Rabbit Skeletal Muscle Microsomes Research Articles

Kinetic basis of quantal calcium release from intracellular calcium stores

The kinetics of Ca2+ release from canine cerebellum and rabbit skeletal muscle microsomes, mediated by the inositol 1,4,5-trisphosphate (IP 3) receptor (IRC) and the ryanodine receptor (RyRC), respectively, were analyzed by a model, which considers that Ca 2+ release channels undergo spontaneous inactivation. We found that: (i) both the initial rate of release (v o) and the rate of inactivation (v 1) were saturable functions of the activating ligand concentration (C L); and (ii) the ratio of v i/v o, termed the relative tendency for inactivation, decreased with increasing C L. Equilibrium [ 3H]-IP 3 binding studies, on the other hand, revealed the presence of one single class of non-co-operative IP 3 sites in cerebellum membranes (K deq = 47 nM and Hill coefficient = 1.1). Based on the above v i−v o relationship and the IP 3-binding data, we propose that quantal Ca 2+ release through IRCs might be a result of spontaneous channel inactivation, whose rate is controlled by the ratio of IP 3-occupied/free monomers in the tetrameric release channel units. Furthermore, because of the kinetic similarities between the IRC- and RyRC-mediated Ca 2+ release processes, as well as between quantal Ca 2+ release and channel adaptation, the same mechanism is also proposed to apply to the RyRC-mediated Ca 2+ release as well as to constitute the basis of release channel adaptation.

A unique fluorescent phenylalkylamine probe for L-type Ca2+ channels. Coupling of phenylalkylamine receptors to Ca2+ and dihydropyridine binding sites.

The first fluorescently labeled phenylalkylamine, DMBODIPY-PAA (5-(3-[3-(4,4-difluoro-5,7-dimethyl-3a, 4a-diaza-4-bora-indacen-3-yl)propionamido] phenethyl-N-methylamino)-2-isopropyl-2-(3,4,5-trimethoxyphenyl)-valer onitrile) has been introduced for L-type Ca2+ channel research. DMBODIPY-PAA binds reversibly to L-type Ca2+ channels purified from rabbit skeletal muscle microsomes by wheat germ agglutinin-Sepharose chromatography. In this preparation DMBODIPY-PAA labels 412 pmol of phenylalkylamine receptors/mg of protein with a Kd of 6.82 nM and a favorable signal-to-noise ratio. Therefore DMBODIPY-PAA has a higher affinity for purified Ca2+ channels than the commonly employed radioligands and consequently has assisted in channel purification after prelabeling by simply monitoring receptor-bound fluorescence. (+)-PN200-110 (which is stimulatory for (-)-[3H]desmethoxyverapamil binding to purified Ca2+ channels) inhibits DMBODIPY-PAA labeling. Since these drug interactions are reciprocal, the phenylalkylamine and dihydropyridine binding sites of the alpha 1-subunit are tightly coupled. Kinetic and equilibrium binding studies with (-)-[3H]desmethoxyverapamil and DMBODIPY-PAA show that phenylalkylamine binding to L-type Ca2+ channels is dependent on Ca2+. Chelation of divalent metal ions converts phenylalkylamine receptors into a very low affinity state. This conversion is temperature- and time-dependent and completely reversible (K0.5 for free Ca2+ = 58 nM). This study demonstrates the utility of fluorescent ligands for binding studies with L-type Ca2+ channels and provides evidence for coupling between Ca2+ binding sites and phenylalkylamine receptors.

Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes.

Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.

Rabbit skeletal muscle microsomes contain two distinct phenylalkylamine‐binding sites

Lu49888, a photoaffinity analog of verapamil, was used to identify specific binding sites for phenylalkylamines of calcium channels present in rabbit skeletal muscle microsomes. Direct binding equilibrium measurements and displacement curves of Lu49888 by its non-radioactive analog yielded an apparent single class of binding sites with Kd and Bmax values of 16.5 nM and 7.5 pmol/mg respectively. Lu49888 was specifically incorporated into three proteins of apparently 165 kDa, and 33 kDa. Incorporation into the 55-kDa protein was blocked by 10--50-fold higher concentrations of unlabeled phenylalkylamines compared to incorporation into the 165-kDa protein, suggesting that the 165-kDa and 55-kDa proteins contain a high and a low-affinity verapamil-binding site respectively. The photoaffinity-labeled proteins were solubilized by 1% digitonin or 1% Chaps in roughly equal amounts. The 165-kDa protein bound to wheat-germ-agglutinin(WGA)--Sepharose and sedimented in sucrose density gradients with the same constant as the purified dihydropyridine receptor, which has been reconstituted to a functional calcium channel. The 55-kDa membrane protein did not bind to the WGA-Sepharose column and sedimented in sucrose density gradients with a lower s value than the 165-kDa protein. The 165-kDa but not the 55-kDa membrane protein was specifically labeled by azidopine, the photoaffinity analogue of dihydropyridines. The 55-kDa protein of the purified dihydropyridine receptor was not significantly labeled by Lu49888 showing that the 55-kDa protein of the membrane is unrelated to the purified high-affinity dihydropyridine receptor.

Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes.

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-110 to an apparent density of 1.5-2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-110. cAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 +/- 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. [3H]PN 200-110 bound at 4 degrees C to one site with apparent Kd and Bmax values of 9.3 +/- 1.7 nM and 2.2 +/- 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200-110 in an apparent allosteric manner. (-)Desmethoxyverapamil inhibited the binding of [3H]PN 200-110 at 37 degrees C and stimulated it at 18 degrees C. In agreement with these results, (-)desmethoxyverapamil increased the dissociation rate of [3H]PN 200-110 from 0.29 min-1 to 0.38 min-1 at 37 degrees C and decreased it threefold from 0.046 min-1 to 0.017 min-1 at 18 degrees C. The (+)isomer of desmethoxyverapamil inhibited PN 200-110 binding at all temperatures tested. d-cis-Diltiazem stimulated the binding of [3H]PN 200-110 at 37 degrees C with an apparent EC50 of 1.4 microM and decreased the dissociation rate from 0.29 min-1 to 0.11 min-1. The stimulatory effect of d-cis-diltiazem was temperature-dependent and was seen only at temperatures above 18 degrees C. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane-bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.

Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes

Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.

Rapid flow chemical quench studies of calcium release from isolated sarcoplasmic reticulum.

45Ca2+ release from a heavy fraction of rabbit skeletal muscle microsomes was induced by chemical depolarization (replacement of 0.15 M K gluconate with 0.15 M choline Cl), or addition of Ca2+ plus caffeine, or both. The time courses of Ca2+ release were investigated with a multimixing chemical quench apparatus by quenching the Ca2+ release reaction using 10 mM EGTA and 5 microM ruthenium red. At low ATP (e.g. 0.2 mM) and low extravesicular [Ca2+] (e.g. 0.1 microM), the time course of depolarization-induced Ca2+ release was similar to that determined by a spectrophotometric method (Ikemoto, N., Antoniu, B., and Kim, D.H. (1984) J. Biol. Chem. 259, 13151-13158). An increase of the extravesicular [Ca2+] up to 5 microM, or addition of high concentrations of ATP (e.g. 7.5 mM), shortened the lag phase that precedes depolarization-induced Ca2+ release and increased the amount of Ca2+ released. On the other hand, upon addition of several millimolars ATP the rate of (Ca2+ plus caffeine)-induced Ca2+ release was increased, resulting in the same time course as that of depolarization-induced Ca2+ release. Induction of Ca2+ release by combined application of chemical depolarization and Ca2+ plus caffeine resulted in the same time course as that induced by either method alone, suggesting that both types of Ca2+ release are mediated by a common channel rather than separate channels.

Rapid calcium release from the isolated sarcoplasmic reticulum is triggered via the attached transverse tubular system.

Rapid replacement of 0.15 M K gluconate with 0.15 M choline Cl led to multiphasic Ca2+ release from a heavy fraction of rabbit skeletal muscle microsomes. Following the initial lag period (0-50 ms), about 15 nmol of Ca2+/mg of protein was rapidly released with first-order rate constants k = 60-140 s-1. Subsequently, a larger amount of Ca2+ (up to 56 nmol/mg) was released at a slower rate (k = 0.8-1.5 s-1). The Ca2+ released in both rapid and slow phases was reaccumulated within 60 s. In agreement with a previous report (Caswell, A. H., Lau, Y. H., Garcia, M., and Brunschwig, J-P. (1979) J. Biol. Chem. 254, 202-208), French press treatment of the tubule/sarcoplasmic reticulum (SR) complex results in dissociation of transverse tubular membrane (T-tubules) from SR. Subsequent incubation with 0.4 M potassium cacodylate results in the reassociation of the complex, as shown by sucrose density-gradient sedimentation. Upon T-tubule dissociation, both rapid and slow Ca2+ release was inhibited. Upon reassociation, the rapid Ca2+ release was completely restored and the slow phase partially restored. The results indicate that the T-tubule associated with SR plays a crucial role in triggering rapid Ca2+ release induced by ionic replacement. Other types of Ca2+ release, e.g. those induced by Ca2+ alone or with drugs such as caffeine and quercetin, are unaffected by T-tubule dissociation, and hence produced by direct stimulation of the SR membrane.

Two-dimensional polyacrylamide gel electrophoresis of rabbit skeletal muscle microsomal proteins

Since the application of polyacrylamide gel electrophoresis to the study of membrane proteins, there have been a number of reports on the protein composition of skeletal muscle microsomes prepared by differential pelleting [l-7] and by density gradient centrifugation [g-10] . These electrophoretic separations of the solubilised membrane proteins have been in one-dimension. There is general agreement that the principal protein component of these preparations is a protein of mol. wt 100 000. Furthermore this component has been shown to be the Ca++ transport ATPase of the sarcoplasmic reticulum [ 1,2,8,9] . Estimates of the amount of this protein present have varied from 3570%. In addition to this major protein five other proteins and a proteolipid have been separated [4,11]. These other proteins have been postulated to play roles associated with the calcium transport function of the sarcoplasmic reticulum [3,4,11,12]. This report describes the use of two-dimensional polyacrylamide gel electrophoresis in the study of membrane proteins from four microsomal fractions prepared by zonal centrifugation [ 10,131. The twodimensional separation consists of electrophoresis in the first dimension in 5.2% gels followed by electrophoresis in the second dimension in gels having a 627% gel gradient. This method is especially useful in the macromolecular mapping of multicomponent mixtures [14,15] and is a novel approach in the study of membrane proteins. The present work is part of a continuing study of rabbit skeletal muscle microsomes. The results reported here show that muscle microsomes contain many more proteins than was previously believed.

