Madoka Makinose

ATP synthesis by the reverse of the sarcoplasmic calcium pump

The result of the release studies described in the preceeding report [l] prompted the search for net ATP synthesis by the sarcoplasmic calcium pump. It is shown that under the conditions where a fast ADP and inorganic phosphate dependent calcium release takes place from calcium loaded sarcoplasmic vesicles, the net outward movement of calcium is stoichiometrically related to a net formation of ATP. The experimental conditions are given in the legend to fig. 1.

Activation of calcium efflux by ADP and inorganic phosphate

The release of calcium from sarcoplasmic vesicles preloaded with calcium oxalate or calcium phosphate has been studied under various conditions such as the addition of high concentration of EGTA to ATP containing calcium uptake media or the depletion of EGTA containing uptake media from ATP [l-3] Under both conditions, the observed initial release rates are unidirectional flux rates because the calcium activity in the solution surrounding the vesicles is very low. When the vesicles were loaded with 300-500 nmoles calcium oxalate/mg of vesicular protein, the rates of release were found to be about 100 times slower than the initial rate of calcium uptake [ 1 ] . It has been shown that the very slow calcium efflux must be attributed to a low calcium permeability of the membranes, since the hydrolysis of membranal lipids or the binding of surface active agents induces a very fast calcium release. The effect of these membrane modifying treatments cannot be reversed. In this report it is shown that in contrast to these unspecific and irreversible permeability changes the calcium efflux is most specifically and reversibly activated by inorganic phosphate in combination with ADP. The experimental conditions are given in the legends to the figures.

Der physiologische Erschlaffungsfaktor und die Muskelgrana

1. 1. Practically the entire granule fraction which inhibits ATP-splitting and contraction is precipitated from crude rabbit-muscle extract by centrifuging at 25,000 × g for 1 h. The diameter of the granules in this fraction is about 1000 A (700–3000 A). 2. 2. The activity of the granules is irreversibly destroyed by SH-reagents, thymol and u.v. light. Inhibition caused by certain detergents, e.g. antarox is partially reversible. 3. 3. The activity of the granules is irreversibly lost when the granules are digested with several times their weight of trypsin in the absence of ATP, but is not affected when ATP is present. 4. 4. Before they can produce inhibition, the granules must be activated by ATP and Mg in the absence of Ca. Activation takes only a few minutes, and occurs normally during the experiments themselves. 5. 5. The inhibition of ATP splitting and contraction of fibrils or actomyosin floccules produced by the granules ceases as soon as the granules are removed. 6. 6. Thus, the granules do not withdraw any functionally essential substances from the contractile proteins, nor do they release any stable contraction-inhibiting substance to the actomyosin or to the solution. 7. 7. Over a wide concentration range, the effect of the granules does not depend on their absolute concentration, but on the granule/fibril ratio, in spite of the fact that the granules are not bound to the fibrils. Hence, the granules can not be identical with the inhibitor which reacts directly with the actin- and myosin-filaments. 8. 8. It seems probable, however, that the granules release a labile inhibitor, since their relaxing effect can be exhausted by repeated washing with fibril suspensions. During such washings, the fibrils remove no granules, but merely take away their activity. 9. 9. It is shown that all relaxing and inhibiting effects of the granules in actomyosin gels are brought about only by dissociation of the actomyosin in the presence of ATP and granules. The L-myosin-ATPase as well as the ability for and the state of polymerisation of the actin are not affected by the relaxing granules.

Calcium efflux dependent formation of ATP from ADP and orthophosphate by the membranes of the sarcoplasmic vesicles

After the cessation of the ATP dependent calcium uptake the sarcoplasmic vesicles can maintain steep concentration gradients across their membranes. In assay media containing 5 mM oxalate or 20 mM phosphate in addition to 5 mM magnesium ATP the final concentration ratio calcium inside/calcium outside was found to be 2 3000.ThisGlci~~adient is maintained as long as ATP is present whereby the calcium inside the vesicles is exchanged continuously with the calcium in the solution outside. The exchange proceeds considerably slower than the net uptake of calcium [ 1,2] . In this report it is shown that during this exchange of calcium inorganic phosphate is steadily incorporated into the ATP fraction. The experimental conditions are given in the legends to the figures.

Possible functional states of the enzyme of the sarcoplasmic calcium pump.

