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Studies on casein kinase from the lactating rabbit mammary gland.

Lennon, David Patrick William; (1990) Studies on casein kinase from the lactating rabbit mammary gland. Doctoral thesis (Ph.D.), University College London. Green open access

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Abstract

The mammary gland specific casein kinase is the enzyme responsible for the in vivo phosphorylation of caseins, the major class of milk proteins. It is distinct from other so-called "casein kinases", which are neither tissue specific nor catalyze the in vivo phosphorylation of caseins. The work here describes the identification, purification and characterization of a casein kinase from the lactating rabbit mammary gland. Initially localization studies, which involved centrifugation of mammary gland sub-cellular fractions through sucrose gradients, showed that whilst a small proportion of the rabbit casein kinase was associated with a golgi enriched fraction, the bulk was present in a fraction shown by electron microscopic analysis to be a homogeneous casein micelle fraction. In order to study further the enzyme from both sources, a sub-cellular fractionation procedure was developed for the co-isolatation of golgi and micelle fractions from mammary gland homogenates. Using these two fractions as starting sources, casein kinase was purified, essentially to homogeneity, by a combination of gel-filtration on Sephacryl-S-300 and affinity chromatography on casein-sepharose. Analysis of the purified material by SDS polyacrylamide gel electrophoresis followed by silver staining showed that in both cases the enzyme comprised a polypeptide doublet of apparent molecular weights 63 and 67 KDaltons. The enzyme was characterized with respect to a number of properties including assay parameters including: temperature, pH, substrate concentration, and the requirement for divalent cations and the effect of a number of activators and inhibitors was studied. It was planned to generate primary sequence data (10-20 amino acids only) which could be used to generate oligonucleotides which in turn could be used in future studies. It was found however that the purification procedure was unsuitable to isolate protein for sequencing since frequently low levels of contaminants were present. To over-come this the purification procedure was modified and an hplc step incorporated. This method was found to be suitable for the isolation of larger quantities (70-100 μg) of pure enzyme. The enzyme was not suceptible to direct sequencing, suggesting the presence of a blocked N-terminus. Treatment of the enzyme by chemical and enzymatic means did not generate any peptide fragments more suitable for sequencing.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Studies on casein kinase from the lactating rabbit mammary gland.
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis Digitised by Proquest.
URI: https://discovery.ucl.ac.uk/id/eprint/10124865
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