A fast passive Ca2+ efflux mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into phospholipid bilayers. The permeability of lipid bilayers to Co2+ and glucose was increased slightly by incorporation of the ATPase, and the permeability of mixed bilayers of phosphatidylethanolamine and phosphatidylcholine increased with increasing content of phosphatidylethanolamine both in the presence and absence of the ATPase. The presence of the ATPase, however, resulted in a marked increase in permeability to Ca2+, the permeability decreasing with increasing phosphatidylethanolamine content. Permeability to Ca2+ was found to be dependent on pH and the external concentrations of Mg2+ and Ca2+, was stimulated by adenine nucleotides but was unaffected by inositol trisphosphate. A kinetic model is presented for Ca2+ efflux mediated by the ATPase. It is shown that the kinetic parameters that describe Ca2+ efflux from vesicles of sarcoplasmic reticulum also describe efflux from the vesicles reconstituted from the purified ATPase and phosphatidylcholine. It is shown that the effects of phosphatidylethanolamine on efflux can be simulated in terms of changes in the rates of the transitions linking conformations of the ATPase with inward- and outward-facing Ca2+-binding sites, and that effects of phosphatidylethanolamine on the ATPase activity of the ATPase can also be simulated in terms of effects on the corresponding conformational transitions. We conclude that the ATPase can act as a specific pathway for Ca2+ efflux from sarcoplasmic reticulum.     

The blocking effect of aliphatic hydrocarbons on Ca2+ leakage channels formed by aggregates of Ca2+-ATPase of the sarcoplasmic reticulum

The effects of aliphatic hydrocarbons within the liposomes on the Ca2+ transport function of isolated sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle, vesiculate preparation of Ca2+ dependent ATPase and proteoliposomes reconstituted from Ca2+-ATPase and egg phosphatidylcholine, were studied. It was shown that liposomes prepared from dipalmitoyl phosphatidylcholine containing aliphatic hydrocarbons increase 2 to 3 times Ca2+ accumulation by Ca2+-dependent ATPase from rabbit skeletal muscle SR. Ca2+ transport by SR vesicles increases in the presence of hydrocarbons by 15--20%. The activating effect of hydrocarbons on Ca2+ transport by proteoliposomes depends on the lipid/protein ratio. The proteoliposomes with a high lipid/protein ratio are practically insensitive to the effects of hydrocarbons. It was suggested that activation of Ca2+ transport by hydrocarbons is due to blocking of Ca2+ leakage channels formed during the aggregation of Ca2+-ATPase molecules. Treatment of membranes by formaldehyde results in the oligomerization of Ca2+-ATPase and decreases 2--4-fold the ATP-dependent accumulation of Ca2+. Subsequent addition of decane restores Ca2+ transport practically completely.     

Silver ions trigger Ca2+ release by interaction with the (Ca2+-Mg2+)-ATPase in reconstituted systems

It has been suggested that vesicles derived from the sarcoplasmic reticulum of skeletal muscle contain Ca2+ channels which can be opened by interaction with sulfhydryl reagents such as Ag+ or Hg2+. We show that, in reconstituted vesicles containing the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum as the only protein, the ATPase can act as a pathway for Ca2+ efflux and that Ag+ induces a rapid release of Ca2+ from such reconstituted vesicles. We also show that Ag+ has a marked inhibitory effect on the ATPase activity of the purified ATPase. We suggest that the (Ca2+-Mg2+)-ATPase can act as a pathway for rapid Ca2+ release from sarcoplasmic reticulum.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

Ca 2+ uptake in reconstituted sarcoplasmic reticulum vesicles

The reconstitution of functional sarcoplasmic reticulum vesicles capable of Ca²⁺ transport has been achieved. Sarcoplasmic reticulum vesicles are first solubilized with deoxycholate and then reassembled into membranous vesicles by removal of the detergent using dialysis. The Ca²⁺ pump protein can, by itself, be reconstituted to form membranous vesicles capable of energized Ca²⁺ binding and uptake. The lipid content of the reconstituted vesicles is about the same as that of the original sarcoplasmic reticulum vesicles. The reconstituted vesicles have an elevated ATPase activity. Ca²⁺ binding and uptake in the presence of ATP are restored to about 25% and 50%, respectively.     

