Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

HQNO-sensitive NADH:quinone oxidoreductase of Bacillus cereus KCTC 3674

The enzymatic properties of NADH:quinone oxidoreductase were examined in Triton X-100 extracts of Bacillus cereus membranes by using the artificial electron acceptors ubiquinone-1 and menadione. Membranes were prepared from B. cereus KCTC 3674 grown aerobically on a complex medium and oxidized with NADH exclusively, whereas deamino-NADH was determined to be poorly oxidized. The NADH oxidase activity was lost completely by solubilization of the membranes with Triton X-100. However, by using the artificial electron acceptors ubiquinone-1 and menadione, NADH oxidation could be observed. The activities of NADH:ubiquinone-1 and NADH:menadione oxidoreductase were enhanced approximately 8-fold and 4-fold, respectively, from the Triton X-100 extracted membranes. The maximum activity of FAD-dependent NADH:ubiquinone-1 oxidoreductase was obtained at about pH 6.0 in the presence of 0.1M NaCl, while the maximum activity of FAD-dependent NADH:menadione oxidoreductase was obtained at about pH 8.0 in the presence of 0.1 M NaCl. The activities of the NADH:ubiquinone-1 and NADH:menadione oxidoreductase were very resistant to such respiratory chain inhibitors as rotenone, capsaicin, and AgNO(3), whereas these activities were sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Based on these results, we suggest that the aerobic respiratory chain-linked NADH oxidase system of B. cereus KCTC 3674 possesses an HQNO-sensitive NADH:quinone oxidoreductase that lacks an energy coupling site containing FAD as a cofactor.     

An isoelectric focusing study of acid phosphohydrolases in rat liver lysosomes.

The differentiation of rat liver lysosomal acid phosphatase, acid ATPase, acid phosphodiesterase, acid ribonuclease, and acid deoxyribonuclease was studied by isoelectric focusing. To prevent autolytic digestion, inhibitors of cathepsins and neuraminidase were used. The proportion of acidic forms of acid phosphatase, acid ATPase and acid phosphodiesterase was increased by the use of extraction medium containing 0.05% Triton X-100. To investigate the identity of acid ATPase and acid phosphodiesterase, the relative activities among the multiple forms of these enzymes, the acid phosphodiesterase/acid ATPase ratio at each activity peak, and the degree of enzyme inhibition by p-chloromercuriphenyl sulfonic acid were estimated. The results suggest that acid ATPase is not identical with acid phosphodiesterase. With extraction medium free of Triton X-100, acid ribonuclease appeared in two forms. However, in addition to these forms, a new form of this enzyme with a more acidic pI (4.22) emerged when extraction medium containing 0.05% Triton X-100 was used. The major peak of acid deoxyribonuclease with pI=8.40-9.39 was obtained regardless of the extracting method.     

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.     

Triton X—100加KI对燕麦根细胞质膜ATP酶的溶解

在酸性条件下,1％ Triton X—100加 0.25mol/L KI能有效地溶解燕麦根细胞质膜ATP酶。溶解的ATP酶水解ATP的最适pH在6.5左右,酶活性受到Na_3VO_4和DES的强烈抑制,而不受Na_2MoO_4和NaN_3的抑制。溶解的酶液经透析后,K~ —ATP酶活性占Mg~(2 ),KCl—ATP酶活性的85％。     

Purification and characterization of a membrane-bound NADPH-cytochrome c reductase capable of catalyzing menadione-dependent O2- formation in guinea pig polymorphonuclear leukocytes

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2-) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983). Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2-. The purified enzyme preparation contained FAD. When assayed by measuring O2--generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 microM, respectively. The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.     

Detergent inhibition of nitric-oxide reductase activity

Gas chromatography revealed that exposure of extracts of the denitrifiers 'Achromobacter cycloclastes', Paracoccus denitrificans, Pseudomonas aeruginosa and Pseudomonas perfectomarina to Triton X-100 inhibited reduction of NO to N2O, and thus concomitantly inhibited reduction of NO2- to N2O. After exposure of extracts to Triton X-100, the ratio of H+ consumed to NO2- added decreased from approx. 2.0 (for untreated extracts) to approx. 1.5, which indicated that NO2- was reduced to NO by the treated extracts. Addition of a CHAPS-soluble extract (devoid of nitrite reductase activity but rich in nitric-oxide reductase activity) to the Triton X-100-treated extract of P. denitrificans restored capacity for reduction of NO2- on to N2O. Exposure to either the NO that accumulated from reduction of NO2- or to enthetic NO transiently inhibited rates of NO2- reduction in Triton X-100-treated extracts. Use of an Oxides of Nitrogen analyzer indicated that only 5-33% of NO2- reduced by untreated extracts appeared in the stripping gas as NO, whereas 80-95% of NO2- reduced by Triton X-100-treated extracts was recovered as NO.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

