In Vivo Effect of Uncoupling Agents on the Incorporation of Calcium and Strontium into Mitochondria and Other Subcellular Fractions of Rat Liver

After injection of ⁴⁵Ca⁺⁺ or ⁸⁹Sr⁺⁺ into rats, the largest part of the radioactivity in the liver cell is associated with the subcellular structures, only negligible amounts of it being found in the soluble hyaloplasm. 50 % or more of the ⁴⁵Ca⁺⁺ and ⁸⁹Sr⁺⁺ in the liver cell is recovered in the mitochondrial fraction. The specific activity of Ca⁺⁺ after injection of ⁴⁵Ca⁺⁺ is far greater in mitochondria than in microsomes. Pretreatment of the rats with uncouplers of oxidative phosphorylation markedly decreases the amount of radioactivity associated with the mitochondrial fraction. The amount of radioactivity recovered in the microsomes and in the final supernatant on the contrary increases. These effects are present only when mitochondrial oxidative phosphorylation is completely uncoupled. The Ca⁺⁺ content of mitochondria from the livers of rats pretreated with uncouplers is sharply decreased with respect to the controls. It is concluded that in the liver cells of the intact animal energy-linked movements of Ca⁺⁺ and Sr⁺⁺ take place in mitochondria.     

Studies on the Active Transport of Calcium in Human Red Cells

The Ca⁺⁺ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca⁺⁺ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of ⁴⁵Ca infused inside red cells indicated that over 95% of all Ca⁺⁺ in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP¹, CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca⁺⁺ transport does not appear to involve exchange of Ca⁺⁺ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca⁺⁺ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca⁺⁺ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.     

A comparative study of the role of mitochondria and the sarcoplasmic reticulum in the uptake and release of Ca++ by the rat diaphragm

The respective importance of mitochondria and of sarcoplasmic reticulum in the uptake and maintenance of Ca⁺⁺ by the isolated rat diaphragm has been compared. Diaphragms were incubated at 30° in conditions optimal for Ca⁺⁺ uptake either by isolated mitochondria or by sarcoplasmic reticulum: more Ca⁺⁺ was taken up from the “mitochondrial” medium. For maximal uptake, Pi and Mg⁺⁺ were necessary; substitution of NaCl and KC1 with sucrose had no effect on the uptake. The uptake was markedly inhibited by uncouplers of oxidative phosphorylation, by respiratory inhibitors, and by lowering the temperature of the incubation medium to 0°; it was not affected by oligomycin, aurovertin, DCCD, nor by inhibitors of Ca⁺⁺ transport in the isolated sarcoplasmic reticulum (ergotamine, ergobasinine, caffeine). The lack of effect of caffeine was not due to lack of penetration into the muscle. Permeability barriers for ergotamine and ergobasinine could not be excluded. The maintenance of Ca⁺⁺ by the diaphragm was optimal in a medium contaming Pi and Mg⁺⁺. Uncoupling agents and respiratory inhibitors accelerated the rate and extent of release of Ca⁺⁺ by the diaphragm. Lowering the temperature of the incubation medium to 0°, or addition of oligomycin, aurovertin, DCCD, had no effect on the release. The release of Ca⁺⁺ was also unaffected by ergotamine, ergobasinine, caffeine. The results suggest a role for mitochondria in the uptake and maintenance of Ca⁺⁺ by the isolated diaphragm.     

Studies on the in Vitro Interaction of Electrical Stimulation and Ca++ Movement in Sarcoplasmic Reticulum

Sarcoplasmic reticulum fragments (S.R.F.) were isolated from skeletal and heart muscles. These fragments were found to take up Ca⁺⁺ very actively from media. When monophasic square waves were passed through the S.R.F. suspension, the Ca⁺⁺ uptake by S.R.F. was decreased. When the suspension was stimulated electrically after the Ca⁺⁺ was taken up by S.R.F., the initiation and the cessation of the stimulation were followed by the release and re-uptake of Ca⁺⁺ by S.R.F., respectively. The degree of inhibition of the Ca⁺⁺ uptake as well as of the Ca⁺⁺ release by electrical stimulation was dependent on the voltage and the frequency of stimulation. The presence of inorganic phosphate or oxalate modified the influence of electrical stimulation on the release and the uptake of Ca⁺⁺ by S.R.F. Attempts were made to observe the release of Ca⁺⁺ by electrical stimulation from unfractionated sarcoplasmic reticulum remaining in myofibers, and the interaction of the released Ca⁺⁺ with myofibrils in vitro. For this purpose, the glycerol-extracted fiber was selected as a muscle model, since it contains both sarcoplasmic reticulum and myofibrils. It was found that electrical stimulation of skeletal and heart glycerol-extracted fibers resulted in the contraction of fibers. It appeared that the contraction of glycerol fibers by electrical stimulation was caused by the Ca⁺⁺ release from sarcoplasmic reticulum by stimulation.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

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.     

