Calcium Binding Capacity Research Articles

Struktur-Eigenschaftsbeziehung in Na-Disilikaten/ Structure — property relationship of Sodium Disilicates

Both crystalline δ-sodium disilicate (SKS-6) and amorphous spray-dried sodium disilicate can be used as the builder substance in detergents and cleansing agents. Differences in the use properties investigated here can be attributed to structural differences in the silicates. On the basis of the unequal solubility properties and the different reactivity towards alkyl-ammonium cations, it can be concluded that softening of water by the two modification takes place in different ways. While the amorphous disilicate removes the water hardening ions via a voluminous alkaline earth metal silicate precipitate, the polyvalent cations are mainly bonded by the crystalline substance via an ion exchange process. The considerably higher calcium-binding capacity of SKS-6 in soft and medium-hard water can be explained by this characteristic at the same time. Both substances can provide the wash liquor with large quantities of alkalinity. The crystalline sodium disilicate has a significantly higher buffer capacity than the amorphous product because of the higher content of active substance. Furthermore, the high structural water content in the amorphous silicate means that during storage the solid forms lumps or deliquesces after only a comparatively low uptake of water.

Distribution and Chemical Characteristics of Cations in Annual Rings of Japanese Cedar

AbstractThe objectives of this study were to investigate the distribution and chemical characteristics of cations in annual rings of Japanese cedar (Cryptomeria japonica D. Don) grown in a healthy stand of an unindustrialized region in Japan and discuss the possibility of using cations in tree‐rings as a chronological index of acidic deposition. Radial distributions of some cations and P at different vertical positions of the stem were analyzed for five trees from the same mountain. Each cation and P in all trees showed a specific distribution in its radial pattern but similar distribution trends were observed at all vertical positions. The cations and P were classified into three groups: (I) constant radial concentrations (Ca2+, Sr2+, Na+, and probably Ba2+), (II) high concentrations in the heartwood and low in the sapwood (Mg2+, K+, Rb+, Cs+), and (III) increasing concentrations in the sapwood (P, Mn2+, Cu2+). The total concentration of cations exceeded the calcium‐binding capacity (CBC) of the wood, and the excess was attributed to K+ in a salt form. The CBC increased from the sapwood/heartwood boundary toward the pith in the heartwood but remained constant in the sapwood. The increase of CBC in the heartwood was consistent with the profile of Mg2+, indicating a transfer of Mg2+ into heartwood and fixation on the acquired binding site. The radial distribution of 90Sr was closely related to the cumulative deposition of fallout from the nuclear weapon tests, but that of 137Cs was unrelated. This suggests immobility of Sr2+ and mobility of Cs+ in the horizontal direction of wood. The conservation of the historical 90Sr/Sr2+ change indicated that no influence of heartwood formation on the chemical environment of Sr2+ in cedar xylem and a possibility of using Sr2+ as a chronological index of nutrient availability. A steady‐state in nutrient availability is speculated for trees growing in this mountain because all of the trees analyzed show constant radial distributions of Sr2+ for decades.

Calbindin28kDa and calmodulin are hyperabundant in rat dental enamel cells. Identification of the protein phosphatase calcineurin as a principal calmodulin target and of a secretion-related role for calbindin28kDa.

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.

Calbindin28kDa and Calmodulin are Hyperabundant in Rat Dental Enamel Cells. Identification of the Protein Phosphatase Calcineurin as a Principal Calmodulin Target and of a Secretion-Related Role for Calbindin28kDa

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin2gkDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33 % of total (SDS-soluble) calbindin2SkDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskele-tal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDil and calcineurin demonstrated here provides the first molecular evidence that enamel cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.

