Intermediate complex of ATP hydrolysis and synthesis by muscle proteins.

Myosin catalyzed exchange between 32Pi and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of 32Pi in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange 32Pi and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of 32Pi incorporation is proposed and the importance of the M-ADP complex for force generation is suggested.     

The possible role of tightly bound adenine nucleotides in oxidative and photosynthetic phosphorylation.

The tightly bound nucleotides of the beff-heart mitochondrial ATPase are released during cold inactivation followed by ammonium sulfate precipitation. During incubation at 0 degrees C the sedimentation coefficient (S20W) of the ATPase first declines from 12.1S to 9S. Prolonged incubation or precipitation with ammonium sulfate leads to dissociation of the 9S component into subunits with S20W of 3.5S. The 9S component still bears bound nucleotides which exchange more extensively and rapidly with added nucleotides than those bound to the active 12.1S component. The bound nucleotides are lost when the 9S form dissociates into the smaller subunits. Thus, firm binding of nucleotides is a property of the quarternary structure of the enzyme. The exchangeability of the nucleotides bound to the ATPase of chloroplast membranes is greatly increased in membranes illuminated in the presence of pyocyanine. Pi can exchange into both the beta and gamma positions of the bound nucleotides when the membranes are energized in the presence of Mg2+. The exchange of the nucleotides and the incorporation of Pi are insensitive to the inhibitor Dio-9 but are inhibited by the uncoupler S13. This inhibition by S13 parallels that of the inhibition of photosynthetic phosphorylation. These findings are discussed with regard to our hypothesis that electron transfer causes release of preformed tightly bound ATP from the ATPase by inducing a conformational change.     

Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography

A method for the determination of adenosine triphosphatase (ATPase) activity of myofibrils of big head carp by using ion chromatography was introduced. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and orthophosphate (Pi) were separated completely. Recoveries for ATP, ADP and Pi were 98+/-5%, 97+/-4% and 98+/-5%, respectively. Pi liberated from ATP during reaction was monitored by ion chromatography using the suggested method. This method was applicable to the determination of myofibrils ATPase activity for quick quality evaluation of surimi.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

Magnetic resonance and kinetic studies of the mechanism of membrane-bound sodium and potassium ion- activated adenosine triphosphatase.

EPR and water proton relaxation rate (1/T1) studies of partially (40%) and "fully" (90%) purified preparations of membrane-bound (Na+ + K+) activated ATPase from sheep kidney indicate one tight binding site for Mn2+ per enzyme dimer, with a dissociation constant (KD = 0.88 muM) in agreement with the kinetically determined activator constant, identifying this Mn2+-binding site as the active site of the ATPase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mM in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar high affinities and decrease 1/T1 of water protons due to a decrease from four to three in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with HPO2-4 and CH3PO2-3 as a function of pH indicates that Na+ induces the phosphate monoanion to interact with enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+, estimated from NMR titrations, agreed with kinetically determined activator constants of these ions consistent with binding to the active site. Parallel 32Pi-binding studies show negligible formation (less than 7%) of a covalent E-P complex under these conditions, indicating that the NMR method has detected an additional noncovalent intermediate in ion transport. Ouabain, which increases the extent of phosphorylation of the enzyme to 24% at pH 7.8 and to 106% at pH 6.1, produced further decreases in 1/T1 of water protons. Preliminary 31P- relaxation studies of CH3PO2-3 in the presence of ATPase and Mn2+ yield an Mn to P distance (6.9 +/- 0.5 A) suggesting a second sphere enzyme-Mn-ligand-CH3PO2-3 complex. Previous kinetic studies have shown that T1+ substitutes for K+ in the activation of the enzyme but competes with Na+ at higher levels. From the paramagnetic effect of Mn2+ at the active site on the enzyme on I/T1 of 205T1 bound at the Na+ site, a Mn2+ to T1+ distance of 4.0 +/- 0.1 A is calculated, suggesting the sharing of a common ligand atomy by Mn2+ and T1+ on the ATPase. Addition of Pi increases this distance to 5.4 A consistent with the insertion of P between Mn2+ and T1+. These results are consistent with a mechanism for the (Na+ + K+)-ATPase and for ion transport in which the ionization state of Pi at a single enzyme active site controls the binding and transport of Na+ and K+, and indicate that the transport site for monovalent cations is very near the catalytic site of the ATPase. Our mechanism also accounts for the order of magnitude weaker binding of Na+ compared to K+.     

Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase.

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.     

Isolation of membrane vesicles with inverted topology by osmotic lysis of Azotobacter vinelandii spheroplasts.

Membrane vesicles were prepared from Azotobacter vinelandii spheroplasts by lysis in either potassium phosphate (pH 7.0) or Tris1-acetate (pH 7.8) buffers. These 2 types of preparations differ considerably in their properties: 1) Examination by scanning electron microscopy reveals that the Pi vesicles consist primarily of closed structures 0.6-0.8 micrometer in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1-0.3 micrometer in diameter, and have a much smoother surface structure. 2) Antisera from rabbits immunized with A. vinelandii lipopolysaccharide antigen will agglutinate Pi vesicles but not Tris vesicles. 3) Tris vesicles have a fourfold higher specific activity of latent H+-ATPase than Pi vesicles. After exposure to Triton X-100 similar ATPase activities are observed for both types of vesicles. 4) Pi vesicles transport calcium in the presence of ATP or lactate at less than 30% of the rats observed for Tris vesicles. 5) Tris vesicles have less than 22% of the transport capacity of Pi vesicles for accumulation of labeled sucrose and less than 3% of the capacity for valinomycin-induced uptake of rubidium observed during respiration. 6) Quinacrine fluorescence intensity is reduced by 30% during lactate oxidation and 20% during ATP hydrolysis by Tris vesicles. Under similar conditions, fluorescence in Pi vesicles is quenched by only 7% and less than 2%, respectively. These findings suggest that Pi vesicles have the normal orientation of the intact cell whereas Tris vesicles have an inverted topology.     

小分子玉米肽Leu-Asp-Tyr-Glu保护线粒体抗氧化损伤的研究

线粒体是细胞内氧代谢的主要场所,其膜上富含不饱和脂肪酸,使得线粒体成为自由基进攻的主要靶部位^[1].自由基作用于线粒体膜发生脂质过氧化损伤反应,引起膜的结构与功能改变,使得膜上一系列重要的酶空间排列紊乱,活性降低,膜的通透性改变,诱发多种疾病^[2].目前,抗氧化肽类作为一种非酶类自由基清除剂的研究正在兴起^[3].来源于玉米醇溶蛋白酶降解物中的低分子量肽具有许多重要的生理功能,如促进乙醇代谢、抗疲劳作用以及降血脂等功效^[4].而这些功能的实现主要依赖于其抗氧化活性^[5].最近,我们从玉米蛋白酶解物中获得了一种新的小分子抗氧化肽Leu-Asp-Tyr-Glu(LD YE,LD YE经文献检索为一种新的寡肽)^[6].     

Experimental and theoretical investigation of the dispersed fluorescence spectroscopy of HC4S

A high-resolution single vibronic level emission study from the A (2)Pi(32) state of the HC(4)S radical is reported. Ground state density functional theory frequencies have been used to assign ground state vibronic levels involving three stretching modes nu(2), nu(3), and nu(5) in the region of 0-3250 cm(-1), while the frequency of nu(4) remains speculative. Tentative assignments are given for the complicated structures arising from Renner-Teller and spin-orbit interactions within the bending energy levels. From analysis of the dispersed emission spectra, Fermi resonances involving pairs of bands have been identified in the A (2)Pi(32)<--X (2)Pi(32) laser induced fluorescence spectrum.     

Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport.

A carrier protein mediating alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE- or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TFo) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.     

The effects of ADP on reverse electron flow and the oxygen exchange reactions catalyzed by bovine heart muscle submitochondrial particles.

1,N6-Ethenoadenosine diphosphate (epsilon-ADP) inhibits reverse electron flow (succinate leads to NAD+ driven by ATP) by competing with ATP, in contrast to ADP which we have shown previously to be a noncompetitive inhibitor. From these and other data it is concluded that the noncompetitive inhibition noted with ADP results from a combination of competitive inhibition plus non- or uncompetitive inhibition, the former occurring at a relatively nonspecific catalytic site and the latter at an extracatalytic site apparently quite specific for ADP. ADP, which stimulates ATP in equilibrium H2O and Pi in equilibrium H2O exchanges appears to be necessary for inhibition by arsenate of these exchanges. It is suggested that the ATP-supported Pi in equilibrium H2O exchange may be predominantly of the medium or intermediate type, depending on the concentrations of the Mg2+ complexes of ADP and Pi. Thus only exchanges involving medium ADP and Pi would be expected to show arsenate sensitivity.     

