Antimicrobial peptides and proteins in the innate defense of the airway surface.

Recent studies have advanced our understanding of innate immune mechanisms that protect the airways and maintain a sterile lung. Multiple antimicrobial peptides and proteins have been identified in airway secretions and their roles are beginning to be established in animal models. Moreover, evidence for coupling between the innate and adaptive immune systems is beginning to emerge. The understanding of the innate airway defense system offers the opportunity for the development of novel therapeutic approaches.

Bactericidal activity of mammalian cathelicidin-derived peptides.

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Activity of abundant antimicrobials of the human airway.

Human airways produce several antimicrobial factors; the most abundant are lysozyme and lactoferrin. Despite their likely importance in preventing infection, and their possible key role in the pathogenesis of cystic fibrosis (CF), we know little about their antibacterial activity in the context of the CF airway. We found that abundant airway antimicrobial factors kill common CF pathogens, although Burkholderia was relatively resistant. To study the antibacterial activity, we developed a rapid, sensitive, and quantitative in vitro luminescence assay. Because NaCl concentrations may be elevated in CF airway surface liquid, we tested the effect of salt on antibacterial activity. Activity of individual factors and of airway lavage fluid was inhibited by high ionic strength, and it was particularly sensitive to divalent cations. However, it was not inhibited by nonionic osmolytes and thus did not require hypotonic liquid. The inhibition by ionic strength could be partially compensated by increased concentrations of antibacterial factors, thus there was no one unique salt concentration for inhibition. CF airway secretions also contain abundant mucin and elastase; however, these had no effect on antibacterial activity of lysozyme, lactoferrin, or airway lavage fluids. When studied at low NaCl concentrations, CF and non-CF airway lavage fluids contained similar levels of antibacterial activity. These results suggest approaches toward developing treatments aimed at preventing or reducing airway infections in individuals with CF.

Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.

While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F- to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl- current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F- increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to AlF4-. F- also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F- increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F- influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F- compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F- interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.

Protein phosphatase 2C dephosphorylates and inactivates cystic fibrosis transmembrane conductance regulator.

cAMP-dependent phosphorylation activates the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelia. However, the protein phosphatase (PP) that dephosphorylates and inactivates CFTR in airway and intestinal epithelia, two major sites of disease, is not certain. We found that in airway and colonic epithelia, neither okadaic acid nor FK506 prevented inactivation of CFTR when cAMP was removed. These results suggested that a phosphatase distinct from PP1, PP2A, and PP2B was responsible. Because PP2C is insensitive to these inhibitors, we tested the hypothesis that it regulates CFTR. We found that PP2Calpha is expressed in airway and T84 intestinal epithelia. To test its activity on CFTR, we generated recombinant human PP2Calpha and found that it dephosphorylated CFTR and an R domain peptide in vitro. Moreover, in cell-free patches of membrane, addition of PP2Calpha inactivated CFTR Cl- channels; reactivation required readdition of kinase. Finally, coexpression of PP2Calpha with CFTR in epithelia reduced the Cl- current and increased the rate of channel inactivation. These results suggest that PP2C may be the okadaic acid-insensitive phosphatase that regulates CFTR in human airway and T84 colonic epithelia. It has been suggested that phosphatase inhibitors could be of therapeutic value in cystic fibrosis; our data suggest that PP2C may be an important phosphatase to target.

PP2C gamma: a human protein phosphatase with a unique acidic domain.

We have cloned a novel cDNA from human skeletal muscle which encodes a protein phosphatase with a unique acidic domain. It is 34% identical to mammalian PP2C alpha and PP2C beta and we call it PP2C gamma. It more closely resembles PP2Cs from Paramecium tetraurelia and Schizosaccharomyces pombe than mammalian PP2Cs. Northern blot analysis shows that PP2C gamma is widely expressed, and is most abundant in testis, skeletal muscle, and heart. Like known PP2Cs, recombinant PP2C gamma requires Mg2+ or Mn2+ for activity. Unlike any other known phosphatase, PP2C gamma has a highly acidic domain: 75% of the 54 residues are glutamate or aspartate.

Contribution of proline residues in the membrane-spanning domains of cystic fibrosis transmembrane conductance regulator to chloride channel function.

