The membrane of both sperm flagella and gill cilia of the scallop Aequipecten irradians may be selectively solubilized in 1% Triton X-100, 30 mM tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), pH8, and 3 mM MgCl2, leaving the axoneme totally intact. This membrane fraction represents about 20% of the total protein of the respective organelle. Analysis of the flagellar membrane by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis revealed one principal protein component, periodic acid-Schiff (PAS) positive and migrating with an apparent molecular weight of 250,000. The remaining minor proteins, none of them PAS positive, accounted for less than one-third of the total flagellar membrane fraction. Analysis of the ciliary membrane also revealed one major protein component, weakly PAS positive and migrating with an apparent molecular weight of 55,000. The remaining minor proteins represented about one-third of the total ciliary membrane fraction; two components with molecular weights of 100,000 and 40,000 predominated. The latter could be substantially reduced by purification of the cilia on a sucrose density gradient and was assumed to be actin, derived by vesiculation of the brush border during deciliation. The principal ciliary membrane protein, that of 55,000 daltons, was resolved into two equimolar components on NaDodSO4-Tris-glycine-polyacrylamide gels, comigrating with the alpha and beta chains of outer fiber tubulin. S-carboxymethylation caused increased splitting of the two components and concomitant migration with similarly treated ciliary tubulin. Preparative gel electrophoresis yielded separate components whose cyanogen bromide cleavage products were virtually identical in size distribution with those obtained from outer fiber alpha and beta chains; tryptic peptides corresponded almost exactly to those of authentic tubulin subunits but certain positional differences indicated possible side chain modification. At 25 degrees C both whole cilia and its solubilized membrane fraction bound colchicine while whole flagella and the 9 + 2 axoneme from either organelle did not. Thus certain molluscan flagellar membranes primarily contain a 250,000-dalton glycoprotein but ciliary membranes have a modified tubulin as the major protein component. At an electron microscopic level, flagellar membranes have a distinct trilamellar "unit membrane" structure while ciliary membranes appear thinner and considerably less distinct, perhaps reflecting the protein compositional differences in the membranes of these other wise morpholobically identical organelles.

Calmodulin has been isolated and characterized from the gill of the bay scallop Aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions shows most of the calmodulin to be localized in the cilia, specifically in the detergent-solubilized membrane-matrix fraction . Calmodulin represents 2.2 ± 0.3% of the membrane-matrix protein or 0.41 ± 0.05% of the total ciliary protein. Its concentration is at least 10 -4 M if distributed uniformly within the matrix . Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper . The ciliary protein is identified as a calmodulin on the basis of its calcium-dependent binding to a fIuphenazine-Sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium . Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine . Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point~0 .2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75% of the major peptides, but at least two major peptides in each show no nearequivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated . However, ciliary arrest will occur at calcium levels >150 uM . Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function . At the concentration in which it is found, it must also at least act as a calcium buffer .There is substantial evidence for the existence of calmodulin in cilia and flagella . However, its specific localization and function in these organelles is uncertain. Satir and co-workers (20; see also reference 17) find Tetrahymena calmodulin to be concentrated considerably more in cilia than in the cell body, using immunofluorescent techniques, whereas Walter and Schultz (27) find a greater concentration of Paramecium calmodulin in cell bodies than in cilia, using SDS-polyacrylamide gel analysis . Gitelman and Witman (10) report that calmodulin from Chlamydomonas occurs in both the cell body and in isolated flagella, with the latter appearing to contain a much greater concentration of calmodulin . About half of the flagellar calmodulin remains tightly bound to the axoneme after deter-622 E. W. STOMMEL, R. E. STEPHENS, H . R. MAS...

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.

The purpose of this study was to stimulate the laryngeal cough reflex using a nebulized, mild chemical irritant and to record an associated laryngeal evoked potential from the internal branch of the superior laryngeal nerve. The laryngeal evoked potential was obtained on ten normal subjects from the right internal branch of the superior laryngeal nerve. The electrodiagnostic setup included an active electrode placed just below the hyoid bone with a 4-cm separation and distal reference. A ground electrode was placed between the active and reference electrodes. The receptors and internal branch of the superior laryngeal nerve were stimulated by inhalation of a nebulized 20% solution of tartaric acid and normal saline. The time line was triggered by a pneumatic switch on initial inspiration of the nebulized tartaric acid. The electrodiagnostic settings were set at a sweep speed of 1 ms/division, a gain of 10 to 20 microV/division, and 20 to 2,000 filters. There were 132 variables recorded from the internal branch of the superior laryngeal nerve of the ten subjects. The mean peak distal latency was 1.66+/-0.42 ms with a 1.6 median, 1.6 mode, and 0.17 variance. The duration was 0.41 ms, and amplitude was 5.19+/-2.91 microV. In conclusion, the laryngeal evoked potential, the afferent component of the involuntary cough reflex, can be recorded from the internal branch of the superior laryngeal nerve after inhalation of tartaric acid-induced cough.

Microtubule proteins have recently been prepared and characterized from a number of diverse sources. Renaud, Rowe & Gibbons (1966, 1968) have obtained a protein from acetone powders of Tetrahymena pyriformis cilia and from isolated outer fibre doublets of this same species. The protein has an actin-like amino acid composition, a minimum subunit weight of 55000, and 7·5 free sulphydryl groups per mole of monomer. Stephens (1968a) has fractionated sea-urchin flagella, following the procedure of Gibbons (1965), to obtain the isolated outer fibres and has found the protein to have properties virtually identical to those of its ciliary counterpart. Both the flagellar and the ciliary outer-fibre proteins contain 1 mole of bound guanine nucleotide per mole of protein subunit (Stephens, Renaud & Gibbons, 1967).