Preparation of highly concentrated super-hot [gamma-32P]ATP using small-scale ion-exchange chromatography

In order to obtain a highly concentrated, pure and super-hot [gamma-32P]ATP, we improved the purification method of super-hot [gamma-32P]ATP which was synthesized by the method of Johnson and Walseth (1979). The super-hot [gamma-32P]ATP was synthesized in a relatively large volume (2 ml) of reaction mixture and purified using semi-micro scale anion exchange chromatography (Dowex 1 X 2, 60-70 microliters column volume). In combination with washing the reaction product with certain organic solvents, this chromatography technique makes it possible to obtain a highly concentrated and pure super-hot [gamma-32P]ATP (approx. 7,000 Ci/mmol; 20-30 mCi/ml) from [32P]Pi of any commercial source in a good yield.     

Laser spectroscopy of NiBr: new electronic states and hyperfine structure

Laser induced fluorescence spectrum of NiBr in the visible region between 604 and 666 nm has been recorded and analyzed. Fourteen bands belonging to three electronic transition systems, namely, [15.1] (2)Delta(52)-X (2)Pi(32), [15.1] (2)Pi(32)-X (2)Pi(32), and [14.0] (2)Delta(52)-X (2)Pi(32) have been observed. Spectra of isotopic molecules were also observed and analyzed. Detailed analysis of the recorded spectra indicated that the two electronic states [15.1] (2)Pi(32) and [15.1] (2)Delta(52) lie about 1 cm(-1) apart from each other and J-dependent perturbation due to spin-uncoupling interaction has been observed. Least squares fitting procedures involving deperturbation matrix elements were used to fit the observed line positions, which yielded accurate molecular constants for the [15.1] (2)Pi(32) and [15.1] (2)Delta(52) states. In addition, the (1,0) band of the [15.1] (2)Delta(52)-X (2)Pi(32) transition shows partially resolved hyperfine structure that was caused by the interaction of unpaired electron with the magnetic moment of the Br nucleus (nuclear spin of I=32) in the excited state. The rapid decrease in hyperfine width as J increases suggests that the hyperfine coupling in the excited state conforms to Hund's case (a(beta)) coupling scheme.     

Phosphorprotein intermediate in the Ca2+-dependent ATPase reaction of macrophage plasma membrane.

ATPase activity and phosphorylation by [gamma-32P] ATP of isolated plasma membrane of alveolar macorphages are stimulated in a parallel fashion by physiologic concentrations of Ca2+, with half-maximal activating effect of this ion at (3--7) X 10(-7) M. For various membrane preparations, a direct proportionality exists between Ca2+-dependent ATPase activity and amount of 32P incorporated. Labeling of membrane attains the steady-state level by 10 sec at 0 degrees C, and is rapidly reversed by adenosine diphosphate (ADP), K+ decreases the amount of membrane-bound 32P, mainly by enhancing the rate of dephosphorylation of the 32P-intermediate. Hydroxylamine causes a release of about 90% of 32P bound to the membrane, thus indicating that the 32P-intermediate contains an acyl-phosphate bond. When the labeled plasma membrane is solubilized and electrophoresed on acrylamide gels in the presence of sodium dodecyl sulphate, the radioactivity appears to be largely associated with a single protein fraction of 132,000 +/- 2,000 aarent molecular weight. These features of the macrophage Ca2+-ATPase suggest that the enzyme activity might be part of a surface-localized Ca1+-extrusion system, participating in the regulation of Ca2+-dependent activities of the macrophage.     

Rotationally inelastic scattering of OH (2Pi 3/2, v=0, J=3/2, f) by HBr (1Sigma, v=0, J<4)

Relative state-to-state cross sections of OH molecules in the (2)Pi(32), v=0, J=32, M(J)=32, f state have been determined for transitions up to (2)Pi(32), v=0, J=112, f and (2)Pi(12), v=0, J=72, e states by collisions with HBr molecules ((1)Sigma, v=0, J<4) at 750 cm(-1) collision energy. In order to investigate features of the anisotropy of the OH-HBr potential energy surface, the steric asymmetries, which account for the effect of the OH orientation with respect to the collision partner, have been measured. A comparison with other systems previously studied shows strong similarities with the OH-HCl system.     

