The susceptibility of MAP-2 to proteolytic degradation increases when bound to tubulin

During experiments studying dietary effects on phosphorylation/dephosphorylation of MAP-2 we found that incubation of microtubules with alkaline phosphatase resulted in extensive proteolysis of MAP-2 but not of tubulin or Tau proteins. In the absence of tubulin, when microtubule-associated proteins (MAPs) were incubated with alkaline phosphatase, MAP-2 was not proteolyzed. This suggests that binding to tubulin induces a conformational change in MAP-2 which makes it more susceptible to proteolysis. The proteolysis of MAP-2 by alkaline phosphatase was prevented by inhibitors of serine proteases, suggesting that the commercial preparation of the enzyme is contaminated by a serine protease and/or that the enzyme also has a weaker proteolytic activity. In addition, selective proteolysis of MAP-2 can be obtained with the metalloprotease collagenase. Brain homogenates are shown to contain a Ca²⁺-dependent protease which selectively degrades MAP-2 bound to tubulin. These results suggest that selective proteolysis of tubulin-bound MAP-2 could play a role in the regulation of microtubule dynamics in response to extracellular signals.     

Studies on calmodulin isolated from Tetrahymena cilia and its localization within the cilium

Tetrahymena calmodulins from cilia, cell bodies and whole cells were isolated separately and compared. These calmodulins showed just the same properties: they co-migrated in SDS-polyacrylamide gel electrophoresis, had a Ca²⁺-dependent electrophoretic mobility change in alkali gel, held the same antigenic determinants in common, and activated brain cyclic nucleotide phosphodiesterase Ca²⁺-dependently with identical activation curves. Distributions of calmodulin and calmodulin-counterpart in Tetrahymena cilium were investigated by using alkali gel electrophoresis in the presence of Ca²⁺ or EGTA, and by immunoelectron microscopy. Calmodulin was detected in the membrane plus matrix fraction and outer-doublet microtubule fraction, and its Ca²⁺-dependent counterpart existed exclusively in the latter fraction. However, neither calmodulin nor its counterpart was detected in the crude dynein fraction. Immunoelectron microscopy revealed that calmodulin was localized along the longitudinal axis of outer-doublet microtubules at regular intervals of about 90 nm. The calmodulin-binding site in the ciliary axoneme was suggested to be interdoublet links.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

The effects of felodipine and bepridil on calcium-stimulated calmodulin binding and calcium pumping ATPase of cardiac sarcolemma before and after removal of endogenous calmodulin

Summary The Ca²⁺ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca²⁺-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca²⁺ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca²⁺ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca²⁺ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca²⁺ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca²⁺ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca²⁺-stimulated binding of calmodulin.Abbreviations Ca²⁺ pumping ATPase Ca²⁺ stimulated Mg²⁺-dependent ATP hydrolyzing activity - Na⁺ pumping ATPase Na⁺-stimulated K⁺- and Mg²⁺-dependent ATP hydrolyzing activity - Tris-maleate tris (hydroxymethyl) aminomethane hydrogen maleate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated,Mg2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca²⁺-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca²⁺-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca²⁺-activated ATPase was calcium dependent and maximal at calculated Ca²⁺ concentrations of 2.0 μM. Cyclic AMP-dependent Ca²⁺-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca²⁺-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K_m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca²⁺-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca²⁺-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca²⁺-activated protease similar to myofibrillar Ca²⁺-activated protease has been described in platelets. To explore the role of Ca²⁺-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca²⁺-activated protease-like activity. The hydrolysis of actin-binding protein by Ca²⁺-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca²⁺-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca²⁺-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca²⁺-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E₁, prostaglandin I₂, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca²⁺-activated protease in platelets and indicate that the source of calcium important in Ca²⁺-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca²⁺-activated protease-like activity as measured by the present analytical technique.     

The microtubule-binding fragment of microtubule-associated protein-2: location of the protease-accessible site and identification of an assembly-promoting peptide

Thrombin cleavage of bovine brain microtubule-associated protein (MAP-2) yields two stable limit polypeptide fragments (28,000 and 240,000 Mr). The smaller cleavage product contains the microtubule-binding domain and is derived from the carboxyl terminus of MAP-2 while the 240,000 Mr fragment is derived from the amino terminus. The amino terminal sequence of the smaller cleavage product is homologous with the microtubule-binding fragment of tau in sequence and in a similar location relative to three imperfect octadecapeptide repeats implicated in microtubule binding. Peptides corresponding to the cleavage site and the three repeats of MAP-2 were synthesized. Only the second octadecapeptide repeat (VTSKCGSLKNIRHRPGGG) was capable of stimulating microtubule nucleation and elongation. Microtubules formed in the presence of this peptide displayed normal morphology and retained the inhibition properties of calcium ion, podophyllotoxin, and colchicine. Our result indicates that a region comprising only approximately 1% of the MAP-2 sequence can promote microtubule assembly.     

