Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.     

Phospholipid-protein interactions in the Ca2+-adenosine triphosphatase of sarcoplasmic reticulum.

Ca2+-adenosine triphosphatase from sarcoplasmic reticulum has been delipidated by gel filtration through a Sephadex G-200 column equilibrated with buffer containing cholate. The delipidated Ca2+-adenosine triphosphatase had negligible adenosine triphosphatase activity, but up to 50% of the ATPase activity was restored when the delipidated enzyme was recombined with phosphilipids. It was shown with the delipidated preparation that the phosphorylation of the enzyme by either ATP or Pi was entirely dependent on phospholipids. Among the purified phospholipids, phosphatidylcholine reactivated the adenosine triphosphatase activity better than phosphatidylethanolamine. Vesicles capable of translocating Ca2+ were reconstituted from delipidated Ca2+-adenosine triphosphatase and phosphatidylethanolamine, but not with phosphatidylcholine alone. We conclude that the firmly bound phospholipids which are purified together with the adenosine triphosphatase protein are not essential for the pump since they can be substituted by phosphatidylethanolamine isolated from soybeans.     

Kinetic changes of the erythrocyte (Mg2+ + Ca2+)-adenosine triphosphatase of rats fed different fat-supplemented diets.

The activation by Mg2+, in the presence of 0.2 mM Ca2+, of the erythrocyte ATPase from rats fed with six different fat-supplemented diets has been studied. A sigmoid kinetic curve was found. The values of the Hill coefficient showed a positive correlation with the membrane fatty acid fluidity, which is expressed as the ratio between double bond index and saturated fatty acid content. The values of the Hill coefficient ranged from 1.0, in animals fed with lard-supplemented diet, to 2.0, in animals fed with corn oil-supplemented diet. When the effect of increasing Ca2+ concentration in these two groups was studied at pH 8.1, an activation with the latter group and an inhibition with the former one were found. The activation by Ca2+ found in corn oil-fed animals was lost after treatment with phospholipase C and restored after the addition of homologous phospholipids. The activation could not be restored by addition of phospholipids from lard-fed animals. In this group, treatment with phospholipase C left the kinetic behavior unmodified, but an activation by Ca2+ could be detected after adding phospholipids from corn oil-fed animals. It is suggested that membrane fatty acid fluidity is involved in the cooperative transitions and cryptic activity of the (Mg2+ + Ca2+)-ATPase.     

Assembly of the sarcoplasmic reticulum. Cell-free synthesis of te Ca2+ + Mg2+-adenosine triphosphatase and calsequestrin

Polyadenylated RNA prepared from neonatal rat muscle was translated in a rabbit reticulocyte cell-free system. Two sarcoplasmic reticulum proteins, the Ca2+ + Mg2+-dependent adenosine triphosphatase (ATPase) and calsequestrin, were isolated from the translation mixture by immunoprecipitation, followed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The [35S]methionine-labeled translation products were characterized by molecular weight, peptide mapping, and NH2-terminal sequence analysis. The ATPase synthesized in the cell-free system was found to have the same molecular weight (Mr = 100,000) and [35S]-methionine-labeled peptide map as the mature ATPase. The methionine residue present at the NH2 terminus of the mature ATPase was donated by initiator methionyl-tRNArMet and it became acetylated during translation. These results suggest that the ATPase was synthesized without an NH2-terminal signal sequence. Calsequestrin (Mr - 63,000) was synthesized as a higher molecular weight precursor (Mr = 66,000) that contained an additional [35S]methionine-labeled peptide when compared to mature calsequestrin. The NH2-terminal sequence of the precursor was different from the mature protein. The precursor was processed to a polypeptide with a molecular weight identical with mature calsequestrin when microsomal membranes prepared from canine pancreas were included during translation. These results show that calsequestrin is synthesized with an NH2-terminal signal sequence that is removed during translation. These data add to the evidence that the ATPase and calsequestrin follow distinctly different biosynthetic pathways, even though, ultimately, they are both located in the same membrane.     