Calcium-binding properties of SSP-5, the Streptococcus gordonii M5 receptor for salivary agglutinin

Streptococcus gordonii M5 expresses a lectin on its surface (SSP-5) which binds to human salivary agglutinin (SAG). This interaction requires sialic acid residues of SAG and divalent cations and may mediate the colonization of oral tissues by this organism. In this report, we show that the binding of SAG to SSP-5 requires calcium and that SSP-5 is a high-affinity calcium-binding protein. SAG-mediated aggregation of S. gordonii M5 was inhibited by 1 mM EDTA, and the restoration of aggregation occurred only upon the readdition of calcium. To ascertain the level at which calcium exerts its effects, the calcium-binding properties of SSP-5 were evaluated by using a 45Ca binding assay. In addition, a kinetic analysis of calcium binding was carried out by using fura2, a fluorescent calcium-binding dye. These analyses showed that SSP-5 is a high-affinity calcium-binding protein that binds 1 mol of calcium per mol of protein and has a dissociation constant of 0.45 +/- 0.2 microM. The calcium-binding capacity of SSP-5 was also calculated independently to be 1.0 +/- 0.2 mol of Ca per mol of SSP-5 by column chromatography on Sephadex G-25 equilibrated with 10 microM 45Ca. To localize the calcium binding site of SSP-5, a series of C-terminal deletion mutants were expressed in Escherichia coli and evaluated for calcium-binding activity. Deletion of the 250 C-terminal residues of SSP-5 had little effect on calcium binding. However, deletion of residues 1168 to 1250 resulted in the loss of calcium-binding activity, suggesting that this region is important for calcium binding by SSP-5.

A Protein Involved in Calcium Sensing of the Human Parathyroid and Placental Cytotrophoblast Cells Belongs to the LDL-Receptor Protein Superfamily

Monoclonal anti-parathyroid antibodies have been utilized to isolate a single-chain glycoprotein of 500 kDa, which apparently acts as a sensor of the extracellular calcium concentration and is expressed on the surface of human parathyroid, placental, and kidney tubule cells. The present contribution reports the isolation of a cDNA clone encoding this protein in human placenta and subsequent Northern blots confirming the mRNA expression also in human parathyroid and kidney cells. Close similarity in sequence as well as in tissue distribution is demonstrated with the rat Heymann nephritis antigen, a kidney tubule glycoprotein with calcium-binding ability. The 500-kDa protein belongs to the LDL-receptor superfamily of glycoproteins, claimed to function primarily as protein receptors and characterized by functionally important calcium-binding capacity. It is proposed that the currently identified protein constitutes part of a common structure for the sensing of extracellular calcium concentrations and influences calcium homeostasis in different organs.

Interference in ionized calcium measurements by heparin salts

We determined the suitability of various heparin salts used for anticoagulation of whole-blood specimens for measurement of ionized calcium (iCa), blood gases, and electrolytes. We were particularly interested in a new heparin product containing both zinc and lithium cations (CNLZ heparin), in which the binding sites with greatest affinity for divalent cations are bound with zinc and low-affinity sites with lithium. In initial experiments Li heparin decreased iCa concentrations 0.07 mmol/L at the lowest heparin concentration (3000 units/L) and progressively lowered them at higher concentrations. Zn heparin initially increased iCa concentrations 0.06 mmol/L but progressively lowered them as the heparin concentration was increased. Li heparin interfered even when present in amounts (9 units per 3-mL syringe) minimally effective in preventing coagulation. Use of CNLZ heparin (36 units per 3-mL syringe; Zn 63-78 g/kg of heparin) largely eliminated interference of heparin in iCa measurements. In studies that included the effects of concentration of heparin through partial filling of syringes, specimens anticoagulated with CNLZ heparin compared well with unheparinized controls in measurements of iCa, blood gases, and electrolytes. Blood gases and iCa results on CNLZ-heparinized specimens from intensive-care-unit patients also compared well with specimens anticoagulated with a preparation of heparin (EB heparin) in which calcium has been added to balance the calcium-binding capacity. However, the presence of calcium in EB heparin significantly increased measured total calcium concentrations, whereas the new CNLZ heparin did not interfere in total calcium determinations.

