Activation of dynein 1 adenosine triphosphatase by organic solvents and by Triton X-100

The presence of low concentrations of methanol or isopropyl alcohol (2-5%, v/v) in the assay medium stabilizes the latency of dynein 1 from sea urchin sperm flagella, with about a 50% decrease in ATPase level compared to that in the absence of solvent. Somewhat higher concentrations (10-20%, v/v) of these solvents in the assay give a 5-10-fold activation of ATPase activity. Dioxane, formamide, and dimethylformamide, on the other hand, always activate the ATPase activity, with a 5-10-fold increase observed at about 15% (v/v). The activation of latent ATPase activity by solvents is reversible for short exposures, especially in the presence of ATP and at low temperature, but the activation becomes irreversible upon more prolonged exposure. The rate constant for irreversible activation by 16% methanol at 21 degrees C is 0.08 min-1, compared to rates of 0.44 and 0.02 min-1 for activation by 0.05% Triton X-100 at 21 and 0 degree C, respectively. The slowness of this reversible activation induced by methanol and by Triton X-100 suggests that it is the result of large-scale conformational changes in the structure of the dynein. However, the activation by methanol occurs without the dissociation of the alpha and beta subunits of dynein that is observed with Triton X-100. The presence of 1 mM MgATP, or of 100 microM MgATP and 10 microM vanadate substantially protects latent dynein from activation by 0.05% Triton X-100.     

Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate

Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier (Lee-Eiford, A., Ow, R. A., and Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342). The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein. These results indicate that Vi acts as a photosensitizing catalyst and suggest that the cleavage proceeds through excitation of Vi bound to dynein at the hydrolytic ATP binding site on each heavy chain, probably in a dynein X MgADP X Vi complex. The exquisite specificity of Vi-sensitized photocleavage will aid the peptide mapping of dynein heavy chains and may be of broader use in studies of protein structure.     

Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.     

A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.

Treatment of demembranated sea urchin sperm axonemes with an extraction solution containing 0.6 M NaCl, pH 7.0 for 10 min at 4 degrees C yields a solution of dynein 1 having a low, latent specific ATPase activity of about 0.25 mumol of Pi mg(-1) min(-1). Exposure of this dynein solution to 0.1% Triton-X-100 for 10 min at 25 degrees C causes an increase in its ATPase activity to about 3 mumol of Pi mg(-1) min(-1). A similar activation can be obtained by treating at 42 degrees C or by reacting with 60 mol of p-chloromercuribenzene sulfonate/10(6) g of protein. The effects of these activating procedures are not additive, suggesting that they lead to a common activated state. Purification of the latent activity dynein 1 by sucrose density gradient centrifugation yields a monodisperse preparation sedimenting at 21 S, and having a molecular weight of 1,250,000 as determined by sedimentation diffusion and sedimentation equilibrium. Activation of the latent dynein 1 with Triton X-100 converts it to a form sedimenting at 10 to 14 S. The 21 S dynein is also converted to a 10 S form by dialysis against 5 mM imidazole/NaOH buffer, 0.1 mM EDTA, 5 mM 2-mercaptoethanol, pH 7, although in this case, the ATPase activity is increased only about 3-fold, with another 3-fold activation being obtainable upon subsequent treatment with Triton X-100. The 21 S latent form of dynein 1 may represent the intact dynein arms that form moving cross-bridges and generate active sliding between adjacent doublet tubules of the flagellar axoneme. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate suggests a model in which the 21 S dynein 1 particle is composed of three subunits of about 330,000 daltons and one of each of three medium weight subunits of 126,000, 95,000, and 77,000 daltons. When latent dynein 1 is added back to NaCl-extracted axonemes in the presence of 0.15 M NaCl, it recombines stoichiometrically and restores the arms on the doublet tubules with a 6-fold activation of its ATPase activity measured in the absence of KCl.     

Chromosome movement in lysed mitotic cells is inhibited by vanadate

Mitotic PtK1 cells, lysed at anaphase into a carbowax 20 M Brij 58 solution, continue to move chromosomes toward the spindle poles and to move the spindle poles apart at 50% in vivo rates for 10 min. Chromosome movements can be blocked by adding metabolic inhibitors to the lysis medium and inhibition of movement can be reversed by adding ATP to the medium. Vanadate at micromolar levels reversibly inhibits dynein ATPase activity and movement of demembranated flagella and cilia. It does not affect glycerinated myofibril contraction or myosin ATPase activty at less than millimolar concentrations. Vanadate at 10-- 100 micron reversibly inhibits anaphase movement of chromosomes and spindle elongation. After lysis in vanadate, spindles lose their fusiform appearance and become more barrel shaped. In vitro microtubule polymerization is insensitive to vanadate.     

