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C M Kay

Publications and source records attributed to C M Kay.

At least 199 records · Page 11Linked to original sources
PubMed1979

Circular dichroism studies of native and chemically modified Ca2+-dependent protein modulator.

The structural features of the native Ca2+-dependent protein modulator and two chemically modified derivatives, namely, nitrotyrosyl modulator and alkylated modulator, were examined by circular dichroism. The binding of Ca2+ to the native molecule was accompanied by an increase in helical content from 40 to 49%, with little effect on the local environments of aromatic residues in the modulator. The Mg2+ and Mn2+ do not elicit the conformational change induced by the binding of Ca2+, which also stabilizes the modulator against urea denaturation. The overall secondary structure of nitrotyrosyl modulator is indistinguishable from that of the native protein and undergoes a similar conformational change upon binding Ca2+. These observations are in agreement with the fact that nitration has no effect on modulator functions. Furthermore, nitrotyrosyl modulator interacts with troponin I only in the presence of Ca2+, as detected by circular dichroism (cd). On the other hand, alkylation of five methionine residues on the modulator with benzyl bromide affects protein conformation, as evidenced by a reduced helical content of only 35%. Alkylated modulator retains the ability of the native protein to bind Ca2+ although the affinity of this derivative for Ca2+ is reduced some three orders of magnitude relative to the native protein, with Kd = 3.2 X 10(-4) M. The results with the alkylated modulator, in conjunction with previous cd studies on N-chlorosuccinimide oxidized modulator are utilized to advance a model for the Ca2+ activation of modulator protein, based on three conformational states of the molecule.

Alkylation↗
PubMed1979

The interaction between beef cardiac troponin T and troponin I as demonstrated by ultraviolet absorption difference spectroscopy, circular dichroism, and gel filtration.

The specific interaction of bovine cardiac troponin T with troponin I has been demonstrated at a 1:1 molar ratio by absorption difference spectroscopy, near and far ultraviolet circular dichroism, and gel filtration chromatography. The maintenance of the sulfhydryl groups of both proteins in the reduced state was essential in order to demonstrate interaction between cardiac troponin I and troponin T using the aforementioned methodology. Carboxamido-methylated troponin I and troponin T samples were prepared by reaction with iodoacetamide. Spectrophotometric titration of the two proteins with 2-chloromercurinitrophenol and amino acid analysis of their carboxamidomethylated derivatives revealed that cardiac troponin I possesses two cysteine residues while cardiac troponin T has one. The modified troponin T possesses properties identical to those of the native molecule. The modification of troponin I is accompanied by an increase in secondary structure and a loss in ability to interact with troponin T at 0.5 M NaCl ionic strength. However, at 0.3 M NaCl the modified troponin I was shown by gel filtration chromoatography to interact very weakly with troponin T. On the other hand, the modified troponin I interacts with troponin C in a manner identical to the native protein, indicating that the troponin T interaction domain of the molecule is distinct from that region which interacts with troponin C.

Animals↗
PubMed1978

Interaction studies of the 165 000 dalton protein component of the M-line with the S2 subfragment of myosin.

The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Sodium dodecyl sulphate electrophoresis revealed that the protein was homogeneous. Circular dichroism measurements indicated that the protein interacts with myosin and heavy meromyosin subfragment 2 (S2). There was an increase in negative ellipticity at 221 nm upon interaction, relative to the calculated values assuming no interprotein interaction. The net increaes in negative ellipticity at 221 nm as a result of interaction of M-protein with myosin and subfragment 2 were 600 degrees and 800 degrees respectively. When the protein was mixed with subfragment 2 in a 1 : 1 mol ratio in 0.5 M KCl/25 mM Tris buffer at pH 8.0, low speed sedimentation equilibrium studies gave a molecular weight of 235 000 +/- 10 000 for the complex, indicative of an interaction of the two components. On a Bio gel A 0.5 m column, M-protein and S2 when applied in 1 : 1 mol ratio, were eluted as a single symmetrical peak and a molecular weight of 230 000 was obtained for the complex from the observed elution volume. Both circular dichroism and sedimentation equilibrium studies indicated no interaction of M-line protein with light meromyosin and subfragment 1. Interaction of the 165 000 component with the flexible hinge region of myosin may have special significance in terms of the mechanism accounting for the reversible expansion of the interfilament distance which occurs during contraction.

