Endogeneous Kinase Activity of Heterogeneous Ribonucleoprotein Particles

McGregor, Catriona W (1986) Endogeneous Kinase Activity of Heterogeneous Ribonucleoprotein Particles. PhD thesis, University of Glasgow.

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Abstract

The aim of this work was to investigate a protein kinase activity found associated with hnRNP particles from rat liver nuclei. The location of this enzyme activity coupled with the known role of protein kinases in controlling other cellular events has led to the suggestion that an hnRNP associated kinase might influence the maturation of hnRNA to mRNA. HnRNP particles were isolated from purified rat liver nuclei by the method of Samarina et al., (1968) and further purified by centrifugation on 15-30% w/v sucrose density gradients. When analysed on a 2-dimensional fractionation system, which employed non-equilibrated pH gradients in the first dimension and SDS polyacrylamide gels in the second, hnRNP particles exhibited a heterogeneous protein profile dominated by the core proteins which appeared as chains of spots showing charge heterogeneity. Isolated hnRNP particles exhibited an endogenous protein kinase activity which was capable of phosphorylating added casein or histone but also phosphorylated hnRNP particle proteins. V/hen the proteins which had been subjected to in vitro phosphorylation were separated on 2-dimensional gels it was seen that the more acidic species in the chains of core proteins were phosphorylated. Evidence/ Evidence for the association of the protein kinase with 40S hnRNP particles came from the fact that a peak of kinase activity coincided with the 40S peak of hnRNP from sucrose density gradients and was still associated with the particles when hnRNP were isolated from one gradient and rerun on a second sucrose gradient. Kinase activity was still found associated with the particles even after gel filtration on a Bio Gel column although losses of kinase activity suggest that the enzyme is only loosely bound. A band of kinase activity corresponding to the stained hnRNP band was detected on non-denaturing polyacrylamide gels. The fractionation of hnRNP particle proteins to yield reasonable quantities of purified proteins is not easy as the proteins have strong affinities for each other. HnRNP particles could be completely dissociated with high salt concentrations. However dissociation was partially reversible and removal of salt led to reaggregation of a substantial percentage of the proteins. In order to try to fractionate hnRNP particle proteins, the particles were treated with 1M NaCl before being loaded on to a Sephadex G100 column in the presence of 1M NaCl. When the fractions from the column were analysed for protein kinase, 2 peaks of activity were seen - 'A' and 'B'. Both peaks contained a different set of polypeptides as seen on SDS polyacrylamide gels, and although 'A' and 'B' had similar pH, Mg2+ , time and temperature profiles their response to Mn2+ and their substrate specificity appeared to differ.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology, Biochemistry
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-76587
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:06
Last Modified: 19 Nov 2019 14:06
URI: http://theses.gla.ac.uk/id/eprint/76587

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