Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.     

Structure and mass analysis of 12S and 19S dynein obtained from bull sperm flagella

The Brookhaven scanning transmission electron microscope (STEM) was used to elucidate the structures and masses of 12S and 19S dynein extracted from bull sperm flagella. The 12S particle was a single globular particle with an average mass of 311 +/- 10 kdaltons. The 19S dynein particles consisted of two globular heads joined to a common base. The average mass of the 19S particle was 1.6 +/- 0.04 X 10(6) daltons. Thus, with the exception of the larger mass, the bull sperm 19S dynein molecule resembles the two-headed 21S dynein obtained from sea urchin sperm flagella and the 18S dynein obtained from Chlamydomonas with the possibility of a third head giving rise to the 12S particle. The structure, mass and polypeptide composition of bull sperm flagella dynein is compared with outer arm dyneins previously obtained from Chlamydomonas, Tetrahymena, and sea urchin sperm flagella.     

Characterization of the ATP-sensitive binding of Tetrahymena 30 S dynein to bovine brain microtubules

Dynein was obtained by high salt extraction of Tetrahymena cilia and purified by DEAE-Sephacel chromatography. This fraction consisted of a mixture of 30 S dynein (80%) and the 14 S ATPase (15%). The column purification effectively removed tubulin and adenylate kinase. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the 30 S dynein was composed of a major heavy chain (approximately 400 kD, three copies), three intermediate chains (70, 85, and 100 kD), and a group of light chains (approximately 20 kD). The binding of the column-purified dynein to bovine brain microtubules was characterized as follows. (i) Titration of the dynein with microtubules showed a linear increase in turbidity up to an equivalence point of 2.7 mg of dynein/mg of tubulin with apparently tight binding; (ii) the addition of ATP caused the turbidity of the solution of decrease to a level equal to the sum of free dynein plus microtubules; (iii) transmission electron microscopy indicated that microtubules were decorated with dynein arms spaced at a 24-nm longitudinal repeat and that the dynein decoration was removed upon addition of ATP; (iv) cross-section images of microtubules that were saturated with dynein showed six to seven dynein arms around a microtubule consisting of 14 protofilaments, corresponding to a molar ratio of one dynein/six tubulin dimers; (v) the dynein arms were bound primarily by their broader end which corresponds to the end normally bound to the B-subfiber in vivo. Experiments with purified 30 and 14 S dyneins indicated that the dynein-microtubule binding activity and the ATP-induced dissociation were the properties of the 30 S dynein alone. These studies demonstrate that the 30 S dynein under our conditions (50 mM PIPES, pH 6.96, 4 mM MgSO4) interacts with bovine brain microtubules through the ATP-sensitive site of the dynein arm.     

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombination of ciliary dynein of Tetrahymena with the outer fibers.

Recombination of ciliary dyneins of Tetrahymena pyriformis with the outer fibers was investigated using turbidimetry, co-sedimentation analysis and electron microscopy. As reported by Gibbons, 30S dynein could recombine with the outer fibers, while 14S dynein did to so a lesser extent. At acidic pH, however, most of the 14S dynein was also rebound to the outer fibers. When an excess of crude dynein fraction was added to the outer fiber fraction at pH 8.2, electron microscopic observations showed that the outer doublet microtubules were decorated not only with arms but also with other electron-dense materials. On the other hand, when crude dynein fraction was mixed with the outer fibers in an appropriate quantity, only arms were reconstituted at the regular positions of A-subfibers. ATP had an inhibitory effect on the recombination of dynein with the outer fibers.     

A squid dynein isoform promotes axoplasmic vesicle translocation

Axoplasmic vesicles that translocate on isolated microtubules in an ATP-dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate-induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.     

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation of Tetrahymena 30 S dynein into 14 S subunit by sonication.

