Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum

The effect of archidonic, oleic and linoleic acid on calcium uptake and release by sarcoplasmic reticulum isolated from longissimus dorsi muscle was investigated using a Ca²⁺ electrode. All three long chain fatty acids stimulated the release of Ca²⁺ from sacroplasmic reticulum when added after exogenous Ca²⁺ was accumulated by the vesicles, and also inhibited Ca²⁺ uptake when added before Ca²⁺. This inhibitory effect on the calcium transport by arachidonic, oleic and linoleic acid was prevented by bovine serum albumin through its ability to bind with the fatty acid. The order of effectiveness of the fatty acids in inhibiting calcium transport by isolated sarcoplasmic reticulum was $\text{arachidonic acid> oleic acid >}$ linoleic acid. Similar inhibition of calcium uptake and induction of calcium release by arachidonic acid was observed in muscle homogenate sarcoplasmic reticulum preparations. Both arachidonic and oleic acid stimulated the (Ca²⁺ + Mg²⁺)-ATPase activity of sarcoplasmic reticulum at low concentrations, but inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity at high concentrations. The maximal (Ca²⁺ + Mg²⁺-ATPase activity observed with arachidonic acid was twice that obtained with oleic acid, but the concentration of arachidonic acid required was 3–4-times greater than that of oleic acid. The concentration of arachidonic acid required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity was 3.6-times greater than that needed for complete inhibition of calcium accumulation by the sacroplasmic reticulum. With oleic acid, however, the concentration required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity inhibited the sarcoplasmic reticulum Ca²⁺ accumulation by 72%. The present data support our hypothesis that, in porcine malignant hyperthermia, unsaturated fatty acids from mitochondrial membranes released by endogenous phospholipase A₂ would induce the sarcoplasmic reticulum to release calcium (Cheah K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84).     

Skeletal muscle mitochondrial phospholipase A2 and the interaction of mitochondria and sarcoplasmic reticulum in porcine malignant hyperthermia

Comparative studies were carried out on the Ca²⁺-transport systems of mitochondria and sarcoplasmic reticulum from longissimus dorsi muscle of genetically selected malignant hyperthermia-prone and normal pigs in order to identify the biochemical lesion responsible for the enhanced release of Ca²⁺ in the sarcoplasm occurring in porcine malignant hyperthermia. Mitochondria isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs contained a significantly (P < 0.001) higher amount of endogenous long-chain fatty acids. Similar amounts of endogenous mitochondrial phospholipase A₂ were observed in both types of pigs, but the total activity in malignant hyperthermia-prone pigs was at least twice that of normal. Spermine, a phospholipase A₂ inhibitor, lowered the activity in both types of mitochondria to a similar final level. Mitochondria of malignant hyperthermia-prone pigs showed a significantly (P < 0.001) higher oligomycin-insensitive (Ca²⁺ + Mg²⁺)-ATPase activity, but the Mg²⁺-ATPase and the (Ca²⁺ + Mg²⁺)-ATPase activities were similar in both types of pigs. Sarcoplasmic reticulum isolated from longissimus dorsi muscle of malignant hyperthermia-prone pigs showed a significantly higher (Ca²⁺ + Mg²⁺)-ATPase activity and a lower rate of Ca²⁺ uptake; the maximal amount and the rate of Ca²⁺ uptake by sarcoplasmic reticulum of malignant hyperthermia-prone pigs were half that of normal. Mitochondria from longissimus dorsi muscle of malignant hyperthermia-prone pigs inhibited the Ca²⁺-transport system of the sarcoplasmic reticulum of longissimus dorsi from both normal and malignant hyperthermia-prone pigs, but mitochondria from normal pigs had no influence on the sarcoplasmic reticulum from either type. Experimental evidence favours the concept that long-chain fatty acids released from skeletal muscle mitochondria by endogenous mitochondrial phospholipase A₂ are responsible for the enhanced release of Ca²⁺ from mitochondria (Cheah, K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84), and also additional release of Ca²⁺ from sarcoplasmic reticulum into the sarcoplasm during porcine malignant hyperthermia syndrome.     

