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C W Slattery

Publications and source records attributed to C W Slattery.

34 records · Page 2Linked to original sources
PubMed1991

Interactions of triply phosphorylated human beta-casein: fluorescence spectroscopy and light-scattering studies of conformation and self-association.

Structural changes of triply phosphorylated human beta-casein, caused by shifts in temperature between 5 and 40 degrees C, were studied using intrinsic and extrinsic fluorescence, fluorescence polarization, turbidity, and light scattering measurements. Intrinsic fluorescence declined between 5 and 20 degrees C then rose between 25 and 40 degrees C, indicative of a shift of the tryptophan fluor toward a more nonpolar environment. The fluorescence of the extrinsic probe, 8-anilino-1-naphthalene-sulfonic acid (ANS), increased only slightly between 5 and 25 degrees C, and then more sharply between 25 and 40 degrees C, suggesting a change in conformation leading to a change in either the dissociation constant, Kd, or the number of ANS binding sites, N. The presence of Ca+2 ions did not significantly alter the pattern of changes of intrinsic and extrinsic fluorescence with changing temperature. For ANS binding, values of Kd and N were calculated by two different procedures, each based upon different assumptions. The results point to increased exposure of hydrophobic surfaces with increased temperature, strongly supportive of conformational changes. Although more opportunity for hydrophobic interaction leads to increased protein-protein association, turbidity and light-scattering also suggest ion bridge formation between protein molecules. A comparison of the primary sequences of beta-caseins from six species reveal residues that are common in all species examined and thus are pivotal in protein folding and conformation, intermolecular hydrophobic interactions and ion bridge formation with Ca+2 and inorganic phosphate.

Amino Acid Sequence↗
PubMed1991

Choline inhibition of prothrombin activation.

A computer-interfaced spectrophotometric kinetic assay for prothrombin activation was developed, coupling the production of thrombin to a thrombin-specific amidolytic chromogenic reaction. As thrombin accumulated initially at constant velocity, the simultaneous release from S-2238 of pNA conformed to an acceleration function. Adherence to the acceleration function of the temporally increasing A400 of pNA was evaluated after transforming the data into linear format which permitted linear regression analysis. High correlation coefficients, routinely greater than 0.99, verified linearity of thrombin production in individual assay mixtures. As prothrombin concentrations were varied, factor Xa exhibited Michaelis-Menten kinetics. Added choline produced a pattern of mixed-type inhibition. Replots of LB slopes and intercepts versus choline concentration gave apparent Ki and Ki' values (mM): 22 +/- 3 and 48 +/- 7 without factor Va, 25 +/- 4 and 41 +/- 4 with factor Va.

Aspirin↗
PubMed1990

Interactions of triply phosphorylated human beta-casein: monomer characterization and hydrodynamic studies of self-association.

The triply phosphorylated form of human beta-casein comprises about 15% of that fraction and is thus a significant component about midway between the two extremes of zero and five phosphoryls. Its partial specific volume, v, of 0.74 +/- 0.01 and absorbancy, E1% 1 cm, 280 nm, of 6.2 +/- 0.2 are almost identical to the other human beta-caseins. Equilibrium dialysis gave an average of 3.1 +/- 0.4 major Ca2+ binding sites at 37 degrees C with Kdiss = 8.6 x 10(-4) M. Sedimentation and viscosity at low temperatures or in 3.3 M urea suggested a prolate ellipsoidal monomer with 1.4 g H2O/g protein, 10 nm in length and 1.4 nm in width. The concentrated charge of the phosphoryls may be near one end of the ellipsoid, allowing the molecules to align with the flow in the viscometer at low concentration but, due to intermolecular electrostatic interactions, not when concentration is high. This would provide a reason for the heretofore unexplained curvature in the plots of reduced viscosity, eta red, vs beta-casein protein concentration. Self-association increased with temperature. At 37 degrees C in low salt buffer, s20,W was 16 S, which increased to about 33 S as ionic strength, I, was increased to 0.2 and above. At the same time, eta red in low salt buffer decreased from about 22 ml/g at 4 degrees C to a constant value of about 5 ml/g above 23 degrees C. A similar value for eta red at 37 degrees C, which was almost independent of protein concentration, was obtained at I greater than 0.25, giving an extrapolated intrinsic viscosity value of [eta] = 4.0 ml/g. Using this value and assuming a spherical aggregate, calculations suggest a radius of 9 nm with about 48 monomers and 0.86 g H2O/g protein.

Calcium↗
PubMed1989

Sheep platelets as a model for human platelets: evidence for specific PAF (platelet activating factor) receptors.