The calcium accumulation performed by the sarcoplasmic vesicles isolated from the skeletal muscle is a unique example for an active ion transport process by biomembranes. The high transport activity on the one hand and the nearly complete absence of other enzymatic activities on the other hand, made it possible to perform detailed kinetic studies of calcium translocation and of the coupled ATP (NTP) hydrolysis. In this respect the sarcoplasmic membrane proved to be superior to most other membranes with transport activity. On the basis of such studies, a number of reaction mechanisms of the sarcoplasmic calcium pump were proposed by several authors with more or less experimental support [1 -8 ] . In the last years, it was demonstrated that the calcium accumulation by the sarcoplasmic vesicles is a reversible process [9 -11 ]. The translocation of calcium in both directions across the membrane is provided by the formation of an activated in t e rmed ia te a phosphoprotein [ 12,13]. Recently, it has been shown that, even in the absence of energy-rich phosphate donators, the vesicle protein can be phosphorylated with orthophosphate after formation of a steep concentration gradient of calcium ions across the membrane [ 12,13]. This phosphoprotein has the same chemical properties as those of the phosphoproteins obtained in the presence of energy rich phosphate donators and was assumed to be an acyl phosphate. These observations indicate directly that the sarcoplasmic membrane acts as an energy converter which transduces osmotic energy into chemical I06

Phosphoprotein formation during osmo-chemical energy conversion in the membrane of the sarcoplasmic reticulum

The isolated vesicles of the sarcoplasmic reticulum accumulate actively calcium from the outside solution using the chemical energy of nucleoside triphosphates or acyl phosphates like acetyl phosphate [ l-31. The process of this active calcium accumulation is obviously the result of a series of reaction steps and one of which is the formation of phosphorylated protein [4, 51. During the calcium uptake, the concentration of ionic calcium inside the vesicles becomes 3000 times (or more) higher than that in the outside milieu of the vesicles. Recently, it was demonstrated [6-81 that. under specific conditions, the accumulated calcium can be released rapidly from the vesicles and that this release of calcium is coupled with the synthesis of ATP from ADP and PO, in the solution. Later, the phosphorylation of ADP during calcium efflux has been reported also by Panet and Selinger [9] although the specific requirements for the calcium release coupled phosphate incorporation obviously were not recognized. Analysis of these observations, especially the strict stoichiometry between the calcium efflux and the ATP synthesis, suggests that these coupled reactions are the reversal of the active calcium accumulation and the so called “extra ATP-splitting” by the transport ATPase. In this paper, evidence is presented that

The inhibition of the sarcoplasmic calcium pump by prenylamine, reserpine, chlorpromazine and imipramine

Zusammenfassung1. Reserpin, Prenylamin, Chlorpromazin und Imipramin hemmen sowohl die Geschwindigkeit der Calcium-Aufnahme als auch die der Calcium-induzierten Extra-ATP-Spaltung an isolierten Vesikelmembranen des sarkoplasmatischen Reticulums von quergestreifter Muskulatur des Kaninchens. Die für eine 50%ige Hemmung erforderlichen Konzentrationen betragen beim Reserpin, Prenylamin und Chlorpromazin 3×10−5M, beim Impiramin ∼3×10−4M.2. Calcium-Efflux und -Influx werden durch die Pharmaka in gleichem Ausmaß gehemmt.3. Das Konzentrationsvermögen der Vesikel für Calcium wird deshalb durch die Hemmstoffe nicht beeinflußt.4. Der erste Reaktionsschritt des aktiven Calciumtransports der Vesikel — die Übertragung des Phosphates des ATP auf das Vesikelprotein — wird durch die Pharmaka nicht beeinflußt.Summary1. The calcium induced increase in ATPase and the rate of calcium uptake of the vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle are reduced by reserpine, prenylamine, chlorpromazine and imipramine. 3×10−5M reserpine, prenylamine, chlorpromazine and ∼3×10−4M imipramine are required to produce 50% inhibition of both activities.2. In the presence of the drugs the rate of calcium exchange at the cessation of calcium uptake is reduced to the same extent as the initial rate of calcium uptake.3. Neither the calcium storing capacity nor the ability to concentrate calcium are impaired by the drugs.4. The drugs do not affect the calcium dependent phosphate exchange between ATP and ADP and the calcium dependent formation of phosphoprotein.