Characterization of Gd3+ and Tb3+ binding sites on Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction between Gd3+ and Tb3+ ions and Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. Three classes of lanthanide-ion binding sites with different affinities were distinguished. Binding of Gd3+ to the site with the highest affinity seemed to occur at less than 10(-6)M free Gd3+ and resulted in severe inhibition of ATPase activity. The reaction rates of both E-P formation and decomposition in the forward direction were inhibited in parallel with this binding, whereas ADP-dependent decay of E-P in the backward direction was not. At these Gd3+ concentrations, Ca2+-binding to the transport site was not inhibited. Binding of Gd3+ and Tb3+ to the Ca2+-transport site did occur, but more than 10(-5)M free Gd3+ or Tb3+ was required for effective competition with Ca2+ for that site. Gd3+ bound to the transport site in place of Ca2+ did not activate the E-P intermediate formation. Addition of 10(-1)M Tb3+ to a suspension of sarcoplasmic reticulum membranes resulted in marked enhancement of Tb3+ fluorescence, which is due to an energy transfer from aromatic amino acid residues of ATPase to Tb3+ ions bound to the low affinity site of the enzyme. Gd3+ and Mn2+ competed with Tb3+ for that site, but Ca2+, Zn2+, and Cd2+ did not.     

Histochemical localization of Mg2+-Na+-K+-adenosine triphosphatase in different stages of the sheep mesonephros.

Mesonephroi of sheep embryos ranging from 12 to 100 mm C.R. length were examined for the occurrence and localization of transport-ATPase. Native cryostat sections were incubated according to the technique of Guth and Albers for demonstrating the nitrophenylphosphatase activity of Mg2+-Na+-K+-adenosine triphosphatase. The basal cytoplasm of the collecting tubule of the narrow segment of the distal tubule exhibit strong activity, the wide segment of the distal tubule is moderately active. Glomeruli, proximal tubule, and Wolffian duct remain unstained. The basal labyrinths of the reactive nephron segments are believed to be the sites of a Na+-K+ exchange pump. In mature and regressing mesonephroi, the findings fully agree with biochemical data; in maturating mesonephroi, whose basal labyrinth is not yet fully established, the biochemical assay proves to be more sensitive. The specifity of the reaction was ascertained by diverse inhibitors and activating ions. The localization of Mg2+-ATPase is different to the above mentioned reaction pattern, as it shows moderate activity in the proximal tubule, too (mature mesonephros). Mesonephroi of very young embryos exhibit strongest Mg2+-ATPase activity in the proximal tubule; here the distal and collecting tubule stain only moderately.     

Assembly of the sarcoplasmic reticulum. Synthesis of calsequestrin and the Ca2+ + Mg2+ -adenosine triphosphatase on membrane-bound polyribosomes.

Membrane-bound and free polyribosomes were isolated from skeletal muscle of neonatal rats and messages were translated in a rabbit reticulocyte lysate treated with Ca2+ -dependent nuclease to reduce endogenous messenger translation. Newly synthesized calsequestrin and adenosine triphosphatase (ATPase) sere isolated by antibody precipitation, followed by separation of the precipitates in SDS-polyacrylamide gels. Radioactivity in calsequestrin and the ATPase were counted in gel slices. Calsewuestrin and the ATPase were both found to be synthesized on membrane-bound polyribosomes. Since calsequestrin is a glycoprotein, localized in Golgi regions in early stages of muscle cell differentiation, it is probable that its synthesis follows the pathway for synthesis of secreted proteins except that its destination is the luminal space of a cellular organelle. The disposition of the ATPase during synthesis is, as yet, unknown.     

Hamster sperm Na+, K+-adenosine triphosphatase: increased activity during capacitation in vitro and its relationship to cyclic nucleotides

These in vitro studies of golden hamster sperm were undertaken to determine whether: Na+, K+-adenosine triphosphatase (ATPase) activity is required for capacitation; Na+, K+-ATPase activity is altered during capacitation; and cyclic nucleotides can control this enzyme activity. Hamster sperm were incubated in a medium in which capacitation occurred in an asynchronous manner and in which acrosome reactions began to occur after approximately 3.5 h of incubation. Inhibition of the hamster sperm acrosome reaction by the Na+, K+-ATPase inhibitor ouabain (1 microM) added at Time (T) = 2 or T = 3 h could be fully reversed by the addition of the ionophore nigericin (0.1 microM) at T = 3.5 h. However, when ouabain was added at T = 0 or T = 1 h, similar nigericin addition could not completely reverse the inhibition. Na+, K+-ATPase activity of hamster sperm increased by 2 h of incubation (compared to that measured initially after 15 min) and this activity remained elevated at 3.5 h. Addition of either monobutyryl cyclic adenosine 3':5'-monophosphate ( BtcAMP ) (12.9 microM) or monobutyryl cyclic guanosine monophosphate ( BtcGMP ) (10.5 microM), or the phosphodiesterase inhibitor SQ20009 (10 microM) at 2 h produced a stimulation of acrosome reactions at 4 and 5 h. However, while BtcGMP and SQ 20009 also induced a further increase in Na+, K+-ATPase activity measured at 3.5 h, BtcAMP had no effect. Intracellular cAMP and cGMP levels measured showed cAMP increased by 2 h and remained elevated when measured at 3.5 h, while cGMP could not be consistently detected at 15 min, 2 h or 3.5 h. However, assays of high numbers of uncapacitated sperm did detect a low level of cGMP. These results suggest that Na+, K+-ATPase activity increases in and is essential for early capacitation [and thereby eventually for the acrosome reaction (AR)] of hamster sperm and that the increase in Na+, K+-ATPase activity occurring during capacitation is probably mediated by intracellular cGMP but not cAMP, although both cyclic nucleotides stimulate the hamster sperm AR.     

