Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus.

Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.

Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium.

The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.

Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation.

Several ion channels are reportedly redox responsive, but the molecular basis for the changes in activity is not known. The mechanism of nitric oxide action on the cardiac calcium release channel (ryanodine receptor) (CRC) in canines was explored. This tetrameric channel contains approximately 84 free thiols and is S-nitrosylated in vivo. S-Nitrosylation of up to 12 sites (3 per CRC subunit) led to progressive channel activation that was reversed by denitrosylation. In contrast, oxidation of 20 to 24 thiols per CRC (5 or 6 per subunit) had no effect on channel function. Oxidation of additional thiols (or of another class of thiols) produced irreversible activation. The CRC thus appears to be regulated by poly-S-nitrosylation (multiple covalent attachments), whereas oxidation can lead to loss of control. These results reveal that ion channels can differentiate nitrosative from oxidative signals and indicate that the CRC is regulated by posttranslational chemical modification(s) of sulfurs.

Isolation and characterization of two types of sarcoplasmic reticulum vesicles.

A purified preparation of sarcoplasmic reticulum from rabbit skeletal muscle has been found to consist of a heterogeneous population of vesicles. Isopycnic centrifugation was used to obtain "light" and "heavy" vesicles from the upper and lower ends of a 25 to 45% (w/w) linear sucrose gradient. Each fraction accounted for about 10 to 15% of the total vesicles. The remainder of the vesicles were of intermediate density and banded between the light and heavy fraction. Light vesicles were composed of about equal amounts of phospholipid and Ca-2+ pump protein which contained approx. 90% of the protein. Heavy vesicles contained in addition to the Ca-2+ pump protein (55-65% of the protein) two other major protein components, the Ca-2+ binding and M55 proteins which accounted for 20-25 and 5-7% of the protein of these vesicles, respectively. The sarcoplasmic reticulum subfractions had 32-P-labelled phosphoenzyme levels proportional to their Ca-2+ pump protein content and contained similar Ca-2+-stimulated ATPase activities. They were capable of accumulating Ca-2+ in the presence of ATP and of releasing the accumulated Ca-2+ when placed into a medium with a low Ca-2+ concentration. The vesicles differed significantly in that heavy vesicles had a greater number of non-specific Ca-2+ binding sites than light vesicles (approx. 220 vs 75 nmol of bound Ca-2+ per mg protein), in accordance with their high content of Ca-2+ binding protein. Electron dense material could be seen within the compartment of heavy but not light vesicles. Removal of Ca-2+ binding and M55 proteins from heavy vesicles resulted in empty membranous structures consisting mainly of Ca-2+ pump protein and phospholipid. Electron micrographs of sections of muscle showed dense material in terminal cisternae but not in longitudinal sections of sarcoplasmic reticulum. These experiments are consistent with the interpretation that (1) the electron dense material inside heavy vesicles may be referable to Ca-2+ binding and/or M55 proteins, and that (2) light and heavy vesicles may be derived from the longitudinal sections and terminal cisternae of sarcoplasmic reticulum, respectively.

Permeability of reconstituted sarcoplasmic reticulum vesicles. Reconstitution of the K+, Na+ channel.

Permeability properties of reconstituted rabbit skeletal muscle sarcoplasmic reticulum vesicles were characterized by measuring efflux rates of [3H]inulin, [3H]choline+, 86Rb+, and 22Na+, as well as membrane potential changes using the voltage-sensitive probe, 3,3'-dipentyl-2,2'-oxacarbocyanine. Native vesicles were dissociated with deoxycholate and were reconstituted by dialysis. Energized Ca2+ accumulation was partially restored. About 1/2 of the reconstituted vesicles were found to be 'leaky', i.e., permeable to choline+ of Tris+ but not to inulin. The remaining reconstituted vesicles were 'sealed', i.e., impermeable to choline+, Tris+ and inulin. Sealed reconstituted vesicles could be further subdivided according to their K+, Na+ permeability. About 1/2, previously designated Type I, were readily permeable to K+ and Na+, indicating the presence of the K+, Na+ channel of sarcoplasmic reticulum. The remaining sealed vesicles (Type II) formed a permeability barrier to K+ and Na+, suggesting that they lacked the K+, Na+ channel. These studies show that the K+, Na+ channel of sarcoplasmic reticulum can be solubilized with detergent and reconstituted with retention of activity. Furthermore, our results suggest that part or all of the decreased Ca2+-loading efficiency of reconstituted vesicles may be due to the presence of a significant fraction of leaky vesicles.

