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J R Horton

Publications and source records attributed to J R Horton.

24 records · Page 2Linked to original sources
PubMed1979

An interaction between lysozyme and mucus glycoproteins. Implications for density-gradient separations.

1. Some mucus glycoproteins form soluble complexes with lysozyme at neutral pH values. 2. The extent of complex-formation was determined, by an ultracentrifugal difference method, for a range of glycoproteins covering the common blood-group specificities. 3. Interaction was strongest with those glycoproteins of blood-group Lea specificity; these were also richest in sialic acid. 4. Interaction diminished with increase of ionic strength, and was not detectable at I 0.50; however, an asialoglycoprotein was found to retain some activity. The interaction is accordingly primarily, but probably not exclusively, coulombic in origin. 5. The buoyant density of lysozyme in CsCl, CsBr, CsI and Cs2SO4 was determined; the values in the last three salts are anomalously high. This finding accounts for the previously noted difficulty of separating free protein from glycoproteins by single-stage centrifugation in CsBr. 6. Conditions for effective separation of glycoproteins from secretions containing lysozyme by density-gradient centrifugation are reported.

Centrifugation, Density Gradient↗
PubMed1977

The separation and characterization of bronchial glycoproteins by density-gradient methods.

1. Sputum samples from a total of 18 asthmatic and chronic bronchitic patients were examined by analytical density-gradient ultracentrifugation. CsBr was used as the dispersal agent and dense electrolyte. 2. The patterns show two main groups of components, banding at about 1.3g/ml and 1.5g/ml; in addition, a few samples showed a further zone at approx. 1.65g/ml. These components were identified as protein, secretory glycoprotein and DNA respectively. The glycoprotein zone was frequently hypersharp, and usually contained two or more partially resolved bands; it was always well resolved from the protein. 3. The glycoprotein components were isolated from nine representative sputum samples by density-gradient ultracentrifugation on a preparative scale. Analytical density-gradient ultracentrifugation was used to monitor the efficiency of the separations. 4. Some sputum samples separated cleanly under these conditions, the glycoprotein being essentially devoid of free protein; in others, separation was apparently incomplete, although computer simulation indicated that the conditions were adequate to ensure separation. Further density-gradient separations in CsCl were necessary with several samples before satisfactory products were obtained; mixtures of CsCl with guanidinium chloride were no more effective than CsCl alone. The reluctance to separate indicates a very strong, but non-covalent, interaction between protein and glycoprotein, probably associated with the gelatinous character of the secretion. 5. The purified glycoprotein components were characterized analytically and physicochemically. They contained N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and N-acetylneuraminic acid, and had an amino acid composition in which serine, threonine and proline predominated; however, aspartic acid, glutamic acid and cystine were also appreciable. The glycoproteins were of very high molecular weight, and usually showed more than one component in sedimentation velocity; their distribution in a density gradient indicated a substantial, but largely monotonic, density heterogeneity. 6. Thiol reduction decreased the molecular weight very substantially, but the products were relatively more homogeneous than the native materials. The amino acid composition was changed significantly and a small and variable proportion of protein or peptide was liberated. It is concluded that the native materials are disulphide-linked aggregates, probably through a cross-linking peptide, in confirmation of earlier studies.

Amino Acids↗
PubMed1977

Macromolecular distribution near the limits of density-gradient columns. Some applications to the separation and fractionation of glycoproteins.

1. Expressions are derived for the distribution at density-gradient equilibrium of macromolecules whose densities are (a) close to the values characterizing the solution limits or (b) outside the span of the gradient. 2. Density-distribution predicted by the expressions agree with those obtained by rigorous methods. 3. The distribution equations are applied to hypothetical mixtures of proteins and glycoproteins in commonly used density-gradient media to simulate separation and fractionation conditions. 4. It is shown that CsBr, although less efficient than CsCl for fractionation, is nevertheless adequate for most purposes; in analytical experiments it may often have advantages over CsCl. Limitations on the use of LiBr are explored. 5. An expression is derived which allows the variance of the partial specific volume of the macromolecular component to be determined from the variance of the buoyant density. It is shown that the relative resolving powers of different salts is expressed by their values of the quantity (formula: see text). 6. The equations are applied to a well-characterized glycoprotein preparation at equilibrium in CsCl and in Cs2SO4:it is shown that the much wider distribution in CsCl than in Cs2SO4 is explicable in terms of the variance in buoyant density and the solvation properties of the salts. 7. Limitations of the expressions arise when dispersity in density is represented by a low apparent molecular weight; realistic simulations can then only be obtained when the component is fully banded.

Centrifugation, Density Gradient↗
PubMedDate not supplied

How is modification of the DNA substrate recognized by the PvuII restriction endonuclease?

In restriction-modification systems, cleavage of substrate sites in cellular DNA by the restriction endonuclease is prevented by the action of a cognate methyltransferase that acts on the same substrate sites. The PvuII restriction endonuclease (R.PvuII) has been structurally characterized in a complex with substrate DNA (Cheng et al., 1994) and as an apoenzyme (Athanasiadis et al., 1994). We report here a structure, determined to 1.9 A resolution by crystallography, of a complex between R.PvuII and iodinated DNA. The presence of an iodine at the 5-carbon of the methylatable cytosine results in the following changes in the protein: His84 moved away from the modified base; this movement was amplified in His85 and disrupts an intersubunit hydrogen bond; and the base modification disturbs the distribution of water molecules that associate with these histidine residues and the area of the scissile bond. Considering these observations, hypotheses are given as to why a similar oligonucleotide, where a methyl group resides on the 5-carbon of the methylatable cytosine, is slowly cleaved by R.PvuII (Rice et al., 1995).

Crystallography, X-Ray↗