Binding capacity of 18 fiber sources for calcium

Eighteen fiber sources were analyzed for protein, phytic acid (PA), soluble (SF) and insoluble (IF) fiber, total dietary fiber, water-holding capacity (WHC), and endogenous calcium concentration. Calciumbinding capacity (CaBC) was determined in acid-washed fiber sources. Protein content varied from 0.0% in cane fiber and cellulose to 22.2% in tomato fiber. Acid-wash treatment significantly (P<0.05) reduced the protein content. PA in acid-washed samples ranged from 0 μg/g of sample in cane fiber, cellulose, corn bran, and orange fiber to 15 312 rgjg of sample in rice bran. SF ranged from 1.9% in corn bran to 28.3% in orange fiber. IF varied from 34.9% in orange fiber to 87.0% in cellulose

Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.

1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane perforation for more than one week. 8. An equation for the effective diffusion constant for total cytoplasmic calcium, D'Ca, was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to fura-2 and some highly mobile buffers.(ABSTRACT TRUNCATED AT 400 WORDS)

New method for discriminating between calcium stone formers and healthy individuals.

A new method for discriminating between the urine of potential calcium stone formers and healthy persons has been proposed, based on determination of the calcium binding capacity of urine (CBC) by titration of early morning urine with a calcium chloride solution. For this purpose a new PVC matrix calcium ion selectrode for measuring calcium ion concentration in whole urine was used. The selectrode has a disposable membrane which can easily be prepared and replaced in the laboratory. Plots of the calcium ion concentration versus the concentration of total added calcium were linear up to a point where precipitation of calcium salts commenced. The slopes of these titration lines were used as criteria for discrimination. Statistical evaluation showed good separation between the urine of healthy and stone forming donors. A 2-sample t test with unequal variances gave mean values of 0.31 for healthy urine (13 samples) and 0.64 for stone forming urine (26 samples). Individual 99% confidence intervals were 0.21-0.40 for the controls and 0.54-0.73 for the patients respectively. Discriminant analysis showed that from a group of treated patients with low Dl (13 samples), 3 were classified as stone formers and 10 were non-formers. Thus there was good correlation with the clinical situation and with the previously proposed Dl test.

Characteristics of Sodium Salts of Agaricic Acid as Detergent

Mono-, di-and trisodium salts of agaricic acid (2-hydroxy-1, 2, 3-nonadecanetricarboxylic acid), a polycarboxylic-type biosurfactant, were prepared by neutralization with sodium methoxide. The properties of salts as detergents such as surface activity, calcium binding capacity, and soil removal ability were examined and compared with those of commercially available synthetic detergents (SDS, LAS) and soaps.The monosodium salt of agaricic acid had the most ability for lowering surface tension. Di-and trisodium salts of agaricic acid exhibited the greatest calcium binding capacity.The soil removal ability of sodium salts of agaricic acid in distilled water was correlated with surface tension. The monosodium salt of agaricic acid was best capable of soil removal. In hard water, the removal ability of trisodium salt of agaricic acid which had the highest calcium binding capacity was retained at the same level as in distilled water although that of SDS, LAS, and soaps in particular decreased remarkably.

A quantitative study of calcium binding and aggregation in selected oral bacteria.

