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C M Kay

Publications and source records attributed to C M Kay.

At least 163 records · Page 9Linked to original sources
PubMed1986

Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.

Animals↗
PubMed1986

Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.

The brain-specific S-100 protein is a mixture of two predominant components, S-100a and S-100b, with subunit compositions of alpha beta and beta beta respectively. In the present study, the alpha-subunit, isolated from S-100a by using anion-exchange chromatography in the presence of 8 M-urea, was homogeneous by the criteria of SDS/polyacrylamide-gel, urea/SDS/polyacrylamide-gel and non-SDS/polyacrylamide-gel electrophoresis. The alpha-subunit underwent a conformational change upon binding Ca2+ and Zn2+ at pH 7.5, as revealed by u.v. difference spectroscopy, c.d. and fluorescence measurements. Far-u.v. c.d. studies indicated the apparent alpha-helical content to fall when the protein bound either Ca2+ or Zn2+. Addition of Ca2+ to the alpha-subunit resulted in exposing to the solvent the single tryptophan residue and one or more tyrosine and phenylalanine residues. Zn2+ induced only a small conformational change, and among the aromatic chromophores only tyrosine residues were affected to a small extent. Ca2+ was able to bind to the alpha-subunit in the presence of Zn2+, and the two metal-ion-binding sites appeared to be different. When the apoprotein was excited at 280 nm, the fluorescence emission maximum was located at 337 nm. In the presence of Ca2+, the emission maximum occurred at 340 nm and was accompanied by a nearly 25% increase in fluorescence intensity. Fluorescence titration with Ca2+ at pH 7.5 revealed only one class of binding site, with a Kd value of 1.26 X 10(-4) M. The effect of K+ on the protein was slightly antagonistic to that of Ca2+, as indicated by u.v. difference spectroscopy and fluorescence titration.

Amino Acids↗
PubMed1986

Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115].

Recent studies using bee and wasp venom peptides have led to the hypothesis that proper complex formation with calmodulin (CaM) requires the presence of a basic amphiphilic helix on the surface of the target protein [Cox, J. A. (1984) Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 3000]. We have tested this hypothesis by examining CaM and troponin C (TnC) complex formation with two basic peptides, the wasp venom tetradecapeptide mastoparan and the physiologically relevant synthetic troponin I (TnI) inhibitory peptide [104-115], using far-ultraviolet circular dichroism as a secondary structure probe. Complex formation between mastoparan and either CaM or TnC results in an increase in helical content, whereas the helical content of TnI inhibitory peptide does not increase when bound to either protein. Significantly, mastoparan is 78% alpha-helical in a 50% solution of the helix-inducing solvent trifluoroethanol and has a high helix-forming potential according to the Chou-Fasman rules while TnI inhibitory peptide contains none and is not predicted to have any. We interpret these data as indicating that these peptides exhibit substantially different secondary structures upon binding to CaM or TnC. The ability of mastoparan to regulate the acto-subfragment 1-tropomyosin ATPase has also been examined. Mastoparan and TnI inhibitory peptide inhibited 31% and 45% of the activity, respectively. TnC and CaM promote differing degrees of Ca2+-sensitive release of inhibition by both peptides. Sequence comparison suggests that the basic residues present in both peptides are important for binding. However, we conclude that an alpha-helical structure is not a prerequisite for the binding of target proteins to CaM and TnC.

Actins↗
PubMed1986

1H NMR spectroscopic studies of calcium-binding proteins. 2. Histidine microenvironments in alpha- and beta-parvalbumins as determined by protonation and laser photochemically induced dynamic nuclear polarization effects.

