Spin-labelling studies of fragmented sarcoplasmic reticulum

Occluded Ca2+ sites in the CrATP-ATPase complex are studied by first forming the complex in the presence of EGTA so that the sites can be occluded while vacant. 45Ca2+ binding to the occluded sites is then studied under equilibrium conditions. Binding curves are produced for two independent Ca2+ sites with Kd(1) = 0.2 microM and Kd(2) = 1.6 microM. When both sites are saturated, only the Ca2+ bound to the lower affinity site can exchange with free Ca2+. On addition of EGTA (15 vs 0.5 mM Ca2+) all bound Ca2+ dissociates, the net dissociation rate of one-half of the Ca2+ being approximately 10-fold greater than that of the other one-half (at 37 degrees C). When Ca2+ is bound only to the higher affinity site, this Ca2+ will exchange slowly if the concentration of free Ca2+ is below the saturation level of the lower affinity site. An ionophore dependency of the rates of binding and dissociation indicates that the access to the sites is through the interior of the vesicle. Solubilization in C12E9 releases the Ca2+ in the higher affinity site. Our observations are consistent with a model of the ATPase where the lower affinity of two transport sites is associated with the interior position (closest to the lumen) in a transmembrane channel. It is further evident that when in the occluded state, the higher affinity site is available without Ca2+ first being bound to the lower affinity site, eliminating cooperativity from the binding mechanism. In turn, this implies a connection between the integrity of the high-affinity binding site and the linking of sections of the catalytic site by CrATP.

Section: Resultssupporting

confidence: 61%

Ca2+ Binding to Occluded Sites in the CrATP-ATPase Complex of Sarcoplasmic Reticulum: Evidence for Two Independent High-Affinity Sites

Coan¹,

Ji²,

Amaral³

1994

“…On the other hand, evidence for two distinct types of ATP binding sites comes from kinetic studies of substrate utilization. It has been well established that the rate of hydrolysis of ATP is biphasic (Inesi et al, 1967; drolysis rate occurs which will produce maximal levels of E-P (4 nM/mg). At millimolar concentrations, the rate increases; however, since maximal levels of phosphoenzyme can be produced at the initial rate, it has been suggested that this increase in rate is due to binding at a secondary activating site (Verjovski-Almeida et al, 1978;Taylor & Hattan, 1979).…”

Section: Discussionmentioning

confidence: 99%

“…The percentage of ATPase in the total protein was obtained from NaDodS04 gel electrophoresis preformed according to the method of Weber & Osborne (1969). Assuming the MT 100000 band to be entirely composed of ATPase units (Inesi & Scales, 1974), we determined 80% of the protein to be ATPase, with an average variation between samples of 5%. This is in good agreement with similar experiments performed as standardizations in earlier studies (Inesi et al, 1980).…”

mentioning

confidence: 99%

Reactivity of sarcoplasmic reticulum ATPase with iodoacetamide spin-label: evidence for two conformational states of the substrate binding site

Coan¹,

Keating²

1982

The labeling kinetics of sarcoplasmic reticulum ATPase with the iodoacetamide spin probe N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide were followed under conditions designed to selectively label all reactive groups. Approximately 1 mol of spin-label reacted per one 100 000-dalton ATPase chain, indicating only one residue on the enzyme had been labeled. One uniform rate of labeling was observed in the presence of Ca2+. When substrate was then added, approximately one-half of the residues showed a 10-fold increase in labeling rate while the remaining residues reacted at the initial, slower rate. Sequential labeling experiments further established that the two labeling rates correspond to the coexistence of two conformational state of the enzyme. Both Ca2+ and substrate are required to obtain an equal distribution between states, and the effect is completely reversed when substrate is removed. The iodoacetamide spin probe is known to be highly sensitive to the conformation of the ATPase binding pocket, and the residue labeled here is the one which generates broadening in the electron paramagnetic resonance spectrum on substrate binding. Due to the unique selectively of the labeling reaction, it is suggested that when both substrate and Ca2+ are bound to the enzyme, conditions which are precursory to enzyme phosphorylation, two specific conformations of the binding pocket exist in approximately at 50:50 ratio.

“…The two spectral components exhibit random redistribution on removal and reintroduction of substrate, indicating that they may represent two forms of a given site. E-P formation by Pi in the absence of Ca2+ does not produce two discernible components in the EPR spectrum, although a small broadening effect is apparent, %e catalytic cycle of sarcoplasmic reticulum (SR)' ATPase includes a phosphorylated enzyme intermediate (Yamamoto & Tonomura, 1967;Makinose, 1969;Inesi et al, 1970) which is formed by incorporation of ATP-terminal phosphate onto an aspartyl residue of the enzyme protein (Bastide et al, 1973;Degani & Boyer, 1973). The cycle is then completed with hydrolytic cleavage and Pi release.…”

Section: Carol Coanmentioning

confidence: 98%

“…The catalytic cycle of sarcoplasmic reticulum (SR)1 ATPase includes a phosphorylated enzyme intermediate (Yamamoto & Tonomura, 1967;Makinose, 1969; Inesi et al, 1970) which is formed by incorporation of ATP-terminal phosphate onto an aspartyl residue of the enzyme protein (Bastide et al, 1973; Degani & Boyer, 1973). The cycle is then completed with hydrolytic cleavage and P¡ release.…”

mentioning

confidence: 99%

Sensitivity of spin-labeled sarcoplasmic reticulum to the phosphorylation state of the catalytic site in aqueous media and in dimethyl sulfoxide

Coan¹

1983

Under controlled conditions, the iodoacetamide spin-label is highly selective for the sarcoplasmic reticulum adenosinetriphosphatase (SR ATPase), labeling 7-8 nmol of reactive residues per mg of SR protein, or approximately two residues per active enzyme unit. The electron paramagnetic resonance (EPR) spectrum of the labeled enzyme exhibits a small but specific broadening on the binding of substrates or inorganic phosphate. Addition of Ca2+ greatly increases the substrate broadening but reverses the effect of Pi. Here we demonstrate that the Ca2+ effect on the enzyme-substrate spectrum is due to the division of the major spectral component into two components, each representing two distinct conformational environments which impart slightly different degrees of mobility to the spin-label. These components are not readily separated in the spectrum of the fully labeled enzyme, and stoichiometric labeling techniques are used to resolve the components and obtain splitting parameters. The two spectral components exhibit random redistribution on removal and reintroduction of substrate, indicating that they may represent two forms of a given site. E-P formation by Pi in the absence of Ca2+ does not produce two discernible components in the EPR spectrum, although a small broadening effect is apparent, and two components can be resolved in the reaction kinetics of N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide with SR. However, addition of dimethyl sulfoxide, which greatly facilitates phosphorylation of the enzyme, induces a two-component EPR spectrum. We conclude that a conformational change occurs in the enzyme specifically under conditions of high phosphate affinity which affects the motional parameters of 3-4 nmol of labeled residues per mg of SR, a number equal to the phosphorylation sites in our preparations. This affinity is induced by substrate and Ca2+ binding at activating sites in the normal mode of the enzymatic cycle, or by dimethyl sulfoxide in a general manner. In addition, we observe the conformational change with non-adenosine substrates in varying degrees, while 5'-adenylyl imidodiphosphate and adenosine 5'-(beta, gamma-methylenetriphosphate) are both fully effective. Since the latter analogues do not actively phosphorylate the enzyme, it is concluded that the conformational change is related to formation of a transition complex which is highly permissive of phosphoryl transfer. This complex is maximized for an adenosine moiety, while Ca2+ remains a stringent requirement.