Charles W. Slattery

A model for the formation and structure of casein micelles from subunits of variable composition

Sedimentation coefficients were determined for association products in systems containing SH-kappa-casein, SH-kappa-casein-alpha(S1) or beta-caseins (1:1, by weight) and SH-kappa-casein-alpha(S1)-casein-beta-casein (2:1:1, by weight) under a variety of environmental conditions. As the concentration of NaCl is increased or, as the concentration of CaCl2 is increased at 0.05 M NaCl, the system containing SH-K-casein alone acts quite differently from those which also contain the Ca2+ -sensitive caseins. It is concluded that electrostatic as well as hydrophobic attractions are important between SH-kappa-casein monomers. Viscosity and sedimentation measurements combined indicate that nearly spherical polymers containing 25-30 monomers each and with radii of approx. 10 nm are formed in all systems. It is considered that these polymers are micelle subunits in which the non-polar portion of each monomer is oriented radially inward while the charged acidic peptides of the Ca2+ -sensitive caseins and the hydrophilic, carbohydrate-containing portion of kappa-casein are near the surface. Asymmetric distribution of kappa-casein in a micelle subunit results in hydrophilic and hydrophobic areas on the subunit surface. In this situation, aggregation through hydrophobic interactions forms a porous micelle. Micelle growth is limited by the eventual concentration, at the micelle surface, of subunits rich in kappa-casein. This model successfully accounts for the properties of equilibrium and nonequilibrium micelle systems, for the destabilization of the micelle by dephosphorylation of Ca2+ -sensitive caseins or by high concentrations of NaCl or CaCl2 and for the events associated with clot by rennin action.

Interactions in human casein systems: Self-association of nonphosphorylated human β-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.

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.

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.

Improvements in human health through production of human milk proteins in transgenic food plants.

Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.

Interactions of triply phosphorylated human β-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.

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.

Interactions of triply phosphorylated human β-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.

Cation binding to αs1-casein B. A comparison of electrostatic models

Abstract The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I , as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [ D ]. Anticipated intrinsic binding constants are k H,P o = 3 × 10 6 , k Ca,P o = 120, k Na,P o = 1, k H,C o = 7 × 10 4 and k Ca,C o = 5.6 Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring k H,P o and k Ca,C o which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed. Discrete charge models accept anticipated k o and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.

Evaluation of PCR-ELISA for determination of telomerase activity in prostate needle biopsy and prostatic fluid specimens

The conventional TRAP assay will determine telomerase activity in tissue or other specimens. However, methodological disadvantages limit its clinical use. We evaluated a modified TRAP assay, the telomerase PCR-ELISA, as a practical clinical system for measuring its activity in conjunction with prostate cancer (PCa). We examined telomerase activity by both TRAP and PCR-ELISA assays in 48 sextant needle biopsy (SNB) specimens from dye-marked areas of the prostate glands of 7 PCa patients. Each specimen was histologically confirmed as cancerous or cancer-free by examining a paired specimen taken from the same marked area. In addition, prostatic fluid (PF) specimens were analyzed from 18 patients, 9 of whom were diagnosed with PCa while 9 were diagnosed as cancer-free but mostly with BPH. The results on individual SNB specimens matched well for the two methods. The sensitivity (91%) and specificity (69%) for the PCR-ELISA measurements were consistent with those for the conventional TRAP assay, 88% and 81%, respectively. Quantitatively, with the PCR-ELISA assay, the mean telomerase activity (24.5+/-28.4 units) per needle core with PCa cells was significantly higher than that in needle cores without PCa cells (7.2+/-2.2 unit), as it was with the conventional TRAP assay, namely 25.6+/-27.8 units and 7.3+/-1.8 units, respectively. In PF specimens from PCa patients, which had a lower mean telomerase than was found in needle cores containing PCa cells (7.1+/-1.5 units in the PCR-ELISA, 7.2+/-1.8 units in the conventional TRAP assay), statistical analysis showed good matching between the results from the two assays, overall. In conclusion, the PCR-ELISA can be considered a reliable method to determine telomerase activity as an adjunct in the diagnosis and treatment of prostate cancer.