The microenvironments of the histidines in three isoforms of Ca(II)-bound parvalbumin (carp, pI = 4.25; pike, pI = 5.00; rat, pI = 5.50) have been examined with 1H NMR techniques to probe their protonation characteristics and photochemically induced dynamic nuclear polarizability (photo-CIDNP). The histidine at position 26 (or 25), present in all three of these proteins, shows absolutely no photo-CIDNP enhancement of its C2H or C5H resonances. Nor does this nonpolarizable histidine possess a normal pKa: values range only from 4.20 for carp to 4.32 for pike to 4.44 for rat. The C2H and C5H resonances of the histidine in this carp isoform split into doublets as the pH is lowered. The magnitude of this splitting depends on the magnetic field strength, temperature, and pH; however, the line intensities within each doublet are temperature-independent. Although the crystal structure of carp parvalbumin indicates that His-26 is exposed to solvent [Kretsinger, R. H., & Nockolds, C. E. (1973) J. Biol. Chem. 248, 3313-3326], we conclude that in solution this residue, in its unprotonated state, is part of the hydrophobic core of the protein. In contrast, His-48 in rat parvalbumin and His-106 in pike III parvalbumin show dramatic photo-CIDNP enhancements of their C2H, C5H, and beta-CH2 1H NMR resonances. Combined with its nearly normal pKa, 6.14, and exchange-broadened C2H resonance, the photo-CIDNP enhancement results for His-48 indicate that its microenvironment differs little from random-coil exposure, consistent with its presumed position on the solvent surface of helix C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

1H NMR spectroscopic studies of calcium-binding proteins. 3. Solution conformations of rat apo-alpha-parvalbumin and metal-bound rat alpha-parvalbumin.

Lacking the extraordinary thermal stability of its metal-bound forms, apo-alpha-parvalbumin from rat muscle assumes two distinct conformations in aqueous solution. At 25 degrees C, its highly structured form predominates (Keq = 5.7; delta G degree = -4.3 kJ X mol-1); as deduced from both 1H NMR and circular dichroism (CD) spectroscopy, this conformation is exceedingly similar to those of its Mg(II)-, Ca(II)-, and Lu(III)-bound forms. The temperature dependences of several well-resolved aromatic and upfield-shifted methyl 1H NMR resonances and several CD bands indicate that the native, highly helical structure of rat apo-alpha-parvalbumin is unfolded by a concerted mechanism, showing no indication of partially structured intermediates. The melting temperature, TM, of rat apo-alpha-parvalbumin is 35 +/- 0.5 degrees C as calculated by both spectroscopic techniques. By 45 degrees C, rat apo-alpha-parvalbumin unfolds entirely, losing the tertiary structure that characterizes its folded form: not only are the ring-current-shifted aromatic and methyl 1H NMR resonances leveled, but the 262- and 269-nm CD bands are also severely reduced. As judged by the decrease in the negative ellipticity of the 222-nm CD band, this less-structured form of rat apo-alpha-parvalbumin shows an approximate 50% loss in apparent alpha-helical content compared to its folded state. Several changes in the 1H NMR spectrum of rat apo-alpha-parvalbumin were exceptionally informative probes of the specific conformational changes that accompany metal ion binding and metal ion exchange. In particular, the line intensities of the ortho proton resonance of Phe-47, the unassigned downfield-shifted alpha-CH resonances from the beta-sheet contacts between the metal-binding loops, the C2H resonance of His-48, and the epsilon-CH3 resonance of an unassigned Met residue were monitored as a function of added metal to determine the stability constants of several metal ion-parvalbumin complexes. We conclude that Mg(II) binds to the CD and EF sites independently, its affinity for the EF site being almost twice that for the CD site. Mg(II)----Ca(II) exchange showed that the CD-site Mg(II) is displaced first, in contrast to Lu(III)'s preferential displacement of the EF-site Ca(II) as determined from the Ca(II)----Lu(III) exchange experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Comparative calcium binding and conformational studies of turkey and rabbit skeletal troponin C.

Troponin C from turkey skeletal muscle has been compared with its chicken counterpart in terms of amino acid composition and fragmentation patterns and with rabbit TN-C by Ca2+ binding and conformational response to Ca2+ as monitored by CD and fluorescence. Cyanogen bromide and tryptic digestion mixtures of chicken and turkey TN-C have been separated by reversed-phase HPLC. The similarity of the elution profiles, along with the almost identical amino acid compositional data, suggest that the sequences are essentially equivalent. Both turkey and rabbit TN-C bound 2 mol Ca2+/mol protein at pH 5.3, while at pH 6.8, this figure was raised to 4 mol/mol protein. Circular dichroism and fluorescence measurements indicated that the conformations of the two proteins responded in a very similar manner to the presence of Ca2+.

