RESUMO
Stimulation of a Ca(2+)-induced Ca(2+)-release channel from skeletal muscle sarcoplasmic reticulum by various adenosine(5')oligophospho(5')adenosines (ApnA, n = 2-6) by a rapid quenching technique using radioactive calcium was studied. Ap4A, Ap5A and Ap6A, as well as adenosine 5'-[beta, gamma-methylene]triphosphate (AdoPP [CH2]P), a non-hydrolyzable ATP analogue, stimulated the Ca(2+)-release channel, whereas Ap2A and Ap3A had no effect. At a concentration of 0.5 mM, the order of stimulation was AdoPP[CH2]P less than Ap4A less than Ap5A much less than Ap6A. As well as having the highest affinity (0.44 mM for half-maximal stimulation), Ap6A showed an extraordinarily high Hill coefficient of 3.3 (1.9 for AdoPP[CH2]P, 2.1 for Ap5A). The stimulating effect of Ap6A was reversible, yet its dissociation proceeded very slowly. Stimulation of Ca2+ release by Ap6A was counteracted by Mg2+ and ruthenium red. A 2',3'-dialdehyde derivative of Ap6A, which is a chemical probe for amino groups, stimulated irreversibly the Ca(2+)-release channel and modified some high-molecular-mass sarcoplasmic reticulum proteins, possibly including the channel protein. Our data suggest that Ap6A stimulates the Ca2+ channel by binding to the activation site of the channel subunit and simultaneously preventing the spontaneous decay of the Ca2+ channel by keeping together two of the four channel subunits by bridging them with its two adenosine groups.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Fosfatos de Dinucleosídeos/farmacologia , Músculos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Cátions Bivalentes , Fosfatos de Dinucleosídeos/antagonistas & inibidores , Eletroforese em Gel de Poliacrilamida , Magnésio/farmacologia , Músculos/efeitos dos fármacos , Coelhos , Rutênio Vermelho/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
The 2',3'-dialdehyde ATP analog (oATP) was synthesized and its ability to activate the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum via the adenosine-nucleotide-binding site was investigated. After reduction by sodium borohydride, oATP binds covalently to the catalytic adenosine-nucleotide-binding site of the enzyme, resulting in 85% loss of acetyl-phosphate-driven Ca2+ uptake and ATP-hydrolysing ability. In the absence of a reducing agent, oATP serves as a substrate for the Ca(2+)-ATPase, as indicated by Pi formation (hydrolysis) and Ca(2+)-uptake ability. oATP binding to the intact light sarcoplasmic reticulum is observed in the absence and presence of the competitive adenosine nucleotide inhibitor, fluorescein isothiocyanate with apparent affinity constants of 1.2 mM and 2.2 mM, respectively. Autoradiography of tryptic fragments from partially purified Ca(2+)-ATPase labeled with [alpha-32P]oATP or [gamma-32P]oATP locates the covalent binding site to the A1 fragment, even in the fluorescein-isothiocyanate-labeled pump protein. With high probability, a lysine residue in the tryptic A1 fragment is labeled by the ribose-modified ATP analog close to the phosphorylation site at Asp351.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Adenosina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Nucleotídeos/metabolismo , Retículo Sarcoplasmático/enzimologia , Marcadores de Afinidade , Animais , Autorradiografia , Sítios de Ligação , Cálcio/metabolismo , Catálise , Cátions Bivalentes , Eletroforese em Gel de Poliacrilamida , Hidrólise , Oxirredução , Coelhos , TripsinaRESUMO
In order to characterize the form of the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum which occludes the calcium bound in the enzyme (Takisawa, H., and Makinose, M. (1981) Nature (Lond.) 290, 271-273), a kinetic method was developed allowing quantitation of the amount of ADP-sensitive and ADP-insensitive phosphoenzyme. The relationships between occluded Ca2+ in the enzyme and the two forms of phosphoenzyme were studied at various concentrations of CaCl2 and MgCl2. The amount of tightly bound Ca2+ in the phosphoenzyme increases concordantly with the increase in the amount of ADP-sensitive phosphoenzyme, suggesting that occlusion of Ca2+ occurs in the ADP-sensitive phosphoenzyme. These results suggest that 1 mol of ADP-sensitive phosphoenzyme occludes 2 mol of Ca2+. Ca2+ is released from the enzyme under conditions which favor the formation of the ADP-insensitive phosphoenzyme (e.g. 5 mM MgCl2 and 50 microM CaCl2). Ca2+ release correlates approximately with the formation of the ADP-insensitive phosphoenzyme. The simulated time course of Ca2+ release, based on the Ca2+-binding properties of the two forms of phosphoenzyme, shows a good fit with the Ca2+ release curves observed, indicating that the ADP-insensitive phosphoenzyme binds no Ca2+ under these conditions.
