Expression and regulation of insulin-like growth factor binding protein-1 in the rat uterus throughout estrous cycle

The role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ sequestering of rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Nuclear Ca²⁺-ATPase activity increased linearly in the range of 10–40 M Ca²⁺ addition. With those concentrations, Ca²⁺ was completely taken up by the nuclei dependently on ATP (2 mM). Nuclear Ca²⁺-ATPase activity was decreased significantly by the presence of arachidonic acid (25 and 50 M), nicotinamide-adenine dinucleotide (NAD⁺; 2 mM) and zinc sulfate (2.5 and 5.0 M). These reagents caused a significant decrease in the nuclear Ca²⁺ uptake and a corresponding elevation in Ca²⁺ release from the nuclei. Moreover, calmodulin (10 g/ml) increased significantly nuclear Ca²⁺-ATPase activity, and this increase was not seen in the presence of trifluoperazine (10 M), an antogonist of calmodulin. The present findings suggest that Ca²⁺-ATPase plays a role in Ca²⁺ sequestering by rat liver nuclei, and that calmodulin is an activator. Moreover, the inhibition of Ca²⁺-ATPase may evoke Ca²⁺ release from the Ca²⁺-loaded nuclei.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Phospholipid requirement of Ca2+-stimulated,Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes

The phospholipid requirement for Ca²⁺-stimulated, Mg²⁺-dependent ATP hydrolysis (Ca²⁺/Mg²⁺-ATPase) and Mg²⁺-stimulated ATP hydrolysis (Mg²⁺-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A₂ (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A₂ caused an increase in the activities of both Ca²⁺/Mg²⁺-ATPase and Mg²⁺-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca²⁺/Mg²⁺-ATPase but Mg²⁺-ATPase activity was inhibited. Inhibition of Mg²⁺-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP₂) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP₂ brought about the reactivation of Ca²⁺/Mg²⁺-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg²⁺-ATPase activity.K _ms for Ca²⁺ (0.47 M) and Mg²⁺ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C₁₂E₈) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca²⁺ only. The membrane fraction has also found to contain Mg²⁺-dependent Ca²⁺-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca²⁺ required for maximum activity is 3 mM for both Mg²⁺-dependent and Mg²⁺-independent Ca²⁺-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca²⁺-uptake study shows that the uptake in the presence of Ca²⁺ and ATP is higher than in the presence of Mg²⁺, Ca²⁺ and ATP. The findings lead us to believe that the Mg²⁺-independent Ca²⁺-ATPase has some role in Ca²⁺ transport like Mg²⁺-dependent enzyme.Abbreviations Tris Tris (hydroxymethyl) amino ethane - Hepes-N 2-hydroxy ethyl piperizine-N¹-2-ethane sulfonic acid - Pipes-Piperizine-N N¹-bis(2-ethane sulfonic acid) - EGTA Ethylene Glycol-bis (-amino ethyl ether) - N, N, N¹, N¹ Tetraacetic Acid, sodium salt - TFP Trifluoperazine - DTNB 5,5¹ Dithiobis (2 nitrobenzoic acid) - TNBS 2, 4, 6-Trinitrobenzene Sulfonate - C₁₂E₈ Octa (ethylene glycol) mono n-dodecyl ether - PMSF Phenylmethyl Sulfonyl Fluoride - PAGE Polyacrylamide Gel Electrophoresis - PME -Mercapto Ethanol     

The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin,ATP and partial proteolysis

(1) Depending on the assay conditions, the ability of the Ca²⁺-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg²⁺ and K⁺ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. (2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca²⁺ so that the activity becomes negligible above 30 μM Ca²⁺. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca²⁺. (3) In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca²⁺ are needed and phosphatase activity is maximal in media without Ca²⁺. This is the first report of an activity sustained by the Ca²⁺-ATPase of red cell membranes in the absence of Ca²⁺. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca²⁺. These results strongly suggest that during activation by calmodulin, Ca²⁺ is needed only to form the calmodulin-Ca²⁺ complex which is the effective cofactor. (4) Protection by ATP of the inhibitory effects of Ca²⁺ and the induction of phosphatase activity by ATP + Ca²⁺ suggests that activation of the phosphatase by Ca²⁺ in media with ATP requires the combination of the cation at sites in the ATPase. (5) Results can be rationalized assuming that E₂, the conformer of the Ca²⁺-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca²⁺ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E₁ and E₂ towards E₂. On the other hand, ATP + Ca²⁺ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E₂ predominates over other conformers of the ATPase.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Comparison of the red blood cell Ca2+-ATPase in ghost membranes and after purification

We have compared properties of the red blood cell Ca²⁺-ATPase in two types of preparations: red cell membrane ghosts (enzyme in unfractionated membranes) and after purification (detergent-soluble enzyme). The Ca²⁺-ATPase activity was studied with respect to its requirement for: calmodulin, calcium, magnesium, monovalent cations, ionic strength, pH, and temperature. Sensitivity of the Ca²⁺-ATPase activity in the two preparations to anticalmodulin drugs and to engineered calmodulins with amino acid substitutions was determined. Finally, stoichiometry of the formation of phosphorylated enzyme intermediate (EP) and titrations of the ATP binding region with fluorescein 5-isothiocyanate (FITC) were characterized. For the first time a high phosphorylation level of 2.0–2.4 mmol EP/mg of purified enzyme is reported.The two enzyme preparations have been found to be very similar with respect to the dependency of all the regulating factors described here. These results complement findings reported from various laboratories on the similarity of other kinetic properties as well as the similarity of modulation of the Ca²⁺-ATPase activity by phospholipids and proteolysis in the membranous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca²⁺-ATPase.     

