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Biomedical subjects

C M Kay

Publications and source records attributed to C M Kay.

At least 181 records · Page 10Linked to original sources
PubMed1982

Physicochemical and optical studies on calcium- and potassium-induced conformational changes in bovine brain S-100b protein.

The brain-specific S-100 protein is a mixture of two components, S-100a and S-100b, with a subunit composition of alpha beta or beta 2, respectively. S-100b, isolated by using hydroxylapatite chromatography in its final purification, is homogeneous by the criteria of gel electrophoresis in the absence and presence of sodium dodecyl sulfate (NaDodSO4) and ultracentrifuge studies. Molecular weight studies by both sedimentation equilibrium in 6 M guanidine hydrochloride and 15% NaDodSO4 gels indicated the subunit molecular weight to be 10 500, and since a molecular weight of 21 000 was obtained in native solvents, the protein exists as a dimer in benign medium. The two subunits are held together by noncovalent forces. The S-100b protein undergoes a conformational change upon binding calcium, as revealed by ultraviolet difference spectroscopy and circular dichroism (CD) studies in the aromatic and far-ultraviolet (UV) range. Far-UV CD studies indicated the apparent helical content drops from approximately 58 to 52% in the presence of Ca2+. The effect of K+ on the protein was antagonistic to Ca2+, and the proteins affinity for calcium was lowered by the presence of K+. The conformational state of the protein is very much dependent upon the metal ions (Ca2+, K+) present, suggesting that changing conformation may be the way S-100 responds to local changes in ionic parameters. Fluorescence studies indicate the presence of an abnormal tyrosine in the protein with the emission maximum centered between 327 and 330 nm when the protein is excited at 280 nm.

Calcium↗
PubMed1982

Hydrodynamic properties of bovine cardiac troponin C.

The size and shape of bovine cardiac troponin C (TN-C) in solution have been examined by gel filtration, ultracentrifugation, and viscosity in the presence and absence of Ca2+. Cardiac TN-C (-Ca2+) has an intrinsic sedimentation coefficient, s020,w, of 1.87 S and a Stokes radius, RS, of 26.3 A as determined by gel chromatography on Sephacryl S-300. In 2 mM Ca2+, S020,w is increased to 2.04 S and RS is decreased to 24.3 A, indicating a conformational change to a more compact structure. Furthermore, the intrinsic viscosity of TN-C in the absence of Ca2+ (6.4 mL/g) is reduced to 5.4 mL/g when Ca2+ is added. Sedimentation equilibrium studies indicate that the effects of Ca2+ are not due to changes in the molecular weight of the protein. The hydrodynamic data suggest that TN-C is a moderately asymmetric protein with an axial ratio of 4--6.

Animals↗
PubMed1982

Evidence for an interaction between the membrane protein of a paramyxovirus and actin.

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.

Actins↗
PubMed1982

Dissociation and characterization of pilin isolated from Pseudomonas aeruginosa strains PAK and PAO.

Pili isolated from Pseudomonas aeruginosa strains PAK and PAO have been dissociated into subunits using the detergent octyl glucoside. Circular dichroism studies indicated that no change in protein conformation occurs as a result of this treatment. Ultracentrifugation measurements showed that both pilins have a Stokes radius of 21 A (1 A = 0.1 nm) corresponding to an axial ratio of between 3:1 and 5:1, when approximated as prolate or oblate ellipsoids. Sedimentation equilibrium measurements show that even at low protein concentrations the pilin-detergent complex exists as a mixture of monomers and dimers.

Bacterial Proteins↗
PubMed1981

Laser photochemically induced dynamic nuclear polarization proton nuclear magnetic resonance studies on three homologous calcium binding proteins: cardiac troponin-C, skeletal troponin-C, and calmodulin.

Laser photo-CIDNP 1H NMR experiments were performed with rabbit skeletal troponin-C (sTn-C), bovine cardiac troponin-C (cTn-C), and bovine brain calmodulin to study the exposure of histidine and tyrosine residues. In cTn-C, tyrosine residues, 5, 111, and 150 were exposed in the apoprotein, becoming buried as Ca2+ was bound. A similar phenomenon was observed for tyrosine residues 10 and 109 of sTn-C. In calmodulin, only tyrosine-99 was accessible in the apoprotein. The lack of exposure of tyrosine-138 observed with this technique correlates with the buried nature of this residue implied by other criteria. In 6 M urea each of the apoproteins were observed to be unfolded from the standpoint of the tyrosine environments. A large tyrosyl CIDNP effect was obtained for each protein which decreased as Ca2+ was bound, with a stoichiometry of one metal ion per protein. This was correlated for cTn-C with the appearance of "native" resonances representing tyrosine residues 111 and 150 in Ca2+-saturated cTn-C, also with a stoichiometry of one. Analysis of our NMR findings, in the light of other spectroscopic and model building studies on these systems, suggests that the sole high-affinity Ca2+ binding site of cTn-C and sTn-C remaining in 6 M urea is site IV.