Animals↗
PubMed1978

Circular dichroism studies on Ca2+-dependent protein modulator oxidized with N-chlorosuccinimide.

The structural features and Ca2+-binding properties of native and N-chlorosuccinimide-oxidized modulator protein were compared by circular dichroism. In the presence of Ca2+,the far-UV spectra of native and oxidized modulator protein are virtually indistinguishable, indicating that oxidation of surface methionine residues does not alter the overall conformation of the molecule. In the absence of Ca2+, however, the circular dichroism spectra of native and oxidized modulator are different with calculated helical contents of 40% and 26%, respectively. As judged by circular dichroism titration studies, the native modulator contains both high-(Kd = 1.9 X 10(-7) M) and low-affinity (Kd = 4 X 10(-4) M) Ca2+-binding sites, whereas the modified modulator appears to possess only low-affinity sites (Kd = 3.8 X 10(-4) M). The reduced secondary structure in Ca2+-free oxidized modulator protein may account for the absence of high affinity Ca2+ binding sites.

Animals↗
PubMed1978

Isolation and characterization of the 165 000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase.

The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Gel electrophoresis, in the presence and absence of sodium dodecyl sulfate, revealed the protein to be homogeneous. Sodium dodecyl sulfate gel electrophoresis and low speed sedimentation equilibrium studies in 0.5 M KCl, 50 mM potassium phosphate gave a molecular weight of 165 000 suggesting the protein to be made up of a single polypeptide chain. Circular dichroism spectra revealed the presence of two negative dichroic bands located at 216 and 208 nm, indicative of the presence of some beta-structure. Ellipticity values at these two wavelengths were --6500 +/- 400 and --7500 +/- 400 deg . cm2 . dmol-1, respectively. Addition of 165 000 component lowered the enzymatic activity of creatine kinase M-line protein and the nature of the inhibition was found to be a competitive one. When the protein was mixed with creatine kinase in a 1 : 1 mole ratio in a medium consisting of 0.2 M KCl, 25 mM Tris, 1 mM dithiothreitol (pH 8.0), low speed sedimentation equilibrium studies gave a molecular weight of 260 000 +/- 10 000 for the complex, indicative of an interaction of the two components of the M-line.

Amino Acids↗
PubMed1978

Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy.

Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+ -binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 x 10(7) and 4.5 x 10(5) M-1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (approximately 30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. & Bothener-By, A. A. (1977) Biochemistry 16,4039-4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium of 2.0 x 10(7) M-1 was calculated while no low-affinity site at this pH was detected by CD. On the other hand, at lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+ -binding site is detected with an apparent binding constant of 3.7 +/- 0.7 x 10(4) M-1. At the lower pH values, protonation of a class of carboxyl groups in each protein which possesses a high pKa (6.2-6.3) elicits the conformational change at the high-affinity sites with a corresponding decrease in the overall magnitude of the Ca2+ -evoked changes. The expression of a conformational change upon Ca2+ binding at the level of the low-affinity sites is enchanced by protonation of a class of carboxyls with a pKa of 6.3 in cardiac TN-C and 6.7-6.8 with the skeletal homologue. In both cases, this contribution is reduced upon protonation of carboxyls with pKa less than or equal to 5.5. It was also observed that the low-affinity sites of skeletal TN-C have a much larger role to play in the total conformational change than the low-affinity sites of cardiac TN-C, a finding probably related to the inability of site 1 in the cardiac protein to bind calcium. In the cardiac protein, the Ca2+ -induced tyrosine difference-spectrum maximum is reduced from deltaepsilonM,287nm =330M-1.cm-1 to 20M-1.cm-1 by protonation of a class of groups with a pKa of 6.4, presumably the same carboxyl groups as those invoved in the CD conformational contribution from the high-affinity binding sites. No such effect was observed for the skeletal protein where deltaepsilonM,287nm was constant at 110M-1 .cm-1 over the pH range studied. The dramatic alterations in the tyrosine environment of cardiac TN-C with pH are attributed to either or both of the tyrosines located in the two high-affinity Ca2+ -binding sites (sites 3 and 4)...

