Dependence Of ATPase Activity Research Articles

Cross-scale analysis of temperature compensation in the cyanobacterial circadian clock system

Circadian clock proteins often reveal temperature-compensatory responses that counteract temperature influences to keep their enzymatic activities constant over a physiological range of temperature. This temperature-compensating ability at the reaction level is likely crucial for circadian clock systems, to which the clock proteins are incorporated, to achieve the system-level temperature compensation of the oscillation frequency. Nevertheless, temperature compensation is yet a puzzling phenomenon, since side chains that make up the clock proteins fluctuate more frequently due to greater thermal energy at higher temperature. Here, we investigated temperature influences on the dynamics of KaiC, a temperature-compensated enzyme (ATPase) that hydrolyzes ATP into ADP in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated by a factor of only 1.2 by increasing the temperature by 10 °C. This temperature insensitivity of the local motions was not necessarily unique to KaiC, but confirmed also for a series of temperature-sensitive mutants of KaiC and proteins other than clock-related proteins. Rather, the dynamics associated with the temperature-compensatory nature of the reaction- and system-level was found in global diffusional motions, which was suggested to regulate the temperature dependence of ATPase activity and dephosphorylation process presumably through changes in the hexamer conformation of KaiC. The spatiotemporal scale at which cross-scale causality of the temperature sensitivity is established is finite, and extends down to picosecond to sub-nanosecond dynamics only in a very limited part of KaiC, not in its entire part.

Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLBM) can compete with wild-type PLB (PLBW) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLBW, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in KCa, measured from the Ca2+-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLBW, while the potency of each of the LOF PLBM was about one third that of PLBW. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLBW and LOF PLBM, inhibitory potency was reduced relative to that of PLBW alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLBW was decreased by both (unlabeled) LOF PLBM. These results show that LOF PLBM can compete both physically and functionally with PLBW, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLBM targeted for gene therapy of heart failure in humans.

Oxidative injury induced by hypochlorous acid to Ca-ATPase from sarcoplasmic reticulum of skeletal muscle and protective effect of trolox

Hypochlorous acid (HOCl) concentration-dependently decreased ATPase activity and SH groups of pure Ca-ATPase from sarcoplasmic reticulum (SERCA) of rabbit skeletal muscle with IC(50) of 150 micromol/l and 6.6 micromol/l, respectively. This indicates that SH groups were not critical for impairment of Ca-ATPase activity. Pure Ca-ATPase activity was analysed individually with respect to both substrates, Ca(2+) and ATP. Concerning dependence of ATPase activity on HOCl (150 micromol/l) as a function of free Ca(2+) and ATP, V(max) of both dependences decreased significantly, while the affinities to individual substrates were not influenced, with the exception of the regulatory binding site of ATP. On increasing HOCl concentration, fluorescence of fluorescein-5-isothiocyanate (FITC) decreased, indicating binding of HOCl to nucleotide binding site of SERCA. A new fragment appeared at 75 kDa after HOCl oxidation of SR, indicating fragmentation of SERCA. Fragmentation may be associated with protein carbonyl formation. The density of protein carbonyl bands at 75 and 110 kDa increased concentration- and time-dependently. Trolox (250 micromol/l) recovered the Ca-ATPase activity decrease induced by HOCl, probably by changing conformational properties of the Ca-ATPase protein. Trolox inhibited FITC binding to SERCA.

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (αβ) 2 dimer

We describe and compare the main kinetic characteristics of the (αβ) 2 form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high ( K 0.5=4 μM) and low ( K 0.5=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate ( K 0.5=0.5 mM) and a lower hydrolysis rate ( V M=42 U/mg). Stimulation of ATPase activity by K + ( K 0.5=0.63 mM), Na + ( K 0.5=11 mM) and Mg 2+ ( K 0.5=0.60 mM) all showed V M's of approximately 600 U/mg and negative cooperativity. K + ( K 0.5=0.69 mM) and Mg 2+ ( K 0.5=0.57 mM) also stimulated PNPPase activity of the (αβ) 2 form. Ouabain ( K 0.5=0.01 μM and K 0.5=0.1 mM) and orthovanadate ( K 0.5=0.06 μM) completely inhibited the ATPase activity of the (αβ) 2 form. The kinetic characteristics obtained constitute reference values for diprotomeric (αβ) 2-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.

