral and acidic regions of the profile. 2., The core polypeptide microheterogeneity was examined in an attempt to determine its possible causes. Four of the detected polypeptides were significantly phosphorylated vitro. These corresponded to the most acidic component of each of four groups of polypeptides. It was suggested that phosphorylation may be the cause of at least some of the core protein heterogeneity. Tryptic peptide mapping of each individual core protein, by situ iodination and trypsin digestion, suggested that core polypeptides consisted of four proteins each of which was modified by post-translational modification. 5. Two enzyme activities, found associated with 4OS hnRNP particles, were investigated. Preliminary charcterisation of the possible RNA processing enzyme, Poly-(A)-polymerase, revealed that the major part of this activity resided in the non-particulate region of the gradient, and was not bound to the hnRNP particles. Evidence for the association of a protein kinase activity with 40s hnRNP particles was obtained by sucrose density gradient analysis and gel exclusion chromatography. In both cases, a clear peak of kinase activity remained associated with the particle, but losses during the purification steps suggested that the enzyme was only loosely bound. The loss appeared to be part of a general degeneration of the complex during purification. The endogenous phosphorylation of hnRNP particles was found to be stimulated by cyclic AMP, polyamines and manganese ions. Examination of the protein profile of hnRNP particles after in vitro phosphorylation in the presence of [V-32p]-ATP, revealed that a number of polypeptides were phosphorylated. These included some components of the core proteins and a number of acidic species. One acidic species of molecular weight 32,000 and pi 3.5, which was not abundant enough to be detected by staining, was particularly strongly phosphorylated. No quantitative differences in protein phosphorylation could be observed in the presence of cyclic AMP, polyamines or Mn++ ions and attempts to show that the phosphorylation occurred in vivo were inconclusive.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J T Knowler
Keywords: Molecular biology
Date of Award: 1980
Depositing User: Enlighten Team
Unique ID: glathesis:1980-72231
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/72231

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