Selective delipidation of the plasma membrane by surfactants : enrichment of sterols and activation of ATPase

The influence of plasma membrane lipid components on the activity of the H⁺-ATPase has been studied by determining the effect of surfactants on membrane lipids and ATPase activity of oat (Avena sativa L.) root plasma membrane vesicles purified by a two-phase partitioning procedure. Triton X-100, at 25 to 1 (weight/weight) Triton to plasma membrane protein, an amount that causes maximal activation of the ATPase in the ATPase assay, extracted 59% of the membrane protein but did not solubilize the bulk of the ATPase. The Triton-insoluble proteins had associated with them, on a micromole per milligram protein basis, only 14% as much phospholipid, but 38% of the glycolipids and sterols, as compared with the native membranes. The Triton insoluble ATPase could still be activated by Triton X-100. When solubilized by lysolecithin, there were still sterols associated with the ATPase fraction. Free sterols were found associated with the ATPase in the same relative proportions, whether treated with surfactants or not. We suggest that surfactants activate the ATPase by altering the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity.     

Phosphorylated cardiac myofibrils and their effect on ATPase activity.

Control guinea pig cardiac myofibrils were isolated in the presence of Triton X-100. Experimental myofibrils, prepared in the presence of Triton X-100, NaF, cyclic AMP and ATP, possessed a reduced myofibrillar ATPase activity. When myofibrils isolated under control conditions were incubated for two hours at 25°C with NaF, ATP and cyclic AMP, the ATPase activity was also decreased; however, the ATPase activity was not reduced as much as that of myofibrils isolated under experimental conditions. Incubation of myofibrils with $\text{E. coli}$ aklaline phosphatase and guinea pig heart phosphoprotein phosphatase resulted in an increase in ATPase activity and a decrease in phosphoprotein phosphate. Thus there appeared to be an inverse relationship between myofibrillar ATPase activity and phosphoprotein phosphate content. The results indicated that a protein kinase is associated with the Triton X-100 purified myofibrils and supports the notion that intact myofibrils can exist in at least two catalytic forms.     

Inhibitory effects of detergents on rat CYP1A1-dependent monooxygenase: comparison of mixed and fused systems consisting of rat CYP 1A1 and yeast NADPH-P450 reductase

Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively. These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1. These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM). Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM Triton X-100. On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems. In the fused system, a mixed-type inhibition was observed with K(i) and K(i)' values of 1.2 and 5.4 mM of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system. Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents.     

Impact of Triton X-100 on alpha 2-antiplasmin (SERPINF2) activity in solvent/detergent-treated plasma

Large-pool solvent/detergent (SD) plasma for transfusion exhibits reduced alpha 2-antiplasmin (alpha2-AP; SERPINF2) functional activity. The reason for the loss of alpha2-AP has not been described and could be due to the SD incubation itself and/or to the processing steps implemented to remove the solvent and the detergent. We have studied alpha2-AP activity during six down-scale preparations of plasma virally-inactivated by 1% (v/v) TnBP combined with two different non-ionic detergents, either 1% Triton X-100 or 1% Triton X-45, at 31 degrees C for 4h. The SD-treated plasmas were then extracted with 7.5% (v/v) soybean oil, centrifuged at 3800 x g for 30 min, and subjected to hydrophobic interaction chromatography (HIC) to remove the SD agents. Control runs without TnBP and Triton were performed to evidence possible impacts of each process step on alpha2-AP activity. TnBP, Triton X-100, and Triton X-45 were measured at all stages of the processes to evaluate potential interferences with the alpha2-AP assay. Alpha 2-AP activity was about 10% that of starting plasma after 1% TnBP-1% Triton X-100 incubation and about 50% after oil extractions, centrifugation, and HIC. By contrast about 73% of the antiplasmin activity was found after the incubation with 1% TnBP and 1% Triton X-45, 88% after removal of the SD agents by oil extractions, 90% after centrifugation and 92% after HIC. The control runs performed without SD agents showed that the process steps did not affect the alpha2-AP activity. In conclusion, the agent altering alpha2-AP activity in SD-plasma is Triton X-100. The choice of detergents for the SD viral inactivation of therapeutic plasma fractions used in patients at risk of fibrinolysis should consider the impact on alpha2-AP activity.     