Interaction of metallochromic indicators with calcium sequestering organelles

Examples are presented of the interaction between cell organelles and metallochromic indicators used in the measurement of ionized Ca²⁺. Sarcoplasmic reticulum was found to sequester murexide type indicators along with Ca²⁺ in the presence of ATP, but not to sequester arsenazo III and antipyrylazo III. The presence of a permeable anion suppresses the sequestration of murexide type indicators by the sarcoplasmic reticulum. In the presence of ruthenium red, both rat liver and beef heart mitochondria release sequestered Ca²⁺ with arsenazo III, but not with murexide.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Ultrastructural distribution of Ca++ within neurons

Summary We used the oxalate-pyroantimonate technique to determine the ultrastructural distribution of Ca⁺⁺ in neurons of the rat sciatic nerve. The content of the precipitate was confirmed by X-ray microanalysis and appropriate controls. In the cell bodies of the dorsal root ganglia, Ca⁺⁺ precipitate was found in the Golgi, mitochondria, multivesicular bodies and large vesicles of the cytoplasm but not in lysosomes, and was prominently absent from regions of rough endoplasmic reticulum and ribosomes. It was seen in the nucleus but not in the nuclear bodies or nucleolus.Within the axon itself, Ca⁺⁺ precipitate was also found sequestered in mitochondria and smooth endoplasmic reticulum. In addition Ca⁺⁺ precipitate found diffusely throughout the axoplasm exhibited a discrete and heterogeneous distribution. In myelinated fibers the amount of precipitate decreased predictably in the axoplasm beneath the Schmidt-Lanterman clefts and in the paranodal regions at the nodes of Ranvier. This correlated with the presence of dense precipitate in the Schmidt-Lanterman clefts them-selves and in the paranodal loops of myelin.Intracytoplasmic ionic Ca⁺⁺ is maintained at 10^–7 M by balanced processes of influx, sequestration and extrusion. The irregular distribution of Ca⁺⁺ precipitate in the axoplasm of myelinated fibers suggests that there may be specific regions of preferential efflux across the axolemma.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

Mechanisms for Intracellular Calcium Regulation in Heart : I. Stopped-Flow Measurements of Ca++ Uptake by Cardiac Mitochondria

Initial velocities of energy-dependent Ca⁺⁺ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca⁺⁺ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca⁺⁺ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca⁺⁺. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca⁺⁺ transport in cardiac mitochondria. Comparable rates of Ca⁺⁺ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca⁺⁺ uptake by frog heart mitochondria were higher at any Ca⁺⁺ concentrations. The half-maximal rate of Ca⁺⁺ transport was observed at 30, 60, 72, 87, 92 µM Ca⁺⁺ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent K_m render mitochondrial Ca⁺⁺ uptake slow below 10 µM. At these concentrations the rate of Ca⁺⁺ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca⁺⁺ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca⁺⁺ by mitochondria is inadequate for regulating the beat-to-beat Ca⁺⁺ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca⁺⁺ uptake are discussed in terms of physiological regulation of intracellular Ca⁺⁺ homeostasis in cardiac cells.     

The subcellular calcium distribution in the smooth muscle cells of the pig coronary artery

In order to complete preliminary investigations on the subcellular calcium localisation in smooth muscle cells, further experiments are presented using smooth muscle cells from the coronary artery of the pig. The methods used were a precipitation technique using potassium oxalate and autoradiography using ⁴⁵Ca. In all cases we were able to reproduce the results obtained in our preliminary study. The preparations clearly show calcium oxalate precipitates in the cell membrane, the sarcoplasmic reticulum, the microvesicles, mitochondria and the nucleus membrane. These findings were supported by silver grain distributions in autoradiograms obtained by means of ⁴⁵Ca. The qualitative results obtained histochemically are in good agreement with estimations of the calcium distribution in subcellular fractions obtained by atomic absorption spectrophotometry.     

Superoxide dismutase does not inhibit the oxidation of cytochrome c and cytochrome oxidase.

