c-Myc and Mxi1 immunoreactivities in the calcifying areas of the epiphyseal-plate cartilage matrix of growing rats.

We looked for the protooncogene protein, c-Myc, its dimerization partner, Max, and the repressors of its transactivation activity, Mad1 and Mxi1, in the epiphyseal-plate cartilage matrix of growing rats by immunocytochemistry in the electron microscope. c-Myc and Mxi1 immunoreactivities were found in the calcifying areas of the cartilage matrix only. There was no immunolabeling in response to anti-Max or anti-Mad1 antibodies. Mxi1 immunoreactivity was mainly in the early calcifying areas, in the calcification front and ahead of it, whereas c-Myc immunoreactivity was essentially in the incompletely calcified regions of the matrix. The two immunolabelings occurred mainly over the large type II collagen fibrils of the cartilage matrix and over the thin filaments connecting them. c-Myc and Mxi1 immunoreactivities were rarely found along the dark cristallites. There was no immunolabeling associated with the matrix vesicles, or in their immediate surroundings. The data suggest that the protooncogene proteins, c-Myc and Mxi1, could be implicated in the calcification involving type II collagen fibrils of the epiphyseal-plate cartilage. The absence of Max immunoreactivity from the calcifying cartilage matrix raises the question of whether there are other c-Myc- and Mxi1-dimerization partners.

Structural characteristics of supramolecular assemblies formed by guanidinium-cholesterol reagents for gene transfection.

We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection in vitro and in vivo. In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 A and 68 A in BGTC/DOPE-DNA and BGTC-DNA aggregates, respectively. In addition, DNA lipoplexes with similar lamellar patterns were detected inside transfected HeLa cells by conventional transmission electron microscopy. These results suggest that DNA condensation by multivalent guanidinium-cholesterol cationic lipids involves the formation of highly ordered multilamellar domains, the DNA molecules being intercalated between the lipid bilayers. These results also invite further investigation of the intracellular fate of the internalized lipid/DNA structures during their trafficking toward the cell nucleus. The identification of the basic features of active complexes should indeed help in the design of improved guanidinium-based vectors.

Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats.

The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation.

Expression and subcellular localization of the Myc superfamily proteins: c-Myc, Max, Mad1 and Mxi1 in the epiphyseal plate cartilage chondrocytes of growing rats.

The changes in the expressions of the protooncogene protein c-Myc, its dimerization partner Max and the competitive inhibitors Mad1 and Mxi1 during the terminal differentiation of chondrocytes in vivo were investigated by immunocytochemistry. The four immunoreactivity patterns in the epiphyseal plate cartilage of growing rats, as they appeared under the light microscope, showed differences in protein expression level and intracellular distribution, with the chondrocyte developmental stage. c-Myc immunoreactivity was intense and mainly in the nuclei of proliferative chondrocytes. It decreased in the nuclei of mature chondrocytes and appeared in the cytoplasm. c-Myc immunoreactivity increased in the fully-differentiated hypertrophic chondrocytes. Immunoreactivity of the c-Myc dimerization partner Max was mainly in the nucleus of proliferative chondrocytes and decreased as the chondrocytes matured. Mad1 immunoreactivity was also concentrated in the nucleus of proliferative chondrocytes, but was mainly in the cytoplasm of mature chondrocytes and almost lost from the hypertrophic chondrocytes. Lastly, there was Mxi1 immunoreactivity in the nucleus and cytoplasm of proliferative, mature and early hypertrophic chondrocytes and the cytoplasm staining was more sustained than in the nucleus. There was little labeling in late hypertrophic chondrocytes. The electron microscope pictures corroborated these findings and showed the subcellular distributions of the immunolabelings. The gold particles reflecting Mad1 frequently formed patches and those for Mxi1 appeared to accumulate within the mitochondria of all chondrocytes. The variations in immuno-patterns and intracellular distributions suggest that each protooncogene protein has specific roles in the functional changes in the chondrocytes at each step of their terminal differentiation.