By means of micro-equilibrium dialysis, calcium binding capacities and affinities were measured in three different oral bacteria, and the effects of extracellular polysaccharide, pH, and aggregation were investigated. Binding capacities of 31.0 +/- 2.1 (C. matruchotii), 34.7 +/- 3.7 (S. sanguis), and 41.5 +/- 5.4 (S. downei) mumol calcium/g wet weight of cells were found at pH 7.0, falling to 21.4 +/- 0.8 mumol calcium/g wet wt. cells at pH 5.0 for S. downei. Dissociation constants were found to vary between 0.78 +/- 0.24 and 1.77 +/- 0.30 mmol/L (at pH 7.0, depending on species), and between 0.62 +/- 0.04 and 1.77 +/- 0.30 (in the pH range 5.0 to 7.0, for S. downei only). Examination suggested that at pH 7.0 calcium-facilitated bacterial association occurs in the streptococci with calcium uptake curves analogous with those of positively cooperative systems. Desorption of calcium from aggregated S. downei suggested that the mechanism of desorption differed from that of uptake. This may be an important factor in plaque formation and in the binding of cells to the surface of formed plaque. Plaque calcium forms a reservoir, readily released by a pH drop, which may increase plaque fluid saturation and reduce demineralization.

Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone.

Calcium binding properties of bone acidic glycoprotein-75, osteopontin, and bone sialoprotein were determined in 10 mM imidazole buffer (pH 6.8), containing either 60 mM KCl or 150 mM NaCl. Proteins assayed were first bound to nitrocellulose to mimic substrate-bound forms in vivo; retention of phosphoproteins was determined through use of radioiodinated tracers. Binding studies were carried out both as a function of calcium concentration and the amount of phosphoprotein. In the presence of 60 mM KCl, bone acidic glycoprotein-75 exhibited the largest calcium binding capacity (139 atoms/molecule at saturation), with bone sialoprotein intermediary (83 atoms/molecule) and osteopontin lowest (50 atoms/molecule). Sites detected for each phosphoprotein exhibited overall binding constants in the 0.5-1.0 mM extracellular range. In 150 mM NaCl and 1-2 mM total calcium, phosphoproteins bound between 72 and 19 mol of calcium/mol with the same relative order. Binding was proportional to amount of phosphoprotein in either salt condition. The presence of 5 mM calcium had a different effect on concentration-dependent binding to type I collagen for each phosphoprotein. Bone acidic glycoprotein-75 alone was found to undergo an unusual calcium-enhanced polymerization reaction, confirmed by light scattering measurements, wherein collagen binding was greatest with polymeric forms. These findings demonstrate that acidic phosphoproteins from bone bind calcium atoms with a range of capacities. Calcium appears to induce conformational changes in bone acidic glycoprotein-75 which influences its self-association and binding to different substrata.

Effects of clonidine on the calcium content and morphology of rat salivary glands

These effects were examined with and without pretreatment of animals with reserpine and the adrenergic antagonists prazosin ( α 1), yohimbine ( α 2) and propranolol (β). The effects of clonidine on glandular concentrations of norepinephrine and dopamine also were examined. These effects were compared with those of xylazine, a presynaptic α 2-adrenergic agonist. A single, high dose of clonidine followed by an overnight fast caused marked increases in calcium content and acinar secretory granules in the submandibular and sublingual glands, similar to those caused by reserpine. However, the calcium content of the parotid gland was not altered by clonidine. although there seemed to be a modest increase in acinar secretory granules. The clonidine-induced increase in submandibular calcium content could not be attributed to any adrenergic receptor activity since it was not blocked by either α- or β-adrenergic antagonists. Unlike reserpine, clonidine did not affect catecholamine concentrations in the parotid and submandibular glands. Pretreatment with reserpine did not significantly alter the clonidine-induced increase in submandibular calcium content. It is likely that the greater accumulation of acinar secretory granules is related to the increased calcium stores of the glands in clonidine- and/or reserpine-treated rats. The large differences in calcium content among the three glands might be attributable, in part, to differences in the calcium-binding capacity of their secretory granules. Possible mechanisms for the clonidine effects on salivary-gland calcium include disturbances in membrane-associated pools or gating mechanisms for calcium, which need further study.

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris.

Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding "vesicles" from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO4, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO4 (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO4 for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).

Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.