Transient kinetics of a Ca2+-induced fluorescence change from membrane-associated and solubilized sarcoplasmic reticulum Ca2+-ATPase

The kinetics and extent of the fluorescence change induced by Ca2+ interaction with the Ca2+-ATPase from sarcoplasmic reticulum have been compared by stopped flow fluorimetry for three preparations: sarcoplasmic reticulum; purified ATPase in membrane vesicles; and solubilized, delipidated ATPase. The kinetics of Ca2+ release and binding for both purified preparations could be described by a single exponential as has been observed for sarcoplasmic reticulum. The rate and extent of the fluorescence change for the solubilized and membrane-associated preparations are shown to be quite similar to those of the sarcoplasmic reticulum. From these results, it is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca2+-ATPase. In addition, since the change in fluorescence is probably result of a conformational change in the ATPase during the Ca2+ pumping cycle, the results provide additional evidence that monomeric Ca2+-ATPase may be capable of Ca2+ transport since the delipidated preparation is monomeric under the conditions used for these experiments. Finally, it is concluded that phospholipid bilayer is not essential for this conformational change.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.     

Interactions of hexachlorocyclohexanes with the (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Hexachlorocyclohexanes have been shown to inhibit the (Ca2+ + Mg2+)-ATPase of muscle sarcoplasmic reticulum reconstituted into bilayers of dioleoylphosphatidylcholine. However, for the ATPase reconstituted into bilayers of dimyristoleoylphosphatidylcholine, a pattern of activation at low concentration followed by inhibition at higher concentration is seen for hexachlorocyclohexanes and alkanes such as decane and hexadecane. The ATPase in sarcoplasmic reticulum vesicles is also inhibited by the hexachlorocyclohexanes. The effects of hexachlorocyclohexanes on activity are largely independent of concentrations of Ca2+ and ATP. Inhibition is more marked at lower temperatures. The hexachlorocyclohexanes quench the tryptophan fluorescence of the ATPase, and the quenching can be used to obtain partition coefficients into the membrane system. As for simple lipid bilayers, partition exhibits a negative temperature coefficient. Binding is related to effects on ATPase activity.     

Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.     

Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum: potential role of the 53-kilodalton glycoprotein

An essential feature of the function of the Ca2+-ATPase of sarcoplasmic reticulum (SR) is the close coupling between the hydrolysis of ATP and the active transport of Ca2+. The purpose of this study is to investigate the role of other components of the SR membrane in regulating the coupling of Ca2+-ATPase in SR isolated from rabbit skeletal muscle, reconstituted SR, and purified Ca2+-ATPase/phospholipid complexes. Our results suggest that (1) it is possible to systematically alter the degree of coupling obtained in reconstituted SR preparations by varying the [KC1] present during cholate solubilization, (2) the variation in coupling is not due to differences in the permeability of the reconstituted SR vesicles to Ca2+, and (3) vesicles reconstituted with purified Ca2+-ATPase are extensively uncoupled under our experimental conditions regardless of the lipid/protein ratio or phospholipid composition. In reconstituted SR preparations prepared by varying the [KC1] present during cholate treatment, we find a direct correlation between the relative degree of coupling between ATP hydrolysis and Ca2+ transport and the level of the 53-kilodalton (53-kDa) glycoprotein of the SR membrane. These results suggest that the 53-kDa glycoprotein may be involved in regulating the coupling between ATP hydrolysis and Ca2+ transport in the SR.     

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice.     