The NH2 terminus of the (Ca2+ + Mg2+)-adenosine triphosphatase is located on the cytoplasmic surface of the sarcoplasmic reticulum membrane

The (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) of sarcoplasmic reticulum contains a cysteine residue at position 12 of its sequence. This sulfhydryl group was 1 out of a total of 10-11 that were labeled by treatment of sarcoplasmic reticulum vesicles with N-[3H]ethylmaleimide under saturating conditions. This was shown by isolating a 31-residue NH2-terminal peptide from a tryptic digest of the succinylated ATPase, prepared from N-[3H]ethylmaleimide-labeled vesicles. Reaction of the vesicles with glutathione maleimide, parachloromercuribenzoic acid, or parachloromercuriphenyl sulfonic acid, membrane-impermeant reagents, prevented further reaction of sulfhydryl groups with N-ethylmaleimide. This result indicates that all sulfhydryl groups that are reactive with N-ethylmaleimide are on the outside of the vesicles. Since Cys12 is located in a hydrophilic NH2-terminal portion of the ATPase, the labeling results suggest that the NH2 terminus of the ATPase is on the cytoplasmic side of the membrane. These results are consistent with earlier observations (Reithmeier, R. A. F., de Leon, S., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 11839-11846) that the (Ca2+ + Mg2+)-ATPase is synthesized without an NH2-terminal signal sequence.     

Interaction of adenosine-5'-O-(3-thiotriphosphate) with Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) with Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30 degrees C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0 degrees C, ATP gamma S showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [3H]ADP in equilibrium ATP gamma S exchange reaction and also by using [35S]ATP gamma S. When the ATPase was reacted with ATP gamma S at 0 degrees C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca2+ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATP gamma S or the thiophosphorylated intermediate was suggested by the fact that 2-mercaptoethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.     

Some characteristics of Ca2+- regulated force production in EGTA- treated muscles from rat heart

McClellan and Winegrad (1980, J. Gen. Physiol., 75:283-295) have reported that in rat ventricular muscles that have reportedly been made "hyperpermeable" to small ions such as Ca2+, CaEGTA2-, and MgATP2- by a soak in EGTA, the maximum Ca2+-regulated force can be permanently increased by a short exposure to positively inotropic drugs, such as epinephrine or cAMP plus theophylline, in the presence of the detergent Triton X-100. The experiments reported here were begun as an attempt to repeat and extend this important observation. However, no evidence could be found for a potentiation of force that was not merely produced by Triton alone. In addition, the thickest muscles used (250-440 microns diameter) exhibited very low values for force per unit cross- sectional area, which suggested that either Ca2+ reached only a fraction of the myofibrils or the myofibrils were in a state of low contractility. The results of further experiments that were designed to test the permeability characteristics of these EGTA-treated muscles indicated that the movement of certain ions into these preparations was restricted, even in thin muscles (80-200 microns diameter). The rate of development of Ca2+-regulated force was slow (t1/2 approximately equal to 1-3 min), but was greatly accelerated after the muscles had been superfused with Triton X-100 (t1/2 approximately equal to 10-20 s). Removal of creatine phosphate (CP) in the presence of MgATP produced a partial rigor contracture in the EGTA-treated muscles. The results were consistent with the suggestion that the EGTA-treated muscles were permeable to some extent to Ca2+ and HCP2- ions but not to CaEGTA2- and MgATP2-. Thus, it would seem unlikely that the [Ca2+], [MgATP2-], and [Mg2+] in the immediate vicinity of the myofibrils in these preparations can be adequately controlled by the solution bathing the muscles.     

