Freeze-fracture study of water-soluble,standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca2+-ATPase

Conventional freeze-fracture electron microscopy was used to study water-soluble proteins and different forms of Ca²⁺-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was close to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca²⁺-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particles sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca²⁺-ATPase. The results can be interpreted on the assumption that the Ca²⁺-ATPase molecule is elongated, with an overall length of about 110 Å and a width in its largest part of about 75 Å. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.     

Photoaffinity spin-labeling of the Ca2+ ATPase in sarcoplasmic reticulum : Evidence for oligomeric structure

A spin labeled fatty acid (16-doxylstearic acid) was linked to a photochemical reacting group (azido derivative). When the molecule is introduced, at a low concentration, into rabbit sarcoplasmic reticulum membranes, the spectrum before illumination is identical to the spectrum obtained with the corresponding spin labeled fatty acid. After illumination, a large immobilized components is seen. It corresponds to about 70% of the ESR signal of the effectively bound label, at room temperature. The fraction of immobilized component varies with temperature, from 100% at 0°C to 50% at 35°C. Addition of a small amount of detergent (dodecyl octaethylene glycol monoether), under non solubilizing conditions, decreases the fraction of signal due to a strongly immobilized probe. A possible interpretation is that the immobilized signal reflects protein bound spin labels trapped in Ca²⁺ ATPase oligomers, which are partially dissociated by detergent addition or temperature increase.     

Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca²⁺-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca²⁺-binding protein and a M₅₅ protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca²⁺-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of ³²P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca²⁺ uptake. The Ca²⁺-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca²⁺-pump and Ca²⁺-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca²⁺-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles ³²P-labelled phosphoenzyme per mg protein. The Ca²⁺-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca²⁺ binding by sarcoplasmic reticulum vesicles and by the Ca²⁺-pump and Ca²⁺-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca²⁺-pump protein contained nonspecific high-affinity Ca²⁺ binding sites with a capacity of 90–100 and 55–70 nmoles Ca²⁺ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca²⁺ per mg protein. The binding constants for nonspecific and specific Ca²⁺ binding by both preparations were approx. 1 μM^?1. The Ca²⁺-binding protein nonspecifically bound 900–1000 nmoles Ca²⁺ per mg protein with a binding constant of about 0.25 μM^?1.     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.     

Dynamic light scattering characterization of the detergent-free,delipidated (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Dynamic light scattering studies have been conducted on the delipidated and detergent-removed (Ca²⁺ + Mg²⁺)-ATPase protein assemblies. Specific characterization of the state of aggregation and the extent of conformation change upon delipidation and detergent removal has been made. The results show that the prominent species are dimers and tetramers of very globular nature, with axial ratios of less than 2 : 1. The hydrodynamic radii of the dimers and the tetramers are, respectively, 57.5 Å and 74.5 Å.The globular nature of these observed entities differ from the delipidated ATPase proteins recently obtained (LeMaire, M., Jorgensen, K.E., Roigaard-Petersen, H. and Moller, J.V. (1976) Biochemistry 15, 5805–5812). Present results suggest that upon the removal of detergents from the lipid-free ATPase protein assembly, only a rather limited degree of aggregation takes place. Such a condition is consistent with models of the membrane protein system which has limited regions of hydrophobic contact. Oligomeric assemblies with aqueous channels is a possible active Ca²⁺ transport model consistent with results of the present data, as well as the data from several other recent studies.     

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.     

