Skeletal and cardiac ryanodine receptors bind to the Ca(2+)-sensor region of dihydropyridine receptor alpha(1C) subunit.

In striated muscles, excitation-contraction coupling is mediated by the functional interplay between dihydropyridine receptor L-type calcium channels (DHPR) and ryanodine receptor calcium-release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross-talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C-terminal cytoplasmic domain of alpha(1C). The interaction is restricted to the CB and IQ motifs involved in the calmodulin-mediated Ca(2+)-dependent inactivation of the DHPR, suggesting functional interactions between the two channels.

Identification of novel bifunctional calmodulin-binding and microtubule-stabilizing motifs in STOP proteins.

Although microtubules are intrinsically labile tubulin assemblies, many cell types contain stable polymers, resisting depolymerizing conditions such as exposure to the cold or the drug nocodazole. This microtubule stabilization is largely due to polymer association with STOP proteins. There are several STOP variants, some with capacity to induce microtubule resistance to both the cold and nocodazole, others with microtubule cold stabilizing activity only. These microtubule-stabilizing effects of STOP proteins are inhibited by calmodulin and we now demonstrate that they are determined by two distinct kinds of repeated modular sequences (Mn and Mc), both containing a calmodulin-binding peptide, but displaying different microtubule stabilizing activities. Mn modules induce microtubule resistance to both the cold and nocodazole when expressed in cells. Mc modules, which correspond to the STOP central repeats, have microtubule cold stabilizing activity only. Mouse neuronal STOPs, which induce both cold and drug resistance in cellular microtubules, contain three Mn modules and four Mc modules. Compared with neuronal STOPs, the non-neuronal F-STOP lacks multiple Mn modules and this corresponds with an inability to induce nocodazole resistance. STOP modules represent novel bifunctional calmodulin-binding and microtubule-stabilizing sequences that may be essential for the generation of the different patterns of microtubule stabilization observed in cells.

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor.

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53.

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.

Tubulin binding sites on gamma-tubulin: identification and molecular characterization.

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.

Separation of tubulin subunits under nondenaturing conditions.

The dissociation and separation of the tubulin alpha- and beta-subunits have been achieved by binding alpha-subunits to an immunoadsorbent gel and selectively inducing release of free beta-subunits. The immunoadsorbent gel was prepared by coupling the monoclonal antibody YL1/2 to Sepharose 4B which specifically recognizes the C-terminal end of tyrosinated alpha-subunits. Extensive tubulin subunit dissociation and separation occurred in Tris buffer at neutral pH but was greatly enhanced at basic pHs (8. 0-8.5). The binding of colchicine to heterodimeric tubulin resulted in a marked protection against dissociation. The dissociation of tubulin subunits was accompanied by loss of colchicine binding capacity, and ability to polymerize into microtubules. As shown by circular dichroism, loss of functional properties was not due to extensive denaturation of tubulin, as tubulin retained most of its secondary structure. Neither of the separated alpha- or beta-subunits was able to bind colchicine, but functional tubulin that was able to bind colchicine could be reconstituted from the dissociated subunits by changing the buffer to a neutral mixture of Tris and Pipes. The yield of reconstitution, as estimated from kinetic measurements of colchicine binding capacity, amounted to about 25%. Such a yield can probably be improved with minor changes in experimental conditions. The quantitative dissociation of tubulin into separated "native" alpha- and beta-subunits should provide a powerful tool for further studies on the properties of the individual tubulin subunits and the structure-function relationships of the tubulins.

Interaction of S100A1 with the Ca2+ release channel (ryanodine receptor) of skeletal muscle.

In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.

Malignant hyperthermia domain in the regulation of ca(2+) release channel (ryanodine receptor).

Malignant hyperthermia (MH) is a potentially lethal condition that is manifested in humans as an acute increase of body temperature in response to stress and exposure to volatile anaesthetics (halothane, enflurane) and muscle relaxants. To date, eight point mutations in the ryanodine receptor gene, the Ca(2+) release channel of the skeletal muscle sarcoplasmic reticulum, segregate with the MH phenotype, yet direct evidence linking altered [Ca(2+)](i) homeostasis to mutation in recombinant RYR has been obtained only for one such mutation. Most of these mutations appear in an "MH domain" that is localized at the NH(2) terminus of the skeletal muscle ryanodine receptor Ca(2+) channel. In this review, we summarize the available data concerning the role of the MH domain in the altered functions of the ryanodine receptor Ca(2+) channel. (Trends Cardiovasc Med 1997;7:312-316). © 1997, Elsevier Science Inc.

Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.

A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.

Localization of the N-terminal and C-terminal ends of triadin with respect to the sarcoplasmic reticulum membrane of rabbit skeletal muscle.