Na+/K+-ATPase activity of different types of striated muscles

Na+/K+-ATPase activity was determined in striated muscles with different aerobic capacities. The underlying hypothesis was that different aerobic capacities are reflective of different contractile activity which imposes greater demands on sarcolemmal ion translocation and may thus set Na pumping capacity. The added ion translocation demands required during exercise-training on Na+/K+-ATPase activity in different muscle fiber types may require an adaptation of this enzyme. The highest and lowest Na+/K+-ATPase activity was in the heart and white gastrocnemius muscle (WG), respectively. A high linear correlation existed between Na+/K+-ATPase activity and succinate dehydrogenase activity in the six muscles studied. Exercise-training did not increase Na+/K+-ATPase activity in any of the muscles, but did increase the aerobic capacity, except in the heart and WG. It was concluded that Na+/K+-ATPase activity has a high positive correlation with the aerobic capacity of striated muscles in the rat and that the Na pump capacity does not adapt to exercise-training of 1 hr X day-1 as does aerobic capacity.     

Biosynthesis of the Ca2+- and Mg2+-dependent adenosine triphosphatase of sarcoplasmic reticulum in cell cultures of embryonic chick heart.

The biosynthesis of the Ca2+- and Mg2+-dependent adenosine triphosphatase of sarcoplasmic reticulum was studied in cell cultures of embryonic chick heart. Rates of synthesis were estimated from the incorporation of tritium-labeled leucine into the ATPase. Newly synthesized ATPase was isolated from cells by immunoprecipitation. Radioactive leucine incorporation into the ATPase was determined by gel electrophoresis of the immunoprecipitates and counting of gel slices containing the ATPase band. Accumulation of the ATPase was estimated from the concentration of Ca2+ and Mg2+-dependent, hydroxylamine-sensitive phosphoprotein in the whole cell membrane fraction of cultured cells. Embryonic heart cells cultured in a medium which permitted cell proliferation showed approximately linearly increasing rates of ATPase synthesis and accumulation/culture plate as the cells proliferated. When cells were cultured in a serum-free medium, cell proliferation was inhibited and there was no sustained increase in the rate of ATPase synthesis or accumulation. Inclusion of isoproterenol or dibutyryl cyclic AMP at concentrations of 10 microM up to 1 mM in serum-free culture medium failed to stimulate significantly ATPase synthesis.     

(Na+ + K+)-adenosine triphosphatase of mammalian brain. Catalytic and regulatory K+ sites distinguishable by selectivity for Li+.

Li+, K+, and Rb+ are compared as activators of the hydrolysis of p-nitrophenylphosphate by beef brain (Na+ + K+)-ATPase. Previous experiments have established two classes of K+ binding sites that are involved in this reaction: "catalytic sites" have the higher affinity, their occupation is essential for catalytic activity, and they appear to correspond to the extracellular binding sites for active K+ transport; regulatory sites appear to have an allosteric function to "unmask" the catalytic sites. A separate set of Na+-binding regulatory sites bring about a similar unmasking of catalytic sites under phosphorylating conditions. Rb+ can activate p-nitrophenylphosphate hydrolysis both in the presence and absence of Na+ and, thus, can interact effectively with both K+ regulatory and catalytic sites. Li+ does not activate p-nitrophenylphosphate hydrolysis at 25 degrees C in the absence of other monovalent ligands. Li+ does activate when the catalytic sites are exposed by Na+ + ATP. Thus, K+ regulatory and catalytic sites differ in their cation selectivity. At temperatures less than 25 degrees C Li+ is able to activate the phosphatase reaction in the absence of other monovalent ligands: maximum activity occurs at 10-12 degrees C. A plot of the ratio, Li+ activation/K+ activation, as a function of temperature shows that the allosteric transition that unmasks catalytic sites occurs spontaneously with decreasing temperatures.     

Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.     

Evidence for a new intermediate state in the mechanism of (Na+ + K+)-adenosine triphosphatase.

A rapid mixing technique was used to follow the intermediate formation of phosphorylated enzyme and liberation of inorganic phosphate by a microsomal preparation of (Na+ + K+)-ATPase. In the presence of 100 mM Na+,but without added K+, phosphorylation reaches a constant level at a rate which is dependent on ATP concentration. Inorganic phosphate production lags during the inital phase of phosphorylation and then accumulates at a constant rate. These observations favor a scheme in which Pi is liberated as the result of turnover of the phosphorylated enzyme. In the presence of 100 mM Na+ and 2.5 mM K+ phosphate production was resolved into two phases consisting of an initial 'burst' and late steady state phase...