Amino Acids↗
PubMed1985

Physicochemical properties of a novel Mr-21 000 Ca2+-binding protein of bovine brain.

The physicochemical properties of a novel Mr-21 000 Ca2+-binding protein isolated from bovine brain were investigated. The protein exhibited a partial specific volume of 0.724 ml/g, a degree of hydration of 0.47 g of water/g of protein and a mean residue weight of 119. Sedimentation equilibrium analysis revealed Mr = 22 600 in the absence of Ca2+; Ca2+ binding appeared to induce dimerization of the molecule. Size-exclusion chromatography indicated a compacting of the molecule on binding of Ca2+: the Stokes radius decreased from 2.75 nm in the absence of Ca2+ to 2.56 nm in its presence. Far-u.v.c.d. spectroscopy showed the apoprotein to be composed of 44% alpha-helix, 18% beta-pleated sheet and 38% random coil. Addition of either KCl (0.1 M) plus Mg2+ (1 mM), or Ca2+ (2 mM), changed the conformation to 49% alpha-helix, 18% beta-pleated sheet and 33% random coil. Near-u.v.c.d. and u.v. difference spectroscopy both indicated perturbations in the environments of all three types of aromatic amino acids on binding of Ca2+. Ca2+ binding also resulted in a 30% enhancement in the tryptophan fluorescence emission intensity. Ca2+ titration of the far-u.v.c.d. and fluorescence enhancement provided KD values of 9.91 microM and 4.68 microM respectively. Finally, the protein was shown to bind Zn2+ with KD = 1.44 microM (no Mg2+) and 1.82 microM (+ Mg2+). These observations strongly support the possibility that this novel Ca2+-binding protein resembles calmodulin and related Ca2+-binding proteins and undergoes a conformational change on binding of Ca2+ which reflects a physiological role in Ca2+-mediated regulation of brain function.

Animals↗
PubMed1985

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits.

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.

Animals↗
PubMed1985

Luminescence studies of lanthanide ion binding to parvalbumin.

Luminescence methods were used to examine the interaction of Eu(III) and Tb(III) with parvalbumin isozyme III from pike (Esox lucius). The bound lanthanide ions were excited both directly, via laser irradiation, and indirectly, via fluorescence energy transfer from adjacent phenylalanine residues. At high (175 microM) protein concentrations, the lanthanide titration curves exhibited pronounced quenching of luminescence at Ln3+:parvalbumin ratios above 2:1, in agreement with earlier reports (Donato, H., Jr., and Martin, R. B. (1974) Biochemistry 13, 4575-4579). However, in experiments performed with lower concentrations (10 microM), the titrations were well behaved and indicated a lanthanide:protein stoichiometry of 2:1. Equilibrium dialysis measurements performed with Eu(III) ruled out the existence of a third strong binding site which could cause the quenching of the luminescence at high protein concentrations. Similarly, careful analysis of the spectrum that results from direct excitation of the 7F0----5D0 transition of parvalbumin-bound Eu3+ ion revealed no peak attributable to a third Ln3+-binding site. The peak which has been construed by others (Rhee, M.-J., Sudnick, D. R., Arkle, V. K., and Horrocks, W. DeW., Jr. (1981) Biochemistry 20, 3328-3334) as evidence for a third site was shown to result from a pH-dependent spectral transition involving the europium ions bound at the CD and EF sites. Luminescent lifetime measurements performed on Tb(III)/parvalbumin solutions follow Stern-Volmer quenching kinetics at terbium:protein ratios in excess of 2:1, suggesting that the quenching results from collisional deactivation of the tightly bound ions by excess terbium ion free in solution.

Animals↗
PubMed1985

Trypsin digestion of bovine cardiac troponin C in the presence and absence of calcium.