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-17) and C-terminal (residues 691-706) ends of rabbit skeletal muscle triadin, a 95 kDa protein located in the sarcoplasmic reticulum membrane at the triad junction. The specificity of the antibodies generated was tested by ELISA and Western blot analysis. These tests demonstrated the ability of the antibodies to react specifically with the proteins. The anti-N-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that the N-terminal end of the membrane-embedded triadin is exposed on the cytoplasmic side of the vesicles. In contrast, the anti-C-terminus antibodies were able to react with sarcoplasmic reticulum vesicles only after permeabilization of the vesicles with a detergent, indicating that the C-terminal end is exposed on the luminal side of the vesicles. These immunological data were complemented by proteolysis experiments using carboxypeptidases and endoproteinase Arg C. A mixture of carboxypeptidases A, B and Y was used to induce degradation of the C-terminal end of triadin in sarcoplasmic reticulum vesicles. This degradation, and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, was observed only when the vesicles were permeabilized, providing further evidence for the luminal localization of the C-terminal end of triadin. Treatment of sarcoplasmic reticulum vesicles with endoproteinase Arg C resulted in the removal of the N-terminal end of triadin, probably due to cleavage after Arg-34. This is a further indication of the cytoplasmic localization of the N-terminal end of triadin (and of its first 34 amino acids). When the proteolysis with endoproteinase Arg C was carried out with permeabilized vesicles, the cleavage occurred after Arg-141 or Arg-157, indicating that at least one of these residues is luminal.

Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle.

Membrane vesicles enriched in both ryanodine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Analysis of the sedimentation behavior of the solubilized proteins showed the existence of a population of alpha 1 subunits of the dihydropyridine receptor which cosedimented with the ryanodine receptor. Solubilized proteins were immunoprecipitated with antibodies directed against either the ryanodine receptor or the alpha 1, alpha 2, or beta subunits of the dihydropyridine receptor. Immunoprecipitated proteins were identified by Western blot analysis and by specific labeling with [3H]ryanodine or [3H]PN200-110. Immunoprecipitation of the solubilized proteins with antibodies directed against the dihydropyridine receptor led to the coimmunoprecipitation of the ryanodine receptor. Conversely, immunoprecipitation with antibodies directed against the ryanodine receptor led to an immune complex containing both receptors, but these antibodies were unable to precipitate purified dihydropyridine receptor. These results demonstrate that ryanodine receptor and dihydropyridine receptor are present in the triad membrane preparation in a complex which may play an important role in excitation-contraction coupling.

Transmembrane orientation of the N-terminal and C-terminal ends of the ryanodine receptor in the sarcoplasmic reticulum of rabbit skeletal muscle.

Antibodies were raised against synthetic peptides corresponding to the N-terminal (residues 2-15) and the C-terminal (residues 5027-5037) parts of the rabbit skeletal muscle ryanodine receptor. The specificity of the antibodies generated was tested by e.l.i.s.a., Western blotting and immunofluorescence. All these tests demonstrated the specificity of the antibodies and their ability to react with both the native and the denaturated ryanodine receptor. Both the anti-N-terminus and the anti-C-terminus antibodies bound to sarcoplasmic reticulum vesicles, indicating that each end of the membrane-embedded ryanodine receptor is exposed to the cytoplasmic side of the vesicles. These immunological data were complemented with proteolysis experiments using carboxypeptidase A. Carboxypeptidase A induced degradation of the C-terminal end of the ryanodine receptor in sarcoplasmic reticulum vesicles and a concomitant loss of reactivity of the anti-C-terminus antibodies in Western blots, providing extra evidence for the cytoplasmic localization of the C-terminal end of the ryanodine receptor.

BisG10, a K+ channel blocker, affects the calcium release channel from skeletal muscle sarcoplasmic reticulum.

The action of bisG10, a potent K+ channel inhibitor, was tested on the Ca2+ release from isolated sarcoplasmic reticulum vesicles of rabbit skeletal muscle. Using a rapid filtration technique, we found that the drug inhibited Ca(2+)-induced Ca2+ release elicited in the presence of extravesicular K+ as counter-ion. This inhibition was not reversed by the addition of valinomycin and still occurred when Cl- was used as co-ion, indicating that not only K+ channels are involved in the inhibiting effect. We found that bisG10 decreased the binding of ryanodine to sarcoplasmic reticulum vesicles, showing that bisG10 is able to block the sarcoplasmic reticulum Ca2+ release channel.

The yeast plasma membrane H(+)-ATPase. An essential change of conformation triggered by H+.

The plasma membrane of Schizosaccharomyces pombe contains an H(+)-ATPase similar to the cation transport ATPases of other eukaryotic organisms. The fluorescence excitation and emission spectra of the purified H(+)-ATPase are characteristic of tryptophan residues. pH reduction from 7.5 to 5.7 produces a 4% decrease in fluorescence intensity, while a further reduction to pH 5.0 leads to an increase of fluorescence. A close correlation is observed between the pH dependence of the intrinsic fluorescence and the pH dependence of (i) ATPase activity, (ii) the fluorescence of Tb-formycin triphosphate bound to the active site, and (iii) inhibition by vanadate of ATPase activity. It is proposed that the effect of pH on intrinsic fluorescence reveals the existence of an H+ induced conformational change of the H(+)-ATPase similar to the E1----E2 transition of the other plasma membrane cation transport ATPases.

