Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.Abbreviations -PP _s1-casein 5P (f59–79) - -PP -casein 4P (f1–25) - ACP amorphous calcium phosphate - Cit citrate - CPN calcium phosphate nanocluster - CPP commercial phosphopeptide - IAP ion activity product - MWCO molecular weight cut-off - PP phosphopeptide - SAXS small-angle X-ray scattering - SCPP secretory calcium-binding phosphoprotein - UF ultrafiltrate     

The Ca2+-sensitive K+-conductance of the human red cell membrane is strongly dependent on cellular pH

The conductance of the Ca2+-sensitive K+-channels in human red cell membranes has been determined as a function of the intracellular pH. A sudden increase in the intracellular concentration of ionized calcium was established by addition of ionophore A23187 to a suspension of cells in buffer-free, Ca2+-containing salt solution. At the various cellular pH-values cellular concentrations of ionized Ca, saturating with respect to activation of the Ca2+-sensitive K+-conductance, were obtained by the use of varied concentrations of extracellular Ca2+ and added ionophore A23187. Changes in membrane potential was monitored as CCCP-mediated changes in extracellular pH. Initial net effluxes of K+, cellular K+ contents and the K+ Nernst equilibrium potentials were calculated from flame photometric measurements. Cellular Ca-contents were determined by aid of 45Ca. With cellular Ca2+ at the saturating level with respect to activation of the K+-channel the K+-conductance calculated from these data was independent of extracellular pH and a steep function of cellular pH with a half maximal conductance of 31 microSeconds/cm2 at a cellular pH of 6.1. The K+-conductance is not a simple function of cellular pH (pHc). From pHc = 6.5 and down to pHc = 6.0 a Hill-coefficient of 2.5 was found, indicating cooperativity between at least two sites regulating the conductance. Below pHc = 6.0 an extremely high Hill-coefficient of 11 was found, probably indicating that the additional titration of the channel protein leads to an increased cooperativity. The importance, as a physiological regulatory mechanism, of a K+-conductance increasing from zero to maximal conductance within less than one unit of pH, is discussed.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

Messenger-specific role for nicotinic acid adenine dinucleotide phosphate in neuronal differentiation

Cells possess several Ca2+-mobilizing messengers, which couple stimulation at the cell surface by a multitude of extracellular cues to the regulation of intracellular Ca2+-sensitive targets. Recent studies suggest that agonists differentially select from this molecular palette to generate their characteristic Ca2+ signals but it is still unclear whether different messengers mediate different functions or whether they act in a redundant fashion. In this study, we compared the effects of nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing messenger, with that of the prototypical messenger inositol trisphosphate on cytosolic Ca2+ levels and differentiation status of PC12 cells. We demonstrate that liposomal delivery of NAADP mediated release of Ca2+ from acidic Ca2+ stores and that this stimulus was sufficient to drive differentiation of the cells to a neuronal-like phenotype. In sharp contrast, cell fate was unaffected by more transient Ca2+ signals generated by inositol trisphosphate-evoked release of endoplasmic reticulum Ca2+ stores. Our data establish for the first time (i) the presence of novel NAADP-sensitive Ca2+ stores in PC12 cells, (ii) a role for NAADP in differentiation, and (iii) that Ca2+-dependent function can be messenger-specific. Thus, differential recruitment of intracellular Ca2+-mobilizing messengers and their target Ca2+ stores may represent a robust means of maintaining stimulus fidelity in the control of Ca2+-dependent cell function.     

A class of parametrically excited calcium oscillation detectors.

Intracellular Ca2+ oscillations are often a response to external signals such as hormones. Changes in the external signal can alter the frequency, amplitude, or form of the oscillations suggesting that information is encoded in the pattern of Ca2+ oscillations. How might a cell decode this signal? We show that an excitable system whose kinetic parameters are modulated by the Ca2+ concentration can function as a Ca2+ oscillation detector. Such systems have the following properties: (1) They are more sensitive to an oscillatory than to a steady Ca2+ signal. (2) Their response is largely independent of the signal amplitude. (3) They can extract information from a noisy signal. (4) Unlike other frequency sensitive detectors, they have a flat frequency response. These properties make a Ca(2+)-sensitive excitable system nearly ideal for detecting and decoding Ca2+ oscillations. We suggest that Ca2+ oscillations, in concert with these detectors, can act as cellular timekeepers to coordinate related biochemical reactions and enhance their overall efficiency.     

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.     

Regenerative release of calcium from functionally discrete subcellular stores by inositol trisphosphate.