Arginine Residue at Position 573 in Enterococcus hirae Vacuolar-type ATPase NtpI Subunit Plays a Crucial Role in Na+ Translocation

The 76-kDa NtpI subunit constitutes the membrane-embedded V(0) moiety of Enterococcus hirae vacuolar type Na+-ATPase with a 16-kDa NtpK hexamer containing Na+ binding sites. In this study, we investigated the role of an arginine residue, which is highly conserved among the corresponding subunits of bacterial vacuolar-type ATPases, at position 573 of NtpI. Substitution of Glu, Leu, or Gln for Arg-573 abolished sodium transport and sodium-stimulated ATP hydrolysis of the enzyme. The conservative replacement of Arg by Lys lowered both activities about one-fifth of those of the wild type enzyme. We have reported previously on ATP-dependent negative cooperativity for Na+ coupling of this enzyme (Murata, T., Kakinuma, Y., and Yamato, I. (2001) J. Biol. Chem. 276, 48337-48340). The negative cooperativity for the Na+ dependence of ATPase activity was weakened by the mutation R573K; the Hill coefficients for the wild type and mutant enzymes at a saturated ATP concentration were 0.22 +/- 0.03 and 0.40 +/- 0.05, respectively. The Hill coefficients of both enzymes at limited ATP concentrations approached 1. These results indicate that NtpI Arg-573 is indispensable for sodium translocation and for the cooperative features of E. hirae vacuolar-type ATPase.

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8.

SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4 degrees C after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4 degrees C. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 microM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 microg/ml) and sodium vanadate (3 microM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphabeta)2 association.

Inhibition of microsomal ATPases by high concentration of Mg 2+ in tracheal epithelial cells

Previous work in our laboratory established the presence of two types of microsomal ATPases, a low-affinity vanadate-sensitive (LAVS) and a high-affinity vanadate-sensitive (HAVS) ATPases, in tracheal epithelial cells. These ATPases were identified as Ca 2+-ATPases by specific inhibitors and microsomal Ca 2+ uptakes. Since the regulatory roles of Mg 2+ on both cellular Ca 2+-signaling and epithelial transports were demonstrated, the effects of Mg 2+ on these ATPases were investigated. Mg 2+-dependence of ATPase activity appeared bell-shaped with a maximal activity at 1–2 mM Mg 2+ and Mg 2+ at higher than 2 mM inhibited these enzymes. In a kinetic analysis of the LAVS ATPase inhibition, high concentration of Mg 2+ appeared to inhibit the binding of ATP to a substrate-binding site. The microsomal 45Ca 2+ uptakes mediated by both ATPases were also inhibited by high concentration of Mg 2+. In order to test whether high concentration of Mg 2+ directly inhibits these enzymes, microsomes were made leaky by the treatment of Triton X-100 and the microsomal ATPases were solubilized with CHAPS. The leaky microsomal ATPases and CHAPS-solubilized ATPases were similarly inhibited by high concentration of Mg 2+, suggesting that Mg 2+ directly inhibit these enzymes. In conclusion, Mg 2+ has two types of modulatory effects on these enzymes, a catalytic effect at low concentration and an inhibitory effect at high concentration.

Covalent Binding of Peptides to the N-terminal Hydrophobic Region of Cardiac Troponin C Has Limited Effects on Function

Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide specific sites for attachment of blocking groups. A synthetic peptide, Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these Cys residues, and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity (70-80% inhibited) resulted when the peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of inhibition (10-25% inhibited) resulted from covalent modification of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the Ca2+ dependence of ATPase activity was essentially identical to the unmodified protein. Thus, a subregion of the N-terminal hydrophobic region in cTnC is sensitive to disruption, while other regions are less important or can adapt to rather bulky blocking groups. The data suggest that Ca(2+)-sensitizing drugs may bind to the N-terminal hydrophobic region on cTnC but not interfere with transmission of the Ca2+ signal.

Properties of the F0F1 ATPase Complex from Rhodospirillum rubrum Chromatophores, Solubilized by Triton X‐100

1. A cold-stable oligomycin-sensitive F0F1 ATPase complex from chromatophores of Rhodospirillum rubrum FR 1 was solubilized by Triton X-100 and purified by gel filtration. 2. The F0F1 complex is resolved by sodium dodecyl sulfate electrophoresis into 14 polypeptides with approximate molecular weights in the range of 58000--6800; five of these polypeptides are derived from the F1 moiety of the complex which carries the catalytic centers of the enzyme. 3. The purified F0F1 complex is homogeneous according to analytical ultracentrifugation and isoelectric focusing. 4. The molecular weight as determined by gel filtration is about 480 000 +/- 30 000. S020,w is 1.45 +/- 0.1 S and the pI is 5.4. 5. The amino acid composition of the F0F1 complex is compared with the data obtained for the F1 moiety of the enzyme. 6. Quantitative data on the sensitivity to N,N'-dicyclohexyl-carbodiimide as well as kinetic parameters, regarding substrate specificity and dependence of ATPase activity on divalent cations, are reported.