Ultrastructural localization of alpha-parvalbumin in the epiphyseal plate cartilage and bone of growing rats.

The distribution of the calcium-binding protein, alpha-parvalbumin, in the epiphyseal plate cartilage and bone of growing rats was examined by electron microscope immunocytochemistry of undecalcified samples. Parvalbumin immunoreactivity, as revealed by gold particles, increased with maturation of chondrocytes and was maximal in the zone of calcification. It was found in the cytoplasm of chondrocytes, osteoblasts and osteocytes, corroborating light microscope observations. The immunolabeling was associated with amorphous electron-dense material in the cytoplasm and not bound to membranes. There was moderate parvalbumin immunolabeling over the dense chromatin in the nuclei of chondrocytes and bone cells, but none in the cell processes of mature and hypertrophic chondrocytes, in the matrix vesicles themselves, or in the cell processes of osteoblasts. However, there was parvalbumin immunoreactivity in the cell processes of the osteocytes of compact cortical bone. The uncalcified and calcified matrix of the epiphyseal cartilage, the osteoid, and the fully mineralized cortical bone were devoid of parvalbumin immunoreactivity. Thus, immunoreactive parvalbumin is confined to the cell bodies of chondrocytes and osteoblasts, and is unlikely to be directly involved in mineral deposition. The maximal parvalbumin immunoreactivity in the last terminal chondrocytes of the zone of calcification suggests that the protein is involved in buffering intracellular Ca2+, preventing the stimulation of degenerative processes by high intracellular calcium. The parvalbumin immunoreactivity in the cell processes of osteocytes of compact cortical bone seems to indicate that this calcium-binding protein may be involved in the regulation of Ca2+ fluxes and hence in calcium homeostasis in bone.

Localization of the Ca(2+)-binding alpha-parvalbumin and its mRNA in epiphyseal plate cartilage and bone of growing rats.

This study describes the localization of alpha-parvalbumin, in undecalcified tibial epiphyseal cartilage and bone of growing rats by immunocytochemistry in the light microscope, and of parvalbumin mRNA by in situ hybridization. They were compared to the distribution of the calbindin-D9K and its mRNA in rat epiphyseal cartilage. All the chondrocytes of the epiphyseal cartilage were parvalbumin-immunopositive, but there was no parvalbumin immunoreactivity in the uncalcified or calcified extracellular cartilage matrix. The intensity of the immunostaining increased from the resting and proliferative to the mature and hypertrophic chondrocytes, with the greatest intensity in the terminal hypertrophic chondrocytes in the calcifying zone. The parvalbumin immunostaining was located in the cytoplasm, but no immunoreactivity was detected in any chondrocyte processes. The parvalbumin mRNA distribution and levels, as revealed by in situ hybridization, exactly mirrored those of the parvalbumin protein. In contrast to parvalbumin, calbindin-D9K and its mRNA appeared in mature chondrocytes and decreased in hypertrophic up to calcifying chondrocytes. Calbindin-D9K was located in the cytoplasm and all along the cell processes. In bone, the osteoblasts and the osteocytes of trabecular and compact cortical bones were immunoreactive for parvalbumin and contained parvalbumin mRNA. Parvalbumin lay in their cytoplasm, but there was no parvalbumin immunostaining in the extracellular uncalcified or mineralized bone matrix. The long processes of osteocytes, in compact bone only, were parvalbumin immunoreactive. Osteoclasts contained cytoplasmic parvalbumin immunoreactivity. Thus, the pattern of immunoreactive parvalbumin distribution indicates that the protein is not involved in the extracellular mineralization of cartilage and bone matrix. It appears to be associated with specific calcium-related intracellular functions in chondrocytes and in osteoblasts, osteocytes, and osteoclasts. As the highest cytoplasmic concentration of parvalbumin is in the terminal hypertrophic chondrocytes, parvalbumin could act as a calcium buffer to delay the death of chondrocytes. In compact bone, parvalbumin could also have a role throughout the osteocyte processes in regulating the fluxes of calcium ions for mineral homeostatis.

