Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Effect of triton X-100 on ultrastructure,reactivation, and motility characteristics of ram spermatozoa

For optimal extraction and reactivation of ram sperm, glutamate, dithiothreitol, magnesium, and cyclic AMP were required in a medium of pH 7.9. On extraction with 0.01 % Triton X-100, ram sperm were only partially demembranated, and extensive areas of plasma membrane remained intact especially in the midpiece region. Treatment with 0.1% Triton X-100 removed all plasma membranes and extracted the mitochondrial membrane and matrix. In the absence of ATP, 16.6% ± 0.4 of the partially demembranated sperm were motile, but sperm extracted with 0.1% Triton X-100 were completely immotile. On adding ATP partially demembranated sperm reactivated better (81.6% ± 2.8) than sperm completely demembranated in 0.1 % Triton X-100 (39.5% ± 4.6). The release of intracellular LDH rose linearly with increasing concentrations of the detergent from 0.01 to 0.05%, at which it plateaued. There was a significant increase in beat frequency and forward velocity of partially demembranated sperm when treated with ATP. Partially demembranated sperm had intact mitochondria that presumably were still able to produce ATP, although the spermatozoan movement was stimulated by exogenous ATP.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

Extraction of 11 beta-hydroxysteroid dehydrogenase from rat liver microsomes by detergents

In these studies our goal was to solubilize the microsomal enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD) as the first step in its purification. Enzyme was extracted from rat liver microsomes with representative detergents (Zwittergents, Tritons, modified sterols). Oxidation-reduction (O-R) ratios of extracts varied with detergent used and ranged from 0.18 (CHAPS) to 3.8 (Zwittergent 3-14) relative to a ratio of 1.7 in intact microsomes. All detergents solubilized 11-HSD using lack of sedimentation during high speed centrifugation as criterion. With Triton DF-18 and Triton X-100, optimum extraction of 11-HSD occurred in the detergent-protein ratio range of 0.1 to 0.2 O-R ratios decreased with increased Triton X-100, but were constant as Triton DF-18 was varied. The pH optimum of enzyme extraction was 9 at a detergent-protein ratio of 0.05 and 7.5-8.0 at a ratio of 0.2. Sodium chloride increased enzyme extraction by detergents; in the absence of detergent, salt extracted protein, but not enzyme. In aqueous solution at 0 degrees C or -15 degrees C, microsomal 11-oxidation activity rose within 24 h, then decreased; reductase activity consistently decreased. Oxidation and reduction activities were inversely related in the microsomal bound enzyme. No relationship between these activities appeared in detergent-solubilized enzymes. Possible mechanisms to account for the unexpected behavior of this enzyme are discussed.     

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.     

Effective lipid‐detergent system for study of membrane active peptides in fluid liposomes

The structure of peptide antibiotic gramicidin A (gA) was studied in phosphatidylcholin liposomes modified by nonionic detergent Triton X‐100. First, the detergent : lipid ratio at which the saturation of lipid membrane by Triton X‐100 occurs (R_e^sat), was determined by light scattering. Measurements of steady‐state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene at sublytic concentrations of detergent showed that after saturation of the membrane by Triton X‐100 microviscosity of lipid bilayer is reduced by 20%. The equilibrium conformational state of gA in phosphatidylcholine liposomes at R_e^sat was studied by CD spectroscopy. It was found that the conformational state of this channel‐forming peptide changed crucially when Triton X‐100 induced transition to more fluid membranes. The gA single‐channel measurements were made with Triton X‐100 containing bilayers. Tentative assignment of the channel type and gA structures was made by correlation of CD data with conductance histograms. Lipid‐detergent system with variable viscosity developed in this work can be used to study the structure and folding of other membrane‐active peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Vaccinia virus-mediated high level expression and single step purification of recombinant Jak2 protein

Jak2 functions as a non-receptor tyrosine kinase and has been linked to pathologies such as cancer and cardiovascular disease. Because of this, many studies have tried to better understand its function. Unfortunately, very little information is known about its catalytic or biochemical properties as purification of significant amounts of functional Jak2 protein has been exceedingly difficult. Here, Jak2 was expressed in BSC-40 cells using a vaccinia virus-mediated expression system. Significant amounts ( approximately 10microg) of Jak2 protein were expressed from a single 100-mm cell culture dish. The protein was first harvested using three different methods of extraction to determine the relative efficiency of each lysis method with respect to Jak2 protein yield and catalytic activity. We found that lysis methods utilizing detergents increased the efficiency of protein extraction about 3-fold when compared to a method lacking detergent. However, with respect to catalytic activity, Jak2 isolated from cells using detergent-containing lysis buffers had significantly less catalytic activity than when compared to the method that was detergent free. Expression was then scaled up and Jak2 protein was purified via a one step immunoaffinity purification scheme using both the detergent-free and a modified detergent-containing method of extraction that maintained catalytic activity. In vitro kinase assays demonstrated that the purified product was highly catalytic as measured by its ability to tyrosine phosphorylate Stat1. Collectively, the results show that (1) Jak2 can be expressed at very high levels in mammalian cells, (2) it can be purified to homogeneity via a single step purification scheme, and (3) the purified product is biologically active.     