Dynein 2. A new adenosine triphosphatase from sea urchin sperm flagella.

A new ATPase electrophoretically and immunologically distinct from the dynein ATPase studied previously has been solublized and purified from sea urchin sperm flagella. This ATPase has properties similar to those of dynein ATPase. Therefore, we propose that the two ATPases be considered as dynein isoenzymes, with previously studied dynein being known as dynein 1, and the newly discovered ATPase as dynein 2. Some physicochemical and enzymatic properties of dynein 2 have been determined. The molecular weight calculated from the sedimentation coefficient (12.3 "/- 1 S) and Stokes radius (12.8 "/- 0.4 nm) is 690,000 +/- 70,000. The molecular weight of the high molecular weight subunit of dynein 2 has been determined to be 325,000 +/- 40,000 by Na dodecyl-SO4-polyacrylamide gel electrophoresis. The enzymatic properties of dynein 1 and dynein 2 are similar in substrate specificity, pH optimum, and Mg2+ requirement for ATPase activity, but they differ in their Michaelis constant and in their dependence of ATPase activity upon salt concentration. Digestion of dynein 2 with trypsin yields an ATPase-containing protein fragment, similar to Fragment A obtained from dynein 1. An antiserum prepared against Fragment A from dynein 1 did not precipitate dynein 2 or inhibit its ATPase activity.

Studies on Myofibrils from the Stored Muscle

Adenosine triphosphatase (ATPase) activity of myofibrils isolated from fresh muscle and the muscle stored at 4°C have been measured.An increase in Mg-activated ATPase activity of myofibrils was caused by lengthened homogenization.With the progress of aging of muscle, Mg-activated ATPase activity of myofibrils increased remarkably.When myofibrils from pre-rigor and rigor muscle in 0.16 m KCl were treated with 0.6 m KCl-18 mm Tris-maleate solution (pH 7.0), Mg-activated ATPase activity of myofibrils at low ionic strength increased markedly. However, the Mg-activated ATPase activity of the myofibril isolated from the muscle stored at 4°C for 8 days (8-myofibril) increased slightly after the similar treatment.The dependence of myofibrillar ATPase activity on KCl concentration became greater with the progress of aging of muscle.These results may show that, as long as ATPase activity and the dependence of ATPase activity on KCl concentration are concerned, 8-myofibril is the most similar to the isolated actomyo...

INTERACTION OF A BACTERIAL ADENOSINE TRIPHOSPHATASE WITH PHOSPHOLIPID BILAYERS

A solubilized nonparticulate adenosine triphosphatase from Streptococcus fecalis spheroplast membranes (Abrams, A., and C. Baron, Biochemistry, 7,501 (1968)) interacts at pH 7.5 with lipid bilayer membranes to produce a 10(2)- to 10(4)-fold increase in the electric conductance of the bilayer. In addition, a small decrease in the electrical capacitance of the interactant system is also observed. Interaction is obtained with bilayers formed from a solution in n-decane of either purified egg phosphatidyl-choline, synthetic diphytanoyl phosphatidyl-choline, or a total extract of the membrane lipid of S. fecalis. The magnitude of the increase in conductance is dependent on the presence of Mg(++) and upon the concentrations of both Na(+) and K(+) in the range of 10(-2) to 10(-1)M. An additional tenfold increase in conductance is obtained when ATP is added to the ambient aqueous phase surrounding the bilayer ATPase interactant system. No conductance change is obtained with ATPase which has been treated with pronase. ATPase activity is dependent upon Mg(++) and upon the concentrations of Na(+) and K(+) in the region of 10(-1)M. The function of the ATPase in the intact bacterial membrane is apparently associated with the active transport of cations. The increased conductance in the bilayer which results from its interaction with the ATPase, together with the similarity between the dependence of this interaction and the dependence of ATPase activity on Mg(++), ATP, and the Na(+), K(+) concentrations suggests that the bilayer-ATPase interactant complex may be similar in structure and properties to the membrane-ATPase complex in the intact organism.