ATPase-rich Fraction Research Articles

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.

A pea NTPase, PsAPY1, recognizes signal molecules from microorganisms

Apyrases (E.C.3.6.1.5; NTP-NDPases) are distributed in the cytosol, nuclei, cytoskeleton, and on the surface of plant cells. Some may play an important role in signal transduction from exogenous stimuli. We previously found a protein of ca. 55-kDa (CWP-55) in an ATPase-rich fraction from the pea cell wall bound to the elicitor and supprescins (suppressors of defense) from pea pathogen Mycosphaerella pinodes. We cloned the cDNA of CWP-55 that coincided with PsAPY1, one of two NTPase clones in a pea cDNA library. An analysis with a green fluorescent protein fusion protein indicated that PsAPY1 was distributed in the cell wall, nucleus, and cytoplasm. The recombinant PsAPY1 expressed in Escherichia coli had ATP-hydrolyzing activity responsive not only to the elicitor and supprescins from the pea pathogen but also to other elicitors such as a bacterial harpin, a yeast extract, and a synthetic glycopeptide. Biotinylated fungal signal molecules were bound to the recombinant PsAPY1 specifically. Resonant mirror detection confirmed such binding characteristics of PsAPY1. Based on these results, we discuss the role of cell-wall-bound NTPases in recognizing and responding to microorganisms on the cell wall surface.

Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa.

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033% w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities having distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5'-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Action of mercurials on the active and passive transport properties of sarcoplasmic reticulum.

The effect of Hg2+ and CH3-Hg+ on the passive and active transport properties of the Ca2+-Mg2+-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10(-5) and 10(-4) M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca2+. The mercurials increase the passive Ca2+ and Mg2+ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca2+-Mg2+-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K+ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the CA2+-Mg2+-ATPase and other SR proteins in black lipid membranes, as presented by others.

Rapid kinetic study of the passive permeability of a Ca2+-ATPase rich fraction of the sarcoplasmic reticulum

A method was developed for the study of divalent cation transport events on the time scale of 20 msec or longer. Passive Ca2+ equilibration across the membranes of the Ca2+-ATPase rich fraction of sarcoplasmic reticulum (SR) was studied. The method makes use of the divalent cation sensitivity of the surface binding of the fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS−). Binding to the inside and outside surfaces is distinguished in fluorescent stopped-flow experiments. The surface binding reactions of the probe are faster than the time resolution of the instrument (ca. 3 msec), while binding reactions requiring transport across the membrane could be resolved. In Ca2+ influx experiments, the time course of fluorescent enhancement was monitored following a Ca2+ jump. The kinetics of Ca2+ efflux were studied by pre-equilibrating Ca2+ across the membrane, removing the external Ca2+ with an EGTA jump, and observing the time course of the fluorescence decrease. Rapid transport of ANS− (coupled to K+) was ensured by the addition of valinomycin. Two processes of Ca2+ influx were observed: (i) a rapid process with small fluorescent amplitudes and at1/2 of 40–60 msec and (ii) a slow process with a large amplitude and at1/2 of 70–100 sec. The rates and extents of the two phases were quantitated in terms of the rates and extents of change in the Ca2+ concentration in the SR lumen. The slow phase accounted for a larger change, in the internal free Ca2+ concentration than did the first phase. For the influx of 10 mM Ca2+, the rapid phase raises the internal Ca2+ concentration to ca. 1 mM within its apparentt1/2 of 20 msec. The slow phase brings about an increase of the internal Ca2+ concentration to 4 mM within its apparentt1/2 of 90 sec. The two phases have average rates of increase of internal free Ca2+ concentration, Δ [Ca] i /sec of ca. 50 mM/sec and ca. 0.02 mM/sec, respectively. The Ca2+ influx rates increased with increasing KCl concentration and with increasing external Ca2+ concentration.

Measurement of surface potential and surface charge densities of sarcoplasmic reticulum membranes.

The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS-) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS- is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(e psi o/kT), where psi o is the membrane surface potential, e is the electronic charge, and kT has its usual meaning. The potential measured is characteristic of the ANS- bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS-, referred to as the aqueous phase bathing the surface, were in the range -10 to -15 mV. This was observed for the outside and inside surfaces of the Ca2+-ATPase-rich fraction of the SR and for both surfaces of the SR fraction rich in acidic Ca2+ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS- binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca2+ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca2+ permeability. The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types of SR. Values for the Ca2+-ATPase rich SR fraction were between 2.9 X 10(3) and 3.8 X 10(3) esu/cm2, (0.96 X 10(-6) and 1.26 X 10(-6) C/cm2) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6 X 10(3) and ca. 2.2 X 10(3) esu/cm2 (2.2 X 10(-6) and 0.73 X 10(-6) C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in the SR. The acidic Ca2+ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of A from the membrane surface. The experiments gave evidence for a Donnan effect on the K+ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in this SR fraction. The equilibrium constant for ANS- was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in the SR lumen.