ATPase activity of Tetrahymena cilia before and after extraction of dynein

Cilia from Tetrahymena pyriformis were extracted twice with Tris-EDTA. The first extraction increased the total ATPase activity by about one-third. No increase in activity occurred as a result of the second extraction, but 40% of the original ATPase activity remained in the pellet. The activity remaining in the pellet differed in its substrate specificity, its thermostability, and its sensitivity to monovalent cation chlorides from the solubilized dynein. Several of the properties of the ATPase activity of whole cilia differed from those computed for a mixture of 40% pellet ATPase + 60% solubilized dynein ATPase. From these differences it was deduced that dynein in situ is more thermostable than is solubilized dynein and, in contrast to solubilized dynein, is slightly inhibited by KCl, NaCl, LiCl, and NH₄Cl. The increase in total activity upon solubilization of the dynein and the changes in thermostability and in sensitivity to monovalent cations indicates that dynein ATPase in situ is modified by interaction with other components of the axonemal bend generating system.The pellet remaining after extraction of dynein by two dialyses against Tris-EDTA was treated with 0.4% Triton X-100 to solubilize ciliary membranes. Less than half of the ATPase activity was solubilized by this treatment. The possibility that the activity remaining in the Tris-EDTA- and Triton X-100-extracted residue represents an additional ATPase of cilia is discussed.     

Purification and properties of dyneins from Paramecium cilia

Dynein ATPases were purified from Paramecium cilia by salt extraction followed by sucrose density gradient centrifugation and anion exchange chromatography. The two major dyneins sedimented in sucrose gradients as species of 22 S and 12 S. After purification by anion exchange chromatography, their specific activities were about 0.4 and 0.5 mumol/min per mg, respectively. The dyneins could be distinguished by subunit composition and immunological crossreactivity. Sucrose density gradient centrifugation revealed additional ATPase activity in the region between the 22 S and 12 S dyneins, including a 19 S activity. Mg2+-ATPase activities of the dyneins and the 19 S activity were inhibited by vanadate and Zn2+, and were activated by Triton X-100. Antibodies against the 22 S dynein from Paramecium reacted on immunoblots with most of the polypeptides of 22 S dynein, and showed that the heavy chains of 22 S dynein are not identical to those that sediment at 19 S and 12 S. Several minor ATPase activities were revealed by anion exchange chromatography of fractions from the 22 S, 19 S and 12 S regions of sucrose gradients. These minor activities were stimulated by Mg2+, inhibited by vanadate, and could be distinguished from each other by their elution positions and polypeptide compositions.     

Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases

The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.     

Inhibition of sperm motility by the volatile anesthetic halothane

The inhalational anesthetic halothane reversibly inhibits the motility of sea urchin sperm dose-dependently at concentrations up to 5 mM. Experiments with Triton X-100 extracted, trypsinized axonemes showed that halothane has no effect on the rate of axonemal disintegration in the presence of ATP. These results suggest that halothane inhibits flagellar activity by acting at a site other than the dynein ATPase component of the flagellum.     

Activation and membrane binding of carboxypeptidase E

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme that is thought to be involved in the processing of peptide hormones and neurotransmitters. Soluble and membrane-associated forms of CPE have been observed in purified secretory granules from various hormone-producing tissues. In this report, the influence of membrane association on CPE activity has been examined. A substantial amount of the membrane-associated CPE activity is solubilized upon extraction of bovine pituitary membranes with either 100 mM sodium acetate buffer (pH 5.6) containing 0.5% Triton X-100 and 1 M NaCl, or by extraction with high pH buffers (pH greater than 8). These treatments also lead to a two- to threefold increase in CPE activity. CPE extracted from membranes with either NaCl/Triton X-100 or high pH buffers hydrolyzes the dansyl-Phe-Ala-Arg substrate with a lower Km than the membrane-associated CPE. The Vmax of CPE present in extracts and membrane fractions after the NaCl/Triton X-100 treatment is twofold higher than in untreated membranes. Treatment of membranes with high pH buffers does not affect the Vmax of CPE in the soluble and particulate fractions. Pretreatment of membranes with bromoacetyl-D-arginine, an active site-directed irreversible inhibitor of CPE, blocks the activation by NaCl/Triton X-100 treatment. Thus the increase in CPE activity upon extraction from membranes is probably not because of the conversion of an inactive form to an active one, but is the result of changes in the conformation of the enzyme that effect the catalytic activity.     

