The kinetics of Ca(2+)-dependent switching in a calmodulin-IQ domain complex

We have performed a kinetic analysis of Ca2+-dependent switching in the complex between calmodulin (CaM) and the IQ domain from neuromodulin, and have developed detailed kinetic models for this process. Our results indicate that the affinity of the C-ter Ca2+-binding sites in bound CaM is reduced due to a approximately 10-fold decrease in the Ca2+ association rate, while the affinity of the N-ter Ca2+-binding sites is increased due to a approximately 3-fold decrease in the Ca2+ dissociation rate. Although the Ca2+-free and Ca2+-saturated forms of the CaM-IQ domain complex have identical affinities, CaM dissociates approximately 100 times faster in the presence of Ca2+. Furthermore, under these conditions CaM can be transferred to the CaM-binding domain from CaM kinase II via a ternary complex. These properties are consistent with the hypothesis that CaM bound to neuromodulin comprises a localized store that can be efficiently delivered to neuronal proteins in its Ca2+-bound form in response to a Ca2+ signal.     

Inhibition of membrane erythrocyte (Ca2+ + Mg2+)-ATPase by hemin

Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme.     

Functional sensitivity of the native skeletal Ca(2+)-release channel to divalent cations and the Mg-ATP complex.

Sarcoplasmic reticulum (SR) vesicles, prepared from rabbit skeletal muscle, were characterized by functional and binding assays and incorporated into planar lipid bilayers. Single-channel activity was recorded in an asymmetric calcium buffer system and studied under voltage clamp conditions. Under these experimental conditions, a large conductance (100 pS in 50 mM Ca2+ trans) divalent cation selective channel displaying high ruthenium red and low Ca2+ sensitivity was identified. This pathway has been previously described as the Ca(2+)-release channel of the SR of skeletal muscle. We now report that in the presence of a Mg-ATP complex, the Ca2+ sensitivity of the open probability of this channel is increased. Furthermore, we show that micromolar cis Sr2+ concentrations also activated the Ca(2+)-release channel. The open probability of the Sr(2+)-activated channel was increased in the presence of a 2 mM Mg-ATP complex and adenine nucleotides on the cytoplasmic face of the Ca(2+)-release channel. These results were confirmed by isotopic flux measurements using passively 45Ca(2+)-loaded vesicles. In the latter case, the presence of extravesicular AMP-PCP (the nonhydrolysable ATP analog) enhanced the percentage of 45Ca2+ release induced either by Ca2+ or Sr2+ activation. In conclusion our findings emphasize the fact that the divalent cation activation of the Ca(2+)-release channel may be induced by Ca2+ and Sr2+, but not by Ba2+, in the presence of adenine nucleotides. Furthermore, they support the view that in situ Ca2+ and Mg-ATP complexes are involved in modulating the gating mechanism of this specific pathway.     

Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin

Many biological processes are now known to be regulated by Ca2+ via calmodulin (CM). Although a general mechanistic model by which Ca2+ and calmodulin modulate many of these activities has been proposed, an accurate quantitative model is not available. A detailed analysis of skeletal muscle myosin light chain kinase activation was undertaken in order to determine the stoichiometries and equilibrium constants of Ca2+, calmodulin, and enzyme catalytic subunit in the activation process. The analysis indicates that activation is a sequential, fully reversible process requiring both Ca2+ and calmodulin. The first step of the activation process appears to require binding of Ca2+ to all four divalent metal binding sites on calmodulin for form the complex, Ca42+-calmodulin. This complex then interacts with the inactive catalytic subunit of the enzyme to form the active holoenzyme complex, Ca42+-calmodulin-enzyme. Formation of the holoenzyme follows simply hyperbolic kinetics, indicating 1:1 stoichiometry of Ca42+-calmodulin to catalytic subunit. The rate equation derived from the mechanistic model was used to determine the values of KCa2+ and KCM, the intrinsic activation constants for each step of the activation process. KCa2+ and KCM were found to have values of 10 microM and 0.86 nM, respectively, at 10 mM Mg2+. The rate equation using these equilibrium constants accurately predicts the extent of enzyme activation over a wide range of Ca2+ and calmodulin concentrations. The kinetic model and analytical techniques employed herein may be generally applicable to other enzymes with similar regulatory schemes.     

