UMLS:C0155339 - FACTA Search

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Query: UMLS:C0155339 (Brown)
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Concanavalin A is known to undergo a first-order conformational transition when metals are added to the demetallized protein at pH 5.6 (Brown, R.D., III, et al. (1977) Biochemistry 16, 3883--3896). The rate constants for this process, which wer have measured using a polarographic technique, are identical when zinc, cobalt, or manganese occupies S1 and calcium occupies S2. The reducible sugar, p-nitrophenyl alpha-D-mannopyranoside, binds only to the locked conformational structure which is formed upon the addition of metals. The affinity of the protein for sugars is dependent upon occupancy of S1 and S2 and quite sensitive to the identity of the metal in S2. The metals may be removed from the locked protein structure and the protein temporarily retains its ability to bind with sugars but with a considerably lower affinity. The locked form of concanavalin A is unstable at a pH near 2 and unfolds to the unlocked structure with a half-life of 25 min resulting in simultaneous loss of metal and sugar binding.
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PMID:Sugar binding properties of various metal ion induced conformations in concanavalin A. 9 52

We have studied the effects of Co2+ and Mn2+ ions on the low-field nuclear magnetic resonance (NMR) spectra of pure class 1 transfer ribonucleic acid (tRNA) species. With 1.2 mM tRNA in the presence of 15 mM MgCl2 discrete paramagnetic effects were observed for Co2+ at concentrations in the range 0.02--0.1 mM and for Mn2+ in the range 0.002--0.01 mM, indicating fast exchange of these cations with tRNA. Both of these cations paramagnetically relax the s4U8--A14 resonance as well as other resonances from proximal base pairs. The Co2+ site appears to be the same site on G15 which was observed crystallographically [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315-328]; the initially occupied tight Mn2+ site is the cation site involving the phosphate of U8. There are three base pairs within 10 A of both sites, namely, G15--C48, A14--s4U8, and C13--G22; this has led to the assignment of the G15--C48 and C13--G22 resonances in the NMR spectrum [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315--328; Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, Sung-Hou (1977) Nucleic Acids Res. 4, 2811--2820; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64--68].
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PMID:Paramagnetic ion effects on the nuclear magnetic resonance spectrum of transfer ribonucleic acid: assignment of the 15--48 tertiary resonance. 38 41

The existence of two conformational states of concanavalin A (Con A) with different metal ion binding properties has been recently demonstrated (Brown, R. D., Brewer, C. F., & Koenig, S. H. (1977) Biochemistry 16, 3883). Introduction of Mn2+ to the S1 site and Ca2+ to the S2 site of apo-Con A was shown to induce a conformational change in the protein, ascribed to a cis-trans isomerization of a peptide bond in the secondary structure, which results in extremely tight binding of the metal ions. This induced conformation is referred to as "locked" and the initial conformation as "unlocked". The locked ternary complex is identical with the native protein. In the present paper, we report evidence for the formation of a relatively stable, locked, ternary Ca2+-Con A complex that possesses properties similar to those of native Ca2+-Mn2+Con A. The experimental technique involves measurement of the magnetic field and time dependence of the nuclear magnetic relaxation rate (1/T1) of solvent water protons in solutions of Ca2+-Con A, after the addition of Mn2+ ion which slowly bind to the protein. The kinetic data can be fit by a model for Ca2+ interactions with Con A which indicates that Ca2+, in the absence of Mn2+, can bind at both the S1 and S2 sites of the protein and, furthermore, can induce the protein to undergo the unlocked to locked conformational transition. In terms of this model, the time-dependent binding of the Mn2+ ions is due to replacement of Ca2+ ions at the S1 sites in the locked protein. The off-rate of Ca2+ from the S2 site of the locked ternary Ca2+-Con A complex is much greater than that from the locked Ca2+-Mn2+-Con A complex. From the effects of added alpha-methyl D-mannopyranoside on the rate of replacement of Ca2+ by Mn2+ at the S1 site of the locked ternary Ca2+-Con A complex, it is concluded that the latter complex binds saccharides as strongly as the locked Ca2+-Mn2+-Con A complex. In addition, analysis of the data indicates that apo-Con A in the locked conformation binds alpha -methyl D-mannopyranoside with approximately 7% of the affinity of the fully metallized locked form of the protein. This strong saccharide-binding activity of locked apo-Con A, compared with that of the unlocked apo-Con A, was further demonstrated by equilibration of unlocked apo-Con A with alpha-methyl D-mannopyranoside, which resulted in the formation of the locked apo-Con A-saccharide complex. These results demonstrate that it is the locked conformation of Con A that is primarily responsible for saccharide-binding activity, and that the function of the bound metals is primarily to maintain the protein in the locked conformation.
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PMID:Conformation as the determinant of saccharide binding in concanavalin A: Ca2+-concanavalin A complexes. 70 10

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.
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PMID:Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver. 157 59