CYTOPLASMIC DYNEIN OF THE SEA URCHIN EGG I. PARTIAL PURIFICATION AND CHARACTERIZATION*

Cytoplasmic ATPase of sea urchin eggs was partially purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, gel-filtration chromatography and sucrose density gradient centrifugation. The specific activity increased to 0.7 μmole/min/mg protein indicating 100 fold purification. The ATPase had a sedimentation constant of 12S and was highly specific for ATP. The enzyme fraction contained neither (Na, K)-ATPase, Ca-ATPase, oligomycin-sensitive ATPase, phosphatases, nor myosin. This cytoplasmic ATPase was inhibited by a low concentration of vanadate (V). Half-maximal inhibition was observed at a vanadate concentration of 1 μM at low ionic strength. The inhibition was almost totally reversed by addition of norepinephrine. The vanadate-sensitivity of cytoplasmic ATPase decreased with increasing KCl concentration. The activation by Mg²⁺ or Ca²⁺, and dependence of the activity on KCl concentration characteristic of dyneins from sea urchin sperm flagella and the embryonic cilia were observed with cytoplasmic ATPase. These results allowed the cytoplasmic ATPase to be classified as a dynein. In addition, this designation was reinforced by the fact that an oligomeric 23S form of cytoplasmic dynein was identified in the cytoplasm as well as in the isolated mitotic apparatus.     

Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane.

Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).     

Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae.

Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5. The enzyme was completely solubilized with 2% Triton X-100. The enzyme was insensitive to oxygen and was stabilized by ATP. There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein. The membrane-bound M. voltae ATPase showed properties similar to those of E1E2 ATPases.     

Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.     

Direct measurement of the passive stiffness of rat sperm and implications to the mechanism of the calcium response

Glass microprobes were used to measure the stiffness of the flagella of Triton X-100-extracted rat sperm models. The sperm models were treated with 50 microM sodium vanadate and 0.1 mM Mg-ATP to evaluate the stiffness of the passive flagellar structure without the influence of the dynein motor proteins. The passive stiffness was determined to be 4.6 (+/- 1.1) x 10(-19) N x m(2). Rat sperm models exposed to greater than 10(-5) M calcium ions exhibit a strong bend in the basal 40 microm of the flagellum, resulting in a fishhook-like appearance. The torque required to bend a passive rat sperm flagellum into the fishhook-like configuration was determined. The result was compared to the previously published measurement of the torque required to straighten the flagella of rat sperm in the Ca(2+)-induced fishhook configuration [Moritz et al., 2001: Cell Motil. Cytoskeleton 49:33-40]. The torque required to induce a fishhook in a passive flagellum was 2.7 (+/- 0.7) x 10(-14) N x m and the torque to straighten an active Ca(2+)-induced fishhook was 2.6 (+/- 1.4) x 10(-14) N x m. These values are identical within the limit of error of the measurement technique. This finding suggests that the fishhook configuration observed in the Ca(2+) response of rat sperm is the result of a Newtonian equilibrium, where active torque produced by dynein is counterbalanced by an equal and opposite passive torque that results from bending the flagellum. Consistent with this mechanism, the Ca(2+)-induced fishhook configuration is progressively relaxed by incremental increases in sodium vanadate concentration. This supports an active role of the dynein motors in producing the torque for the response. When rat sperm respond to Ca(2+), the bend in the flagellum always forms in the direction opposite the curvature of the asymmetric sperm head. Based on this polarity, the bending torque for the Ca(2+) response must result from the action of the dyneins on outer doublets 1 through 4.     

Cytoplasmic dynein of the sea urchin egg. II. Purification, characterization and interactions with microtubules and Ca-calmodulin

Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein A beta chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KC1 as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCl2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+, and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.     