The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III

Release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca²⁺. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca²⁺ + Mg²⁺)-ATPase in the presence of Ca²⁺ causes the rapid phosphorylation of the enzyme at which time the bound Ca²⁺ becomes inaccessible to external EGTA. The release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded from the Ca²⁺-binding site which precedes the release of Ca²⁺ into the vesicle. The rate of Ca²⁺ accumulation by sarcoplasmic reticulum vesicles is increased by K⁺ (5–100 mM) and ATP (50–1000 μM) but the initial rate of Ca²⁺ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca²⁺ was not influenced by these concentrations of K⁺ and ATP.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Isolation and Partial Characterization of an Acetylcholine Receptor-Enriched Membrane Fraction from Skeletal Muscle

Abstract

A procedure for purification of the bungarotoxin-binding fraction of sarcolemma from rabbit skeletal muscle is described. Muscle is homogenized in 0.25M sucrose without high salt extraction and membrane fractions separated initially by differential centrifugation procedures. An ultracentrifugation pellet enriched in cell surface and sarcoplasmic reticulum markers is further fractionated on a dextran gradient (density = 1.0 to 1.09). Two fractions are identified as sarcolemma according to high specific activities for lactoperoxidaseiodination, Na⁺, K⁺-ATPase and α-bungarotoxin-binding. No Ca⁺⁺, Mg⁺⁺-ATPase activity is found in these fractions. A third fraction, the dextran gradient pellet, is enriched in Ca⁺⁺, Mg⁺⁺-ATPase activity and lactoperoxidase iodinatable material and characterized by low bungarotoxin binding. This fraction represents a mixture of sarcoplasmic reticulum and transverse tubules with some sarcolemma contamination.     

Effects of fatty acids on calcium-stimulated adenosine triphosphatase in rat submandibular gland microsomes.

Effects of fatty acids on Ca²⁺-ATPase and Mg²⁺-ATPase in the microsomal fraction of rat submandibular gland have been investigated. Saturated fatty acids had almost no effect, but unsaturated fatty acids inhibited both ATPases. Modes of inhibition by linoleic acid were as follows: competitive for calcium and ATP with Ca²⁺-ATPase; non-competitive for magnesium and ATP with Mg²⁺-ATPase     

Heterogeneity of SH groups in sarcoplasmic reticulum.

The incorporation of [¹⁴C] N-ethylmaleimide reveals fast and slow-reacting sulfhydryl groups in sarcoplasmic reticulum. Two proteins react with the label: a fast-reacting glycoprotein recently isolated (Ikemoto, Cucchiaro and Garcia (1976) J. Cell Biol.$\text{70}$, 290a), and the Ca²⁺-ATPase. Labeling sarcoplasmic reticulum with a maleimide spin label gives a similar pattern. The spectra of maleimide-spin-labeled sarcoplasmic reticulum have both ‘strongly’ and ‘weakly’ immobilized components. Maleimide-spin-labeled purified Ca²⁺-ATPase, or sarcoplasmic reticulum labeled first with N-ethylmaleimide, and then with maleimide spin label, show spectra devoid of the ‘weakly’ immobilized component; the latter is enhanced in partially purified glycoprotein obtained from spin-labeled sarcoplasmic reticulum. This indicates that spectra from maleimide-spin-labeled sarcoplasmic reticulum do not reflect exclusively the state of the Ca²⁺-ATPase enzyme.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Ionophorous properties of the 20,000-dalton fragment of (Ca2++Mg2+)-ATPase in phosphatidylcholine: Cholesterol membranes

Summary The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca²⁺+Mg²⁺)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem.251:4147) to have Ca²⁺-selective ionophoric activity. The Ca²⁺-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca²⁺-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same,P _Ba>P _Ca>P _Sr>P _Mg>P _Mn. This selectivity sequence is the same as that for the intact (Ca²⁺ +Mg²⁺)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except thatP _Mn>P _Mg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca²⁺-ionophoric activity of the (Ca²⁺+Mg²⁺)-ATPase.     