The bulk of ATP is associated to proteins in the living cell: a release kinetics study

The analysis of the nonionic detergent induced release kinetics of intracellular components is a promising approach to obtain information on the possible molecular interactions inside the living cell. The molecular interactions of ATP were studied by monitoring the nonionic detergent-induced release kinetics of ATP from cultured human cells. The observed rapid mobilization rate in the presence of Triton X 100 and the retarded release in the presence of Brij 58 were in good agreement with the detergent-induced release kinetics of proteins and K+ reported earlier. These data are consistent with the idea that the bulk of ATP is associated to the protein macromolecules of the living cell.     

Lipid Raft Association and Cholesterol Sensitivity of P2X1-4 Receptors for ATP: CHIMERAS AND POINT MUTANTS IDENTIFY INTRACELLULAR AMINO-TERMINAL RESIDUES INVOLVED IN LIPID REGULATION OF P2X1 RECEPTORS*

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20–23 and 27–29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.     

The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System

Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I_core–hydrophobin I (EGI_core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI_core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI_core–HFBI was quantitatively back-extracted (K_{EGIcore–HFBI}=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGI_core–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI_core–HFBI into a water phase.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone.

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH3 pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent:protein ratios from 0.5 to 20 led to a progressive loss of hormone . receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone . receptor complex was not retained by 0.22 micron filters and remained soluble after ultracentrifugation. Following incubation with high (2.5--10%) concentrations of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent:protein ratio of 0.033. The hormone . receptor complex was included in Sepharose 6B and exhibited an apparent Stoke radius of 46 A in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0 degrees C, while the membrane hormone . receptor complex was stable for up to 5 at 0 degrees C.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH₃ pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C.     

The interaction of liposomes containing intrinsic erythrocyte membrane proteins with lipid monolayers at air/water and oil/water interfaces

The main intrinsic membrane proteins of the human erythrocyte membrane, glycophorin and the anion transporter, were isolated by extraction with Triton X-100 and ion-exchange chromatography. After removal of detergent the extract consisted of proteolipid vesicles with a lipid:protein molar ratio in the range 50-60 and a diameter of the order of 200 nm. The interaction between these vesicles and dipalmitoylphosphatidylcholine (DPPC), cholesterol and cholesterol:DPPC (2:1 molar ratio) monolayers at air/water and n-decane/water interfaces has been studied. The vesicles interact with the monolayers, rapidly causing large increases in surface pressure. Limiting values of surface pressure, 39.4-43 mN . m-1 at air/water and 31.5-33.4 mN . m-1 at the n-decane/water interface, were reached at protein levels above 1 microgram . ml-1. At the air/water interface, and probably at the n-decane/water, surface pressure increases were limited by monolayer collapse. Compression isotherms and surface potential measurements indicated that material from the proteolipid vesicles entered the monolayer phase. In contrast to proteolipid vesicles, injection of protein-free liposomes beneath the monolayer resulted in smaller, slower increases in surface pressure. Thus, the presence of intrinsic membrane proteins in vesicles greatly facilitated the transfer of material into the lipid monolayer.     

Extraction and determination of adenosine 5'-triphosphate in bovine milk by the firefly luciferase assay

The release of ATP from somatic cells in milk with the detergent Triton X-100 was optimized for assay with firefly luciferase. A small volume of milk (40 microliters) is added to 0.8 ml of 0.2% Triton X-100 in 100 mM Tris, 4 mm EDTA, pH 7.8. After approximately 1 min, 0.2 ml of luciferase reagent is added and the emission of light is measured in a luminometer. Results are calibrated with an ATP standard. This single method gave high yields of ATP from somatic cells in milk without interference from bacterial ATP. Extracts could be stored or transported prior to assay without deterioration of results. A close correlation was found between somatic cell count and ATP in milk samples collected at a farm as well as in milk samples from a cow with experimental mastitis. Results are promising for future use for diagnosis of mastitis but further work and field testing has to be done before it can be used on a wider scale.     

