The binding interactions for the three primary reactants of the fibroblast growth factor (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the cofactor heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<==>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<==>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<==>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed growth factor and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

The purpose of this study was to explore in rats the in vivo metabolism of phytate, using [U-14C]phytate and to assess the effects of calcium intake on phytate and myo-inositol metabolism. Labeled phytate was prepared from wheat following injection of the plants at the milk stage with [U-14C]myo-inositol. Groups of 30-day-old male rats were adapted for at least 6 weeks either to a low-calcium (2.9 mM/100 g dry diet) or a high-calcium (30.6 mM/100 g dry diet) cereal-based diet. They were then transferred to metabolic cages, continued on their respective diets and given an oral dose of [14C]phytate or [14C]myo-inositol. Appearance of radioactivity in expired air, feces and urine was monitored for 48 hours; the rats were then killed for determination of 14C-activity in liver, kidneys, blood and femur. The high-calcium diet significantly increased the output of radioactivity in feces (54% of dose) and reduced the appearance of radioactivity in expired air (25% dose) and in body tissues following 14C-phytate administration. The high-calcium intake did not affect the fate of [14C]myo-inositol (96% of dose). These results suggest that phytate or a derivative(s) is almost quantitatively absorbed (94% of dose) when calcium intake is low (Ca/P molar ratio; 0.21) and it is extensively oxidized to CO2 (60% of dose). Thus, a high-calcium intake (Ca/P molar ratio; 2.24) inhibits the utilization of [14C]phytate. Hence, it is suggested that the impact of dietary phytate on trace mineral bioavailability will depend upon the presence of factors, including excess calcium, that alter the absorption and utilization of phytate.

Various aspects of selenium metabolism and nutrition in relation to the question of selenium bioavailability in foods and the diet of man are reviewed. Few published studies exist on selenium metabolism in human subjects, particularly those representative of healthy individuals in the United States. Animal studies reveal that various factors, including the source and chemical form of selenium in foods and feeds, influence selenium bioavailability. However, the quantitative significance of animal assay data for human nutrition is not known. The limited number of published studies in man suggest that the metabolic fate and physiological function of dietary selenite may differ from that of selenomethionine or of food selenium. However, much additional research will be required to establish an adequate picture of the significance of dietary selenium bioavailability in human nutrition and health. Based on initial human experiments carried out at the Massachusetts Institute of Technology, use of stable isotopes of selenium offers promising opportunities for closing the gap of knowledge that now exists concerning the role and significance of factors that determine how the selenium present in foods is used to meet the physiological requirements of the consumer.

PHYTATE CONCENTRATION IN WHEATweight less than 30,000 (the cut-off point in the ultrafiltration experiments of Knuckles et al., 1975) since it is absent in the diafiltered reteníate. This same impurity is also noncoagulable from neutral or alkaline solutions since it is absent in the heat coagulated protein of Edwards et al. (1975) (see Table II).Reducing agents, such as sodium bisulfite and 2-mercaptoethanol, are frequently used when attempting to isolate soluble active enzymes and undenatured protein fractions (Anderson and Rowan, 1967; Wolf, 1972). In attempting to improve the solubility of the protein precipitated at room temperature, several reducing agents were added to the CBJ (before precipitation) at the following levels: sodium bisulfite, 0.156 M; sodium dithionite, 0.078 M; and mercaptoethanol, 0.40 M. The samples were precipitated and washed at pH 3.5 at room temperature, and the protein reslurried in pH 8.5 borate buffer (0.1 M) for 1 hr. There was no increase in solubility when using the reducing agents compared to the untreated control. In related work, Betschart (1974) reported that mercaptoethanol was ineffective in increasing the solubility of freeze-dried, acid-precipitated alfalfa protein.By acid precipitating the alfalfa protein at 2°, the native solubility is preserved. This protein concentrate, being free from the dark green color and most of the grassy flavor of the typical LPC, and still retaining its native solubility, should have many uses in the food industry. The protein can be further purified by membrane filtration if necessary.