ADP-ribosylation of Ca2+-dependent ATPase in vitro suppresses the enzyme activity

We investigated the effect on the Ca2+-dependent ATPase activity of ADP-ribosylation of the enzyme from the rabbit skeletal muscle sarcoplasmic reticulum. A reconstituted ADP-ribosylation system of Ca2+-dependent ATPase in which the enzyme and ADP-ribosyltransferase, both were partially purified from the vesicles, and poly L-lysine were contained, was preincubated with 1 mM NAD, and the Ca2+-dependent ATPase activity was assayed. The NAD-dependent suppression of the enzyme activity depended on both the concentration of NAD and preincubation-time for the ADP-ribosylation, and was reversed by adding 20 mM arginine during the preincubation. These results taken together with the findings that Ca2+-dependent ATPase is a major acceptor protein for the modification in rabbit skeletal muscle sarcoplasmic reticulum [Hara et al. (1987) Biochem. Biophys. Res. Commun. 144; 856-862] suggest that Ca2+-transport in the sarcoplasmic reticulum may be regulated through changes in the rate of ADP-ribosylation of Ca2+-dependent ATPase.     

Ca2+ binding,ATP-dependent Ca2+ transport,and total tissue Ca2+ in embryonic and adult avian dystrophic pectoralis

Summary Avian muscular dystrophy is an autosomal recessive genetic disease characterized by early hypertrophy and loss of function of the pectoralis major. The disease is progressive, ultimately resulting in atrophy and heavy lipid deposition.Previous investigators have noted a decrease in the ability of the dystrophic sarcoplasmic reticulum to concentrate Ca²⁺. More recently, other investigators have shown an abnormal calcium uptake in avian dystrophic sarcoplasmic reticulum. They indicated, using freeze-fracture techniques, that a 90 Å particle of the vesicle membrane exhibited a decreased population and suggested that they might be the ATPase involved in calcium transport.Our studies confirm the earlier observations of a decreased rate of Ca²⁺ uptake and Ca²⁺ binding capacity of dystrophic fragmented sarcoplasmic reticulum vesicles which are isolated from both embryonic and adult pectoralis. These observations correlate in turn with a 75% drop in the Ca: ATP transport efficiency of the dystrophic sarcoplasmic reticulum determined by measuring the rate of³²P_i liberation from -ATP³² during active calcium transport by the isolated sarcoplasmic reticulum SR.In addition, we have found a quantitative deficiency in a 65,000 dalton component of the dystrophic fragmented SR at the time of myoblast fusion by measuring³⁵S-Methionine incorporation into the SR, coupled to high resolution polyacrylamide gel electrophoresis and radioautography. Analysis of total tissue calcium by atomic absorption spectroscopy revealed a decrease in the total calcium content of dystrophic muscle.     

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading). Freeze-fracture analysis showed that the reconstituted vesicles had a remarkably narrow size distribution (diameter 794 +/- 77 A (S.D.], suitable for stereological analysis. Intramembranous particles were dispersed and occurred with a low frequency in the fractured shells, also before sucrose fractionation. It was calculated that the number of intramembranous particles corresponded to the number of Ca2(+)-ATPase polypeptide/vesicle. A ratio of unity between particles and polypeptide chains was also obtained from the density of particle distribution on flat surfaces of fused vesicles, prepared by sucrose fractionation. The size of the particles formed a broad distribution, having a peak value around 60-67 A, both in the reconstituted preparation and sarcoplasmic reticulum vesicles. No evidence for protein-protein interactions was found in chemical cross-linking experiments. It is concluded that the intramembranous particles in the reconstituted preparations are referable to monomeric Ca2(+)-ATPase which is capable of transporting Ca2+ inside the vesicles. The implications of the observations for the associational state of Ca2(+)-ATPase at high protein concentration are considered in relation to previous ultrastructural investigations of membranous Ca2(+)-ATPase in native and two-dimensional-crystalline forms.     

