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J A Sophianopoulos

Publications and source records attributed to J A Sophianopoulos.

9 recordsLinked to original sources
PubMed1986

Effect of the conformation of concanavalin A on its affinity for manganous ion.

The stoichiometry of Mn2+ binding to concanavalin A at pH 6.4-7 which had been established in two independent studies [J.A. Sophianopoulos, A.J. Sophianopoulos, and W.C. MacMahon (1983) Arch. Biochem. Biophys. 223, 350-359; D.J. Christie, G.R. Munske, and J.A. Magnuson (1979) Biochemistry 18, 4638-4644] was challenged [C.F. Brewer, R.D. Brown, III, and S.H. Koenig (1983) Biochemistry 22, 3691-3702] on grounds of possible experimental errors. Additional evidence is presented in this study in support of the previous finding that at pH 6.4 only one Mn2+ binds per concanavalin A monomer of Mr 25,550. Also, evidence is presented showing that the results of Sophianopoulos et al. could not have been due to contamination by Ca2+. A comparison is made of the results in the three studies cited above which indicates that the concanavalin A used by Brewer et al. had decreased affinity for Mn2+ and it contained an appreciable fraction of concanavalin A incompetent of binding saccharides.

Apoproteins↗
PubMed1983

Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration.

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.

Calcium↗
PubMed1981

Preparation of homogeneous concanavalin A.

Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6-8 hr at pH 3.0-3.2 in 1 M NaCl, 0.08 M glycine and 3 mM each Ca2+ and Mn+2, heat treating at 45 degrees C for 2 hr and centrifuging. The supernatant was neutralized to pH 5 and stored in the cold. Te overall yield was 70-80%. Some of the properties of Con A at pH 5 are: The absorption coefficient of a l g/dl solution is 13.7 at 280 nm; the mean residue ellipticity at 224.5 nm is -9,300 degrees to -9,800 degrees; by sedimentation equilibrium, its molecular weight is 53,000 between pH 3.0 and pH 5.2. Con A solutions standing at room temperature at pH 7 for ten days lose through precipitation only 5-8% of the protein in 0.2 M NaCl and 15% of the protein in 0.1 M NaCl. In the solution conditions of SDS and urea-SDS gels, Con A not only unfolds slowly and incompletely, but it also forms high molecular weight aggregates. Thus, electrophoresis of Con a in such gels is unsuitable for tests of homogeneity. However, as judged by sedimentation equilibrium in 6.5 M quanidine at pH 8.1, purified Con A was monodisperse.

Chromatography, Affinity↗