Batch purification of ovomucoid and characterization of the purified product

Davis, John Gorton; Mapes, Carol J.; Donovan, John W.

doi:10.1021/bi00777a006

Cited by 267 publications

(30 citation statements)

References 24 publications

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“…However, as indicated by intrinsic viscosity and gel filtration behavior [22], the protein molecule seems to exist in an asymmetric conformation. Earlier results on intrinsic viscosity [ 171, diffusion constant [19], gel filtration behavior [21] and time of escape of dialysis [20] obtained on other preparations of ovomucoid also suggested marked asymmetry in the protein molecule. Changes in AA: :m, relative fluorescence and reduced viscosity of ovomucoid produced by the increasing concentrations of urea showed definite conformational transitions involving three observable stable states: N, X and D. The intermediate state, X, of ovomucoid obtains at 6 M urea, whereas the fully denatured state, D, exists near and above 9 M urea.…”

Section: Discussionmentioning

confidence: 88%

“…Since the intrinsic viscosity of a protein measures its hydrodynamic volume and depends both on asymmetry and hydration of the protein molecule, a relatively high intrinsic viscosity of ovomucoid would mean a significant deviation from a spherical shape and/or an increased hydration. Asymmetry in the native ovomucoid molecule is also indicated by the frictional ratio (1.33) calculated from sedimentation and diffusion data [19] and by other physicochemical data [19- Table 1). …”

Section: % and 42% Earlier Studies Of Herskovits Andmentioning

confidence: 88%

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Characterization of Stable Conformational States in Urea-Induced Transition in Ovomucoid

1977

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The urea-induced transition of ovomucoid, which was studied by viscosity, difference-spectral and fluorescence measurements in 0.06 M phosphate buffer, pH 7.0, has been shown to occur in two steps involving at least three conformational states, that is, the native, the intermediate and the fully denatured states. Equilibrium and kinetic studies showed that the two transitions were completely reversible. These states have been characterized by solvent perturbation of the absorption and fluorescence spectra of ovomucoid as well as by viscosity measurements. Perturbation results with ethylene glycol and propylene glycol suggested an exposure of about three phenolic residues in the native ovomucoid. As judged by the intrinsic viscosity (5.3 ml/g), the native protein seems to exist in a conformation more expanded than that of a typical globular protein. In the intermediate state, which obtains at 6 M urea, the intrinsic viscosity of ovomucoid was measured to be 6.44 ml/g. As a result of this small but significant unfolding, the exposure of tyrosyl residues was increased ; probably an additional phenolic group became exposed. The fully denatured state occurs near and above 9 M urea. As measured by intrinsic viscosity (7.2 ml/g), the effective hydrodynamic volume increased by 12 % upon transition of the intermediate state to the denatured state. Solvent perturbation data indicated exposure of nearly all of the tyrosine residues of ovomucoid in the denatured state. These along with the intrinsic viscosity of ovomucoid (14.3 ml/g) in 9 M urea containing 0.1 M 2-mercaptoethanol have shown that the protein probably loses almost all the elements of its native structure in 9 M urea. Analysis of the preliminary kinetic results indicated that the refolding is faster than the unfolding process for both the transitions; native + intermediate s denatured.The final step in protein biosynthesis is the folding of polypeptide chain into a native and functional conformation. Useful information about the mechanism of the overall process of protein folding can be obtained by characterizing the different conformational states involved in the folding-unfolding process [l]

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Section: Discussionmentioning

confidence: 88%

Section: % and 42% Earlier Studies Of Herskovits Andmentioning

confidence: 88%

Characterization of Stable Conformational States in Urea-Induced Transition in Ovomucoid

1977

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“…Ovomucoid (molecularweight 27 300 [16], Boehringer, Mannheim, Germany) was used as an acceptor without any modification or purification, at a concentration of 17.5 mg/ml (0.64 mM).…”

Section: Acceptorsmentioning

confidence: 99%

Detergent Influence on Rat‐Liver Galactosyltransferase Activities towards Different Acceptors

Bretz

Stäubli

1977

European Journal of Biochemistry

147

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1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high-and low-molecular-weight acceptors. Transfer to N-acetylglucosamine was measured after the separation of the reaction product N-acetyllactosamine from all other radioactive molecules (including galactose) on an ionexchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.2. Both enzymic activities were enriched 60 -80-fold (compared with the post-nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 9573, presumably because both compete for the same enzyme.3. The transferase activities were enhanced by the nonionic detergent Triton X-100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the wjv concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation : Half-maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50

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“…of fragments LS and M gives 25 500. These values may be compared with the value of 27100 obtained by sedimentationequilibrium analysis of ovomucoid (Davis et al, 1971).…”

Section: Size and Composition Offragmentsmentioning

confidence: 99%

“…of approx. 27000 (Davis et al, 1971), contain families of closely related species which differ in carbohydrate content (Beeley, 1971).…”

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confidence: 99%

Active fragments obtained by cyanogen bromide cleavage of ovomucoid

Jg¹

1976

Biochemical Journal

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Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant 01, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56 % of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol

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Batch purification of ovomucoid and characterization of the purified product

Cited by 267 publications

References 24 publications

Characterization of Stable Conformational States in Urea-Induced Transition in Ovomucoid

Characterization of Stable Conformational States in Urea-Induced Transition in Ovomucoid

Detergent Influence on Rat‐Liver Galactosyltransferase Activities towards Different Acceptors

Active fragments obtained by cyanogen bromide cleavage of ovomucoid

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