Subcellular fractionation of pig coronary artery smooth muscle

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

An analytical approach to the preparation and characterization of subcellular membranes from canine mesenteric arteries

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.     

Target size of Ca-pumps in pig coronary artery smooth muscle

The target sizes of the oxalate-independent Ca uptake by the plasma membrane enriched fraction F2, and the oxalate-stimulated Ca uptake by a fraction F3 slightly enriched in the endoplasmic reticulum were determined by radiation inactivation. The oxalate-independent Ca uptake was inactivated with a D37 value of 1.96 +/- 0.30 Mrad but the oxalate-stimulated Ca uptake had a D37 value of 0.45 +/- 0.07 Mrad. Thus, in the smooth muscle the oxalate-stimulated Ca uptake appeared to be due to a structure 3 to 6 times larger than was the oxalate-independent Ca uptake. The subcellular site of the ATP-dependent azide insensitive Ca uptake in the smooth muscle has been disputed in the past. It has been suggested to be plasma membrane (PM) by several workers, and endoplasmic reticulum (ER) by others. Recently, however, there has been substantial evidence to support the hypothesis that one Ca uptake system, unaffected by oxalate, resides in the PM and another, stimulated by oxalate, is located in the ER of the smooth muscle. The evidence has been reviewed recently. Here, we show that the two modes of Ca uptake differ in their target sizes as well. To our knowledge, this is the first report on the use of radiation inactivation to distinguish between the two modes of Ca uptake in any tissue.     

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.     

Effect of digitonin on rat myometrium subcellular membrane fractions

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.     

Ca2+ transport by rat liver plasma membranes: the transporter and the previously reported Ca2+-ATPase are different enzymes

A rat liver plasma membrane fraction showed an ATP-dependent uptake of Ca2+ which was released by the ionophore A23187. This activity represents a plasma membrane component and is not due to microsomal contamination. The Ca2+ transport displayed several properties which were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209-11215; Birch-Machin, M.A. and Dawson, A.P. (1986) Biochim. Biophys. Acta 855, 277-285). These observations have shown that Ca2+-ATPase does not require added Mg2+ whereas we have demonstrated that, in the same membrane preparation, Ca2+ uptake required millimolar concentrations of added Mg2+. The Ca2+-ATPase has a broad specificity for the nucleotides ATP, GTP, UTP and ITP while Ca2+ uptake remains specific for ATP. Ca2+ uptake also displayed different affinities for free Ca2+ and MgATP compared to Ca2+-ATPase activity, with apparent Km values of 0.25 microM Ca2+, 0.15 mM MgATP and 1.0 microM Ca2+, 4 microM MgATP respectively. The apparent maximum rate of Ca2+ uptake was about 150-fold less than Ca2+-ATPase activity. These features suggest that the high-affinity Ca2+-ATPase is not the enzymic expression of the ATP-dependent Ca2+ transport mechanism.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.     

Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets

Human platelet membrane vesicles that accumulated Ca2+ in the presence of ATP were isolated on an isoosmotic KCl-Percoll gradient. ATP-dependent Ca2+ uptake was stimulated by oxalate and phosphate to steady-state levels of greater than 100 nmol/mg protein, and the accumulated Ca2+ could be largely released by ionophore A23187. Inositol 1,4,5-trisphosphate, in a dose-dependent manner (0.5-5.0 microM), caused the rapid release (less than 5 s) of 40-70% of the total A23187-releasable store of accumulated Ca2+. The membrane vesicles that release accumulated Ca2+ in response to inositol 1,4,5-trisphosphate were enriched in enzymes characteristically found in smooth endoplasmic reticulum. These results support the hypothesis that inositol 1,4,5-trisphosphate, produced by the hydrolysis of phosphatidylinositol 1,4-bisphosphate in response to stimulation of cell surface receptors, is a second messenger mediating the release of Ca2+ from intracellular storage sites.     

Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.     

Aggregation of smooth muscle membranes and its use in the preparation of plasma membrane enriched fraction from gastric fundus smooth muscle

Microsomal membranes isolated from rat gastric fundus smooth muscle by differential centrifugation aggregate substantially in the presence of the divalent metal ion Mg2+ or Ca2+. The magnitude of cation-induced membrane aggregation is higher for Ca2+ than for Mg2+, but the ion concentration required for half-maximum membrane aggregation (K0.5 value) is similar for Mg2+ and Ca2+. Cation-induced membrane aggregation is suppressed by high ionic strength and low pH of the medium. Cation-induced membrane aggregation of mitochondrial membrane and plasma membrane enriched fractions differ in the rate of aggregate formation, metal ion concentration dependence, and pH dependence. Such different properties of membrane aggregation were used to prepare a plasma membrane enriched fraction by conventional differential centrifugation. Subfractionation of the heterogeneous microsomal membranes by free-flow electrophoresis indicated that smooth muscle plasma membranes showed a higher electrophoretic mobility than the intracellular membranes. These results suggest that ionic interactions on the cell membrane surfaces differ from those on the intracellular membrane surfaces and that induction of membrane aggregation by Ca2+ or Mg2+ is a useful procedure for an effective and rapid preparation of plasma membrane enriched fraction from smooth muscle.     