Calbindin-D28K and -D9K and 1,25(OH)2 vitamin D3 receptor immunolocalization and mineralization induction in long-term primary cultures of rat epiphyseal chondrocytes.

Rat epiphyseal plat chondrocytes were grown on glass slides, as nonadhering monolayer cultures for up to 6 weeks. Chondrocyte growth, differentiation and maturation, matrix formation and mineralization, and the temporospatial distribution of the vitamin D-dependent calcium-binding proteins, calbindin-D9K and -D28K, and the 1,25(OH)2D3 receptor (VDR), were all monitored. Chondrocytes became confluent in 2.5 weeks, differentiated to acquire a chondrocyte (polygonal) morphology, produced extracellular matrix, and finally formed a true monolayer mineralizing cartilaginous tissue, with all the stages of chondrocyte development within a single culture. beta-Glycerophosphate promoted initial matrix mineralization in 4 weeks and accelerated cell differentiation. High nominal calcium and ascorbic acid were needed for abundant matrix formation. VDR occurred at all differentiation stages, in the nuclei and nucleoli and in the cytoplasm. Calbindin-D28K and -D9K were not coexpressed. Calbindin-D28K was found in prechondroblasts, chondroblasts, and in newly differentiated chondrocytes. It was cytoplasmic in prechondroblasts and subsequently also in the perinuclear region and in nuclei, suggesting migration to the nuclear chromatin. Calbindin-D28K was nuclear only in newly differentiated chondrocytes in vitro and was not found in mature chondrocytes. In contrast, calbindin-D9K was present in the cytoplasm of mature and hypertrophic chondrocytes only. It was first in the cell body and eventually migrated within and to the far end of long cell processes with a decreasing cytoplasmic concentration showed by decreased immunostaining intensity, and ultimately hypertrophy of chondrocytes in culture. These in vitro patterns of calbindins-D and VDR accurately reflect their in vivo distributions. The genomic action of vitamin D, in vitro, resulted in the synthesis of nuclear VDR and calbindins-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Zonal variations of types II, IX and XI collagen mRNAs in rat epiphyseal cartilage chondrocytes: quantitative evaluation of in situ hybridization by image analysis of radioautography.

The spatial-temporal distribution of the mRNAs for type IX and type XI collagens were compared to that of type II collagen mRNA in the tibial epiphyseal plate cartilage of normal growing rats. The mRNAs were detected by in situ hybridization with radio-labelled specific probes and visualized by radioautography. The areas covered by the resulting silver grains were quantified by computer assisted image analysis. The areas in chondrocytes of each zone of the epiphyseal plate cartilage, which correspond to the stages of chondrocyte development and function were determined. Types II, IX and XI mRNAs were present to some extent in chondrocytes of all zones. The distributions of type II and type IX collagen mRNAs were similar with the highest concentrations in the proliferative zone, and the lowest in the resting and calcifying zones chondrocytes. In contrast, type XI collagen mRNA had a different distribution, with the lowest concentration in the resting zone chondrocytes and a significant decrease in the calcifying zone chondrocytes. These patterns correlates with the changes in chondrocyte function, and may reflect the roles of the type IX and type XI collagens. The data show that computer assisted image analysis of in situ hybridization radioautographic images is a precise, rapid tool for analysing differences in gene expression.

Relationship between vitamin D status and deposition of bound calcium in skeletal muscle of the rat.