Resistance of Ca2+-ATPase to dilution by excess phospholipid in reconstituted vesicles

Ca²⁺-ATPase and other membrane proteins of the sarcoplasmic reticulum membrane from rabbit skeletal muscle have been reconstituted into lipid vesicles with increasing amounts of phosphatidylcholine. The protein composition and phospholipid concentration of these vesicles were analyzed by determining the density of the reconstituted membrane vesicles on linear H₂O-²H₂O gradients, in a constant concentration of sucrose. In all combinations of the Ca²⁺-ATPase with a weight excess of phosphatidylcholine, the reconstituted vesicles had a phospholipid-to-protein ratio similar to that of the native sarcoplasmic reticulum membrane, even though both solubilization and mixing had occurred. These vesicles of low phospholipid and high protein content exhibited all the original Ca²⁺-ATPase activity and ATP-stimulated calcium transport. The Ca²⁺-ATPase, and the calcium-binding proteins to a lesser extent, may order the lipid in such a manner so as to maintain the initial stoichiometry of lipid to protein observed in the native sarcoplasmic reticulum membrane.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase and other ATPases.

Vanadate is a potent inhibitor of the Ca²⁺-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca²⁺ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca²⁺Mg²⁺-ATPases of the ascites and human red cell plasma membranes, Mg²⁺-ATPase of the ascites plasma membrane, and the K⁺-ATPases of $\text{E.}\text{coli}$ and hog gastric mucosal cell membranes. The ascites plasma membrane Ca²⁺-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate.     

Effect of jasmonic acid on Ca<Superscript>+2</Superscript> transport through the plasmalemma of potato tuber cells

The rate of Ca²⁺ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10^?5–10^?10 M jasmonic acid on the accumulation of Ca⁺² in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca⁺²,Mg⁺²-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca⁺²,Mg⁺²-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.     

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.     

Environmental cadmium is not sequestered by metallothionein in rainbow trout

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca²⁺ transport which is mediated by a (Ca²⁺ + Mg²⁺)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-M_r in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-M_r phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca²⁺ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.     

Influence of monovalent cations on the Ca2+-ATPase of sarcoplasmic reticulum isolated from rabbit skeletal and dog cardiac muscles. An interpretation of transient-state kinetic data

Transient-state kinetics of phosphorylation and dephosphorylation of the Ca²⁺-ATPase of sarcoplasmic reticulum vesicles from rabbit skeletal and dog cardiac muscles were studied in the presence of varying concentrations of monovalent and divalent cations. Monovalent cations affect the two types of sarcoplasmic reticulum differently. When the rabbit skeletal sarcoplasmic reticulum was Ca²⁺ deficient, preincubation with K⁺ (as compared with preincubation with choline chloride) did not affect initial phosphorylation at various concentrations of Ca²⁺, added with ATP to phosphorylate the enzyme. This is in contrast to preincubation with K⁺ of the Ca²⁺-deficient dog cardiac sarcoplasmic reticulum, which resulted in an increase in the phosphoenzyme level. When Ca²⁺ was bound to the rabbit skeletal sarcoplasmic reticulum, K⁺ inhibited E ~ P formation; but under the same conditions, E ~ P formation of dog cardiac sarcoplasmic reticulum was activated by K⁺ at 12 μM Ca²⁺ and inhibited at 0.33 and 1.3 μM Ca²⁺. Li⁺, Na⁺ and K⁺ also have different effects on E ~ P decomposition of skeletal and cardiac sarcoplasmic reticulum. The latter responded less to these cations than the former. Studies with ADP revealed differences between the two types of sarcoplasmic reticulum. For rabbit skeletal sarcoplasmic reticulum, 40% of the phosphoenzyme formed was ‘ADP sensitive’, and the decay of the remaining E ~ P was enhanced by K⁺ and ADP. Dog cardiac sarcoplasmic reticulum yielded about 40–48% ADP-sensitive E ~ P, but the decomposition rate of the remaining E ~ P was close to the rate measured in the absence of ADP. Thus, these studies showed certain qualitative differences in the transformation and decomposition of phosphoenzymes between skeletal and cardiac muscle which may have bearing on physiological differences between the two muscle types.     

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current.On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca²⁺ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca²⁺, Mg²⁺, or Mn²⁺ are detected. A23187 produces fluorescent transients under conditions of Ca²⁺ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca²⁺ content. Changes in A23187 fluorescence due to mitochondrial Ca²⁺ accumulation are much smaller by comparison and fluorescence transients are not detected.Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca²⁺ and Mg²⁺, and less than 2 nsec in the presence of excess EDTA.The present results are consistent with a model of ionophore-mediated cation transport in which free M²⁺ binds with A23187 at the membrane surface to form the complex M(A23187)⁺. Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187)₂, which carries the divalent cation through the membrane.These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.