The rate of tryptic digestion of cardiac troponin C (cTNC) has been shown to be dependent on Ca2+ as was noted earlier for skeletal TNC (sTNC). Two representative peptides have been characterized on the basis of amino acid composition and partial amino terminal sequence analysis. Circular dichroism and fluorescence studies monitored their response to the presence of Ca2+. Their ability to form complexes with the ATPase inhibitory subunit of cardiac troponin (cTNI) was determined by urea - polyacrylamide gel electrophoresis and fluorescence experiments. The ability of these peptides to substitute for whole cTNC in restoring the ATPase activity of a partially inhibited synthetic actomyosin system was also explored. The N-terminal peptide 1-88 already contains a large amount of ordered structure, which indicates that the alpha-helices flanking binding site II of cTNC exist independently of Ca2+. Consequently this peptide shows limited increase in structure in the presence of Ca2+. It binds to cTNI independently of the presence of Ca2+ and could substitute for whole cTNC by relaxing the inhibitory effect of cTNI. The C-terminal peptide 103-158 has a low amount of secondary structure in the absence of Ca2+ but this increases dramatically in the presence of this cation. This peptide could only form a stable complex with cTNI in the presence of Ca2+ and was unable to release the inhibitory effect of cTNI.

Adenosine Triphosphatases↗
PubMed1984

Tricarboxylate-binding proteins of Salmonella typhimurium. Purification, crystallization, and physical properties.

Citrate transport in Salmonella typhimurium involves inducible periplasmic components. Two forms of a tricarboxylate-binding protein, C1 and C2, were isolated, in high yield, from the periplasm of a cyclic AMP phosphodiesterase mutant. These immunologically cross-reactive Mr = 29,000 proteins were crystallized using ammonium sulfate. CD measurements indicated considerable secondary structure: 24% a helix, and 12% beta structure. The amino acid compositions of C1 and C2 were identical. The NH2-terminal sequence of C1 was determined; C2 was found to have a blocked NH2 terminus (pyroglutamate). C1 and C2 are products of the same gene (Somers, J. M., and Kay, W. W. (1983) Mol. Gen. Genet. 190, 20-26). C1 and C2 bound a variety of citrate analogues and organic acids, with a predominant specificity for tricarboxylates (citrate KD 1.4 X 10(-7) M), and both required a deprotonated central carboxyl group for binding. Citrate was not bound to C protein as either a salt or metal ion complex.

Amino Acids↗
PubMed1984

Hydrodynamic properties of bovine brain S-100 proteins.

The size and shape of S-100a and S-100b proteins in solution have been examined by gel filtration and ultracentrifugation in the presence and absence of Ca2+. S-100a and S-100b proteins, in the absence of Ca2+, have an intrinsic sedimentation coefficient, so20,w of 2.20 and 2.15 S, respectively and in 1 mM Ca2+ their so20,w values were decreased to 2.05 and 1.95 S, respectively, indicating an unfolding of the protein molecules. The Stokes radii of S-100a and S-100b (-Ca2+) were 23.4 A and 24.0 A and they decreased to 22.2 A and 22.3 A in the presence of Ca2+. The Ca2+ effect on S-100b greater than S-100a was in agreement with our earlier CD observations. Among the monovalent cations tested (K+, Na+ and Li+) K+ had the maximum effect on the Stokes radii and so20,w values of S-100 proteins. Since certain functions of the nervous system are accompanied by local changes in ionic concentrations of Ca2+, Na+ and K+, it is conceivable that these respective conformational changes induced in S-100 proteins by these metals may be related to their function in the brain.

Animals↗
PubMed1983

Spectral properties of three quaternary arrangements of Pseudomonas pilin.

Pseudomonas aeruginosa possess multisubunit, filamentous appendages called pili which are involved in adhesion, twitching motility, and bacteriophage adsorption. The spectral properties of three forms of pili have been compared. These are native pili, pilin dimers in octyl glycoside, and an in vitro assembled form of pilin which we call reassembled pilin filaments. Alkaline pH titrations, solvent perturbation, quenching of tryptophan fluorescence with acrylamide, and circular dichroism were used to demonstrate that tyrosines-24 and -27 are at a dimer/dimer interface in both native pili and in the reassembled pilin filaments. Dissociation of pili by octyl glucoside results in exposure of the two tyrosines and in partial exposure of a least one tryptophan in pilin.

Amino Acid Sequence↗
PubMed1983

Spectral studies on the calcium binding properties of bovine brain S-100b protein.

