Kinetics of Mg2+-ATPase inhibition by benthiocarb and its recovery by oximes and L-cysteine in brain of albino rat (Wistar strain)

Effects in vitro of benthiocarb on rat brain Mg2+-ATPase were studied to elucidate the interaction of benthiocarb with Mg2+-ATPase. Based on IC50 values, it was evident that Mg2+-ATPase was sensitive to benthiocarb. Non-competitive inhibition with respect to activation by ATP was indicated by decreased maximal velocity (V) without change in Michaelis Menten constant (Km). It was also noted that pyridine-2-aldoxime (30 microM), pyridine-4-aldoxime (35 microM) and L-cysteine (90 microM) neutralized the inhibition of benthiocarb (8 microM).     

Mechanism of regulation of phosphorylation-dephosphorylation of Ca2+, Mg2+ and Ca2+ -Atpases by modulator proteins isolated from rat brain cytosol

Two proteins of molecular mass 13 kDa, a specific inhibitor of Na+, K+ -ATPase and another of 12 kDa, which can distinguish between Ca2, Mg2+ and Ca2+ -ATPase activities have been obtained from the pooled fractions isolated from rat brain, using Sephadex G-100 chromatography. In order to determine the key step(s), which is affected by the modulators, we have designed an in vitro experiment of phosphorylation and dephosphorylation of these ATPases in the absence and presence of the modulators. The results suggest that the phosphorylation step of Mg2+ -independent Ca2+ -ATPase is inhibited, while in Mg2+ -dependent Ca2 -ATPase, the dephosphorylation step is stimulated by the modulators. The findings support our earlier observation that the modulators are able to distinguish between Mg2+ -independent and dependent Ca2+ -ATPases activities.     

Kinetic characteristics of Ca2+, Mg2+-ATPases in cells of the submandibular salivary gland of rats

Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.     

Inhibition of microsomal ATPases by high concentration of Mg2+ in tracheal epithelial cells.

Previous work in our laboratory established the presence of two types of microsomal ATPases, a low-affinity vanadate-sensitive (LAVS) and a high-affinity vanadate-sensitive (HAVS) ATPases, in tracheal epithelial cells. These ATPases were identified as Ca2+-ATPases by specific inhibitors and microsomal Ca2+ uptakes. Since the regulatory roles of Mg2+ on both cellular Ca2+-signaling and epithelial transports were demonstrated, the effects of Mg2+ on these ATPases were investigated. Mg2+-dependence of ATPase activity appeared bell-shaped with a maximal activity at 1-2 mM Mg2+ and Mg2+ at higher than 2 mM inhibited these enzymes. In a kinetic analysis of the LAVS ATPase inhibition, high concentration of Mg2+ appeared to inhibit the binding of ATP to a substrate-binding site. The microsomal 45Ca2+ uptakes mediated by both ATPases were also inhibited by high concentration of Mg2+. In order to test whether high concentration of Mg2+ directly inhibits these enzymes, microsomes were made leaky by the treatment of Triton X-100 and the microsomal ATPases were solubilized with CHAPS. The leaky microsomal ATPases and CHAPS-solubilized ATPases were similarly inhibited by high concentration of Mg2+, suggesting that Mg2+ directly inhibit these enzymes. In conclusion, Mg2+ has two types of modulatory effects on these enzymes, a catalytic effect at low concentration and an inhibitory effect at high concentration.     

Modulation of benthiocarb in vitro inhibited neonate rat (Wistar strain) brain Na+K+-ATPase by norepinephrine

Effect in vitro of benthiocarb, an organocarbamate herbicide on neonate rat (3 day old) brain was studied to understand the interaction of benthiocarb with Na+K+-ATPase. Na+K+-ATPase of the developing rat brain was selected as an index enzyme since alterations in the Na+K+-ATPase activity leads to neuronal dysfunction. The assay of Na+K+-ATPase in the presence of 1-8 mu moles of benthiocarb showed decreased activity and a concentration dependent inhibition of Na+K+-ATPase was noticed upto 7 mu moles of benthiocarb. Based on IC50 values (median concentration), 50% inhibition of the enzyme was observed with 5 mu moles of benthiocarb. Norepinephrine (NE) was selected to study the modulation of benthiocarb inhibited enzyme. Maximum increase (76.7%) of Na+K+-ATPase was noticed with 35 mu moles of NE and effective concentration (EC50) of NE which produced 50% activation of the enzyme was found to be 20 mu moles. This study suggests that NE acts as a protective agent in reversing the benthiocarb in vitro inhibited neonate rat brain Na+K+-ATPase.     

Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat-kidney mitochondria

The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat kidney mitochondria were found to be essentially similar to those described previously for other mammalian tissues; in particular each enzyme could be activated severalfold by Ca2+ with half-maximal effects (K0.5 values) of about 1 microM and effective ranges of approx. 0.1-10 microM Ca2+. In intact mitochondria prepared from whole rat kidneys incubated in a KCl-based medium containing respiratory substrates, the amount of active, nonphosphorylated pyruvate dehydrogenase could be increased severalfold by increases in extramitochondrial [Ca2+]; these effects could be blocked by ruthenium red. Similarly, Ca2+-dependent activations of NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase could be demonstrated in intact, fully coupled, rat kidney mitochondria by either following O2 uptake (in the presence of ADP) and NAD(P)H reduction (in the absence of ADP) on presentation of non-saturating concentrations of either threo-Ds-isocitrate or 2-oxoglutarate, respectively, under appropriate conditions, or for the latter enzyme only, also by following 14CO2 production from 2-oxo[1-14C]glutarate (in the absence or presence of ADP). Effects of Na+ (as a promoter of egress) and Mg2+ (as an inhibitor of uptake) on Ca2+-transport by rat kidney mitochondria could be readily demonstrated by assaying for the Ca2+-sensitive properties of the intramitochondrial Ca2+-sensitive dehydrogenases within intact rat kidney mitochondria. In the presence of physiological concentrations of Na+ (10 mM) and Mg2+ (2 mM), activation of the enzymes was achieved by increases in extramitochondrial [Ca2+] within the expected physiological range (0.05-5 microM) and with apparent K0.5 values in the approximate range of 300-500 nM. The implications of these results on the role of the Ca2+-transport system of kidney mitochondria are discussed.     

Purification of the Ca2+-and Mg2+-requiring ATPase from rat brain synaptic plasma membrane.

A Ca2+-ATPase (Ca2+- and Mg2+-requiring ATPase) was purified from a synaptic plasma-membrane fraction of rat brain. This enzyme had properties similar to those of plasma-membrane Ca2+-ATPases from other organs: its splitting of ATP was dependent on both Ca2+ and Mg2+, it bound in a Ca2+-dependent fashion to calmodulin-Sepharose and it cross-reacted with specific antibodies raised against human erythrocyte-membrane Ca2+-ATPase. It had an apparent Mr of 138 000, similar to those of plasma-membrane ATPases from human erythrocyte and from dog heart sarcolemma. Previous high-Ca2+-affinity ATPases observed in brain had Mr 100 000; in at least one case, such an ATPase probably represented a different type of enzyme, derived from coated vesicles.     

Polyunsaturated fatty acids inhibit Mg2+ -ATPase in basolateral membranes from rat enterocytes.

It is known that certain polyunsaturated fatty acids of the n-6 family, for example linoleic and arachidonic acids, can activate both Na+, K+-ATPase and Ca2+-ATPase. These enzymes drive active absorption processes in the duodenal enterocyte. This study presents data which show a 30-50% inhibition of Mg2+-ATPase activity in enterocyte basolateral membrane preparations by linoleic and gamma-linolenic acids (also a member of the n-6 family.) Mg2+-ATPase activity has several possible roles in the enterocyte: involvement in Mg2+ and Ca2+ absorption (as part of Ca2+-ATPase and also myosin I activity) as well as control of phospholipid distribution in the membrane by a class of Mg2+-ATPases called 'flippases'. The action of linoleic and gamma-linolenic acids on basolateral membrane Mg2+-ATPase may thus modulate several cellular transport processes.     