Isolation and characterization of cytoskeletons from cotton fiber cytoplasts

Summary Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca²⁺-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.     

Protein analysis of mammalian cells in monolayer culture using the bicinchoninic assay

We have applied the bicinchoninic acid (BCA) protein assay to rat brain primary astrocyte monolayer cultures growing in multiwell culture plates. The BCA method provides a more rapid and sensitive procedure with greater stability of color than is obtained using the Lowry method. Also, large numbers of samples can be read rapidly at the available wavelengths on an enzyme-linked immunosorbent assay microtiter plate reader. We found, however, artifactually high readings when using isotonic buffered sucrose to wash the cultures followed by sodium hydroxide to solubilize the cell protein. Such a procedure is commonly used for washing monolayer cell cultures in transport and binding studies. This effect was found to be due to hydrolysis of sucrose to the reducing sugar glucose. Use of Triton X-100 eliminated this problem, but this agent only solubilized about 80% of the protein that could be solubilized with sodium hydroxide. Furthermore, the high viscosity of Triton X-100 makes it more difficult to use. We found that washing the cells with isotonic mannitol solution followed by solubilization with sodium hydroxide gave reliable results. The sensitivity and speed of this method makes it suitable for multiple protein determinations in experiments using large numbers of cell culture samples.     

Cell surface proteins of encapsulated Streptococcus cremoris: identification and immunochemical characterization

Cell surface proteins of two slime-forming, encapsulated Streptococcus cremoris strains, MLS96 and T₅ from the fermented milk product viili, were extracted with the non-ionic detergent Triton X-100. The isolated protein antigens were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immuno-blotting with antisera produced against whole Strep. cremoris cells. When protein profiles of these strains were compared, seven prominent polypeptides were found common to both and were recognized by both antisera. Five of these polypeptides with molecular weights of 70000, 54000, 50000, 47000 and 40000 were identified as cell wall components. The remaining two polypeptides with molecular weights of 42000 and 26000 are being studied further in connection with slime formation for which modified Triton X-100 extraction provides a suitable method for isolation of the surface-associated antigens of lactic streptococci.     

Protein estimation in tissues containing high levels of lipid: modifications to Lowry's method of protein determination

A method to estimate protein in detergent-solubilized homogenates of lipid-rich biological samples (e.g., adipose tissue, myelin-enriched fractions of sheep brain) is described. The method is also suitable for samples in which protein is present as a protein-detergent complex. The method involves homogenization of tissue in the presence of a suitable detergent and KCl. Protein is then estimated in an aliquot of this homogenate by Lowry's method in the presence of excess sodium dodecyl sulfate, the solutions being clarified by extraction with ethyl acetate. Protein solubilization by Triton X-100 from adipose tissue was biphasic, extracting two to three times more protein under optimum conditions [1.7 +/- 0.1% (v/v) Triton X-100 and 0.75 M KCl], compared with homogenization without salt and detergent. Unlike adipose tissue, protein solubilization from myelin-enriched fractions of sheep brain peaked at 1% (v/v) Triton X-100, resulting in the extraction of approximately three times more protein than homogenization in the absence of detergent and salt.     

Presence of abundant filaments in apical caps of the nonciliated bronchiolar epithelial (Clara) cells

The nonionic detergent Triton X-100 has often been used for the extraction of cytoplasmic materials. We used the detergent in a vascular perfusion medium when preparing rat lung in order to observe the cytoskeleton of the nonciliated bronchiolar epithelial (Clara) cells. To eliminate some cytoplasmic materials selectively and to maintain good fine cell structure simultaneously, the lungs were perfused sequentially with the detergent (0.2% Triton X-100) alone for 2 min, with a mixture of low-concentration (0.1%) glutaraldehyde and detergent (0.2% Triton X-100) for 15 min, and finally with 2.5% glutaraldehyde for 5-10 min. After fixation, the nonciliated bronchiolar epithelial (Clara) cells were observed by scanning and transmission electron microscopy. At the apical region of the cells, there were central cytoplasmic protuberances (apical caps) filled with microfilaments. These filaments were bound at one end to the cytoplasmic side of the cell membrane and ran into the interior of the cytoplasm at the other end. As a control, the Clara cells were observed by transmission electron microscopy after perfusion with 2.5% glutaraldehyde solution. The luminal surfaces of the cells were covered with short, thick microvilli. The apical caps also had microvillus-like protrusions. These results suggest that the apical cap is not an apocrine droplet but rather is a stable structure involved in the function of the Clara cells.