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.

T-tubule depolarization-induced SR Ca2+ release is controlled by dihydropyridine receptor- and Ca(2+)-dependent mechanisms in cell homogenates from rabbit skeletal muscle.

In vertebrate skeletal muscle, the voltage-dependent mechanism of rapid sarcoplasmic reticulum (SR) Ca2+ release, commonly referred to as excitation-contraction (EC) coupling, is believed to be mediated by physical interaction between the transverse (T)-tubule voltage-sensing dihydropyridine receptor (DHPR) and the SR ryanodine receptor (RyR)/Ca2+ release channel. In this study, differential T-tubule and SR membrane monovalent ion permeabilities were exploited with the use of an ion-replacement protocol to study T-tubule depolarization-induced SR 45Ca2+ release from rabbit skeletal muscle whole-cell homogenates. Specificity of Ca2+ release was ascertained with the use of the DHPR antagonists D888, nifedipine and PN200-110. In the presence of the "slow" complexing Ca2+ buffer EGTA, homogenates exhibited T-tubule depolarization-induced Ca2+ release comprised of an initial rapid phase followed by a slower release phase. During the rapid phase, approximately 20% of the total sequestered Ca2+ (approximately 30 nmol 45Ca2+/mg protein), corresponding to 100% of the caffeine-sensitive Ca2+ pool, was released within 50 ms. Rapid release could be inhibited fourfold by D888. Addition to release media of the "fast" complexing Ca2+ buffer BAPTA, at concentrations > or = 4 mM, nearly abolished rapid Ca2+ release, suggesting that most was Ca2+ dependent. Addition of millimolar concentrations of either Ca2+ or Mg2+ also greatly reduced rapid Ca2+ release. These results show that T-tubule depolarization-induced SR Ca2+ release from rabbit skeletal muscle homogenates is controlled by T-tubule membrane potential- and by Ca(2+)-dependent mechanisms.

Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel.

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)-conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.

Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum. Activation by Ca2+ and ATP and modulation by Mg2+.

A high-conductance (100 pS in 53 mM trans Ca2+) Ca2+ channel was incorporated from heavy-density skeletal muscle sarcoplasmic reticulum (SR) fractions into planar lipid bilayers of the Mueller-Rudin type. cis Ca2+ in the range of 2-950 microM increased open probability (Po) in single channel records without affecting open event lifetimes. Millimolar ATP was found to be as good as or better than Ca2+ in activation; however, both Ca2+ and ATP were required to fully activate the channel, i.e., to bring Po = 1. Exponential fits to open and closed single channel lifetimes suggested that the channel may exist in many distinct states. Two open and two closed states were identified when the channel was activated by either Ca2+ or ATP alone or by Ca2+ plus nucleotide. Mg2+ was found to permeate the SR Ca channel in a trans-to-cis direction such that iMg2+/iCa2+ = 0.40. cis Mg2+ was inhibitory and in single channel recordings produced an unresolvable flickering of Ca- and nucleotide-activated channels. At nanomolar cis Ca2+, 4 microM Mg2+ completely inhibited nucleotide-activated channels. In the presence of 2 microM cis Ca2+, the nucleotide-activated macroscopic Ba conductance was inhibited by cis Mg2+ with an IC50 equal to 1.5 mM.

Imperatoxin A induces subconductance states in Ca2+ release channels (ryanodine receptors) of cardiac and skeletal muscle.

Single-channel and [3H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTxa), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTxa in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current-voltage relationship. The chord conductance at -40 mV was approximately 43% of the full conductance, whereas it was approximately 28% at a holding potential of +40 mV. The substate formation by IpTxa was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTxa reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTxa binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was approximately 1.5. The IpTxa binding site was calculated to be located at approximately 23% of the voltage drop from the cytosolic side. IpTxa induced substates in the ryanodine-modified skeletal CRC and increased or reduced [3H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTxa induces subconductance states in skeletal and cardiac muscle Ca2+ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.

Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle.