Determination of the calcium binding capacity to discriminate between urines of calcium stone formers and healthy persons

Calcium binding by undiluted fasting urine has been tested as a means of demonstrating the capacity of urine from control subjects and calcium stone patients to hold spontaneous precipitation of stone forming compounds. Preliminary data are promising with respect to the possibility that controls and patients can be separated.

Protective effects of R 56 865 against ischemic damage in the isolated rabbit heart

We examined the role of calcium in the pathogcnesis of ischemie cardiac cell death in the isolated working rabbit heart subjected to normothermic global ischemia followed by rcperfusion. Apart from measurements of cardiodynamic function and ultrastructural examination, we also used a cytochemical procedure to localize exchangeable calcium pools at the ultrastructural level. The effects of verapamil (1.5 × 10 −8 M, 3 × 10 −8 M) (high affinity for L-type calcium channels) were compared with those of R 56 865 (4 × 10 −7 M) (Ca 2+ overload blocker with low affinity for the L-type calcium channels). A severe depression of cardiac function was observed after solvent or verapamil pretreatment and 25 min of ischemia followed by reperfusion. R 56 865 treatment resulted in a significantly improved postischemic recovery when compared to solvent and verapamil treatment groups. The ultrastructural and cytochemical results corroborated the hcmodynamic findings. In solvent and verapamil-treated hearts, irreversible damage was observed mainly in mid- and cndocardial areas. Ultrastructural changes were accompanied by shifts in calcium localization: i.e. loss of sarcolcmmal calcium binding capacity, accumulation of calcium precipitate in the mitochondria. In the R 56 865 treatment group, damage was limited to some cells scattered in the midcardial areas. In conclusion, R 56 865, which has little affinity for the slow channels was highly effective in protecting against ischemie damage, indicating that, in this experimental set-up, the calcium responsible for cellular Ca 2+ overload is not entering via L-typc calcium channels.

The effects of heating, alone or in the presence of calcium or lactose, on calcium binding to milk proteins

The binding of calcium by different milk proteins and the effects of heat were investigated. The influences of calcium and lactose were also investigated. It was shown that the caseins of cow's milk have a high calcium binding capacity, when treated with calcium at 25°C for 30 min, pH 7·00, due to the high number of calcium binding sites. The order of binding capacities is α s1 -casein > β-casein > κ-casein. For whey proteins the order is β-lactoglobulin (β-lg) > α-lactalbumin (α-la). Lactose, in general, when present at 25°C, did not appreciably affect the calcium binding of the major milk proteins. Heating of whole casein at 80 or 95°C for 30 min caused conformational changes of the protein resulting in an increase in its calcium binding ability. The presence of lactose during heating with or without calcium reduced its calcium binding. The calcium binding of α s1 -casein was not affected by heating, throughout the temperature range studied, either in the presence or in the absence of calcium, an indication that α s1 -casein may undergo physical changes at high temperatures, but the accessibility and/or the number of calcium binding centres are not increased. β-Casein, when heated with calcium, was subject to conformational changes which were reflected in changes in the degree of calcium-binding. Heating of β-lg in the presence of calcium did not much affect the amount of calcium bound to it. However, in the absence of calcium an increase of 21·5% in calcium binding was observed after heating at 80°C and a decrease of 40·0% after heating at 95°C, compared to unheated samples.

Cloning and sequencing of a cDNA encoding human milk β-casein

A cDNA of 1065 bp encoding the human milk β-casein was cloned and sequenced using a synthetic oligodeoxyribonucleotide probe and a human mammary gland library. The nucleotide (nt) sequence contained an open reading frame sufficient to encode the entire amino-acid (aa) sequence of a β-casein precursor protein consisting of 210 aa and a signal peptide of 15 aa. The nt sequence shows 45–62% homology to those of bovine, ovine, rat, and mouse β-caseins. The highly phosphorylated site, which is responsible for the calcium-binding capacity of β-casein, the signal peptide, and a sequence encoding for an inhibitor to the angiotensin-converting enzyme seem highly conserved among the β-caseins with known sequences.