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).     

Changes in Soybean Fruit Ca2+ (Sr2+) and K+ (Rb+) Transport Ability during Development

Mineral uptake by soybean (Glycine max [L.] Merrill) seeds during development can significantly affect seed quality and value. Little is known about seed mineral transport mechanisms and control processes, although it is clear that each mineral displays a characteristic accumulation pattern. Ion-specific accumulation patterns could result from changes in source availability, in transport kinetics through the seed pod and seed coat, or in the mineral uptake capability of the embryo. Ca2+ and K+ have negligible and high phloem mobilities, respectively. Ca2+ accumulation lags behind dry matter (C and N) and K+ accumulation in soybean embryos. To eliminate source availability influences, the Ca2+ and K+ uptake ability of isolated embryos and of seeds in pod culture was examined during seed development. Sr2+ and Rb+ were used as transport analogs of Ca2+ and K+, respectively. Sr2+ and Rb+ uptake rates by isolated embryos increased with seed fresh weight, indicating that the embryo was not limiting Ca2+ accumulation. However, the pod-cultured embryo Sr2+ and Rb+ uptake rate trends differed: Rb+ uptake increased with seed fresh weight, whereas Sr2+ uptake rates remained constant or decreased slightly. Ovule Sr2+ influx data suggest that the pod and seed coat impose a transport barrier that could account for the relative decline in embryo Ca2+ content during development.     

Ca(2+)-responsive extensible monolayer membrane of calmodulin-albumin conjugate.

A Ca(2+)-responsive monolayer protein membrane was prepared by developing calmodulin and bovine serum albumin at the air-water interface and by conjugating them with a bifunctional agent. In the case of the BSA monolayer, complex formation with Mg2+ generated a larger change in surface pressure than that with Ca2+. On the other hand, a drastic change in surface pressure was observed for the conjugated thin membrane associated with Ca2+ than that associated with Mg2+. Due to a drastic change in the conformation of calmodulin, the conjugated protein film changes its morphology (STM image), depending on Ca2+ concentration: the extended structure in the presence of Ca2+ transforms to a shrinked structure in the absence of Ca2+. The largest surface pressure change was detected when calmodulin was mixed with an equimolar amount of bovine serum albumin.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

Linker-gating ring complex as passive spring and Ca(2+)-dependent machine for a voltage- and Ca(2+)-activated potassium channel

Ion channels are proteins that control the flux of ions across cell membranes by opening and closing (gating) their pores. It has been proposed that channels gated by internal agonists have an intracellular gating ring that extracts free energy from agonist binding to open the gates using linkers that directly connect the gating ring to the gates. Here we find for a voltage- and Ca(2+)-activated K+ (BK) channel that shortening the linkers increases channel activity and lengthening the linkers decreases channel activity, both in the presence and absence of intracellular Ca2+. These observations are consistent with a mechanical model in which the linker-gating ring complex forms a passive spring that applies force to the gates in the absence of Ca2+ to modulate the voltage-dependent gating. Adding Ca2+ then changes the force to further activate the channel. Both the passive and Ca(2+)-induced forces contribute to the gating of the channel.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

Ehrlich tumour cells: Ca(2+)-uptake modification by aluminium lactate.

Aluminium lactate provokes the same modification of 45Ca(2+)-uptake by Ehrlich ascites tumour cells as does ferric lactate. The increase of uptake is metal-complex concentration-dependent, and in contrast to ferric lactate, is only partially inhibited by albumin. Under the same experimental conditions, chromic lactate provoked no modification of that uptake. A plasma membrane rigidity increase provoked by coordination binding of aluminium to phospholipids is likely to be the main cause of Ca(2+)-uptake modification by aluminium lactate.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Caffeine activates a Ca(2+)-permeable, nonselective cation channel in smooth muscle cells