Electron spin echo envelope modulation (ESEEM) experiments have been used to investigate the Mn(2+)-binding site in a series of lectins including concanavalin A, pea lectin (Pisum sativum), isolectin A from lentil (Lens culinaris), soybean agglutinin (Glycine max), Erythrina indica lectin, and Lotus tetragonolobus isoelectin A. Together with model studies, the results provide direct evidence for a single nitrogen atom of a conserved residue bonded directly to Mn2+ in all of them. ESEEM measurements of the lectins exchanged with deuterium oxide, together with model studies, provide evidence for the presence of two water molecules coordinated to the Mn2+ in all of the proteins. In contrast to concanavalin A, the absence of solvent exchange at the Mn2+ site in the pea and lentil lectins demonstrated by nuclear magnetic relaxation dispersion measurements [Bhattacharyya, L., Brewer, C.F., Brown, R. D., III, & Koenig, S. H. (1985) Biochemistry 24, 4985-4990] must therefore be due to slow exchange of the water ligands of the bound Mn2+. Binding of saccharides was observed to have little effect on the structural features of the Mn2+ site in the lectins as determined by ESEEM.
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PMID:Electron spin echo envelope modulation studies of lectins: evidence for a conserved Mn(2+)-binding site. 185 Jun 25

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.
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PMID:Effect of the conformation of concanavalin A on its affinity for manganous ion. 375 9

Measurements of the magnetic field dependence of the longitudinal magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons led to the discovery of two classes of solvent binding sites in Ca2+-Mn2+-concanavalin A (CMPL) [Koenig, S. H., Brown, R. D., III, & Brewer, C. F. (1985) Biochemistry (second of three papers in this issue)]. In this paper, we compare proton and deuteron NMRD profiles of Ca2+-Mn2+-lentil lectin (CMLcH) and Ca2+-Mn2+-pea lectin (CMPSA) with those of CMPL. All three metalloproteins are D-mannose/D-glucose-specific lectins that have a high degree of structural similarity and require the metal ions for their biological activities. We have developed a method for the preparation of fully active metal ion derivatives of lentil lectin (LcH) and pea lectin (PSA), including the diamagnetic derivatives Ca2+-Zn2+-LcH and Ca2+-Zn2+-PSA [Bhattacharyya, L., Brewer, C. F., Brown, R. D., III, & Koenig, S. H.(1984) Biochem. Biophys. Res. Commun. 124, 857-862]. The behavior of these two lectins with regard to their NMRD profiles is essentially identical, for both the paramagnetic and diamagnetic forms. Together with CMPL, all three lectins have a common paramagnetic contribution with a negative temperature dependence of the rates, while CMPL contributes an additional component with a positive temperature dependence. The common contribution derives from the class of fast exchanging water molecules observed in the proton NMRD profile of CMPL (Koenig et al., 1985); their protons are calculated to be relatively remote from the Mn2+ ions (4.4 A for CMPL and 5.5 A for LcH and PSA).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proton and deuteron nuclear magnetic relaxation dispersion studies of Ca2+-Mn2+-lentil lectin and Ca2+-Mn2+-pea lectin: evidence for a site of solvent exchange in common with concanavalin A. 407 70

Mouse kidney carnosinase (aminoacyl-L-histidine hydrolase, EC 3.4.13.3) has been isolated, the amino acid composition determined and antiserum prepared against it. The apparent subunit molecular weight is 58 000, which increases to 112 000 on crosslinking. Carnosinase is sensitive to chelating agents and is 50% inhibited by 0.3 microM EDTA, 35 microM o-phenanthroline, or 35 microM 8-hydroxyquinoline-5-sulfonic acid. The Km for carnosine is 60 microM. Anserine is a poor substrate and homocarnosine a non-substrate, with Ki values of 37 and 17 microM, respectively. Mn2+ shifts the Km for carnosine to approx. 2 mM and increases the Vmax about 50%. The specific antiserum discriminates between this carnosinase and a second carnosinase activity which is absolutely dependent on Mn2+ for activity (Margolis, F.L., Grillo, M., Brown, C.E., Williams, T.H., Pitcher, R.G. and Elgar, G.J. (1979) Biochim. Biophys. Acta 570, 311-323). Immunocytochemistry with this antiserum has demonstrated carnosinase to be localized in proximal tubules of kidney, glandular cells of uterus and nasal olfactory mucosa and in vomeronasal and certain other nerve pathways.
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PMID:Purification, characterization and immunocytochemical localization of mouse kidney carnosinase. 634 77

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn2+ and Ca2+, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a "locked" form and an "unlocked" form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn2+ and Ca2+. They also demonstrated that Mn2+ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25 degrees C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin. Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (approximately 22 kcal M-1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied. We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn2+ and saccharide, and have determined both metal ion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Metal ion binding and conformational transitions in concanavalin A: a structure-function study. 640 Sep 8

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.
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PMID:Stoichiometry of manganese and calcium ion binding to concanavalin A. 661 93

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