In a number of animals, the platelet response to Platelet Activating Factor (PAF) has been shown to differ considerably from that in humans. However, aggregation, release and particularly shape change were quite similar for human and sheep platelets. In this study, aggregation and shape change analysis were used to measure the response of sheep platelets to various synthetic analogs of PAF. Response is greatly reduced with no alkyl group in position 2 of PAF and decreases progressively as the number of carbons of the alkyl gets larger than three. A reduction of activity is also seen as the ether linkage at position 1 of PAF is replaced by an ester linkage. These changes are indicative of a specific membrane receptor for PAF in sheep platelets and confirm the usefulness of sheep platelets as a model for PAF-platelet interaction in humans.

Animals↗
PubMed1989

Measuring platelet function with platelet shape change, an early event in aggregation.

Platelet shape change precedes aggregation and is sensitive to significantly less stimulant. In this study, differences in percent light transmission (%T) through stirred suspensions of spheroid or discoid platelets are exploited for studying the response of platelets to Platelet Activating Factor (PAF). Changes in %T between stirred and non-stirred suspensions of unstimulated platelets in Platelet Rich Plasma (PRP) varied with the starting concentration. Aggregation was blocked by the presence of citrate while activation by ADP was minimized by apyrase. However, phosphatidic acid formation, ATP release and minimal serotonin release occurred. A shape change parameter (SCP) was defined which varied with the concentration of PAF. Its dependence on PAF was shifted to lower concentration with washed platelets. The dose-response curves for the SCP were found to be virtually identical for both sheep and human platelets. Human platelets were then used to correlate morphology, as determined by phase contrast microscopy, with spectrophotometric data in the form of the SCP. A linear regression analysis of SCP and percent activation revealed a virtual 1:1 correspondence with a slope of 1.066 and an intercept of 0.009 with the standard error of the estimate equal to 0.053. The correlation coefficient was 0.990, making it significant at p less than 0.001. This method provides an alternative to platelet aggregation assays as a convenient, sensitive and reliable measure of platelet activation.

Adenosine Triphosphate↗
PubMed1989

Hydrophobic interactions in human casein micelle formation: beta-casein aggregation.

The association of non-phosphorylated (0-P) and fully phosphorylated (5-P) human beta-caseins was studied by fluorescence spectroscopy and laser light scattering. The tryptophan fluorescence intensity (FI) level increased between 20 and 35 degrees C, indicating a change in the environment of that residue. A similar transition occurred when ANS was used as a probe. Transition temperatures were slightly lower in 10 mM-CaCl2 but were not affected by an equivalent increase in ionic strength caused by NaCl. The magnitude of the FI change was less for the 5-P than the 0-P protein but was increased for both by CaCl2 addition. These FI data were characteristic of a conformational change and this was supported by fluorescence polarization which indicated that with CaCl2, tryptophan and ANS mobility increased at the transition temperature even though the extent of protein association also increased. Light scattering suggested that protein association proceeded with the primary formation of submicellar aggregates containing 20-30 monomers which then associated further to form particles of minimum micelle size (12-15 submicelles), and eventually larger. The temperature of precipitation of the 5-P form in the presence of CaCl2 was lower than the conformational transition and suggested that both hydrophobic interactions and Ca bridges between phosphate esters on adjacent molecules are important in micelle formation.

Caseins↗
PubMed1988

Interactions in human casein systems: self-association of nonphosphorylated human beta-casein.

Since caseins were originally defined as phosphoproteins, nonphosphorylated beta-casein, comprising nearly 5% of the total beta-casein in the isoelectric precipitate from human milk, appears to be unique. Despite the relatively small amount present, its properties suggest that it may play an important role in micelle formation and structure. It has a partial specific volume, v, of 0.749 +/- 0.008 and an absorbance, E1% 1 cm,280 nm of 6.2 +/- 0.2. Sedimentation and viscosity data yield a solvation of 3 g H2O/g protein and an axial ratio of about 5 for the monomer. This would be consistent with a prolate ellipsoid of 10 nm length and 2 nm width. Equilibrium in the system is attained quite slowly and the temperature-dependent polymerization was found to be reversible. With calcium, the solubility behavior reflects an increased hydrophobicity and lower electrostatic repulsion in the molecule. There is essentially no strong calcium binding to this protein but there is evidence which strongly suggests that calcium binds to nonphosphate groups at higher concentrations. Increasing the temperature from 4 to 37 degrees C causes an apparent conformational change and an increase in protein aggregation which is further increased by addition of NaCl at 37 degrees C until a limiting size is reached at about 0.1 M NaCl. This limiting size polymer contains about 75 monomers and is nearly spherical with a radius of about 12 nm and a solvation of 1.5 g H2O/g protein. Laser light scattering measurements on the solution in 0.25 M NaCl revealed a relatively homogeneous particle size with a corrected diffusion coefficient, D20,w, of 2.8 X 10(-7) cm2/s.