Animals↗
PubMed1976

Physicochemical studies on the creatine kinase M-line protein and its interaction with myosin and myosin fragments.

The M-line protein which is identical to the muscle form of creatine kinase was purified from rabbit skeletal muscle using ion exchange chromatography. Gel electrophoresis in the presence and absence of sodium dodecyl sulfate revealed the protein to be homogeneous. Sodium dodecyl sulfate gel electrophoresis gave 44 000 +/- 2000 as the minimum molecular weight while low speed sedimentation equilibrium experiments yielded a molecular weight of 84 000 +/- 4000, suggesting that the parent molecule is a dimer. Circular dichroism spectra revealed the presence of two negative dichroic bands located at 218 and 208 nm suggesting the presence of some beta-structure. Ellipticity values at these two wavelengths were -8000 +/- 400 and -9000 +/- 400 deg-cm2-dmol-1. Circular dichroism measurements indicated the protein to interact with myosin, heavy meromyosin and heavy meromyosin subfragment 1 (S1). The Ca2+-activated ATPase activities of myosin, heavy meromyosin and subfragment 1 were inhibited by the addition of M-line protein. When the protein was mixed with subfragment 1 in a 1:1 mole ratio in 0.15 M KC1, 50 mM Tris pH 8, low speed sedimentation equilibrium studies gave a molecular weight of 205 000 +/- 10 000 for the complex, indicative of an interaction of the two components. Both circular dichroism and sedimentation equilibrium studies indicated no interaction of M-line protein with light meromyosin.

Amino Acids↗
PubMed1976

Circular dichroism of intermediate subviral particles of reovirus. Elucidation of the mechanism underlying the specific monovalent cation effects on uncoating.

1. Circular dichroic (CD) spectra of purified intermediate subviral particles of reovirus were determined in the presence of different monovalent cations. 2. The CD spectra reveal that reo intermediate subviral particles can exist in two conformationally different forms. The two forms are readily distinguished by comparison of their ellipticities in the wavelength regions 210 nm and 220 nm, with a Na+-induced form exhibiting a reduced negative ellipticity relative to a Cs+-induced form. 3. The transition between the Na+- and Cs+-induced forms is reversible by manipulation of the species of monovalent cation present and appears to be temperature independent. 4. Temperature variation studies on dilute suspensions of particles indicate that the Na+-induced form is stable, whereas the Cs+-induced from undergoes a second transition, temperature dependent and irreversible, to become a viral core. 5. A model is presented relating these observations to the known properties of reovirus uncoating and transcriptase activation.

Cesium↗
PubMed1976

Circular dichroism study of the interaction of glutamyl-tRNA synthetase with tRNAGlu2.

The interaction of glutamyl-tRNA synthetase with tRNAGlu2 has been studied. The enzyme was purified to apparent homogeneity, and consists of a single chain with a molecular weight of 59 000. The sedimentation coefficient (sdegrees20,w) was found to be 3.7 S and suggests this enzyme is quite asymmetric. The enzyme binds 1 mol of tRNAGlu2 and has a binding constant of 5 X 10(6) M-1 at pH 7.0 in 0.1 M sodium chloride. A circular dichroic study of the interaction under the same solvent conditions implied both the synthetase and tRNAGlu2 underwent a change in conformation as the complex was formed. In the case of the enzyme there appears to be some loss of alpha-helical structure. The tRNAGlu2 results can be interpreted to indicate a change in the conformation of one or more of the helical regions of this molecule. A residue in the anticodon loop, 5-methylaminomethyl-2-thiouridine, has a distinct circular dichroic band at 340 nm in the free tRNAGlu2. As the complex is formed this band is shifted to the blue. This was interpreted to indicate that the enzyme forms a hydrogen bond with this residue in the anticodon loop, with a change in the conformation of the loop possibly also having occured.

Amino Acids↗
PubMed1976

The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin.

The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 +/- 2 000, consistent with a value of 37 000 +/- 1 000 determining by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent alpha-helical content of 43 +/- 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.

Amino Acids↗
PubMed1976

Circular dichroism and difference spectra of members of the troponin-tropomyosin system in cetyltrimethylammonium bromide.

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The delta epsilonM values at their difference maxima were found to be 1300 at 292 nm for Ac-Trp-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studies by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.

Cetrimonium Compounds↗