Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes

1998 ◽  
Vol 76 (4) ◽  
pp. 395-400 ◽  
Author(s):  
Chikako Katagiri ◽  
Hiro-Hide Ishikawa ◽  
Masamichi Ohkura ◽  
Osam Nakagawasai ◽  
Takeshi Tadano ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Specific Activity ◽  
Specific Binding ◽  
High Specific Activity ◽  
Single Class ◽  
Whole Brain ◽  
Brain Microsomes ◽  
Myotoxin A

Myotoxin a, a small basic polypeptide from prairie rattlesnakes (Crotalus viridis viridis), induces myonecrosis and binds to a single class of binding sites in skeletal muscle sarcoplasmic reticulum. In the present study, [125I]myotoxin a with a high specific activity was prepared and it was shown to bind mainly to microsomes in rat whole brain. [125I]Myotoxin a was further shown to bind to microsomes prepared from all regions tested in brain. Its specific binding to whole brain microsomes was of approximately 1.9 times lower affinity (KD = 0.76 µM; Bmax = 13.1 nmol/mg) than that to skeletal muscle sarcoplasmic reticulum. [125I]Myotoxin a binding to brain microsomes was displaced by unlabeled myotoxin a with an IC50 value of 4.5 µM. [125I]Myotoxin a binding was markedly reduced by treatment of microsomes with trypsin, suggesting that the binding site of [125I]myotoxin a is partially proteins. The binding was significantly inhibited by Mg2+ at concentrations above 1 mM. Having looked at several drugs, we noted that [125I]myotoxin a binding was noncompetitively inhibited by spermine, whereas it was enhanced by heparin. On the other hand, the i.c.v. injection of myotoxin a in mice induced potent convulsive effects at 0.05 nmol/mouse or more. This paper is the first to show that the specific binding site of myotoxin a is present in mouse brain and that myotoxin a is a novel peptidic convulsant in mice.Key words: myotoxin a, specific binding site, brain microsomes, powerful convulsion, central nervous system.

scholarly journals Cross-reactivity of amylin with calcitonin-gene-related peptide binding sites in rat liver and skeletal muscle membranes

1991 ◽  
Vol 277 (1) ◽  
pp. 139-143 ◽  
Author(s):  
A Chantry ◽  
B Leighton ◽  
A J Day
Keyword(s):  
Skeletal Muscle ◽  
Binding Site ◽  
Rat Liver ◽  
Specific Binding ◽  
Calcitonin Gene Related Peptide ◽  
Cross Reactivity ◽  
Scatchard Analysis ◽  
Human Calcitonin ◽  
Related Peptide ◽  
Calcitonin Gene

This study examines whether the high degree of sequence identity between amylin and calcitonin-gene-related peptide (CGRP) is reflected in their cross-reactivity at the level of membrane receptor binding. Rat liver plasma membranes contain a specific saturable binding site for 125I-labelled human CGRP-1. Binding reached equilibrium within 30 min and was rapidly reversed by re-incubating membranes in the presence of 1 microM human CGRP. In addition, the presence of 50 mM- or 500 mM-NaCl lowered specific binding by 30% and 77% respectively. Scatchard analysis was consistent with a single high-affinity site with a dissociation constant (Kd) of 0.125 nM and binding capacity (Bmax.) of 580 fmol/mg of membrane protein. Specific binding of 125I-labelled human CGRP-1 to both liver and skeletal muscle membranes was inhibited by human CGRP-1 [IC50 (concn. causing half-maximal inhibition of binding) 0.1-0.3 nM], and rat amylin (IC50 10 nM), but not by human calcitonin. Covalent cross-linking of 125I-CGRP to its binding site in rat skeletal muscle and liver membranes resulted in labelling of a major species of about 70 kDa under reducing conditions and about 55 kDa under alkylating conditions, as visualized on SDS/PAGE. These radiolabelled species were absent in the presence of CGRP or amylin at 1 microM. These results are indicative of a common binding site for both CGRP and amylin in liver and skeletal muscle, and it is suggested that both peptides mediate their actions through the same effector system. The normal physiological importance and the relevance to the pathology of type 2 diabetes of these data are discussed.