The effects of vitamin D on the intramuscular distribution of total and bound calcium, phosphate and on available cytosolic calcium, were investigated in skeletal muscle. Total calcium and phosphorus were measured on ashed subcellular fractions of muscles from vitamin D-repleted and vitamin D-deprived rats. The variations in available calcium were followed by determining the activities of calcium-sensitive enzymes in isolated cytosol. Bound-calcium was revealed ultra-microscopically by pyroantimonate. In vitamin D-repleted muscles, the pyroantimonate method revealed specific areas of intense bound-calcium deposition: the myofibrils, where they formed pronounced lines parallel to the Z-bands. In vitamin D-deficient muscles, the calcium-pyroantimonate deposits appeared clearly reduced. This loss was accompanied by a marked reduction in total calcium and phosphorus in all the subcellular fractions, as compared to vitamin D-repleted muscles. Unexpectedly, the activity of the Ca(+)-activated isocitrate-dehydrogenase was increased in the cytosol, while that of the Ca2(+)-inhibited pyruvate-kinase decreased. Prolonged vitamin D-administration to vitamin D-repleted rats led to an intensification of calcium-pyroantimonate deposits and a general increase in total calcium and phosphorus, but no change in the cytosolic Ca2(+)-sensitive enzyme activities. Cessation of vitamin D-administration to vitamin D-repleted rats produced a regression of calcium-pyroantimonate deposits, a general decrease of total calcium and phosphate levels, and stimulation of the Ca2(+)-activated isocitrate-dehydrogenase accompanied by lowering of the Ca2(+)-inhibited pyruvate-kinase. The results clearly indicate a correlation between vitamin D-repletion and the total and bound calcium content of skeletal muscle. In addition, they demonstrate an apparent contradiction between the decrease of total and bound calcium, and the activities of cytosolic Ca2+ sensitive enzymes during vitamin D-deprivation, which can only be explained by an increase in available calcium. It is suggested that vitamin D stimulates intramuscular mechanisms tending to lower available calcium by inactivating the cation via the formation of calcium chelates.

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria.

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c1. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we notices a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone.

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.

Stimulation of oxygen consumption at the cytochrome A3 level inhibits aldosterone biosynthesis from 18-hydroxycorticosterone.

A mitochondrial preparation from duck adrenal gland was used, under aerobic conditions, to show that the oxygen requirement for the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) is at the cytochrome P-450 level only. Vitamin C and tetramethyl-p-phenylene-diamine (TMPD) were used to increase oxygen consumption at the cytochrome a3 level, thereby decreasing its availability to cytochrome P-450. The vitamin C plus TMPD system acts as an 'oxygen trap'. Results show that despite reducing equivalents provided by L-malate, vitamin C plus TMPD strongly inhibits aldosterone biosynthesis from 18-hydroxycorticosterone (89%). Moreover, we used KCN in order to block oxygen consumption, even in the presence of vitamin C plus TMPD. Under these conditions, the inhibition of aldosterone biosynthesis from 18-hydroxycorticosterone is reduced by 51%. The reversal of this inhibition by KCN was evident but only partial. According to polarographic and electron microscopy studies, the reversal of inhibition can only be explained by an increased availability of oxygen at the cytochrome P-450 level. Experiments performed under aerobic conditions, without a nitrogen atmosphere, show that oxygen is required in the transformation of 18-hydroxycorticosterone into aldosterone, at the cytochrome P-450 level. This suggests that a classical hydroxylating mechanism is involved.

Degenerative processes in skeletal muscle of Cd2+-treated rats and Cd2+ inhibition of mitochondrial Ca2+ transport.

In rat skeletal muscle, chronic exposure to 50 ppm Cd2+ in drinking water produced both ultrastructural and functional damage, which took place successively and increased gradually with duration of treatment. Ultrastructurally, the first effect was a regression of the sarcoplasmic reticulum, mitochondrial cristae, and glycogen granules. Then, the number of mitochondria diminished and a degeneration of myofilaments appeared. During the 9-month course of treatment, however, the terminal cisternae and the lateral saccules of sarcoplasmic reticulum remained, and the Z lines maintained their tight and rigorously parallel appearance. Functionally, the activities of two cytosolic Ca2+-sensitive enzymes (lactate dehydrogenase and malate dehydrogenase) decreased. Their partial restoration by EGTA and EDTA suggested the presence of inhibitory divalent cations in the cytosol of treated rats. Cd2+ also inhibited Ca2+ transport in mitochondria through the uncoupling of oxidative phosphorylation. Hence, an increase of Ca2+ concentration of the cytosol of Cd2+-treated rats, activating degradative enzymes such as phospholipases, proteases and phosphorylase b kinase, can be incriminated. A direct inhibitory action of Cd2+ on the activities of lactate dehydrogenase and malate dehydrogenase also occurred. Direct action of Cd2+ on certain other Ca2+-sensitive enzymes, thereby aggravating the indirect damage induced through increasing the Ca2+ concentration in cytosol, is hypothesized. In mitochondria, a Cd2+ activation of phospholipase A2 and/or a Ca2+-sensitive protease is a unique possibility, since Ca2+ accumulation was prevented by cytosolic Cd2+.