To understand the nature of the transmission process of excitation-contraction (EC) coupling, the effects of the anion perchlorate were investigated on the voltage sensor (dihydropyridine receptor, DHPR) and the Ca release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum (SR). The molecules, from rabbit skeletal muscle, were either separated in membrane vesicular fractions or biochemically purified so that the normal EC coupling interaction was prevented. Additionally, the effect of ClO4- was investigated on L-type Ca2+ channel gating currents of guinea pig ventricular myocytes, as a native DHPR not in the physiological interaction of skeletal muscle. At 20 mM, ClO4- had minor effects on the activation of ionic currents through Ca channels from skeletal muscle transverse tubular (T) membranes fused with planar bilayers: a +7-mV shift in the midpoint voltage, V, with no change in kinetics of activation or deactivation. This is in contrast with the larger, negative shift that ClO4- causes on the distribution of intramembrane charge movement of skeletal muscle. At up to 100 mM it did not affect the binding of the DHP [3H]PN200-110 to triad-enriched membrane fractions (TR). At 8 mM it did not affect the kinetics or the voltage distribution of gating currents of Ca channels in heart myocytes. These negative results were in contrast to the effects of ClO4- on the release channel. At 20 mM it increased several-fold the open probability of channels from purified RyR incorporated in planar bilayers and conducting Ba2+, an effect seen on channels first closed by chelation of Ca2+ or by the presence of Mg2+. It significantly increased the initial rate of efflux of 45Ca2+ from TR vesicles (by a factor of 1.75 at 20 mM and 4.5 at 100 mM). ClO4- also increased the binding of [3H]ryanodine to TR fractions. The relative increase in binding was 50-fold at the lowest [Ca2+] used (1 microM) and then decayed to much lower values as [Ca2+] was increased. The increase was due entirely to an increase in the association rate constant of ryanodine binding. The chaotropic ions SCN- and I- increased the association rate constant to a similar extent. The binding of ryanodine to purified RyR protein reconstituted into liposomes had a greater affinity than to TR fractions but was similarly enhanced by ClO4-. The reducing agent dithiothreitol (5 mM) did not reduce the effect of ClO4-, and 5% polyethylene glycol, with an osmolarity equivalent to 20 mM ClO4-, did not change ryanodine binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Ryanodine sensitive calcium release channel from left ventricle, septum, and atrium of canine heart.

OBJECTIVE: The aim was to consider the possibility that functionally distinct forms of the ryanodine sensitive calcium release channel are expressed in different regions of heart. METHODS: Membranous fractions enriched in ryanodine binding activity were isolated from canine left ventricular free wall, interventricular septum, and atrium. Ryanodine receptors (RyR) were purified by sucrose density gradient centrifugation, following solubilisation of sarcoplasmic reticular membranes with the detergent Chaps. Single channel currents were measured, upon reconstitution of sarcoplasmic reticular vesicles and the purified RyR into planar lipid bilayers. RESULTS: Ryanodine sensitive Ca2+ release channels from three different regions of canine heart displayed the same [3H]ryanodine binding and single channel characteristics. CONCLUSIONS: The left ventricular free wall, septum, and atrium of canine heart may express functionally related, if not identical, ryanodine receptor/Ca2+ release channels.

Evidence for a role of C-terminal amino acid residues in skeletal muscle Ca2+ release channel (ryanodine receptor) function.

The effects of deleting 1, 3 and 15 amino acid residues from the highly conserved C-terminus of the tetrameric skeletal muscle ryanodine receptor (RyR) complex were determined. Immunoblot analysis indicated similar expression levels in HEK293 cells for full-length and mutant proteins. Full-length and RyR lacking the last amino acid showed [3H]ryanodine binding and single channel activities typical of native receptors. Deletion of 3 amino acids resulted in decreased activities, whereas deletion of 15 amino acids yielded an inactive RyR. These results suggest that the most 15 C-terminal amino acids are important for the expression of a functional RyR complex.

Reconstitution of the solubilized cardiac sarcoplasmic reticulum potassium channel. Identification of a putative Mr approximately 80 kDa polypeptide constituent.

Recent evidence has indicated that potassium ion movement through sarcoplasmic reticulum (SR) K+ channels is an important countercurrent for Ca2+ release from SR. We used Chaps-solubilized SR vesicles and sucrose density gradient centrifugation to identify components of the canine cardiac SR K+ channel. To overcome the difficulty of the absence of a high-affinity specific ligand, we have successfully applied the planar lipid bilayer reconstitution technique to identify and functionally assay for the solubilized SR K+ channel. We found that Chaps solubilization of the channel did not change the protein's functional properties. The cardiac SR K+ channel sediments as a 15-20S protein complex. A polypeptide of Mr approximately 80 kDa was found to specifically comigrate with the 15-20S gradient fractions and might be a major constituent of the cardiac SR K+ channel.