Decreased Ca2+-binding and Ca2+-ATPase activities in heart sarcolemma upon phospholipid methylation

Heart sarcolemma has been shown to possess three catalytic sites (I, II and III) for methyl transferase activity (Panagia V, Ganguly PK and Dhalla NS. Biochim Biophys Acta 792: 245–253, 1984). In this study we examined the effect of phosphatidylethanolamine N-methylation on ATP-independent Ca²⁺ binding and ATPase activities in isolated rat heart sarcolemma. Both low affinity (1.25 mM Ca²⁺) and high affinity (50 µM Ca²⁺) Ca²⁺ binding activities were decreased following incubation of sarcolemmal membranes with AdoMet under optimal conditions for site II and III. Similarly, Ca²⁺ ATPase activities measured at 1.25 mM and 4 mM Ca²⁺ were depressed by phospholipid N-methylation. S-adenosyl homocysteine, a specific inhibitor of phospholipid N-methylation, prevented the depression of low affinity Ca²⁺ binding and Ca²⁺ ATPase activities, whereas the methylation-induced effect on the high affinity Ca²⁺ binding was not influenced by this agent. Pretreatment of sarcolemma with methyl acetimidate hydrochloride, an amino group blocking agent, also prevented the methylation-induced inhibition of both Ca²⁺ binding and Ca²⁺ ATPase. A further decrease in Ca²⁺ binding and Ca²⁺ ATPase activities together with a marked increase in the intramembranal level of PC was seen when membranes were methylated under the site III conditions in the presence of phosphatidyldimethylethanolamine as exogenous substrate. There was no effect of phospholipid methylation on sarcolemmal Na⁺-K⁺ ATPase and Mg²⁺ ATPase activities. These results indicate a role of phospholipid N-methylation in the regulation of sarcolemmal Ca²⁺ ATPase and low affinity ATP-independent Ca²⁺ binding.     

Annular and non-annular binding sites on the (Ca + Mg)-ATPase

Quenching of the fluorescence of the (Ca²⁺ + Mg²⁺)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Interrelationships between thermal-, N-ethylmaleimide-, and Ca2+-calmodulin-mediated activation/inactivation of dynein ATPase activities

Interactive effects between calmodulin activation of 30 S dynein ATPase activity and activation by heat or N-ethylmaleimide (NEM) have been studied. Addition of calmodulin during the heat treatment caused a larger increment in ATPase activity (above that caused by heating alone) than did addition of calmodulin after the heat treatment. Similar results were obtained in experiments where activation was caused by NEM treatment. For both the heat and NEM treatments, the synergistic effect of calmodulin when present during the treatment was Ca²⁺ dependent although activation of ATPase activity by either treatment alone was not Ca²⁺ dependent. Heating 14 S dynein inhibited its ATPase activity and reduced the effectiveness of calmodulin as an activator. The activating effect of calmodulin added after heat or NEM treatments was about the same as if the calmodulin was present during the treatment, i.e., interactive effects were minimal. Concentrations of NEM that had little effect on the ATPase activity of 14 S dynein largely eliminated the ability of calmodulin to activate its ATPase activity. Chromatography of the heat-treated 14 S dynein on calmodulin-Sepharose 4B indicated that the loss of sensitivity of 14 S dynein ATPase to calmodulin was not due to loss of ability of the dynein to bind to calmodulin. Retention of calmodulin binding ability was also shown for heat-treated 30 S dynein. These results suggest that calmodulin and heat/NEM activate solubilized 30 S dynein ATPase by separate mechanisms which may include a common process.     

(Ca2+ + Mg2+) ATPase activity in kidney basolateral membrane in diabetes: role of atrial natriuretic peptide

Summary The (Ca²⁺ + Mg²⁺) ATPase which serves as a Ca²⁺ pump in the kidney basolateral membranes is essential to the maintenance of an intracellular Ca²⁺ concentration optimal for kidney function. Since atrial natriuretic peptide (ANP) is known to participate in the Ca²⁺ homeostasis mechanism, altered levels of ANP in diabetes may vary the pump activity and consequently the kidney function. In order to examine the modulatory role of ANP on (Ca²⁺ + Mg²⁺) ATPase in short- (6 weeks) and long-term (6 months) diabetes, rats were injected with streptozotocin (65 mg/kg body wt, i.v.). At 6 weeks, the plasma ANP was decreased whereas, ANP-receptor binding in the kidney basolateral membrane was increased. In contrast, there was an increased plasma ANP and decreased ANP receptor binding at 6 months. Insulin treatment to diabetic animals normalized these parameters. The (Ca²⁺ + Mg²⁺) ATPase activity was unchanged both at 6 weeks and 6 months. Our results demonstrate that the unchanged Ca²⁺ pump activity in short-term and long-term diabetes serves to maintain the Ca²⁺ homeostasis in the kidney cells and thus may maintain the hyperfiltration state in diabetes. Unaltered (Ca²⁺ + Mg²⁺) ATPase is achieved by the initial up-regulation and subsequent down-regulation of the ANP receptors.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

Ca2+/H+ exchange,lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca²⁺ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca²⁺ in intracellular membrane bound compartments, thereby lowering cytosolic Ca²⁺ to induce relaxation. The stored Ca²⁺ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca²⁺, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca²⁺. We review here biochemical and biophysical evidence demonstrating that release of bound Ca²⁺ into the lumen of SR requires Ca²⁺/H⁺ exchange at the low affinity Ca²⁺ sites. Rise of lumenal Ca²⁺ above its dissociation constant from low affinity sites, or reduction of the H⁺ concentration by high pH, prevent Ca²⁺/H⁺ exchange. Under these conditions Ca²⁺ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca²⁺pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.