Mechanisms of the acido- and thermophily of Cyanidium caldarium Geitler III. Loss of these characteristics due to detergent treatment

The photochemical reactions of Cyanidium cells treated withvarious concentrations of Triton X-100 or digitonin were examined.As the concentration of Triton X-100 was increased, the following4 responses were observed: inhibition of endogenous O₂ evolution(above 0.005%), stimulation of the p-BQ Hill reaction (above0.01%), loss of thermophily (above 0.03%) and loss of acidophily(above 0.5%). In the presence of Triton X-100 (0.1% of finalconcentration), the p-BQ Hill reaction showed optimum activityat 30?C and was completely inactivated at temperatures over45?C, though the optimum activity in the absence of Triton X-100was 45?C. The pH activity curve, however, was unchanged by treatmentwith Triton X-100. The loss of heat tolerance caused by TritonX-100 was observed not only in the Hill reaction but also inthe photosystem I reaction. The thermophily of Triton X-100-treatedcells was completely recovered after washing with distilledwater. The acidophily of the alga was lost after digitonin (0.01% offinal Concentration) treatment without any loss of thermophilyor the inactivation of photosystem I and II reactions. The p-BQHill activity of digitonin-treated cells was optimum at pH 7and completely lost in acid pH regions, while the temperaturedependency was unchanged by this treatment. The irreversibleloss of acid tolerance of the photosystem I and II activitiesdue to digitonin was confirmed by various acid and alkalinetreatments. (Received November 20, 1976; )     

A sensitive, continuous spectrophotometric method for assaying alpha-glycerophosphate dehydrogenase: activation by menadione

By use of a more sensitive method than standard assays, it is demonstrated that alpha-glycerophosphate dehydrogenase activity can be monitored continuously in rat liver mitochondria; maximum activity was obtained by sonication in the presence of Triton X-100. Vitamin K3 (menadione) seems to enhance the activity of the enzyme. The assay in the presence of menadione is linear over a much greater mitochondrial concentration (up to 250 micrograms of protein in the reaction mixture).     

Interactions between sarcoplasmic reticulum calcium adenosintriphosphatase and nonionic detergents

The interaction of Triton X-100 and other nonionic detergents with a delipidated preparation of the Ca2+ ATPase from sarcoplasmic reticulum has been studied. Binding of radiolabeled Triton X-100 was determined by column chromatography at 6 degrees C, and two classes of binding sites were observed. Below the critical micelle concentration (cmc), binding of Triton occurred at 35-40 equivalent sites on the delipidated ATPase with a binding constant of 2.7 X 10(4) M-1. Near the cmc cooperative binding of an additional 70 molecules of the detergent was observed. The binding of monomeric Triton X-100 below the cmc was associated with a parallel activation of over half of the ATPase activity, and the remainder of the activity was recovered after the detergent concentration was increased to the cmc. The ability to reactivate ATPase activity was more dependent on the polar poly(oxyethylene) portion of nonionic detergents than on the hydrocarbon portion. Generalizing for all amphiphiles, these results suggest that there are discrete binding sites on the Ca2+ ATPase for phospholipid molecules in the native membrane and that the polar head groups of phospholipids interact more strongly with the protein than the hydrophobic acyl chains. Perturbations in micelle structure were observed for several nonionic detergents by measurement of cis-parinaric acid fluorescence and differential scanning calorimetry, and discontinuities in Arrhenius plots occurred at the transition temperature of the detergent used for reactivation of ATPase activity. It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity.     

Binding, activation, and solubilization of the Ca2+-ATPase from sarcoplasmic reticulum by nonionic detergents

Interactions between delipidated Ca2+-ATPase from sarcoplasmic reticulum and four nonionic detergents--dodecyl octaoxyethyleneglycol monoether (C12E8), Triton X-100, Brij 58, and Brij 35--were characterized with respect to activation of ATPase activity, binding, and solubilization. C12E8 and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C12E8. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C12E8 and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C12E8 are more useful when bulk solubilization is the goal.     

Inactivation of the calcium-transport ATPase in the sarcoplasmic reticulum by the combined effect of lasolocid and Triton X-100

The calcium-transport ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately, 0.2 mg X ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. Phosphoprotein formation is less affected than phosphate liberation. The influence of the ATPase protein on the fluorescence intensity of Lasolocid passes a distinct maximum at the most effective Triton X-100 concentration of 0.2 mg X ml-1.     

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.