Assuntos
Difosfato de Adenosina/farmacologia , Adenosina Trifosfatases/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Cloreto de Cálcio/farmacologia , Ácido Egtázico/farmacologia , Cinética , Magnésio/farmacologia , Cloreto de Magnésio , Matemática , Músculos/enzimologia , Fosfoproteínas/metabolismo , CoelhosRESUMO
1. Based on a detailed reaction scheme of the phosphorylation process of the sarcoplasmic transport ATPase the inhibition mechanisms of benzoctamine, DIO 9, AMP-PNP and of Ca2+-ions at relatively high concentrations (1 approximately 100 microM) were determined. 2. The inhibition mechanisms were analyzed by measuring the gamma-phosphate exchange between ATP and ADP and evaluated by applying conventional and an extended Dixon plot procedures. 3. The kinetic patterns of the inhibition were shown to be compatible with the assumed reaction scheme. 4. Each inhibitor combines with definite intermediates: Benzoctamine with the intermediate species Ca2MgE and Ca2Mg2E-ATP; AMP-PNP with Ca2Mg2E approximately P; DIO 9 with E and MgE and Ca2+ at relatively high concentrations with E. 5. The central intermediate blocked by benzoctamine can partially exist as Ca2Mg2E ADPP-benzoctamine which is detected as phosphoprotein after acid denaturation.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Nucleotidiltransferases/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Cinética , Matemática , Músculos/enzimologia , CoelhosRESUMO
The Ca2+ + Mg2+-activated ATPase of the sarcoplasmic reticulum is responsible for the active Ca2+ transport of this membrane system, the key feature of which is the formation of an energy-rich phosphorylated transport enzyme (EP) and its conversion. To understand the Ca2+-transport mechanism, it is essential to clarify the behaviour of this intermediate in relation to such ligands as ATP, ADP, Mg2+ and, particularly, Ca2+. Recent kinetic studies on the phosphate turnover of this system suggested a relatively slow rate of Ca2+ dissociation from the phosphorylated enzyme, which possibly indicated Ca2+ binding in some occluded form with the intermediate. Here we report direct measurements of the binding and release of Ca2+ during phosphorylation of the sarcoplasmic transport enzyme. The results indicate an occlusion of the Ca2+ binding, accompanying an initial configurational change of the enzyme induced by the energy-rich phosphoryl transfer.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Sítios de Ligação , Transporte Biológico Ativo , Ativação Enzimática , Fosforilação , Retículo Sarcoplasmático/enzimologiaAssuntos
Antagonistas Adrenérgicos beta/farmacologia , Cálcio/antagonistas & inibidores , Retículo Sarcoplasmático/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzofuranos/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Cálcio/metabolismo , Nucleotídeos de Citosina/farmacologia , Etanolaminas/farmacologia , Nucleotídeos de Guanina/farmacologia , Hidrólise , Técnicas In Vitro , Nucleotídeos de Inosina/farmacologia , Cinética , Músculos/citologia , Músculos/enzimologia , Oxprenolol/farmacologia , Practolol/farmacologia , Propranolol/farmacologia , Propilaminas/farmacologia , Coelhos , Sotalol/farmacologia , Nucleotídeos de Uracila/farmacologiaAssuntos
Cálcio/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Retículo Sarcoplasmático/enzimologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Transporte Biológico Ativo , Radioisótopos de Cálcio , Cinética , Músculos/citologia , Músculos/enzimologia , Fosfatos/metabolismo , Ligação Proteica , Retículo Sarcoplasmático/efeitos dos fármacos , Fatores de TempoRESUMO
The phosporyl group of acetylphosphate is transferred to the membranal protein of the sarcoplasmic vesicles during active calcium transport. Although the phosphoprotein formed cannot be distinguished from that obtained in the presence of ATP, the conditions for ATP and acetylphosphate hydrolysis are different from each other.