Fluorescence imaging was used to determine the spatial and temporal patterns of subcellular calcium (Ca2+) liberation induced in Xenopus oocytes by photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. Increasing levels of InsP3 evoked Ca2+ release that began in a graded manner but, at varying threshold levels of InsP3, localized sites then showed transient and asynchronous 'puffs' of Ca2+ release. With higher levels of InsP3, Ca2+ from adjacent sites formed a focus for initiation of a propagating Ca2+ wave. The results show that InsP3-sensitive Ca2+ stores are arranged as distinct and functionally independent units, and that Ca2+ is released in both graded and regenerative fashions.     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.     

Cytoplasmic calcium oscillations: a two pool model

Cytosolic calcium oscillations induced by a wide range of agonists, particularly those which stimulate phosphoinositide metabolism, are the result of a periodic release of stored calcium. The formation of inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) seems to play an important role because it can initiate this periodic behaviour when injected or perfused into a variety of cells. A two pool model has been developed to explain how Ins(1,4, 5)P3 sets up these calcium oscillations. It is proposed that Ins(1,4,5)P3 acts through its specific receptor to create a constant influx of primer calcium (Ca2+p) made up of calcium released from the Ins(1,4,5)P3-sensitive pool (ISCS) together with an influx of external calcium. This Ca2+p fails to significantly elevate cytosolic calcium because it is rapidly sequestered by the Ins(1,4,5)P3-insensitive (IICS) stores of calcium distributed throughout the cytosol. Once the latter have filled, they are triggered to release their stored calcium through a process of calcium-induced calcium release to give a typical calcium spike (Ca2+s). In many cells, each Ca2+s begins at a discrete initiation site from which it then spreads through the cell as a wave. The two pool model can account for such waves if it is assumed that calcium released from one IICS diffused across to excite its neighbours thereby setting up a self-propagating wave based on calcium-induced calcium release.     

Casein kinase activity in rat mammary gland Golgi vesicles. Demonstration of latency and requirement for a transmembrane ATP carrier.

A Golgi vesicle-enriched preparation from mammary tissue of lactating rats has been used to investigate the phosphorylation of caseins in vitro. Casein kinase, together with its casein substrates, is enclosed within the lumen of Golgi membrane vesicles and has a requirement for Ca2+ and ATP. The permeability characteristics of the Golgi membrane to ATP and Ca2+ therefore have a possible regulatory influence on casein kinase activity. This influence has been investigated by alteration of the permeability characteristics by using several agents having differing degrees of selectivity. The ionophore A23187, which permits loss of Ca2+ from the vesicles, caused a decrease in casein phosphorylation which could be reversed by externally supplied Ca2+. Alamethicin, an ionophore that creates larger transmembrane channels, caused an increase in casein phosphorylation. This increase showed a requirement for divalent metal ions which could be satisfied by either Ca2+ or Mn2+. Under the same conditions, La3+ was inhibitory. Triton X-100 caused loss of intravesicular Ca2+, yet this was accompanied by an increase in phosphate incorporation into the caseins. We conclude from these results that the binding site on casein kinase for ATP is within the Golgi membrane barrier and that they imply the presence of a transmembrane ATP-transport mechanism. Inhibition of casein phosphorylation by atractyloside and carboxyatractyloside lends support to this concept.     

Induction of Na+ channel voltage sensitivity in Xenopus oocytes depends on Ca2+ mobilization

An unusual inward current which is slowly elicited in the Xenopus oocyte membrane during sustained depolarization is reportedly carried by Na+. It is thought that Na+ selective channels are in some way induced to become voltage-sensitive by the depolarization. Earlier studies report that the induction process involves a phospholipase C and a protein kinase C as well as calcium ions. The present work investigated the origins of this calcium in the oocyte. We show that injection of the powerful Ca2+ chelator (BAPTA) in the oocyte, before induction of the Na+ channels, prevented the appearance of the Na+ current, confirming an important role for [Ca2+]i. However, in oocytes perfused with Ca2+ -free medium, induction of the channels could still be obtained, indicating that induction did not depend upon the entry of external Ca2+. Downmodulation of Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores with caffeine and with a low molecular weight heparin resulted in decreased or no Na+ currents. The results are discussed in terms of the contributions from other endogenous calcium-dependent conductances which can influence the Na+ current amplitudes and time courses. The results presented support the idea that intracellular Ca2+ increase principally due to Ca2+ released from InsP3-sensitive stores is needed by the enzyme systems to produce the depolarization-induced activation of the Na+ conductance in the Xenopus oocyte.     