Mitochondria alterations in Cd2+-treated rats: general regression of inner membrane cristae and electron transport impairment.

Young adult rats absorbed 50 p.p.m. Cd2+ added to drinking water. After 6 weeks, 3, 6 and 9 months of treatment, the ultrastructural condition of liver, kidney and muscle was observed by electron microscopy. The choice of these tissues was determined by their differences in the capacity to accumulate Cd2+: the liver is able to concentrate a considerable amount of metal, but redistributes it throughout the entire organism, while the kidney collects it in view of its elimination. Muscle contains the least Cd2+. A general regression in mitochondria cristae accompanied by a vesiculation and a fragmentation of endoplasmic reticulum appeared simultaneously in the three tissues, at as early as 6 weeks of treatment, and extended progressively with its continuation supporting evidence of a general attack of the intracellular membrane systems. Cd2+ stimulation of membrane-degrading enzymes such as phospholipases and proteases was suggested. A concomitant diminution in glycogen stores was noted. Active synthesis of neutral lipids, especially cholesterol esters, took place in liver mitochondria of treated rats in collaboration with rough endoplasmic reticulum, and progressively generated a multiplication of electron-transparent inclusions in cytoplasm. Isolated mitochondria from liver, kidney and muscle of Cd2+-treated rats maintained partial energy coupling, but displayed a rapid early fall in cytochrome oxidase followed by a partial restoration after 6 months of treatment, and a progressively slackening of succinate dehydrogenase. Isolated vesicles of liver mitochondria inner membrane of treated rats behaved as intact mitochondria, indicating changes inside the membrane itself. Addition in vitro of the metal ion to mitochondria and also to inner membrane vesicles isolated from control rats revealed that Cd2+ was able to stop completely succinate dehydrogenase, but was totally ineffective on cytochrome oxidase. Membrane fixation of Cd2+ on the flavoprotein or SH associated with succinate dehydrogenase is proposed. Considering the close parallelism of the extensive depression of microsomal NADPH cytochrome c reductase and the rapid fall in mitochondrial cytochrome oxidase, it is suggested that an indirect inhibition process occurs, through Cd2+-induced diminution of a constituent common to all cytochromes in the cell.

Relation between energy metabolism in mitochondria and conversion of 18 hydroxycorticosterone to aldosterone in adrenals.

The purpose of this study was to see whether there was any link between conversion of 18 hydroxycorticosterone to aldosterone and mitochondrial energy metabolism. In vitro incubations of duck adrenal mitochondria with 18 OH B were used in this study. Results show that 18 oxidation is inhibited by compounds blocking electron transport (antimycin A, cyanide, rotenone, amytal). Inhibition induced by cyanide and antimycin A is reversed with ATP. 2,4 dinitrophenol, oligomycin and DCCD inhibit 18 oxidation but guanidine stimulate this reaction. Thus aldosterone synthesis from 18 OH B depends on energy metabolism in mitochondria. This is a very new aspect related to the last step of aldosterone synthesis.

[In vitro calcium binding by isolated brush border vesicles of rat jejunum and ileum]. / Fixation du calcium in vitro par des vésicules de bordures en brosse isolées de jéjunum et d'ileum de rat.

A new procedure for isolation of the Rat jejunum and ileum brush border vesicles is described. Several calcium transport conditions have been established. A previous incubation with ATP induces an increased calcium binding. The role of a magnesium ATPase is discussed.