EPR and fluorescence depolarization studies on bovine cardiac myosin.

Abstract

SUMMARY To test for possible differences in local conformation and SI flexibility, bovine cardiac and rabbit skeletal myosins were labeled with a fluorophore (1,5-IAEDANS*) and a spin label having iodoacetamide reactivity. The marked activation of the Ca 1+ -ATPase (G-to 8-fold) and inhibition of the K + (EDTA)-ATPase (80-90%) by both labels indicated specific labeling of the fast-reacting thiols (SH,) of both myosins. Fluorescence depolarization studies of 1,5-IAEDANS-labeled cardiac myosin indicated that, like skeletal myosin, the SI moieties of cardiac myosin exhibit considerable segmental flexibility with respect to the rod portion of the molecule. This indicates that segmental flexibility may be a property of all myosins. Cardiac and skeletal myosins immobilized spin labels to approximately the same extent, indicating a similarity in eteric restraints around the SHj thiol of the two myosins. The magnitude of the changes in spin label mobility accompanying binding of MgADP and hydrolysis of MgATP was reduced in cardiac myosin relative to skeletal myosin. This suggests that the lower catalytic center activity of cardiac myosin is associated with more restricted conformational changes accompanying formation of M*«ADP and M**"ADP»P,. From measurements of spin label mobility, the affinity of cardiac and skeletal myosin for ADP were similar: Kj (ADP) -7 UM, n -1.8. The EPR spectrum of spin labels attached to cardiac and skeletal myosin showed similar saturation effects upon actin binding indicating immobilization of myosin heads occurs with both proteins. Circ Res 49: 677-6S4, 1981

Use of Electron Paramagnetic Resonance Spectroscopy to Evaluate the Redox StateIn Vivo

“…Maleimide and iodoacetamide spin labels react (22,94) with thiols at or below room temperature in the physiological pH range (pH 6.5-8.0) to yield chemically stable thio-ethers. Among the applications are specific thiol labeling of sarcoplasmic reticulum ATPase (111), human and horse hemoglobin (66), sugar-H(+) symporter, GalP, in cell membranes of Escherichia coli (65,94), and bovine cardiac and rabbit skeletal myosins (95). However, at an elevated pH they can react with amines or may be hydrolyzed to non-reactive products, therefore compromising labeling specificity.…”

A Role of Bovine Cardiac Myosin-Hydrogel Polymere to Accelerate Wound Healing of Autograft Skin in Rabbits

“…Re-vascularization and vascular permeability are occasionally depend on myocin phosphorylation and calcium C 2+ -calmodulin CaM activation to stimulate myosin chain bioactivity in permeablized endothelium, ATP activity, and C 2+ motion, these processes are called myosin chain phosoryltion (MCP) are absolute requirements for bio-reaction and intercellular gap formation in the wound (10). The activity of myosin motor is modulated by phosphorylation of regulatory myosin chain kinase and phosphotase (11). Myosin ligand-based C 2+ signaling may important in fibroblast-mediated wound repair, but the mechanism supporting transiet changes in C 2+ are unkown (11).…”

“…The activity of myosin motor is modulated by phosphorylation of regulatory myosin chain kinase and phosphotase (11). Myosin ligand-based C 2+ signaling may important in fibroblast-mediated wound repair, but the mechanism supporting transiet changes in C 2+ are unkown (11). Magnesium (Mg +2 ) play an important role in myosin bioactivity, ATPase activity of myosin is activated by C…”

Internal flexibility of cardiac myosins

“…The experimental data could be fitted by curves predicted for either one or two independent ADP 9 xl z t binding sites and association constant in the range of 10-12.4 0 M- [22]. Since the reactive sulfhydryl sites are near to the nucleotide binding pocket in the crystal structure of myosin [23], changes are expected in the environment of the probe molecules.…”