Interaction of alkaline metal ions with Ca(2+)-binding sites of Ca(2+)-ATPase of sarcoplasmic reticulum: 23Na-NMR studies.

The analysis of the 23Na-NMR signal shape variations in the presence of vesicles of light sarcoplasmic reticulum (SR) shows the existence of sodium sites on the membranes with Kd values of about 10 mM. Other monovalent cations displace Na+ from SR fragments in a competitive manner according to the row K+ greater than Rb+ greater than Cs+ greater than Li+. Calcium ions also reduce Na+ binding, the Na+ desorption curve being of a two-stage nature, which, as suggested, indicates the existence of two types of Ca(2+)-sensitive Na+ binding sites (I and II). Sites of type I and II are modified by Ca2+ in submicromolar and millimolar concentrations, respectively. Analysis of sodium (calcium) desorption produced by calcium (sodium) allowed us to postulate the competition of these two cations for sites I and identity of these sites to high-affinity Ca(2+)-binding ones on the Ca(2+)-ATPase. Sites I weakly interact with Mg2+ (KappMg approximately 30 mM). Reciprocal effects of sodium and calcium on binding of each other to sites II cannot be described by a simple competition model, which indicates nonhomogeneity of these sites. A portion of sites I (approximately 70%) interacts with Mg2+ (KappMg = 3-4 mM). The pKa value of sites II is nearly 6.0. The number of sites II is three times greater than that of sites I. In addition, sites with intermediate affinity for Ca2+ were found with Kd values of 2-5 microM. These sites were revealed due to the reducing of the sites II affinity for Na+ upon Ca2+ binding to SR membranes. It can thus be concluded that in nonenergized SR there are binding sites for monovalent cations of at least three types: (1) sites I (which also bind Ca2+ at low concentrations), (2) magnesium-sensitive sites II and (3) magnesium-insensitive sites II.     

Ca2+ entry in T cells is activated by emptying the inositol 1,4,5-triphosphate sensitive Ca2+ pool

Using alpha-linolenic acid (ALA), one of several polyunsaturated fatty acids (PUFAs) that have previously been shown to both mobilize intracellular Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool independently of IP3 production and inhibit Ca2+ influx, the relationship between Ca2+ mobilization from intracellular stores and Ca2+ influx in T cells (JURKAT) was studied. JURKAT cells were treated with 30 microM ALA to deplete the IP3-sensitive Ca2+ pool. When the intracellular free Ca2+ concentration [( Ca2+]i) returned to basal level, fatty acid free bovine serum albumin (BSA) was added to remove extracellular and membrane bound ALA. This resulted in a sustained increase in [Ca2+]i in the absence of inositol phosphates' formation. This sustained increase in [Ca2+]i was insensitive to protein kinase C activation but was inhibited by Ni2+ ions. The extent of Ca2+ influx was found to be correlated to the amount of Ca2+ initially discharged from the IP3-sensitive Ca2+ pool by sub-optimal concentrations of ALA. Ligation of the CD3 complex of the T cell antigen receptor with an anti-CD3 antibody (OKT3) during the sustained [Ca2+]i increased (induced by a sub-optimal concentration of ALA), produced a greater response. No increase in the sustained response was observed when the CD3 complex was activated in cells pretreated with an optimal concentration of ALA. In summary, Ca2+ entry in T cells is activated by emptying of the IP3-sensitive Ca2+ pool which can be dissociated from inositol phosphate production. The rate of Ca2+ influx appears to be closely correlated to the initial discharge of Ca2+ from the IP3-sensitive Ca2+ pool, suggesting that Ca2+ may first enter the depleted pool and then is released into the cytosol.     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk

An equilibrium thermodynamic model of the interaction of calcium, phosphate and casein in milk is described in which the micellar calcium phosphate is assumed to be in the form of calcium phosphate nanoclusters. A generalized empirical formula for the nanocluster is used to define the molar ratios of small ions (Ca, Mg, P_i and citrate) to a casein phosphorylated sequence (phosphate centre, PC). From this model, a method of calculating the partition of milk salts into diffusible and non-diffusible fractions is obtained. No arbitrary assumptions are made, no fitting of adjustable parameters is done and the PCs in the caseins are defined by inspection of their primary structures. In addition to the salt partition, the mole fractions of the individual caseins not complexed to the calcium phosphate through one or more of their PCs are computed and a generic stability rule for milks is derived. The use of the model is illustrated by calculations of the partition of salts in a standard milk and by comparison with experimental data on the partition of salts in the milk of individual cows. The generic stability rule is applied to the individual milks to determine whether the micellar calcium phosphate is thermodynamically stable. According to the calculations, compositions that might lead to pathological calcification in the lumen of the mammary gland were seldom found in primiparous healthy cows in early or mid lactation but occurred more often in multiparous animals, in late lactation and during mastitic infection.Abbreviations ACP amorphous calcium phosphate - Cit citrate - CN casein - CPN calcium phosphate nanocluster - DCPD dicalcium phosphate dihydrate - HA hydroxyapatite - IAP ion activity product - MCP micellar calcium phosphate - MWCO molecular weight cut-off - OCP octacalcium phosphate - PC phosphate centre - TCC tricalcium citrate     