Effects of cAMP- and cGMP-dependent protein kinases, and calmodulin on Ca2+ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle

Sarcolemmal fractions of vascular smooth muscles were prepared from porcine thoracic aortae by differential and sucrose density gradient centrifugation. In these fractions, there was a high activity of 5'-nucleotidase, a putative marker enzyme of plasma membrane, and a low activity of rotenone insensitive NADH-cytochrome c reductase a marker of sarcoplasmic reticulum. In these fractions, the Ca2+ uptake was ATP-dependent. A low concentration of saponin which inhibited Ca2+ uptake by the plasma membrane but not by the sarcoplasmic reticulum, inhibited 65% of the Ca2+ uptake of this fraction. The Ca2+ uptake of this fraction was enhanced by cAMP- and cGMP-dependent protein kinases, and by calmodulin. The cAMP-dependent protein kinase enhanced the phosphorylation of 28 and 22 kDa proteins, while the cGMP-dependent protein kinase phosphorylated the 35 kDa protein. The phosphorylation of 100, 75, 65, 41 and 22 kDa proteins was enhanced by Ca2+ and calmodulin. These results indicate that cAMP- and cGMP-dependent protein kinases as well as calmodulin play important roles in Ca2+ transport in the sarcolemma, and that the phosphorylated proteins may be associated with an enhancement of Ca2+ transport in the sarcolemma.     

The asymmetric effect of lanthanides on Na+-gradient-dependent Ca2+ transport in synaptic plasma membrane vesicles

Lanthanides (La3+, Pr3+ and Tb3+) inhibit Na+-gradient-dependent Ca2+ influx into synaptic plasma membrane vesicles. 50% inhibition is obtained by 7 microM lanthanide concentration. The inhibition of the Na+-gradient-dependent Ca2+ uptake exhibits competitive kinetic behaviour. The apparent Km of the Ca2+ influx is increased from 50 microM in the absence of lanthanides to 118 microM in the presence of La3+, 170 microM in the presence of Pr3+ and 130 microM in the presence of Tb3+. The maximal reaction velocity is not altered (8.35 nmol Ca2+ transported per mg protein per min in the absence of lanthanides and 8.16 nmol/mg per min in the presence of lanthanides). Lanthanides also inhibited Na+-gradient-dependent Ca2+ efflux from synaptic plasma membrane vesicles that were preloaded with Ca2+ in a Na+-gradient-dependent manner. Introduction of La3+ into the interior of the synaptic plasma membrane vesicles by rapid freezing of the vesicles in liquid N2 and slow thawing had no effect on either Na+-gradient-dependent Ca2+ influx or efflux. Synaptic plasma membrane vesicles can be preloaded with Ca2+ also in an ATP-dependent manner. This form of Ca2+ uptake is also inhibited by La3+ though at higher concentrations than the Na+-gradient-dependent Ca2+ uptake. Na+-gradient-dependent efflux from synaptic plasma membrane vesicles preloaded in an ATP-dependent fashion ('inside-out' vesicles) unlike efflux from synaptic plasma membrane vesicles preloaded in a Na+-gradient-dependent manner was not inhibited by La3+. These findings suggest that the inhibition by La3+ is manifested asymmetrically on both sides of the synaptic plasma membrane. Lanthanides are probably not transported via the Na+-Ca2+ exchanger since Tb3+ entry measured by fluorescence of Tb3+-dipicolinic acid complex formation occurred at high Tb3+ concentrations only (1.5 mM or above) and was not Na+-gradient dependent.     

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Calmodulin-sensitive ATP-dependent Ca2+ transport across adipocyte plasma membranes

An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.     

Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction ( SF2 ) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction ( SF1 ) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.     

ATP-dependent Ca2+-uptake by the plasma membrane fraction of the myometrium

The specific activities of Mg2+, Ca2+-ATPase in the plasma membrane fraction of rabbit and cattle myometrium are 8.30 +/- 0.80 and 2.36 +/- 0.48 mkmoles of Pi per mg of protein, respectively. This fraction possesses a higher (in comparison with other subcellular fractions) capacity for ATP-dependent uptake of 45Ca2+ (9.37 +/- 1.66 and 6.86 +/- 0.96 nmoles of 45Ca2+ per mg of protein in 15 min for rabbit and cattle myometrium, respectively); the ratio of ATP-dependent uptake of Ca2+ to adsorbed Ca2+ is also high. Phosphate increases Ca2+ uptake in the presence of ATP and Mg2+. The ionophore A-23187 added to the incubation mixture without ATP and Mg2+ sharply increases Ca2+ binding. An addition of the ionophore at the 15th min of the ATP-dependent Ca2+ uptake causes a complete and rapid release of the accumulated Ca2+. The release of Ca2+ can be also caused by an addition of Na-DS or EGTA to the incubation mixture. This suggests that Ca2+ is accumulated through the plasma membrane inside the closed structures. It was assumed that myometrial sarcolemma plays an essential role in regulation of intracellular Ca2+ concentration in the uterus at rest and that the active Ca2+ efflux from the cells is controlled by the Mg2+, Ca2+-ATPase system.