An electrometric system was used to measure Ca⁺⁺ uptake by sarcoplasmic reticulum vesicles (SR). The method permits continuous recording of Ca⁺⁺ uptake and thus the valuation of kinetic parameters. Furthermore, the ultrasensitivity of the method permits to follow changes in Ca⁺⁺ concentration below 10^?6 M.     

Ca2+ uptake by hyperpermeable mouse heart cells: Effects of inhibitors of mitochondrial function

The relative importance of heart mitochondria in regulating intracellular [Ca²⁺] in cardiac muscle is controversial. In a new approach to the question, we have measured the energy-linked ⁴⁵Ca uptake of an unusual myocardial tissue preparation in which the cells appear to be intact yet the sarcolemmae are highly permeable to exogenous solutes. Inhibitors of mitochondrial energy metabolism were used to estimate the mitochondrial contribution to rate and extent of total cell uptake. At 6.6μM Ca, which is close to the probable intracellular [Ca] range, inhibitors of mitochondrial energy metabolism did not diminish initial rates of ⁴⁵Ca uptake by myocardial fragments, if ATP was present to drive Ca²⁺ sequestration by the sarcoplasmic reticulum. The ultimate extent of uptake was reduced somewhat, however. Similar uptake profiles were obtained in the presence of carbonyl cyanide m-chlorophenyl-hydrazone, CN^?, and atractyloside, each of which acts at a different locus to inhibit mitochondrial Ca²⁺ transport. These data suggest that the mitochondria cannot control beat-to-beat [Ca²⁺] oscillations, because at μM Ca concentrations, the Ca²⁺ uptake rate of mitochondria $\text{in}$$\text{situ}$ is slow in comparison to the extra-mitochondrial (sarcoplasmic reticulum) uptake rate.     

Adenosine triphosphate-induced rapid calcium release from fragmented sarcoplasmic reticulum

Calcium efflux from skeletal muscle fragmented sarcoplasmic reticulum was studied using a dilution technique and Millipore filtration. In the absence of Mg⁺⁺ and external Ca⁺⁺, addition of lmM adenosine triphosphate to the suspension resulted in an immediate loss of 26–55% of total vesicular calcium. The amount of calcium released was calculated to be sufficient to effect muscle contraction. After separation of the sarcoplasmic reticulum into light, intermediate and heavy vesicles, the light and heavy fractions were found to be only weakly responsive to adenosine triphosphate, whereas the intermediate fraction lost nearly half of its calcium. The significance of these results with respect to excitation-contraction coupling in muscle is discussed.     

The migration of divalent cations in mitochondria visualized by a fluorescent chelate probe

Summary The use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca⁺⁺ and Mg⁺⁺. The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca⁺⁺ in coupled mitochondria and Mg⁺⁺ in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn⁺⁺ indicate that the probe is primarily visualizing actively accumulated Ca⁺⁺ on the inner surface of the inner membrane. By employing the Ca⁺⁺ transport inhibitor, Tb⁺⁺⁺, the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.     

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.     

Adenylate cyclase system of human skeletal muscle: Characteristics of catecholamine stimulation and nucleotide regulation

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

The effect of anti-ATPase antibodies upon the Ca++ transport of sarcoplasmic reticulum

Sheep or guinea pig antisera against the purified Ca⁺⁺ transport ATPase of sarcoplasmic reticulum inhibit Ca⁺⁺ transport due to a complement-dependent damage of the membrane, which causes massive leakage of Ca⁺⁺. The Ca⁺⁺-activated ATPase activity is only slightly affected even at ten times higher antibody concentration than that required for inhibition of Ca⁺⁺ transport. Antibodies prepared against the Ca⁺⁺ binding protein (C₁ protein) have no influence upon either ATPase activity or Ca⁺⁺ transport and ferritin-labeled anti-C₁ antibodies do not bind to microsomes.     

Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++- ATPase from pig heart sarcoplasmic reticulum

Vanadate inhibits the Ca⁺⁺-ATPase of sarcoplasmic reticulum from pig heart half maximally at about 10^?5 M. Mg⁺⁺ promotes this inhibition by vanadate whereas increasing Ca⁺⁺-concentrations protect the enzyme against vanadate inhibition. Keeping the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 × 10^?3 M ATP. Whenever the ratio $\text{Mg}{}^{\mathrm{++}}\text{ATP}$ was higher than 1:1 the inhibitory effect of vanadate on the Ca⁺⁺-ATPase was increased.