Proline residues located in membrane-spanning domains of transport proteins are thought to play an important structural role. In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro99, Pro205, Pro324, and Pro1021. These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. To evaluate the contribution of these prolines to CFTR Cl- channel function, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A). We also constructed the two cystic fibrosis-associated mutations. cAMP agonists stimulated whole cell Cl- currents in HeLa cells expressing the individual constructs that resembled those produced by wild-type CFTR. However, the amount of current was decreased in the rank order: wild-type CFTR = Pro324 > Pro1021 > Pro99 >/= Pro205 mutants. The anion selectivity sequence of the mutants (Br- >/= Cl- > I-) resembled wild-type except for P99L (Br- >/= Cl- = I-). Although the Pro99, Pro324, and Pro1021 mutants produced mature protein, the amount of mature protein was much reduced with the Pro205 mutants, and the P-Quad-A made none. Because the Pro99 constructs produced mature protein but had altered whole cell currents, we investigated their single-channel properties. Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR >/= P99G > P99L >/= P99A. These results suggest that proline residues in the transmembrane segments are important for CFTR function, Pro205 is critical for correct protein processing, and Pro99 may contribute either directly or indirectly to the Cl- channel pore.

Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.

Despite an increased understanding of the cellular and molecular biology of the CFTR Cl- channel, it is not known how defective Cl- transport across airway epithelia causes chronic bacterial infections in cystic fibrosis (CF) airways. Here, we show that common CF pathogens were killed when added to the apical surface of normal airway epithelia. In contrast, these bacteria multiplied on CF epithelia. We found that bactericidal activity was present in airway surface fluid of both normal and CF epithelia. However, because bacterial killing required a low NaCl concentration and because CF surface fluid has a high NaCl concentration, CF epithelia failed to kill bacteria. This defect was corrected by reducing the NaCl concentration on CF epithelia. These data explain how the loss of CFTR Cl- channels may lead to lung disease and suggest new approaches to therapy.

Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl- channels.

A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPi and three nonhydrolyzable PPi analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPi affected two distinct steps in channel regulation. First, PPi increased the rate at which channels opened. Second, once channels were open, PPi delayed their closure. PPi could only stimulate channels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPi or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.

The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel contains two cytoplasmic nucleotide-binding domains (NBDs). After phosphorylation of the R domain, ATP interacts with the NBDs to regulate channel activity. To learn how the NBDs regulate channel function, we used the patch-clamp technique to study CFTR and variants which contained site-directed mutations in the conserved Walker A motif lysine residues in either NBD1 (K464A), NBD2 (K1250A and K1250M), or both NBDs simultaneously (K464A/K1250A). Studies in related proteins suggest that such mutations slow the rate of ATP hydrolysis. These mutations did not alter the conductive properties of the channel or the requirement for phosphorylation and ATP to open the channel. However, all mutations decreased open state probability. Mutations in NBD1 decreased the frequency of bursts of activity, whereas mutations in NBD2 and mutations in both NBDs simultaneously prolonged bursts of activity, as well as decreased the frequency of bursts. These results could not be attributed to altered binding of nucleotide because none of the mutants studied had reduced 8-N3ATP binding. These data suggest that the two NBDs have distinct functions in channel gating; ATP hydrolysis at NBD1 initiates a burst of activity, and hydrolysis at NBD2 terminates a burst.

Phosphate stimulates CFTR Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cytosolic nucleotides and by cAMP-dependent phosphorylation. In excised membrane patches, CFTR Cl- channel activity requires hydrolyzable nucleotides and Mg2+, and is inhibited by ADP. We examined the interactions between CFTR and nucleotides using 8-azidoadenosine 5'-triphosphate (8-N3-ATP), a photoactivatable ATP analog. Because CFTR functions as a membrane ion channel, we studied CFTR in membranes of Sf9 insect cells. We found that [alpha-32P]8-N3ATP specifically photolabeled CFTR, with half-maximal labeling at 10 microM 8-N3ATP in the presence of Mg2+ and 100 microM in the absence of Mg2+. The 8-N3ATP also substituted for ATP in activating CFTR Cl- channels, indicating that it interacts with the active site(s). Both ATP and GTP prevented photolabeling with half-maximal inhibition at 1 mM. ADP and adenyl-5'-yl imidodiphosphate (AMP-PNP) prevented photolabeling but at much higher concentrations, whereas AMP did not inhibit photolabeling at concentrations of up to 100 mM. Phosphorylation of CFTR was not a prerequisite for nucleotide binding. These results demonstrate that CFTR interacts directly with nucleotides at concentrations that regulate CFTR Cl- channel activity.