Intravesicular calcium transient during calcium release from sarcoplasmic reticulum.

The time course of changes in the intravesicular Ca2+ concentration ([Ca2+]i) in terminal cisternal sarcoplasmic reticulum vesicles upon the induction of Ca2+ release was investigated by using tetramethylmurexide (TMX) as an intravesicular Ca2+ probe. Upon the addition of polylysine at the concentration that led to the maximum rate of Ca2+ release, [Ca2+]i decreased monotonically in parallel with Ca2+ release. Upon induction of Ca2+ release by lower concentrations of polylysine, [Ca2+]i first increased above the resting level, followed by a decrease well below it. The release triggers polylysine, and caffeine brought about dissociation of calcium that bound to a nonvesicular membrane segment consisting of the junctional face membrane and calsequestrin bound to it, as monitored with TMX. No Ca2+ dissociation from calsequestrin-free junctional face membranes or from the dissociated calsequestrin was produced by release triggers, but upon reassociation of the dissociated calsequestrin and the junctional face membrane, Ca2+ dissociation by triggers was restored. On the basis of these results, we propose that the release triggers elicit a signal in the junctional face membrane, presumably in the foot protein moiety, which is then transmitted to calsequestrin, leading to the dissociation of the bound calcium; and in SR vesicles, to the transient increase of [Ca2+]i, and subsequently release across the membrane.

Polylysine induces a rapid Ca2+ release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot protein.

The addition of polylysine to a heavy fraction of sarcoplasmic reticulum (SR) vesicles produces a rapid Ca2+ release with no appreciable lag period. The polylysine concentration for half-maximal activation (C1/2) is approximately 0.99 micrograms/ml, or 0.3 microM, the lowest C 1/2 for Ca2+ release-inducing reagents reported in the literature. The time course and the [Ca2+] dependence of polylysine-induced release are similar to those of caffeine-induced Ca2+ release. At higher concentrations of polylysine (e.g., 10 micrograms/ml), however, little or no Ca2+ release occurs. Upon photolysis of SR vesicles with the photocrosslinkable radiolabeled polylysine derivative, [3H]succinimidyl azido benzoate polylysine, 0.28 and 0.52-1.2 mol polylysine were bound to 1 mol of the 400-kDa foot protein at activating (3 micrograms/ml) and inhibitory (10 micrograms/ml) concentrations of polylysine, respectively. On the other hand, the amounts of polylysine bound to the other SR proteins (mol/mol) were negligible (e.g., less than or equal to 0.0127 mol polylysine/mol calsequestrin). This suggests that the binding of polylysine to the foot protein is responsible not only for the induction of release but also for inactivation. These results provide direct evidence that the receptor for the chemical trigger of Ca2+ release is localized within the foot protein. Ruthenium red, which inhibits polylysine-induced Ca2+ release, does not inhibit polylysine binding to the foot protein, suggesting that the polylysine binding domain of the foot protein is different from the channel domain.

Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum.

Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.

Kinetic analysis of excitation-contraction coupling.

Recent studies of isolated muscle membrane have enabled induction and monitoring of rapid Ca2+ release from sarcoplasmic reticulum (SR)5 in vitro by a variety of methods. On the other hand, various proteins that may be directly or indirectly involved in the Ca2+ release mechanism have begun to be unveiled. In this mini-review, we attempt to deduce the molecular mechanism by which Ca2+ release is induced, regulated, and performed, by combining the updated information of the Ca2+ release kinetics with the accumulated knowledge about the key molecular components.

Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex.

The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H2O by D2O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.

The remotor muscle of the lobster antenna: sarcoplasmic reticulum and skinned fiber experiments.

The striated remotor muscle of the lobster antenna has an extraordinarily profuse sarcoplasmic reticulum as shown by electron microscopy. Gel electrophoresis reveals a simple protein composition in which the Ca2+-ATPase predominates. Vesicles of sarcoplasmic reticulum (SR) from this remotor are shown to operate Ca2+ binding, Ca2+ transport, and Ca2+-activated hydrolysis of ATP with an usual efficiency (2 Ca2+ transported per ATP hydrolysed, 4 mumol ATP hydrolysed/mg protein/min). Skinned fiber experiments were performed. They indicate behaviour of the remotor expected from observations by EM and gel electrophoresis: contraction of low maximal intensity under Ca2+ excitation, long internal diffusion time due to the large volume of SR to be crossed, and large Ca2+ content released in a caffeine-sensitive manner.