The effects of caffeine on cytoplasmic [Ca2+] ([Ca2+]i) and plasma membrane currents were studied in single gastric smooth muscle cells dissociated from the toad, Bufo marinus. Experiments were carried out using Fura-2 for measuring [Ca2+]i and tight-seal voltage-clamp techniques for recording membrane currents. When the membrane potential was held at -80 mV, in 15% of the cells studied caffeine increased [Ca2+]i without having any effect on membrane currents. In these cells ryanodine completely abolished any caffeine induced increase in [Ca2+]i. In the other cells caffeine caused both an increase in [Ca2+]i and activation of an 80-pS nonselective cation channel. In this group of cells ryanodine only partially blocked the increase in [Ca2+]i induced by caffeine; moreover, the change in [Ca2+]i that did occur was tightly coupled to the time course and magnitude of the cation current through these channels. In the presence of ryanodine, blockade of the 80-pS channel by GdCl3 or decreasing the driving force for Ca2+ influx through the plasma membrane by holding the membrane potential at +60 mV almost completely blocked the increase in [Ca2+]i induced by caffeine. Thus, the channel activated by caffeine appears to be permeable to Ca2+. Caffeine activated the cation channel even when [Ca2+]i was clamped to below 10 nM when the patch pipette contained 10 mM BAPTA suggesting that caffeine directly activates the channel and that it is not being activated by the increase in Ca2+ that occurs when caffeine is applied to the cell. Corroborating this suggestion were additional results showing that when the membrane was depolarized to activate voltage-gated Ca2+ channels or when Ca2+ was released from carbachol- sensitive internal Ca2+ stores, the 80-pS channel was not activated. Moreover, caffeine was able to activate the channel in the presence of ryanodine at both positive and negative potentials, both conditions preventing release of Ca2+ from stores and the former preventing its influx. In summary, in gastric smooth muscle cells caffeine transiently releases Ca2+ from a ryanodine-sensitive internal store and also increases Ca2+ influx through the plasma membrane by activating an 80- pS cation channel by a mechanism which does not seem to involve an elevation of [Ca2+]i.     

Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca(2+)-ATPase.

The mechanism of inhibition of the sarcoplamc reticulum (SR) Ca(2+)-ATPase by the fluoroaluminate complexes was investigated. First, AlF4- was shown to bind to the Ca(2+)-free conformation of the enzyme by a slow quasi-irreversible process. The rate constants of the reaction are k+ = 16 x 10(3) M-1 s-1 and k- < 1.5 10(-3) s-1. We directly measured a stoichiometry of about 4.8 nmol of AlF4- bound/mg of protein. Mg2+ was a necessary cofactor for the reaction with a dissociation constant of 3 mM. It was demonstrated (Dupont, Y., and Pougeois, R. (1983) FEBS Lett. 156, 93-98) that phosphorylation by P(i) induced a dehydration of the catalytic site. The same process has been shown here to occur upon AlF4- binding either by the use of Me2SO or by demonstration of an increase of bound 2',3'-O-(2,4,6-trinitrocyclohexadienyldene)adenosine triphosphate fluorescence. Phosphorylation by P(i) is inhibited by the binding of AlF4-. Second, a fluoroaluminate complex, presumably AlF4-, was also shown to bind to the Ca(2+)-bound conformation of the Ca(2+)-ATPase in the presence of ADP and stabilize a E1.Ca2.ADP.AlFx complex. The dissociation constant of the nucleotidic site for ADP was shifted to the micromolar range. The Ca2+ ions bound on the external high affinity sites became occluded upon binding of (ADP + AlFx). We propose that AlF4- mimics P(i) binding to the Ca(2+)-free conformation of the ATPase and stabilizes an intermediate similar to the acyl-phosphate derivative; it also acts as an analogue of the gamma-phosphate of ATP and stabilizes an E1.[Ca2].ADP.AlF4 complex where the Ca2+ ions are occluded.     