Vanadate inhibition of brain (Ca+Mg)-ATPase

Vanadate was a potent inhibitor of the membrane-bound (Ca+Mg)-ATPase from rat brain, the concentration required for 50% inhibition under conditions optimal for enzymatic activity being 3 M. Vanadate inhibition increased with the MgCl₂ concentration, half-maximal inhibition occurring at 2 mM MgCl₂, near the MgCl₂ concentration required for half-maximal activation of the ATPase activity. MnCl₂ could substitute for MgCl₂, and at concentrations of 1 mM (Ca+Mn)-ATPase activity was greater than (Ca+Mg)-ATPase activity, although sensitivity to vanadate was less. Vanadate inhibition increased also with the KCl concentration, half-maximal inhibition occurring at 8 mM, again near the concentration required for half-maximal activation of ATPase activity. By contrast, NaCl stimulated (Ca+Mg)-ATPase activity without potentiating vanadate inhibition. These effects of cations on ATPase activity and vanadate inhibition resemble properties of certain transport ATPases and thus suggest mechanistic and functional similarities.     

Reversible conversion from Ca(2)+-ATPase activity to Mg(2)+- and Mn(2)+-ATPase activities of coupling factor purified from acetone powder of Rhodospirillum rubrum chromatophores.

It is known that the coupling factor purified from the acetone powder of chromatophores from Rhodospirillum rubrum shows ATPase activity in the presence of Ca(2)+, but not in the presence of Mg(2)+ or Mn(2)+. The present study deals with conditions, under which the Ca(2)+-ATPase activity is reversibly converted into Mg(2)+- and Mn(2)+-ATPase activites with the purified coupling factor. 1. Of the pH indicators tested, 6 kinds coverted the Ca(2)+-ATPase activity into Mg(2)+- and Mn(2)+-ATPase activities in the order, ethyl orange greater than tropaeolin 000 greater than or equal to metanil yellow greater than tropaeolin 00 greater than ethyl red greater than or equal to bromthymol blue. 2. Of the detergents tested, those other than Triton X-100 and Brij 58 caused the conversion described above; dodecylsulfonate was most effective, whereas dodecylpyridinium chloride was moderately effective. 3. 2,4-Dinitrophenol stimulated approximately two-fold the Ca(2)+-ATPase activity, but not the Mg(2)+- or Mn(2)+-ATPase activity at all. However, in the presence of dodecylpyridinium chloride, the pH indicator remarkably stimulated the Mg(2)+- and Mn(2)+-ATPase activities, accompanied with a partial inhibition of the Ca(2)+-ATPase activity. Methyl red and ethyl red showed similar effects. 4. All the nucleoside triphosphates tested can serve as the substrate. ATP was most effective for the Ca(2)+-ATPase activity, whereas dATP was most effective for the Mg(2)+- and Mn(2)+-ATPase activities induced by ethyl orange. 5. In the presence of ethyl orange, the ATPase activity was induced by various divalent cations in the following order of effectiveness, Mg(2)+ greater than Zn(2)+ greater than CO(2)+ greater than Mn(2)+ greater than Ni(2)+. 6. The mechanism of the reversible conversion from the Ca(2)+-ATPase activity to the Mg(2)+- and Mn(2)+-ATPase activities by pH indicators and detergents is discussed.     

Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.     

Purification and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8.

Cell-free extracts of Thermus thermophilus HB8 catalyze the in vitro, NADH-dependent reduction of potassium tellurite (K2TeO3). Three different protein fractions with tellurite-reducing activities were identified. Two exhibited high molecular weight and were composed of at least two different polypeptides. The protein in the third fraction was purified to homogeneity and had a single polypeptide chain of 53 to 54 kilodaltons, with an isoelectric point of 8.1. Each enzyme was thermostable, the temperature optimum was 75 degrees C, and 30 mM NaCl, 1.5 M urea, or 0.004% sodium dodecyl sulfate caused 50% inhibition of the enzymes. However, 2% Triton X-100 did not have an inhibitory effect. The enzymes were also able to catalyze the reduction of sodium selenite and sodium sulfite in vitro. NADH was replaceable by NADPH. Divalent cations, such as Ca2+ and Ba2+, had no effect on the activity, while similar concentrations of Zn2+, Ni2+, and Cu2+ abolished the activity. This reductase activity could enable these bacteria both to reduce K2TeO3 and to increase their tolerance toward this salt.