Interaction of hydroxyapatite with sodium chondroitin sulfate and calcium chondroitin sulfate in an aqueous phase

The suspension pH at a given concentration of hydroxyapatite (HAP) decreased with the concentration of calcium chondroitin-6-sulfate (CaChs), whereas it increased with the concentration of sodium chondroitin-6-sulfate (Na2Chs). The former effect is due to the increase in the concentration of H+ by ion exchange between H+ on the surface of HAP and Ca2+ of CaChs, and the latter is due to the protonation of phosphate ion (Pi) released from the surface of HAP. The absorbed amount of chondroitin-6-sulfate anion (Chs) by HAP was higher with CaChs than with Na2Chs over the concentration range examined. The equilibrium concentration of Pi decreased with increasing concentration of added CaChs because the concentration of free Ca2+, which dissociates from CaChs, regulates the free concentration of Pi through the restriction of the solubility product of HAP (Ksp). In contrast, that in the presence of Na2Chs increased with increasing concentration of added Na2Chs owing to the anion exchange between Chs and Pi of the HAP surface. The total concentration of Ca2+, which was released from HAP into the solution phase, increased after passing through a minimum with increasing concentration of added Na2Chs. That is, the concentration of Ca2+ free from Chs decreased with an increase in the concentration of released Pi owing to the restriction of the solubility product, whereas that of Ca2+ bound by Chs increased with increasing concentration of added Na2Chs through the ion exchange of Na+ with Ca2+. It was confirmed by the dialysis method that the value of Ksp was almost constant around 10, although HAP dissolves incongruently in the presence of Na2Chs.     

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.

We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.     

Shuttles for translocation of NADH in isolated liver cells from fed rats during oxidation of xylitol.

Translocation of xylitol-derived NADH via malate-aspartate and alpha-glycerophosphate shuttles was studied in liver cells isolated from fed rats. In bicarbonate medium amino-oxyacetate, rotenone and antimycin A, were equally efficient in depressing the xylitol removal. Incubation of cells in nonbicarbonate medium did not affect the rate of xylitol removal. In this medium amino-oxyacetate and antimycin A, but not rotenone, inhibited xylitol removal. Xylitol inhibited the lactate accumulation found when the cells were incubated without any exogenous substrates. Glucose was the main end product of xylitol oxidation. In nonbicarbonate medium ketogenesis was high, whereas in bicarbonate medium a low rate of ketone body formation was found. Xylitol had no effect on the rate of ketone body formation in either medium tested. Xylitol markedly decreased the ATP and Pi contents of the cells, but no change in the ATP/ADP x Pi ratio or the rate of oxygen consumption was found. The results suggest that NADH formed during xylitol oxidation is translocated to the mitochondria mainly through the malate-aspartate shuttle and only when this shuttle is inhibited does the alpha-glycerophosphate shuttle transfer NADH. Intramitochondrial reactions which form NADH and FADH2 are also suggested to be important regulators of the activity of the alpha-glycerophosphate shuttle.     

Laser induced fluorescence spectroscopy of NC3O

Laser induced fluorescence spectra of the NC(3)O radical in a supersonic jet have been observed. The radical was produced in a pulsed electric discharge of HC(3)N and O(2) diluted to 0.3% with Ar. A total of 17 vibronic bands with a radiative lifetime of approximately 30 ns have been observed in a region from 27 000 to 27 500 cm(-1). The observed vibronic bands are classified as (2)Pi(12)-(2)Pi(12), (2)Pi(32)-(2)Pi(32), and (2)Sigma-(2)Sigma types. The upper states of the (2)Sigma-(2)Sigma bands have large spin-rotation constants, which should be denoted as Sigma((+)) and Sigma((-)). From high-level ab initio calculations and rotational analyses, the observed transition was assigned to the B (2)Pi-X (2)A(") transition. Dispersed fluorescence spectra from the upper (2)Sigma and (2)Pi vibronic levels have also been observed, yielding fundamental vibrational frequencies for the nu(1), nu(2), nu(3), and nu(7) modes of the ground state.