Calcium↗
PubMed1985

Interactions in human casein systems: self-association of fully phosphorylated human beta-casein.

Human beta-casein was separated according to the extent of phosphorylation and the fully phosphorylated moiety was characterized. Fully phosphorylated human beta-casein makes up to 13-15% of the beta-casein fraction. It has a partial specific volume, v, of 0.754 +/- 0.008 and an absorbancy, E1(1%)cm,280 nm of 6.4 +/- 0.2. Sedimentation and viscosity data yield a solvation of 2.9 g H2O/g protein and an axial ratio of about 5 for the monomer. This would be consistent with a prolate ellipsoid of 10 nm length and 2 nm width. There is one strong binding site for Ca2+ for each organic phosphate ester in the molecule. The protein will precipitate at room temperature upon the addition of either 10 mM Ca2+ or greater than 1 M NaCl. Increasing the temperature from 4 to 37 degrees C causes an apparent conformational change and an increase in protein aggregation which is further increased by the addition of NaCl at this temperature until a limiting size is reached at about 0.25 M NaCl. This limiting size polymer contains 95-105 monomers and is nearly spherical with a radius of about 15 nm and a solvation of 3 g H2O/g protein. If this polymer were the submicelle of human casein, it could account for the abnormally high solvation of human casein micelles but their small average size would be more difficult to reconcile without additional information concerning K-casein association. The addition of Ca2+ to the system introduces association patterns which are more complex and not easily assessed.

Amino Acids↗
PubMed1976

Model calculations of casein micelle size distributions.

A previously proposed model for the formation and structure of casein micelles from subunits of variable composition is used to calculate theoretical micelle size distributions. Using the fractional content of k-casein as the only variable but with a value near that observed in a sample of milk serum, the model successfully reproduces experimentally determined distributions. Predicted size distributions are quite sensitive to the value of the variable and shift toward smaller average size as the assumed fractional content k-casein gets larger. Also, there is a discontinuity in the distributions which predicts that there will be essentially no micelles with radii smaller than 25-30 nm. These predictions are all in accord with experimental observations. The good agreement between theory and experimenet supports the micelle structure suggested by the model.

Binding Sites↗
PubMed1976

Review: Casein micelle structure; an examination of models.

The casein micelle system of bovine milk is unique in that protein aggregates of similar spherical shape but extreme variability of size are formed by the self-assembly of three major nonidentical subunits. The monomeric subunits appear to be approximately the same size and shape with similar amphiphilic natures, the chief difference in properties being in the carbohydrate-containing kappa-casein which acts to stabilize the system against precipitation by calcium ion. Micelle models with kappa-casein exclusively in the interior lack a stabilization mechanism and can be eliminated. Statistical considerations of a chain polymer model also lead to its rejection. Electron microscopy reveals spherical submicellar aggregates which at present can be accounted for by only three models. Of these three, the experimental data are predicted only by one in which, alphas 1-, beta-, and kappa-casein subunits are associated into spherical soap micelle-like particles with the kappa-casein segregated into one portion, giving these submicelles an amphiphilic nature. The alphas 1- and beta-caseins are hydrophobic while the kappa-casein portion of the submicelle surface is hydrophilic. Of particular interest is the ability of this micelle model to explain the formation of a minimum micelle which is larger than a submicellar particle.

Amino Acid Sequence↗
PubMed1975

Cation bindint to alpha-sl-casein B. A comparison of electrostatic models.

System characteristics which determine calcium binding to and subsequent proton release from alpha-sl-casein B are reported at pH 6.6 and [Na-plus] equal to 0.04, 0.0, and 0.16M. Values of protein solvation, G, site bound calcium, Ca,S, and net monomer charge, Z, permitted distributed charge models to be constructed. The models examined proved inadequate in that it was impossible to keep the dielectric constant, D, within acceptable limits and/or predict measured proton release. Three discrete charge models were constructed. At D equals 4, all three gave good agreement between predicted and experimental data as Ca, S increased. The known amino acid sequence was used to make rodlet models for the whole molecule andfor just the phosphate-containing acidic peptide portion. A comparison of these shows the electrostatic dominance of the acidic peptide and suggests that the electrostatic environment for the remainder of the binding sites is essentially constant as Ca, S increases during addition of calcium ion. The third discrete charge model bends the acidic peptide rodlet into a torus. In this case, data were matched with less assumed bond strain under conditions of high molecular charge than with the other two models. This indicates that conformation and association may be important factors to consider when constructing discrete charge models to calculate electrostatic free energy.

Amino Acid Sequence↗