Animals↗
PubMed1981

Hydrogen-1 nuclear magnetic resonance investigation on bovine cardiac troponin C. Comparison of tyrosyl assignments and calcium-induced structural changes to those of two homologous proteins, rabbit skeletal troponin C and bovine brain calmodulin.

The effect of Ca2+ binding on the 270-MHz proton nuclear magnetic resonance spectrum of bovine cardiac troponin C (cTnC) has been examined. Assignment of resonances in the aromatic spectral region to tyrosine residues 10, 111, and 150 has been made for apo-cTnC and calcium-bound cTnC on the basis of decoupling experiments, pH titrations, temperature-induced changes, and gadolinium broadening experiments. The sequence homology which these tyrosine residues display with residues in two previously studied proteins, rabbit skeletal troponin C (sTnC) [Seamon, K. B., Hartshorne, D. J., & Bothner-By, A. A. (1977) Biochemistry 16, 4039] and bovine brain calmodulin [Seamon, K. B. (1980) Biochemistry 19, 207], was also used in assignments. High-affinity calcium binding (up to 2 mol/cTnC) causes large alterations in the environments of tyrosines-10 and -150, indicating that the N terminus is probably buried in the protein interior. The evidence suggests that the environment of tyrosine-150 in calcium-saturated cTnC must closely resemble that of tyrosine-138 in calmodulin in that it experiences the hydrophobic core of the protein. However, there is no similarity between these environments in the apoproteins. Dramatic alterations in phenylalanine resonances are seen during the binding of the third mole of calcium, corresponding to filling the sole low affinity site. Comparison of the spectral calmodulin reveals many structural similarities which stem from their high degree of primary sequence homology.

Animals↗
PubMed1980

The effect of temperature on some calcium-binding properties of troponin C and calmodulin.

Some calcium-binding properties of skeletal and cardiac troponin C (TnC) have been measured as functions of temperature employing several physical and spectroscopic techniques. The degree of exposure of the tyrosine residues in brain calmodulin has also been determined by a new approach. Circular dichroism thermal unfolding profiles have been established for the three cases: metal-free protein, high-affinity sites filled, and fully saturated. In addition some thermodynamic parameters have been calculated for these reversible melting process. It was found that the calcium-binding parameters n and K, where n is the fraction of the total conformational change and K is the apparent association constant, for both skeletal and cardiac TnC, did not vary significantly over the temperature range 10-38 degrees C, but at 50 degrees C differences became quite apparent, dramatically so in the case of the skeletal protein. The technique of thermal perturbation difference spectroscopy was applied to determine the degree of exposure of aromatic chromophores for the TnC(s) and calmodulin in the absence and presence of calcium. For skeletal TnC and calmodulin the results were in good agreement with previous observations, but the reduced degree of exposure of the tyrosine residues in cardiac TnC, in the absence of Ca2+, was contrary to the earlier work. Calcium-induced difference absorption spectra have been measured for the TnC(s) over the temperature range 10-70 degrees C. Cardiac TnC showed greater heat stability than its skeletal counterpart, in terms of the rate and the amount of change of the difference spectral maxima.

Animals↗
PubMed1979

Circular dichroism of lipoxygenase-1 from soybeans.

The circular dichroism spectra of the three forms of lipoxygenase-1 from soybeans show characteristic differences in the region between 300 and 600 nm. Native lipoxygenase-1 only shows a negative dichroic band around 330 nm. Yellow lipoxygenase-1, obtained by addition of an equimolar amount of 13-F-hydroperoxylinoleic acid to the native enzyme, shows a positive Cotton effect at 425 nm, while the negative band band at 330 nm has increased in intensity. The blue enzyme, representing a complex of yellow enzyme with 13-L-hydroperoxylinoleic acid exhibits a negative dichroic band at 580 nm and positive bands at 410 and 391 nm. The near-ultraviolet CD spectra of the three forms of lipoxygenase are very similar, showing several well resolved positive dichroic bands at 0 degrees C. Using the method of Chen et al. (Chen, Y.-H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131) the contents of alpha-helix, beta- and unordered form of native lipoxygenase-1 were estimated to be 34, 27 and 39% respectively.

Circular Dichroism↗
PubMed1979

Interaction of studies of a 100,000 dalton chymotryptic fragment of rabbit skeletal M-line protein with the S2 subfragment of myosin.

Controlled chymotryptic digestion of the 165,000 dalton component of the M-line of rabbit skeletal muscle, followed by Biogel P-150 chromatography of the digest, has led to the isolation of a homogeneous 100,000 dalton species. This fragment was found, by both sedimentation equilibrium and gel filtration chromatography, to interact with heavy meromyosin subfragment 2 (HMM-S2). The persistence of this fragment, after chymotryptic digestion, to bind HMM-S2, along with the known insensitivity of the M-line to proteolysis, suggests a structural role for the parent 165,000 dalton component along the lines of the M-filament, as suggested by the Knappeis and Carlsen model for M-line structure.

Amino Acids↗