The stability properties of the iron(II)-dioxygen bond in myoglobin and hemoglobin are of particular importance, because both proteins are oxidized easily to the ferric met-form, which cannot be oxygenated and is therefore physiologically inactive. In this paper, we have formulated all the possible pathways leading to the oxidation of myoglobin to metmyoglobin with each required rate constant in 0.1 M buffer (pH 7.0) at 25 degrees C, and have set up six rate equations for the elementary processes going on in a simultaneous way. By using the Runge-Kutta method to solve these differential equations, the concentration progress curves were then displayed for all the reactive species involved. In this complex reaction, the primary event was the autoxidation of MbO2 to metMb with generation of the superoxide anion, this anion being converted immediately and almost completely into H2O2 by the spontaneous dismutation. Under air-saturated conditions (PO2 = 150 Torr), the H2O2 produced was decomposed mostly by the metMb resulting from the autoxidation of MbO2. At lower pressures of O2, however, H2O2 can act as the most potent oxidant of the deoxyMb, which increases with decreasing O2 pressures, so that there appeared a well defined maximum rate in the formation of metMb at approximately 5 Torr of oxygen. Such examinations with the aid of a computer provide us, for the first time, with a full picture of the oxidation reaction of myoglobin as a function of oxygen pressures. These results also seem to be of primary importance from a point of view of clinical biochemistry of the oxygen supply, as well as of pathophysiology of ischemia, in red muscles such as cardiac and skeletal muscle tissues.
The membrane-bound Ca 2ϩ -ATPase (110 kDa) of the sarcoplasmic reticulum (SR) 1 is a calcium pump (1-3) and a P-type ATPase (4, 5). The Ca 2ϩ -ATPase transports 2 mol of calcium across the SR membrane by hydrolytic coupling with 1 mol of ATP, accompanying the transition of the ATPase from E 1 (high affinity state for calcium) to E 2 (low affinity state for calcium) (1-3, 5). The ATP hydrolysis cycle has been shown to be accelerated by ATP binding to a putative regulatory site (1,3,6,7) at concentrations higher than those required for saturation of the catalytic site (8, 9). ATP regulation consists of the acceleration of several intermediate steps in the catalytic reaction (10 -14). Recently, Toyoshima et al. (15) determined the atomic structure of the pump enzyme at a resolution of 2.6 Å and showed that the ATPase molecule has one nucleotide-binding site. However, the molecular basis of the regulatory site has not been clarified (16 -23).Calcium binding to the two calcium transport sites of the enzyme is required for phosphorylation of the enzyme with ATP; the phosphorylation drives the calcium transport reaction (1-3). It is therefore crucial to determine whether calcium binding is regulated by ATP binding. Based on observations of the velocities of calcium transport that are supported by various kinds of high energy phosphate compounds, Ogawa (24) and Ogawa and Ebashi (25) reported that ATP increases the calcium affinity of the enzyme depending on the ATP concentration (0.3 M to 2.7 mM). On the other hand, the calcium dependence of equilibrium calcium binding in the absence of ATP (26) has been shown to exhibit a cooperative profile (Hill number (n H ) of ϳ2) with K 0.5 ϳ 0.4 M. Such a cooperative profile of kinetic calcium binding (n H ϳ 2, K 0.5 ϳ 0.1 M), which was obtained from the calcium dependence of Ca 2ϩ -ATPase activity at a saturating concentration (5 mM) of ATP, has also been observed (27). To clarify whether ATP changes calcium binding of the ATPase, kinetic calcium binding to the enzyme should be examined in the presence and absence of ATP. Recently, the enzyme molecules in the SR membrane have been suggested to exist as two conformational variants of the chemically equivalent enzyme molecules at a ratio of 1:1 (Fig. 1) on the basis of the following observations (28). (i) At 0°C, about half of the calcium-binding sites of the enzyme molecules are in a slow (t1 ⁄2 Ն 2 s)/rapid (t Ͻ2 s) binding state dependent on pH, and the other half are in a slow binding state independent of pH. The enzyme molecules are slowly or rapidly phosphorylated by ATP, concurrent with slow or rapid calcium binding at a ratio of ϳ1:2, indicating that each of the two pools of the calcium sites belongs to one of the two different types (A and B forms) of the enzyme molecules, which are in pH-dependent equilibrium between E 1 and E 2 and predominantly in E 2 independent of pH, respectively, before calcium binding (Fig. 2). (ii) The amino acid sequence of lysyl endopeptidase peptides of the enzyme preparation is homogeneous ...
The Ca2+-ATPase is an integral transmembrane Ca2+ pump of the sarcoplasmic reticulum (SR). Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca2+]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca2+] (≤1.3 µM). The arrays were classified as ‘’tetramer’’, “two-rail” (like a railroad) and ‘’monomer’’. Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca2+] of ~0.002 µM, “two-rail” arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable “two-rail” forms in the presence of ATP at low [Ca2+].
The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.
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