Cytochemical study of the effect of aluminium on cultured brain microvascular endothelial cells

Summary The cytotoxic effect of aluminium was studied on cultured goat brain microvascular endothelial cells used as an in vitro model of the blood—brain barrier. Confluent monolayers of these cells were exposed for 4 days to aluminium maltol and, for control purposes, to maltol alone, and also to cadmium chloride as a known cytotoxic substance. The localization of plasmalemma-bound enzymatic activities of 5-nucleotidase and Ca²⁺-ATPase and the distribution of sialic acid residues were studied at the ultrastructural level.It was observed that the reaction for 5-nucleotidase activity was only insignificantly affected, indicating its resistance to the cytotoxic action of both substances used. On the contrary, the activity of Ca²⁺-ATPase was evidently suppressed, especially in the interendothelial clefts where junctional complexes are presumably to be formed. Aluminium also affects the density of sialic acid residues, as shown by their redistribution, leading to the appearance of relatively long segments of unlabelled apical cell surface.The data obtained suggest that observed changes in the localization of Ca²⁺-ATPase and sialic acid residues can lead ultimately to impairment of the formation and maintenance of intercellular junctions and to disturbances in the negatively charged domains of the endothelial cell surface. Whether these alterations, induced in vitro, contribute to in vivo disturbances of blood—brain barrier function requires further experimental study.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide

Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na⁺+K⁺)-ATPase and K⁺-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na⁺+K⁺)-ATPase is impeded by either Na⁺ or K⁺ whereas only K⁺ impedes protection by ATP of K⁺-activated phosphatase. On the other hand, Na⁺ or K⁺ slightly protects Ca²⁺-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca²⁺-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca²⁺. This effect is half-maximal at less than 1 m Ca²⁺ and does not require ATP, which suggests that sites with high affinity for Ca²⁺ exist in the Ca²⁺-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K⁺ or Na⁺ in the presence of Ca²⁺ parallels that of the Ca²⁺-dependent activities, suggesting that the Ca²⁺-ATPase system possesses sites at which monovalent cations bind to increase its activity.     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

Effect of nuclear Ca2+ uptake inhibitors on Ca2+-activated DNA fragmentation in rat liver nuclei

The effect of nuclear Ca²⁺ uptake inhibitors on the Ca²⁺-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca²⁺ (40 M) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca²⁺-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca²⁺ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 M arachidonic acid, 2.0 mM NAD⁺, 10 M zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca²⁺-ATPase activity and Ca²⁺ uptake in the nuclei, produced a significant inhibition of the Ca²⁺-activated DNA fragmentation in the nuclei. The results show that the Ca²⁺-activated DNA fragmentation is involved in the uptake of Ca²⁺ by the nuclei, suggesting a role of Ca²⁺ transport system in the regulation of liver nuclear functions.     

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca²⁺-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca²⁺-transporter showed high affinity for Ca²⁺ (K_Ca ²⁺ = 0.04 ± 0.01 M), an apparent requirement for Mg²⁺ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca²⁺ or Mg²⁺ but showed a low affinity for these cations. At low concentrations, Mg²⁺ appeared to inhibit the Ca²⁺-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg²⁺ concentration (0.5–1 mM), a high affinity Ca²⁺-dependent ATPase activity was observed (K_Ca ²⁺ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca²⁺-transport nor high affinity Ca²⁺-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca²⁺ (or Mg⁺)-ATPase activity, the presence of both a high affinity Ca²⁺-pump and high affinity Ca²⁺-dependent ATPase. However, the high affinity Ca²⁺-ATPase activity does not appear to be the biochemical expression of the Ca²⁺-pump.Abbreviations Ca²⁺-ATPase calcium-activated, magnesium-dependent adenosine triphosphatase - CaM calmodulin - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate - EDTA ethylene-diaminetetraacetate - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate - NADPH reduced form of nicotinamide adenine dinucleotide phosphate     

Evidence that both Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

• 1.1. Evidence was obtained that activities of both low-affinity Ca²⁺-ATPase and high-affinity (Ca²⁺ + Mg²⁺)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme.
• 2.2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant.
• 3.3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca²⁺-ATPase activity comigrated with that of (Ca²⁺ + Mg²⁺)-ATPase.
• 4.4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated.
• 5.5. These findings suggest that Ca²⁺-ATPase and (Ca²⁺ + Mg²⁺)-ATPase are a single enzyme.