The use of a model water-lipid system for the study of the electric function of Ca2+, Mg2+-ATPase of the sarcoplasmic reticulum

The method of dynamic capacity in the model organic phase-water system was used to investigate a possibility of studying the electrical function of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum of the rabbit hind limb skeletal muscles. Decane and decane solution of azolectin were used as an organic phase. It is stated that in the model systems the sarcoplasmic reticulum Ca2+,Mg2+-ATPase did not cause ATP-dependent changes in the boundary Volta potential (delta phi) irrespective of the presence of polyvalent cation chelates in the organic phase. The fragmented sarcoplasmic reticulum is able of realizing Mg-ATP, Ca2+-dependent generation of delta phi only with phospholipids present in the organic phase. It is supposed that generation of delta phi of the fragmented sarcoplasmic reticulum is due to the active transport of calcium ions by the reticulum Ca2+,Mg2+-ATPase.     

Infrared spectroscopic characterization of the structural changes connected with the E1----E2 transition in the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-transporting ATPase (EC 3.6.1.38) of sarcoplasmic reticulum alternates between several conformational states during ATP-dependent Ca2+ transport. The E1 conformation is stabilized by 0.1 mM Ca2+ and the E2 conformation by vanadate in a Ca2+-free medium. Fourier transform infrared spectroscopy reveals significant differences between the two states that indicate differences in the protein secondary structure. The two states and the corresponding spectra can be interconverted reversibly by changing the Ca2+ concentration of the medium. The infrared spectral changes indicate the appearance of a new alpha-helical substructure connected with the E1----E2 conversion accompanied by small changes in beta-turns, while the beta-sheet content remains essentially unchanged. There are also differences between the E1 and E2 states in the C = O stretching vibrations of the ester carbonyl groups of phospholipids in intact sarcoplasmic reticulum that are not observed under identical conditions in isolated sarcoplasmic reticulum lipid dispersions. These observations imply an effect of proteins on the structure of the interfacial regions of the phospholipids that is dependent on the conformational state of the Ca2+-ATPase. The CH2- and CH3-stretching frequencies of the membrane lipids are not affected significantly by the E1----E2 transition. The Fourier transform infrared spectra of sarcoplasmic reticulum vesicles in the presence of 20 mM Ca2+ suggest the stabilization of a protein conformation similar to the E2 state except for differences in the behavior of COO- and phospholipid ester C = O groups that may reflect charge effects of the bound Ca2+.     

Rapid reconstitution and characterization of highly-efficient sarcoplasmic reticulum Ca pump

The Ca pump was reconstituted from the purified sarcoplasmic reticulum ATPase and excess soybean phospholipids by the freeze-thaw sonication procedure in the presence of cholate. In the absence of Ca precipitating agents, the reconstituted proteoliposomes accumulated Ca2+ at an initial rate of up to 0.7 mumol/mg per min at 25 degrees C, and a value of 1.54 was obtained for the coupling ratio between Ca uptake and Ca2+-dependent ATPase activities. The proteoliposomes were mainly unilamellar vesicles but were heterogeneous with respect to their size. When reconstituted at a lipid/protein ratio of 40, proteoliposomes had a buoyant density of about 1.04 and their average internal volume was 1.4-1.6 microliters/mg of phospholipids. More than 95% of the ATPase was incorporated randomly into these proteoliposomes and the fraction of proteoliposomes that represented about 50% of the total intravesicular isotope space contained right-side-out oriented enzyme. 86Rb efflux from the 86Rb-loaded proteoliposomes was found to be slow even at 25 degrees C. Therefore, the proteoliposomes prepared by the present simple method should be useful for the study of the side-specific interaction of ions such as alkali metal cations with the sarcoplasmic reticulum Ca pump.     

Cause of increase in the efficiency of Ca2+ transport by fragments of sarcoplasmic reticulum from fast skeletal muscles induced by protein kinase

The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.