An endogenous inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase

An inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was purified to apparent homogeneity from rat cerebrum by a molecular weight cut followed by chromatography of cytosol proteins with molecular weights between 10 000 and 3500 on DEAE-Sephadex at pH 5.2. The inhibitor could be partially inactivated by proteinases and dithiothreitol, but was heat-stable. Gel filtration gave a molecular weight of about 6000. Like the (Ca2+ + Mg2+)-ATPase inhibitor protein isolated from erythrocytes, the inhibitor from brain contains a characteristic high proportion of glutamic acid (36%) and glycine (37%) residues. Synaptic plasma membrane Mg2+-ATPase and microsomal membrane (Ca2+ + Mg2+)-ATPase did not respond to the inhibitor. Synaptic plasma membrane and erythrocyte membrane (Ca2+ + Mg2+)-ATPases, however, were affected. Inhibitory influence on synaptic membrane (Ca2+ + Mg2+)-ATPase was reversible, since inhibition could be relieved upon removal of inhibitor from saturable sites on the membrane. The inhibitor is not a calmodulin-binding protein, since the concentration of calmodulin for half-maximal activation of the ATPase was unaffected by its presence. Mode of inhibition of the (Ca2+ + Mg2+)-ATPase by the inhibitor was non-competitive.     

Comparison of Properties of the Plasma Membrane Ca2+ -ATPase from Wheat Root and Leaf

The properties of plasma membrane Ca2 + -ATPases from wheat ( Triticum aestivum L. cv. Lengchun No. 13) root and leaf were compared, and their different properties were analyzed in association with the differentia of the functions of these two organs and their relevant environments. Root plasma membrane Ca2 + -ATPase showed a high activity in a broad range of pH and an optimum reaction temperature of 45 ℃, while the leaf enzyme activated in a narrow range of pH and an optimum reaction temperature of 50 ℃. Hill coefficient of root plasma membrane Ca2 + -ATPase for ATP was 1.6, revealing an obvious positive cooperativity. In contrast, that of leaf plasma membrane Ca2 +-ATPase was 1.0, being in keeping with Michaelis-Menten dynamics. For Ca2 + activation, Hill coefficient of plasma membrane Ca2 + -ATPases from both organs were less than 1, suggesting that both had negative cooperativity. The enzymes were activated by calmodulin and inhibited by Mg2+.     

A high-affinity plasma membrane Ca2+-ATPase in Dictyostelium discoideum: its relation to cAMP-induced Ca2+ fluxes

Chemotactic stimulation of Dictyostelium discoideum induces an uptake of Ca2+ by the cells followed by a release of Ca2+. In this study we investigated the mechanism of Ca2+ release and found that it was inhibited by La3+, Cd2+ and azide. Ca2+ release occurred in the absence of external Na+, indicating that an Na+/Ca2+ exchange was not involved. Plasma membranes contained high- and low-affinity ATPase activities. Apparent K0.5 values were 8 microM for the major Mg2+-ATPase and 1.1 microM for the high-affinity Ca2+-ATPase, respectively. The Mg2+-ATPase activity was inhibited by elevated concentrations of Ca2+, whereas both Ca2+-ATPases were active in the absence of added Mg2+. The activities of the Ca2+-ATPases were not modified by calmodulin. The high-affinity Ca2+-ATPase was competitively inhibited by La3+ and Cd2+; we suggest that this high-affinity enzyme mediates the release of Ca2+ from D. discoideum cells.     

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+-dependent ATPases in the basolateral membrane of rat kidney cortex

The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established.     

Localization of Mg2+-ATPase in the membranes of the skeletal muscles and myocardium of the frog Rana temporaria

Using differential centrifugation in sucrose density gradient, from muscles of the frog fractions were obtained which contain fragments of sarcolemma, as well as membranes of T-system tubules and sarcoplasmic reticulum. In isolated membrane fractions, studies were made on the activity of cation-stimulated ATPases (Na+, K+-, Ca2+, Mg2+- and Mg2+-ATPases). Enzymic and electrophoretic analyses showed that the highest content of Mg2+-ATPases is typical of the fractions which are located on the surface of 35% sucrose. The data obtained indicate that Mg2+-ATPase is the enzyme which is specific for the membranes of T-system tubules in skeletal muscles of not only birds but amphibians as well. From cardiac muscle of the frog, membrane fraction was isolated which is similar (with respect to its predominant content of Mg2+-ATPase) to the membranes of T-system tubules. It is suggested that the presence of Mg2+-ATPase in these membranes is a common property of phasic striated muscle fibers in all mature vertebrate animals.     

The animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases--approaches to molecular arrangements of functional parts and oxidative changes.