The binding of the calcium transport inhibitors reserpine, chlorpromazine and prenylamine to the lipids of the membranes of the sarcoplasmic reticulum

Zusammenfassung1. Reserpin, Chlorpromazin, Prenylamin und Imipramin werden sowohl von den isolierten Vesikeln des sarkoplasmatischen Reticulums (quergestreifte Muskulatur von Kaninchen) als auch von den aus diesen mit Chloro-form-Methanol (2:1) extrahierten Lipiden gebunden.2. Bei einer Konzentration von 10−4 M werden Reserpin, Chlorpromazin und Prenylamin von dem intakten Lipoproteinkomplex der Vesikelmembran in Mengen von 0,1 μmol/mg Vesikelprotein, von 0,2 μmol/mg vesikulärem Total-Lipid und von ∼0,4 μmol/mg Phosphatidylcholin (Lecithin) gebunden. Lipidfreies Vesikelprotein hat keine Bindungsfähigkeit.3. Die Bindung der Pharmaka ist reversibel und temperaturunabhängig.4. Die Bindungsfähigkeit der Phospholipide ist nicht beeinträchtigt, wenn diese durch Phospholipase A, C oder D hydrolysiert werden. Sie wird jedoch aufgehoben durch Hydrierung der isolierten bzw. der membrangebundenen Lipide. Nach Hydrierung ist auch die Calcium-Aufnahme, die ATP-Extraspaltung und die Phosphatübertragung aufgehoben.5. Die lipidfreien Proteine Myokinase und Actin werden durch den Hydrierungsvorgang funktionell nicht beeinflußt.Summary1. Reserpine, chlorpromazine, prenylamine and imipramine are bound to vesicles isolated from the sarcoplasmic reticulum of rabbit striated muscle cells as well as by the lipids which can be extracted from the vesicular preparations.2. At concentrations of 5×10−7 M reserpine, chlorpromazine and prenylamine, drug binding can just be detected. At drug concentrations of 10−4 M the drug uptake amounts to 0.1 μmol per mg vesicular protein, 0.2 μmol drug per mg vesicular lipid and ∼0.4 μmol drug per mg phosphatidylcholine.3. Drug binding is reversible and temperature independent.4. Drug binding is not diminished when the phospholipids are hydrolysed by phospholipase A, C or D.5. Drug binding is abolished by hydrogenation of the isolated lipids as well as of the lipids bound to the membranes.6. In addition calcium uptake the Ca++ induced increase in ATP breakdown and the phosphate transfer reaction are inhibited when the lipids in the membranes are hydrogenated.7. The normal ATPase as well as the lipid free proteins myokinase and actin are not affected by hydrogenation.

Phosphorylation of skeletal muscle microsomes by acetylphosphate

The phosporyl group of acetylphosphate is transferred to the membranal protein of the sarcoplasmic vesicles during active calcium transport. Although the phosphoprotein formed cannot be distinguished from that obtained in the presence of ATP, the conditions for ATP and acetylphosphate hydrolysis are different from each other.

Mechanism of calcium‐independent phosphorylation of sarcoplasmic reticulum ATPase by orthophosphate

Magnesium affects several reaction steps of the calcium transport cycle in sarcoplasmic reticulum membranes [ 1,2]. Recent studies on the effect of magnesium on sarcoplasmic reticulum function [3] indicate a dual role of magnesium on the phosphorylation of sarcoplasmic reticulum transport ATPase from ATP, as evaluated from analysis of the exchange rate of y-phosphate between ATP and ADP [4] in the presence of saturating calcium concentrations: (i) That magnesium activates the enzyme directly; (ii) That it represents part of the substrate MgATP for the phosphorylation reaction [3]. From studies on the role of magnesium in calciumindependent and calciumdependent phosphorylation of sarcoplasmic reticulum transport ATPase from orthophosphate [5-191, the formation of a magnesium-phosphoprotein in calcium-independent phosphorylation was suggested [ 181. Based on the good fit of a reaction scheme to the data the following features were proposed: (i) The phosphoprotein (Mge E-P,), in which the phosphate is covalently bound to the enzyme, is in equilibrium with the Michaelis complex (Mg . E . Pi), the concentration of which is determined by the concentration of free orthophosphate and free magnesium; (ii) The binding of orthophosphate and magnesium appears to be interdependent, both ligands apparently bind randomly [ 181.