Isolation and partial cloning of ryanodine-sensitive Ca2+ release channel protein isoforms from human myometrial smooth muscle.

Partial cDNAs of the ryanodine receptor were cloned using PCR analysis from reverse transcribed total and mRNA, extracted from freshly isolated pregnant, non-pregnant, and cultured human myometrial smooth muscle. The identity of these clones was confirmed by nucleotide sequencing of the fragments and indicate the expression of both the skeletal and brain ryanodine receptor isoforms in these preparations. In freshly isolated non-pregnant myometrial tissue, membrane fractions displaying specific [3H]ryanodine binding activities were isolated using density gradient centrifugation. SDS-PAGE of the sucrose gradient fractions indicated the specific comigration of a polypeptide with a molecular mass of approximately 544 kDa with the ryanodine binding activity.

Evidence for a role of C-terminus in Ca(2+) inactivation of skeletal muscle Ca(2+) release channel (ryanodine receptor).

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity.

Identification and functional characterization of a novel ryanodine receptor mutation causing malignant hyperthermia in North American and South American families.

Malignant hyperthermia is a pharmacogenetic disorder associated with mutations in Ca(2+) regulatory proteins. It manifests as a hypermetabolic crisis triggered by commonly used anesthetics. Malignant hyperthermia susceptibility is a dominantly inherited predisposition to malignant hyperthermia that can be diagnosed by using caffeine/halothane contracture tests. In a multigenerational North American family with a severe form of malignant hyperthermia that has caused four deaths, a novel RYR1 A2350T missense mutation was identified in all individuals testing positive for malignant hyperthermia susceptibility. The same A2350T mutation was identified in an Argentinean family with two known fatal MH reactions. Functional analysis in HEK-293 cells revealed an altered Ca(2+) dependence and increased caffeine sensitivity of the expressed mutant protein thus confirming the pathogenic potential of the RYR1 A2350T mutation.

Cytochemical localization of a "basic" ATPase to canine myocardial surface membrane.

Enzymatic properties of a canine cardiac muscle microsomal fraction were determined to localize in situ a "basic," divalent cation dependent adenosine triphosphatase (ATPase) by ultrastructural cytochemistry. The microsomal fraction had a buoyant density of 1.08--1.13 (20--30% [w/w] sucrose) and hydrolyzed adenosine triphosphate in the presence of Mg2+, Ca2+, Mn2+, or Co2+, but not in that of Sr2+ or Ni2+, under conditions that inhibited interfering (Na+ + K+)-ATPase and sarcoplasmic reticulum Ca2+-ATPase activities. "Basic" ATPase was localized in paraformaldehyde-fixed tissue in a medium containing Mg2+ or a high Ca2+ concentration (4 mM). A free Pb2+ concentration of less than 1 microM was used to capture enzymatically released phosphate anions. Electron-dense lead precipitates were present at the plasmalemma, T-system, and intercalated disc membranes with the exception of the nexus. These studies suggest that "basic" ATPase activity is associated with surface membrane structures of canine cardiac muscle.

Monoclonal antibody specific for the T-tubule of skeletal muscle.

Monoclonal antibodies were raised against a triad-enriched (sarcoplasmic reticulum-T-tubule complex) microsomal membrane fraction of rabbit skeletal muscle. The avidin-biotin complex (ABC) immunoperoxidase staining method was used to screen hybrid colonies. Positive antibodies exhibited a granular doublet pattern at the A-I junction, consistent with the location of triads in rabbit muscle. One monoclonal antibody, M171, was further characterized by ultrastructural and immunoadsorption techniques. Postembedding electron immunocytochemistry was performed on tissue sections embedded in Lowicryl K4M. Goat anti-mouse immunoglobulin absorbed to 10 nm colloidal gold particles was used as an ultrastructural label. In these studies, M171 recognized an epitope at the triads and at periodic openings along the plasmalemma. Immunoadsorption on protein transfers of isolated sarcoplasmic reticulum, surface membrane (plasmalemma and T-tubule), and triad-enriched fractions showed that M171 reacts with a surface membrane component. Taken together, these studies suggest that M171 recognizes an epitope associated with the T-tubule at the triad and at the "mouth" of the T-system at the plasmalemma.