Kinetics of chlorotetracycline permeation in fragmented, ATPase-rich sarcoplasmic reticulum.

Conditions are defined under which the fluorescent chelate probe chlorotetracycline (CTC) may be quantitatively used as a kinetic indicator of CA2+ transport by the membranes of the ATPase-rich fraction of sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle. The fluorescence enhancement accompanying Ca2+ transport results from binding of Ca-CTC complexes to the inside surface, a process requiring transport of CTC across the membrane.Slow permeation of the dye results in a time course of fluorescence mat is slower than the transport reaction. When the probe was added after attainment of maximally-accumulated Ca2+ the rate of fluorescence rise (t1/2 ≈ 30 sec) was on the same order as the rate when added before Ca2+ uptake had begun (t1/2 ≈ 35 sec). In both cases, the rate of the fluorescence was limited by the rate of transport of the probe. However, when A23187 was added to the SR following active Ca2+ accumulation in the presence of CTC, decreases in fluorescence with 1/2 values less than 3 s...

An ATPase activity associated with brain microtubules

A persistent ATPase/GTPase activity has been found to be associated with highly recycled bovine brain microtubules. A GTP regeneration system was introduced to minimize the inhibitory effects of this hydrolase on microtubule polymerization. The characteristics of the ATPase indicate that it is not involved in GTP-induced mictrotubule polymerization, but is directly involved in ATP-induced polymerization. ATP-induced polymerization was also shown to require stoichiometric amounts of GDP, but higher levels of GDP inhibited both microtubule formation and the ATPase activity. An ammonium sulfate fractionation procedure was devised to separate microtubule protein into an ATPase-rich fraction and a pure tubulin fraction. The pure tubulin fraction polymerized in the presence of GTP, but not in the presence of ATP and GDP. In contrast, the ATPase-rich fraction polymerized with either ATP or GTP. It is still not known whether the microtubule associated ATPase plays a significant role in cellular microtubule function.

A high activity divalent cation ATPase from the distal, caudal epididymis of bulls.

A membrane fraction rich in divalent cation ATPase is sedimented at high centrifugal force from medium flushed through distal, caudal bovine epididymides after sperm and cytoplasmic droplets are removed. This membrane fraction rapidly liberates equimolar amounts of inorganic phosphate from ATP, CTP, ITP, CTP, UTP, and TTP. Inorganic phosphate is slowly hydrolyzed from nucleoside diphosphates by these membranes, but nucleoside monophosphates, pyrophosphate, phosphoenolpyruvate, glucose-6-phosphate, dihydroxyacetone phosphate, or p-glycerophosphate are not substrates. ADP and AMP are weak inhibitors of Mg’-ATPase activity. ATPase is optimal at pH 7, and the effectiveness of divalent cations to activate the ATPase is Mg2*> Ca2*> Mn’�> Co’�> Cd’. High concentrations of K�, Cs, Rb, Na, and choline produce a ouabain-insensitive stimulation of Mg’�-ATPase. Potassium also increases the maximal velocity of ATPase activated by Mn’ and Co’, but not Ca’�. Ouabain, ethacrynic acid, and dihydroethacrynic acid, in concentrations up to 1 mM, have no appreciable effect on Mg�- or Ca’�-ATPase. Mersalyl (0.1 mM) effectively inhibits both ATPase activities, whereas N-ethylmaleimide only weakly inhibits the enzyme. Very high concentrations of rutamycin and aurovertin have no effect on epididymal ATPase, while venturicidin and A23187 produce a slight inhibition. Mg�-ATPase is effectively inhibited by 50 mM sodium fluoride and sodium azide, whereas Ca’�-ATPase is less affected by these inhibitors. Electron micrographs of thin sections of the ATPase-rich fraction show numerous vesicles and long profiles, which appear to be microvilli detached from epididymal principal cells. Negatively stained preparations show numerous 3-4 nm particles covering the surface of these membranes. Histochemical studies indicate that both microvilli of distal, caudal epididymis and the membrane fraction from epididymal flushing medium contain an active magnesium-stimulated ATPase. The origin of the ATPase-rich fraction and possible roles of this enzyme in epididymal function are discussed.