Inhibition of calmodulin-activated Ca2+-ATPase by propranolol and nadolol

Propranolol, at concentrations ranging from 0.05 to 0.5 mM, inhibits the calmodulin-activated Ca²⁺-ATPase of human erythrocyte membranes. In the same concentration range it is without effect on the basal Ca²⁺-ATPase. The inhibition is competitive and appears to be due to membrane binding, rather than to combination with cytoplasmic calmodulin as is the case for phenothiazines. This effect of propranolol may explain its ability to open the calcium-gated potassium channel, and could also be related to its action as a β-adrenergic blocker. Nadolol, another β-adrenergic blocker, is also an inhibitor of calmodulin-activated Ca²⁺-ATPase.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles I. Ca2+ outflow in the non-energized state of the calcium pump

Passive Ca²⁺ permeability of sarcoplasmic reticulum vesicles has been studied after maximal loading with Ca²⁺ (150–200 nmol/mg protein) in the presence of Ca²⁺, MgATP and an ATP generating system of limited capacity. Outflow of accumulated Ca²⁺ in the non-energized state of the system was studied by depletion of the medium of one of the substrates, either MgATP (by complete consumption) or Ca²⁺ (by complexation with EGTA). It was found that Ca²⁺ outflow under these conditions is relatively slow and independent of the medium concentration of Ca²⁺ (5·10^?9–5·10^?5 M) or MgATP (0.7–730 μM). Outflow curves were steep at the beginning of the outflow phase (30–60 nmol/min per mg protein), and outflow proceeded at a much lower rate below 100 nmol Ca²⁺/mg protein. Outflow could be completely inhibited by La³⁺. The Ca²⁺ release curves are not compatible with simple diffusion, and cannot be accounted for by Ca²⁺ binding inside the vesicles. Neither are our observations consistent with permeation mediated via the Ca²⁺ translocation sites involved in active transport. We suggest that non-energized Ca²⁺ outflow may proceed by a process of ion-exchange through negatively charged, water-filled channels in the membrane, the properties of which are altered by a high intravesicular concentration of Ca²⁺.     

Ionophorous properties of the 13 000-Da fragment from sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

The 25 000-Da tryptic fragment from rabbit muscle sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase was subjected to cyanogen bromide digestion, and the four fragments isolated. Only the 13 000-Da fragment induced ionophorous activity in planar thin lipid membranes made with 5:1 (w/w) phosphatidylcholine/cholesterol in decane. The membranes became cation selective, with a selectivity sequence among divalent of Mn²⁺ > Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺. This is different from that of the 25 000-Da fragment (A.E. Shamoo, 1978, J. Memb. Biol. 43, 227–242), it's ‘parent’ 55 000-Da fragment, and the intact enzyme, all of which have the same selectivity sequence. The inhibitory effects of Hg²⁺, Cd²⁺ and Zn²⁺ were also examined. All were inhibitory, with Zn²⁺ being the most effective of these. The heavy-metal-induced inhibition of Ca²⁺ conductance could be reversed by selective chelation of the heavy metals by EDTA. From changes in the selectivity as well as changes in heavy-metal-induced inhibition behavior, we conclude that the ion transport site of the 13 000-Da fragment may not be the same site as that of the parent fragment. It is either a different site altogether or has been physically modified by peptide cleavage.     

Reversible shift between two states of Ca2+-ATPase in human erythrocytes mediated by Ca2+ and a membrane-bound activator

The (Ca²⁺ + Mg²⁺)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) from human erythrocytes occurred in two different states, A-state and B-state, depending on the membrane preparation.The A-state showed low maximum activity (V) and the Ca²⁺ activation was characterized by a Hill coefficient, n_H, of about 1 and a Michaelis constant, K_Ca, about 30 μM.The B-state showed high V, a n_H above 1, which indicates positive cooper-activity of Ca²⁺ activation, and a K_Ca of about 1 μM.With varying ATP concentrations, both the A-state and the B-state showed negative cooperativity and slightly different values of K_m.The B-state was shifted to the A-state when the membranes were exposed to low Ca²⁺ concentrations. The shift reached 50% at approx. 0.5 μM Ca²⁺. At the low Ca²⁺ concentrations an activator was released from the membranes.The A-state was shifted to the B-state when the membranes were exposed to Ca²⁺ in the presence of the activator. The shift reached 50% at about 30 μM Ca²⁺. The recovery of high V was time dependent and lasted several minutes. Increasing concentrations of Ca²⁺ and activator accelerated the recovery.It is suggested that the A-state and the B-state correspond to enzyme free of activator and enzyme associated with activator, respectively. Furthermore, the two states may represent a resting and an active state, respectively, of the calcium pump.     