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a regulated Cl- channel; in secretory epithelia, it is located in the apical membrane where it regulates transepithelial Cl- secretion. Previous studies have shown that cAMP-dependent protein kinase (PKA) can phosphorylate and activate CFTR Cl- channels. We asked whether other kinases would phosphorylate CFTR in vitro and activate CFTR Cl- channels in excised, inside-out patches of membrane from NIH 3T3 fibroblasts stably expressing recombinant CFTR. We found that both Ca(2+)-independent and Ca(2+)-dependent isoforms of protein kinase C (PKC) activated the CFTR Cl- channel. Consistent with this finding, PKC also phosphorylated CFTR in vitro. In contrast, the multifunctional Ca2+/calmodulin-dependent protein kinase failed to either activate or to phosphorylate CFTR Cl- channels, suggesting that this enzyme has no direct effect on CFTR. We found that cGMP-dependent protein kinase (cGK) (purified from bovine lung) phosphorylated CFTR in vitro. However, cGMP failed to increase the apical membrane Cl- permeability in human airway epithelia, and addition of cGMP, ATP, and cGK failed to activate CFTR Cl- channels. These results suggest that if cGK phosphorylates CFTR in vivo, it does so at sites not involved in CFTR Cl- channel activation. Because cAMP-dependent activation of CFTR Cl- channels and Cl- secretion in intact cells is reversible, we asked whether specific phosphatases can dephosphorylate and inactivate CFTR Cl- channels. Addition of protein phosphatase 2A (PP2A) decreased PKA-activated current by 67% within 10 min. The phosphatase inhibitor calyculin-A blocked the effect of PP2A. In contrast, neither protein phosphatases 1, 2B, nor two preparations of alkaline phosphatase inactivated PKA-phosphorylated CFTR Cl- channels. The effects of protein phosphatases on CFTR function were paralleled by their ability to dephosphorylate CFTR in vitro. Our data indicate that CFTR Cl- channels can be phosphorylated and activated by PKA as well as by Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC and can be dephosphorylated and thus inactivated by PP2A.

Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.

We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.

Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia.

We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides.

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.

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.

Characterization of Ca2+- or Mg2+-ATPase of the excitable ciliary membrane from Paramecium tetraurelia: comparison with a soluble Ca2+-dependent ATPase.

We have characterized divalent-cation-stimulated nucleoside triphosphate hydrolase activity of the excitable ciliary membrane and compared it with a soluble Ca2+-ATPase released upon deciliation of Paramecium. The membrane-bound activity is strongly dependent on a divalent cation; calcium stimulates the basal activity of this enzyme at least 10-fold; magnesium and manganese stimulate less well, and strontium and barium, although less effective, also give measurable stimulation. This membrane-bound activity prefers ATP and GTP as substrates but also hydrolyzes UTP and CTP at measurable rates. The maximum velocity at saturating ATP concentrations and optimal calcium concentrations is 0.3 mumol/min per mg. The pH optimum for the membrane-bound activity is broad and centers around pH 7. From the temperature dependence of ATP hydrolysis, we calculate activation energies of 14 and 11 kcal/mol for the Ca2+- and Mg2+-stimulated activities, respectively. The Arrhenius plot is linear over the temperature range of 4 to 25 degrees C. The membrane ATPase is relatively insensitive to ouabain, oligomycin, N,N'-dicyclohexylcarbodiimide, vanadate, Ruthenium red and two calmodulin antagonists. Polyclonal antisera raised against the purified soluble ATPase from the deciliation supernatant show low reactivity with the membrane-bound ATPase. We conclude from the comparison of properties of the two activities that the ciliary membrane-bound ATPase is distinct from the soluble ATPase released by deciliation.