Oxalate dependence of calcium uptake kinetics of rabbit skeletal muscle microsomes (fragmented sarcoplasmic reticulum)

The initial rate of oxalate-facilitated Ca 2+ uptake by skeletal microsomes depends on both Ca 2+ and oxalate concentrations in the medium. The apparent K m for Ca 2+ increases with increasing oxalate concentration, indicating that Ca 2+ uptake can involve a carrier-mediated transport system.

Calcium transport by rabbit skeletal muscle microsomes (“fragmented sarcoplasmic reticulum”)

Ca 2+ binding by skeletal muscle microsomes in 5 mM ATP exhibited saturation kinetics in the range of Ca 2+ concentrations between 3 · 10 −8 and 10 −5 M. Approximately 140 nmoles binding sites per mg protein were found. These had a Ca 2+ binding constant of approximately 4.5 · 10 6 M −1 with half saturation at 2.2 · 10 −7 M Ca 2+. In the presence of oxalate, much larger amounts of Ca 2+ (approx. 6 μmoles/mg protein) were taken up by the microsomes (Ca 2+ uptake), but the rate of Ca 2+ uptake increased linearly with [Ca 2+] when ionized Ca 2+ concentrations were below 3 · 10 −6 M. At Ca 2+ concentrations above 3 · 10 −6, Ca 2+ uptake was inhibited. Double reciprocal plots of the Ca 2+ dependence of the initial rates of Ca 2+ uptake in the concentration range between 3 · 10 −7 M and 10 −5 M, unlike those of Ca 2+ binding, did not demonstrate saturation kinetics, but could be interpreted as representing a non-saturable system with inhibition at higher Ca 2+ concentrations. In view of these differences, and because Ca 2+-binding sites were almost fully saturated at 10 −6 M Ca 2+, whereas Ca 2+ uptake rate increased linearly with increasing [Ca 2+] to approximately 3 · 10 −6 M, the Ca 2+-binding sites are not shown kinetically to participate in oxalate-dependent Ca 2+ uptake.

Isolation of Ca ++-sequestering sarcotubular membranes from rat skeletal muscle

A method is described for the preparation of relatively pure sarcotubular membranes from rat skeletal muscle which have a high capacity to sequester Ca ++. It involves the subfractionation of crude muscle microsomes by the sequential use of two sucrose density gradient centrifugation systems. Subfraction SF 1 contains membranous vesicles which on the basis of both morphological and functional evidence appear to be as free of other cellular components as those obtained from rabbit skeletal muscle microsomes by currently available fractionation methods.

Steroid effects on the calcium ion transport of rabbit skeletal muscle microsomes

The effect of steroids on the active Ca 2+ transport of rabbit skeletal muscle microsomes was studied. The inhibitory effect of steroids appears connected with the cis conformation of rings A and B and with the presence of at least two hydrophilic groups in the molecule. Steroids which inhibit the Ca 2+ transport of skeletal muscle microsomes activate the microsomal ATPase activity and promote the hemolysis of human red blood cells in similar concentrations.

STUDIES ON THE EFFECT OF SODIUM AND POTASSIUM ON THE ATPase ACTIVITY AND CALCIUM UPTAKE OF THE SKELETAL MUSCLE MICROSOMES

1. Calcium uptake by rabbit skeletal muscle microsomes was stimulated by the addition of Na+ or K+ alone or both, but the synergistic action of Na+ and K+ was not remarkable.2. The stimulation of calcium uptake was the highest at the concentration of 20 mM of Na+ or 40mM of K+.3. The pH optimum of calcium uptake was 6.8. The pH optimum of ATPase activity was about 78 in the case of Mg2+ alone or Mg2++Na+, and about 7.4 in the case of Mg2++ Na++K+4. ATPase activity was also stimulated by the addition of Na+ or K+, but the synergistic action of Na+ and K+ was not observed. Ouabain did not affect the stimulated ATPase activity.5. ATPase activity was stimulated by a low concentration of Ca2+ in the presence of Na+ and K+ alone or both regardless of the presence of oxalate. In the presence of Mg2+ alone the ATPase activity was stimulated slightly by the addition of Ca2+, but in the presence of oxalate the ATPase activity with Mg2+ alone was not stimulated by the presence of Ca2+.6. The addition of salyrgan depressed the ATPase activity to a certain level. The ATPase which links to Ca2+ uptake was considered to be Na+-K+ stimulated, Ca2+ stimulated ouabain insensitive and salyrgan sensitive enzyme.