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M., Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194–6198), which in contrast to Ca²⁺ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca²⁺ and Na⁺ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na⁺ and Ca²⁺ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca²⁺ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca²⁺ efflux from sarcoplasmic reticulum vesicles which had been loaded with Ca²⁺ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H⁺, OH^?, or Cl^?.     

Short-time effects of neuroactive steroids on rat cortical Ca2+-ATPase activity

Recent experimental evidence indicates that some steroid hormones, apart from their well-documented genomic actions, could produce non-genomic rapid effects, and are potent modulators of the plasma membrane proteins, including voltage- and ligand-operated ion channels or G protein-coupled receptors. Neuroactive steroids, 17β-estradiol, testosterone, pregnenolone sulfate and dehydroepiandrosterone sulfate, after a short-time incubation directly modulated the activity of plasma membrane Ca²⁺-ATPase purified from synaptosomal membranes of rat cortex. The sulfate derivatives of dehydroepiandrosterone and pregnenolone applied at concentrations of 10^?11–10^?6 M, showed an inverted U-shape potency in the regulation of Ca²⁺-ATPase activity. At physiologically relevant concentrations (10^?8–10^?9 M) a maximal enhancement of the basal activity reached 200%. Testosterone (10^?11–10^?6 M) and 17β-estradiol (10^?12–10^?9 M) caused a dose-dependent increase in the hydrolytic ability of Ca²⁺-ATPase, and the activity with the highest concentration of steroids reached 470% and 200%, respectively. All examined steroids decreased the stimulatory effect of a naturally existing activator of the calcium pump, calmodulin. The present study strongly suggests that the plasma membrane calcium pump could be one of the possible membrane targets for a non-genomic neuroactive steroid action.     

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.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+-ATPase and calmodulin insensitive (Na+ + K+)- and Mg2+-ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca²⁺-ATPase and calmodulin-insensitive (Na⁺ + K⁺)- and Mg²⁺-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca²⁺-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca²⁺-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Kinetic studies of Ca2+ release from sarcoplasmic reticulum of normal and malignant hyperthermia susceptible pig muscles

The time-course of Ca²⁺ release from sarcoplasmic reticulum isolated from muscles of normal pigs and those of pigs susceptible to malignant hyperthermia were investigated using stopped-flow spectrophotometry and arsenazo III as a Ca²⁺ indicator. Several methods were used to trigger Ca²⁺ release: (a) addition of halothane (e.g., 0.2 mM); (b) an increase of extravesicular Ca²⁺ concentration ([Ca₀²⁺]); (c) a combination of (a) and (b), and (d) replacement of ions (potassium gluconate with choline chloride) to produce membrane depolarization. The initial rates of Ca²⁺ release induced by either halothane or Ca²⁺ alone, or both, are at least 70% higher in malignant hyperthermic sarcoplasmic reticulum than in normal. The amount of Ca²⁺ released by halothane at low [Ca₀²⁺] in malignant hyperthermic sarcoplasmic reticulum is about twice as large as in normal sarcoplasmic reticulum. Membrane depolarization led to biphasic Ca²⁺ release in both malignant hyperthermic and normal sarcoplasmic reticulum, the rate constant of the rapid phase of Ca²⁺ release induced by membrane depolarization being significantly higher in malignant hyperthermic sarcoplasmic reticulum ($\text{k = 83}\text{s}{}^{\mathrm{?1}}$) than in normal ($\text{k = 37}\text{s}{}^{\mathrm{?1}}$). Thus, all types of Ca²⁺ release investigated (a, b, c and d) have higher rates in malignant hyperthermic sarcoplasmic reticulum than normal sarcoplasmic reticulum. These results suggest that the putative Ca²⁺ release channels located in the sarcoplasmic reticulum are altered in malignant hyperthermic sarcoplasmic reticulum.