The effect of Ca2+ binding on the circular dichroism (CD) and 270-MHz proton nuclear magnetic resonance (NMR) spectra of brain-specific S-100b calcium binding protein has been examined at two pH values, 8.5 and 7.5. At pH 8.5, S-100b protein binds two Ca2+ per monomer with Kd values of 6 x 10(-5) and 2 x 10(-4) M, whereas at pH 7.5, the protein binds only one Ca2+ per monomer with a Kd of 2 x 10(-4) M. The presence of K+ inhibits the binding of Ca2+ to the higher affinity site at pH 8.5, and the affinity for calcium is lowered to Kd = 8.5 x 10(-4) M. Mg2+ has no effect on protein conformation. In the absence of Ca2+, S-100b undergoes a conformational change when the protein is titrated from pH 8.6 to 6.0. Addition of Ca2+ perturbed the environment of tyrosine and phenylalanine residues as measured by ultraviolet difference spectroscopy and 1H NMR. CD melt experiments and far-ultraviolet CD studies at alkaline pH and NMR experiments suggest that the protein is more stable in the presence of Ca2+. The single tyrosine residue in the protein ionizes only after the protein is denatured by exposure to high pH.

Animals↗
PubMed1983

Hydrodynamic properties of bovine cardiac troponin-I and troponin-T.

Bovine cardiac troponin-I (TN-I) and troponin-T (TN-T) have been examined in solution using ultracentrifugation, gel filtration, and viscosity. A new method of purifying TN-T, employing hydroxylapatite chromatography in 6 M urea, is reported. Cardiac TN-T (Mr = 36,000) undergoes a reversible, concentration-dependent association in nondenaturing buffers, probably to a tetramer. The Stokes radius (Rs) of aggregated TN-T, determined by sedimentation velocity and gel chromatography on Sephacryl S-300, is 80 A and the reduced viscosity of the subunit ranges from 20 to 25 ml/g at protein concentrations between 0.5 and 2.5 mg/ml. These data suggest that TN-T forms highly asymmetric aggregates in solution. Bovine cardiac TN-I also has a tendency toward self-association, but is essentially monomeric (Mr = 23,000) at protein concentrations below 1 mg/ml. The presence of reducing agent is necessary to avoid intermolecular disulfide bond formation. From gel filtration experiments, the value of Rs is 29 A indicating that TN-I is a moderately asymmetric protein (frictional ratio = 1.5). Similar properties are observed when both sulfhydryl groups of TN-I are modified by carboxamidomethylation.

Animals↗
PubMed1982

Purification and characterization of troponin C from pike muscle: a comparative spectroscopic study with rabbit skeletal muscle troponin C.

The conformation of troponin C (TN-C) isolated from the white muscle of pike (Esox lucius), in the Ca2+ and metal-free states, was studied by circular dichroism, absorption difference spectroscopy, solvent perturbation difference spectroscopy, intrinsic fluorescence, thiol titration, and 1H nuclear magnetic resonance spectroscopy. In addition, the molecular weight of the protein was determined by sedimentation equilibrium and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The composition of the protein was established by amino acid analysis. The resulting data were compared with those from the widely studied analogue isolated from rabbit skeletal muscle. The results indicate near equivalence in many of the properties of pike and rabbit TN-C, such as molecular weight, the magnitude of the calcium-induced conformational change, and urea- or thermal-induced denaturability. However, the pike protein has five additional potential carboxyl groups, and there is good evidence from NMR, solvent perturbation, and fluorescence studies for the presence of a buried tyrosine residue in the apo state.

Amino Acids↗
PubMed1982

Bovine cardiac troponin subunits: binary complexes and reconstitution of whole troponin.

The reconstitution of bovine cardiac troponin from its subunits has been investigated using hydrodynamic techniques. Gel filtration (Sephacryl S-300) and sedimentation velocity experiments indicate that troponin-C and troponin-I form a stable binary complex (1:1 mole ratio) with an apparent Stokes' radius of 36 A (frictional ratio = 1.6). Troponin-C and troponin-T do not interact significantly while troponin-I and troponin-T undergo partial complex formation. The effect of subunit ratio on the reconstitution of whole troponin has been examined by SDS-polyacrylamide gel electrophoresis and gel filtration and the results suggest that native troponin contains the subunits in an equimolar ratio.

Animals↗