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

1986 ◽  
Vol 246 (1) ◽  
pp. 90-97 ◽  
Author(s):  
Pompeo Volpe ◽  
Ernesto Damiani ◽  
Andreas Maurer ◽  
Anthony T. Tu
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Myotoxin A

scholarly journals Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles

2003 ◽  
Vol 160 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Paola Bagnato ◽  
Virigina Barone ◽  
Emiliana Giacomello ◽  
Daniela Rossi ◽  
Vincenzo Sorrentino
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Physiological Activity ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Structural Function ◽  
Striated Muscles

Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of ∼800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

Abstract P622: Characterization of 125-I-Angiotensin (1-7) Binding to Rat Kidney

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Filipe F Conti ◽  
Andrea Linares ◽  
Leena E Couling ◽  
Mariana Morris ◽  
Katia De Angelis ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Binding Sites ◽  
Specific Activity ◽  
Specific Binding ◽  
Rat Kidney ◽  
Albino Rats ◽  
Angiotensin Peptides ◽  
Putative Receptor ◽  
Wet Weight

Despite the plethora of data indicating beneficial effects of angiotensin (1-7) (Ang 1-7) on the cardiovascular system, its putative receptor, Mas, has not been characterized in tissue membrane preparations other than single concentration demonstrations of the localization of 125 I-Ang 1-7 binding sites in rat kidney. This does not indicate the specificity of 125 I-Ang 1-7 binding nor does it indicate the actual densities of the binding sites, i.e., B max (fmoles/mg tissue), or dissociation constant (K D ) to indicate binding affinity of 125 I-Ang 1-7 for its putative receptor. To characterize 125 I-Ang 1-7 binding in the kidney we prepared a low specific activity, monoradioiodinated Ang 1-7 using a 1:19 mix of 125 iodine : 127 iodine which allows for assessment of the B max and K D with concentrations of radioligand up to 100 nM. Frozen kidneys from adult male albino rats were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:MgCl 2 (50:1) pH 7.2 and incubated with 12 concentrations of 125/127 I-Ang 1-7 ranging from ~3-100 nM for 30 min at 22 C, after which bound 125/127 I-Ang 1-7 was resolved from unbound 125/127 I-Ang 1-7 by filtration and measured with a gamma counter. Specific binding (defined as 100 μM Ang 1-7 displaceable binding) of 125/127 I-Ang 1-7 showed a moderate binding affinity (K D = 14.7 ± 1.8 nM) and binding site density (B max = 24.5 ± 9.9 fmoles/mg initial wet weight). The B max value tended to be lower than that in the liver (B max = 62.3 ± 20.1 fmoles/mg initial wet weight) and the K D value was significantly greater (lower affinity) than that in the liver ( K D = 5.7 ± 0.6 nM, p = 0.0085). Of note, competition for 125/127 I-Ang 1-7 binding Ang 1-7 indicated that the IC 50 for Ang 1-7 competition for 125/127 I-Ang 1-7 binding was 42.5 μM. Moreover, the ability of a variety of angiotensin peptides to inhibit 125/127 I-Ang 1-7 binding at 100 μM, Ang 1-7 was less potent that the other angiotensin peptides: Ang III > Ang II > Ang I ~ Ang IV > Ang 2-7 > Ang 1-7 ~ Ang 3-7. These studies suggest that the binding site for 125/127 I-Ang 1-7 is not specific for the putative Ang 1-7 receptor mas, and may represent a low affinity binding to the AT 1 or AT 2 receptor

scholarly journals Identification of 30 kDa protein for Ca2+ releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum

1999 ◽  
Vol 1451 (1) ◽  
pp. 132-140 ◽  
Author(s):  
Yutaka Hirata ◽  
Norimichi Nakahata ◽  
Masamichi Ohkura ◽  
Yasushi Ohizumi
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Myotoxin A

scholarly journals A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 713-721 ◽  
Author(s):  
J M McWhirter ◽  
G W Gould ◽  
J M East ◽  
A G Lee
Keyword(s):  
Experimental Data ◽  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Adenine Nucleotides ◽  
Contraction Coupling ◽  
High Affinity

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.

scholarly journals Crystallization of the Ca2+ -ATPase of skeletal muscle sarcoplasmic reticulum Inhibition by myotoxin a

FEBS Letters ◽  
1987 ◽  
Vol 224 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Andreas Maurer ◽  
Anthony T. Tu ◽  
Pompeo Volpe
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Myotoxin A

scholarly journals Synthesis of a high specific activity 125I-labeled analog of PK 11195, potential agent for SPECT imaging of the peripheral benzodiazepine binding site

1989 ◽  
Vol 16 (4) ◽  
pp. 423-429 ◽  
Author(s):  
David L. Gildersleeve ◽  
Tay-Yean Lin ◽  
Donald M. Wieland ◽  
Brian J. Ciliax ◽  
James M.M. Olson ◽  
...  
Keyword(s):  
Binding Site ◽  
Specific Activity ◽  
High Specific Activity ◽  
Spect Imaging ◽  
Benzodiazepine Binding

Properties of specific binding site of myotoxin a, a powerful convulsant, in brain microsomes

1998 ◽  
Vol 76 (4) ◽  
pp. 395-400
Author(s):  
Chikako Katagiri ◽  
Hiro-Hide Ishikawa ◽  
Masamichi Ohkura ◽  
Osam Nakagawasai ◽  
Takeshi Tadano ◽  
...  
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Brain Microsomes
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