Biosynthesis of ovomucoid

McCairns, Eric (1973) Biosynthesis of ovomucoid. PhD thesis, University of Glasgow.

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Abstract

Ovomucoid was isolated from egg white or the homogenates of oviduct tissue by adsorbtion on and elution from an insolubilised trypsin derivative (Sephadex G-200-trypsin). Ovomucoid bound to the insolubilised trypsin at pH 7, but could be eluted by washing the complex at pH 1.5. The method was shown to be selective for ovomucoid if a preliminary precipitation of other components with 5% (w/v) trichloroacetic acid, pH 3.5, was included in the isolation. Ovomucoid isolated using insolubilised trypsin had the same trypsin inhibiting activity and the same hexosamine, hexose and sialic acid content as ovomucoid prepared by conventional methods, such as ethanol precipitation. Immunochemical studies suggested that ovomucoid isolated by adsorbtion on and elution from G-200-trypsin was immunologically homogenous, while ovomucoid prepared by ethanol precipitation, following a preliminary pH 3.5 trichloroacetic acid precipitation of other components, contained more than one antigenic species. Ovomucoids from egg white or the homogenates of washed oviduct tissue were indistinguishable in the above studies. Ovomucoid was fractionated into differently charged variants by sulphoethyl-Sephadex chromatography or isoelectric focusing. The 5 variants of egg white ovomucoid observed on isoelectric focusing were present in oviduct tissue before secretion making it unlikely that the multiplicity of species arose from degradation following secretion. The isoelectric points of 4 of the variants were determined to be 4.45, 4.30, 4.15 and 4.0 and the isoelectric point of the most acidic species estimated to be less than 4. All bad the same molecular weight, estimated by sodium dodecyl sulphate gel electrophoresis to be 30,000 +/- 2,500. After neuraminidase treatment the number of variants was reduced from 5 to 3 suggesting that part of the charge heterogeneity resided in the sialic acid residues. The insolubilised trypsin isolation method was shown to be suitable for isolating labelled ovomucoid from tissue incubation studies. Ovomucoid isolated from the tissue after a 7 hour incubation period in vitro was shown to be indistinguishable from ovomucoid from an unincubated piece of tissue or from egg white ovomucoid. Radiological purity was indicated by the lack of change in specific radioactivity (d.p.m./mg of ovomucoid) when additional purification steps, e.g. gel filtration or ethanol precipitation were included in the isolation. [3H] Lysine was incorporated linearly into intracellular ovomucoid over a 5 hour period in vitro. After about an hour a small amount of newly synthesised ovomucoid appeared in the medium. There was a lag of approximately an hour before the linear incorporation of [14C] glucosamine into intracellular ovomucoid. The incorporation of [14C] glucosamine into extracellular ovomucoid was lower over an 8 hour incubation period than its incorporation into intracellular ovomucoid. Although only a very small amount of newly synthesised material appeared in the medium, about 30% of the total ovomucoid isolated from the tissue was in the extracellular fraction after 8 hours. There was no combination of progesterone and estrogen tested that could stimulate ovomucoid synthesis or secretion in vitro. However the rate of synthesis of ovomucoid varied when compared to the synthesis of the total trichloroacetic acid precipitable protein fraction when the oviduct was excised at different times post-oviposition. Ovomucoid which was released from the 105,000g pellet of oviduct homogenates by deoxycholate had a slightly higher, though fairly similar, specific radioactivity in terms of both lysine and glucosamine labelling to that of intracellular ovomucoid over a 5 hour incubation period. The deoxycholate extracted ovomucoid seemed to have different solubility properties from intracellular ovomucoid. Only a very small proportion of the total ovomucoid isolated from the tissue (approximately 7%) was released by deoxycholate. Fractionation of oviduct homogenates by centrifugation suggested that there was no clear separation of subcellular components. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J G Beeley
Keywords: Biochemistry
Date of Award: 1973
Depositing User: Enlighten Team
Unique ID: glathesis:1973-72325
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: http://theses.gla.ac.uk/id/eprint/72325

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