Modulation of intestinal absorption of calcium]

1. Absorption of ingested calcium (2 ml of a 10mM CaCl2 solution + 45Ca) by the adult rat was shown to be facilitated by the simultaneous ingestion of an active carbohydrate, L-arabinose. As the carbohydrate concentration is increased from 10 to 200 mM, the adsorption of calcium is maximized at a level corresponding to about twice the control adsorption level. 2. A similar doubling of calcium adsorption is obtained when a 100 mM concentration of any one of a number of other carbohydrates (gluconic acid, mannose, glucosamine, sorbitol, lactose, raffinose, stachyose) is ingested simultaneously with a 10 mM CaCl2 solution. 3. Conversely, the simultaneous ingestion of increasing doses (10 to 100 mM) of phosphate (NaH2PO4) with a 10 mM CaCl2 solution results in decreased 45Ca absorption and retention by the adult rat. 4. The maximum inhibition of calcium adsorption by phosphate is independent of the concentration of the ingested calcium solution (from 5 to 50 mM CaCl2). 5. The simultaneous ingestion of CaCl2 (10 mM) with lactose and sodium phosphate (50 and 10 mM, respectively) shows that the activating effect of lactose upon 45Ca adsorption may be partly dissimulated by the presence of phosphate. 6. These various observations indicate that, within a large concentration range (2 to 50 mM CaCl2), calcium adsorption appears to be a precisely modulated diffusion process. Calcium absorption varies (between minimum and maximum levels) as a function of the state of saturation by the activators (carbohydrates) and inhibitors (phosphate) of the calcium transport system.     

Fusion of erythrocyte ghosts induced by calcium phosphate. Kinetic characteristics and the role of Ca2+, phosphate and calcium-phosphate complexes

Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.     

Casein kinase activity in rat mammary gland Golgi vesicles. Phosphorylation of endogenous caseins

A Golgi vesicle preparation isolated from the mammary tissue of rats in mid-lactation has been shown to contain the caseins of rat milk. These proteins were phosphorylated when the Golgi vesicles were incubated in the presence of [gamma-32P]ATP. Although this phosphorylation occurred when the physical integrity of the vesicles was maintained, it was markedly increased when the membrane structure was disrupted by hypoosmotic conditions or by use of detergents. The kinase responsible has been shown to be responsive to the intravesicular concentration of Ca2+ and to the extravesicular concentration of Mg2+. These results have been interpreted in terms of a model suggesting a transmembrane location for the enzyme with binding sites on the cytosolic membrane face for Mg2+ and possibly also for ATP and on the luminal surface for Ca2+ and the caseins. Others have postulated that the assembly of caseins into micelles occurs in Golgi vesicles and requires both prior phosphorylation of the proteins and the presence of Ca2+. In this investigation we demonstrate that treatments which increase the intravesicular casein phosphorylation also alter the Ca2+ balance within the vesicle lumen. These results are discussed in relation to the ATP-dependent accumulation of Ca2+ by the mammary gland Golgi vesicles.     

Three kinetically different inorganic phosphate entities in bovine casein micelles revealed by isotopic exchange method and compartmental analysis

Much controversy exists concerning the way calcium phosphate is linked to milk phosphoproteins including caseins. Homoionic exchange of inorganic phosphate between micellar calcium phosphates (MCP) of casein micelles and solute phosphates in cows' milk was investigated using H(32)PO(4)(2-) as radiotracer. Compartmental analysis and modelling revealed the presence of three MCP-related inorganic phosphate compartments each representing a separate phosphate entity. The relative phosphate quantities per compartment, i.e. the quantities of kinetically identical phosphate ions per MCP-ion cluster, and their mean residence times are 2:1:1 and 818, 0.24 and 23 h, respectively. Hence each MCP-ion cluster comprises four inorganic phosphate ions divided over three intra-MCP binding sites each characterised by a mean residence time for homomolecular phosphate exchange at solution/MCP interface.