The molecular structures of animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases are not completely understood in part due to the fact that no suitable single crystal is available. The elucidation of the two-dimensional structure is in progress. The amino acid sequences of human erythrocyte and rat plasma membrane Ca2+ pump isoforms as well as of the pig smooth muscle plasma membrane Ca2+ pump are already known. This article reviews the present state of the knowledge in (Ca(2+)+Mg2+)-ATPase research of animal and human plasma membranes performed in the past few years, concerning in particular arrangements of proteolytically cleaved fragments, and relations between the erythrocyte (Ca(2+)+Mg2+)-ATPase in situ and the purified red cell enzyme, oxidative changes. Results of different experimental approaches concerning the structure of (Ca(2+)+Mg2+)-ATPases rather than the applications of the methods used are emphasized.     

A high affinity Ca2+-stimulated and Mg2+-dependent ATPase in rat corpus luteum plasma membrane fractions

Plasma membrane fractions from rat corpus luteum contain two kinds of Ca2+-stimulated ATPase, one having a high affinity for Ca2+, the other a low affinity for Ca2+. The high affinity ATPase had a specific Ca2+ requirement with a K 1/2 of 0.2 to 0.3 microM; it had a Vmax of 105 nmol min-1 mg-1 and distributed, upon subcellular fractionation, with recognized plasma membrane enzymes. The properties of this enzyme indicate that it is a CA2+ extrusion pump. The low affinity pump (K 1/2 for Ca2+, about 15 microM) was nonspecific, being stimulated equally well by Ca2+ of Mg2+; its function is unknown. Although the high affinity ATPase resembled the erythrocyte Ca2+-pumping ATPase in the properties mentioned above, it differed in that it failed to respond to Mg2+ or calmodulin. The lack of response to Mg2+ was due to the enzyme's retention of endogenous Mg2+; it did, after incubation with chelators, show a Mg2+ requirement. However, we were unable to show any effect of added calmodulin or trifluoperazine. This failure may be related to the high content of tightly bound calmodulin in these membranes. Much of this calmodulin could not be extracted even by washing with 1 mM EGTA and/or 0.1% (w/v) Triton X-100. This enzyme, the erythrocyte enzyme, and the adipocyte plasma membrane Ca2+ ATPase all belong to the class of Ca2+ ATPases with plasma membrane distribution and high affinity for Ca2+, indicating that they are Ca2+ extrusion pumps. However, the data indicate that tissue-specific differences exist within this class, with the enzyme from adipocytes and rat corpus luteum belonging to a subclass in which the requirement for Mg2+ and any response to calmodulin are difficult to demonstrate.     

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

Effects of spermine on mitochondrial Ca2+ transport and the ranges of extramitochondrial Ca2+ to which the matrix Ca2+-sensitive dehydrogenases respond.

1. Spermine has previously been reported to be an activator of mitochondrial Ca2+ uptake [Nicchitta & Williamson (1984) J. Biol. Chem. 259, 12978-12983]. This is confirmed in the present studies on rat heart, liver and kidney mitochondria by using the activities of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) as probes for matrix Ca2+, and also, for the heart mitochondria, by using entrapped fura-2. 2. As also found previously [Damuni, Humphreys & Reed (1984) Biochem. Biophys. Res. Commun. 124, 95-99], spermine activated extracted pyruvate dehydrogenase phosphate phosphatase. However, it was found to have no effects at all on the extracted NAD+-isocitrate or 2-oxoglutarate dehydrogenases. It also had no effects on activities of the enzymes in mitochondria incubated in the absence of Ca2+, or on the Ca2+-sensitivity of the enzymes in uncoupled mitochondria. 3. Spermine clearly activated 45Ca uptake by coupled mitochondria, but had no effect on Ca2+ egress from mitochondria previously loaded with 45Ca. 4. Spermine (with effective Km values of around 0.2-0.4 mM) caused an approx. 2-3-fold decrease in the effective ranges of extramitochondrial Ca2+ in the activation of the Ca2+-sensitive matrix enzymes in coupled mitochondria from all of the tissues. The effects of spermine appeared to be largely independent of the other effectors of mitochondrial Ca2+ transport, such as Mg2+ (inhibitor of uptake) and Na+ (promoter of egrees). 5. In the most physiological circumstance, coupled mitochondria incubated with Na+ and Mg2+, the presence of saturating spermine (2 mM) resulted in an effective extramitochondrial Ca2+ range for matrix enzyme activation of from about 30-50 nM up to about 800-1200 nM, with half-maximal effects around 250-400 nM-Ca2+. The implications of these findings for the regulation of matrix and extramitochondrial Ca2+ are discussed.