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

The effect of lipid intermediates on Ca2+ and Na+ permeability and (Na+ + K+)-ATPase of cardiac sarcolemma. A possible role in myocardial ischemia

The effect of fatty acid and acylcarnitine on Ca2+ and Na+ transporting enzymes and carriers was studied in sealed cardiac sarcolemma vesicles of mixed polarity. Palmitoylcarnitine markedly reduced the Na+ gradient-induced Ca2+ uptake. Half-maximal reduction was obtained at 15 microM of the carnitine derivative. In a same concentration range palmitoylcarnitine caused a rapid release of accumulated Ca2+ when added to Ca2+-filled vesicles, which suggests that palmitoylcarnitine increases the permeability of the sarcolemma vesicles to Ca2+. A rapid release of Ca2+ was also observed if Ca2+ was taken up by action of the Ca2+ pump. The (Ca2+ + Mg2+)-ATPase, which most likely drives this active Ca2+ uptake, was 90% increased by 50 microM palmitoylcarnitine and evidence was presented that the acylcarnitine effect again was linked to an alteration of Ca2+ permeability of the vesicles. At the same concentration acylcarnitine was not able to unmask the latent protein kinase, so that probably the sarcolemma ATP permeability was not affected. Palmitoylcarnitine at 25 microM did not affect the ouabain-sensitive (Na+ + K+) -ATPase in native sarcolemma vesicles, however, it inhibited markedly if the enzyme was measured in SDS-treated vesicles. The effect of increased free fatty acid concentration on some of the sarcolemma transporting properties was tested by adding oleate-albumin complexes with different molar ratios to the sarcolemma vesicles. In contrast to molar ratios 1 and 5, the ratio of 7 was able to induce a rapid Ca2+ release and to inhibit (Na+ + K+)-ATPase in either native or SDS-treated vesicles markedly. 22Na release from 22Na-preloaded sarcolemma vesicles was shown to be stimulated by either palmitoylcarnitine (50 microM) or oleate-albumin complex (with a molar ratio of 7). The possible significance of the observed effects of lipid intermediates on ion permeability and (Na+ + K+)-ATPase activity in isolated sarcolemma vesicles for the derangement of cardiac cell function in ischemia is discussed.     

Modulation of the matrix redox signaling by mitochondrial Ca~(2+)

Mitochondria sense,shape and integrate signals,and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance,the molecular nature of the proteins involvedin mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes energy metabolism through the activation of matrix dehydrogenases and downstream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)+ ratio,but at the same time will increase reactive oxygen species(ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state,which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redoxsensitive sensors,real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca2+ combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca2+ and redox signals and their impact on cell function. In this review,we describe mitochondrial Ca2+ handling,focusing on a number of newly identified proteins involved in mitochondrial Ca2+ uptake and release. We further discuss our recent findings,revealing how mitochondrial Ca2+ influences the matrix redox state. As a result,mitochondrial Ca2+ is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease.     

Ca~(2+)通过膜脂影响肌质网Ca~(2+)-ATP酶的活性与Ca~(2+)转运功能

重建在大豆磷脂脂质体上的兔骨骼肌肌质网Ca~(2+)—ATP酶在ATP驱动下可将溶液中的Ca~(2+)转运到脂酶体内部;外加EGTA则可除去脂酶体外部的Ca~(2+),由此可得到四种含Ca~(2+)状态不同的脂酶体:(1)内、外都无Ca~(2+);(2)仅外部有Ca~(2+);(3)内、外都有Ca~(2+);(4),仅内部有Ca~(2+).用DPH和AS系列萤光探针对这四种含Ca~+状态不同的脂酶体的膜脂流动性进行了测定,结果表明:脂酶体外部加入Ca~(2+),脂双层外表面的流动性降低.当Ca~(2+)进入脂酶体内部后,内表面膜脂的流动性也降低,而且外层膜脂流动性进一步降低.脂酶体内、外的Ca~(2+)含量不同时,Ca~(2+)—ATP酶功能状态也不同.转运到脂酶体内部的ca~(2+)积累到一定浓度后,通过Ca~(2+)泵向内转运的Ca~(2+)及Ca~(2+)—ATP酶活力都受到了抑制.转运进行到第四分钟时的酶活只有第一分钟的9%.但在相同的实验条件下,失去了完整的膜结构的纯化的Ca~(2+)—ATP酶蛋白没有被抑制.这提示完整的膜结构是这种抑制作用所必需的,而且膜两侧Ca~(2+)浓度的梯差可通过影响膜脂来调节Ca~(2+)—ATP酶的功能.