The sonication of 30 S dynein obtained from Tetrahymena cilia induced dissociation into 14-S subunits, some of the enzyme still remaining as intact 30 S dynein and partially dissociated dynein (21 S) in a minor amount. It was demonstrated that the enzymatic properties of the 14 S subunit are quite similar to those of 30 S dynein except for the Ca2+:Mg2+ ratio. ATPase (EC 3.6.1.3) (ATP phosphohydrolase activity of the 14 S subunit was steadily enhanced by increasing concentrations of Mg2+. It was also activated by Ca2+ with an optimum at 6 mM but inhibited by a further increase in concentration. The Ca2+:Mg2+ ratio at 1 mM was about 0.62. 0.6 M KCl stimulated ATPase activity of the 14 S subunit two-fold. The Mg2+-ATPase had an optimum at pH 6.2 and revealed a high activity over pH 10. The Ca2+-ATPase showed two optima at pH 6.2 and 9.5. The Km for ATP was 10 muM. Only 10% of the 14 S subunit recombined with the outer fibers in the presence of Mg2+. The 14 S subunit was shown to have the same mobility as that of 30 S dynein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.     

Interaction of proteins with the mRNA for ribosomal protein L1 in Xenopus: structural characterization of in vivo complexes and identification of proteins that bind in vitro to its 5'UTR.

Xenopus r-protein mRNAs are known to be coordinately regulated at the translational level. To find out if RNA/protein interactions are involved in this control mechanism, we have characterized the particles containing the translationally repressed rp-mRNA and we have investigated the proteins that specifically bind to this type of mRNA. By sedimentation analysis and isopycnic centrifugation we have found that the repressed rp-mRNAs are assembled in slow sedimenting complexes where the RNA is prevalent over the protein mass (2.3 to 1). This composition is maintained also after in vitro reconstitution of the particle. We carried out also a detailed analysis of in vitro RNA/protein complex formation by focusing our attention on the 5'UTR, very similar in different rp-mRNAs and important in the translational regulation. We describe specific interactions of L1 mRNA with four proteins. The binding site of two of them, 57 kD and 47 kD, is in the typical pyrimidine sequence at the 5' end and is position dependent. Proteins of the same size interact also with the analogous region of r-protein S1 and L14 mRNA, not with unrelated RNAs. Binding of two other proteins, 31 kD and 24 kD, in the downstream region of the 5'UTR was also observed. The most evident 57 kD protein has been partially purified. Although the binding of these proteins to the r-protein mRNA 5'UTR is specific, their involvement in the translation regulation remains to be proved.     

Characteristics of defective lysogeny in Streptomyces chrysomallus

When Streptomyces chrysomallus 2703 grows on solid media, lytic zones appear in the form of negative colonies which are not caused by the virulent phage. The material from these colonies and the cultural broth of S. chrysomallus 2703 were examined by electron microscopy. Four different morphological types of particles were revealed, three of which were defective phage particles (tails). Particles of the fourth type had a regular hexagonal shape and a diameter of 200A. The particles prevailed in all of the preparations. Their origin is discussed. S. chrysomallus in considered as a poly- and defective lysogenic culture.     

New Axonemal Dynein Heavy Chains from Tetrahymena thermophila

Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.     

Effect of spin-labeled maleimide on 14S and 30S dyneins in solution and on demembranated ciliary axonemes.

The effects of N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide(SLM) on the pellet height response and ATPase activity of glycerinated Triton X-100 extracted cilia of Tetrahymena pyriformis have been studied. Preincubation of cilia with SLM caused complete inhibition of the pellet height response and an initial increase in ATPase activity followed upon longer exposure to SLM by inhibition of ATPase. The effect of SLM on extracted 30S dynein was the reverse of that for whole cilia: ATPase activity was increased when 30S dynein was added to a mixture of ATP and SLM and inhibited when the 30S dynein was preincubated with SLM. The activity of 14S dynein was only inhibited by SLM. Electron spin resonance spectra of ciliary axonemes that had reacted with SLM for various times showed that much of the covalently bound SLM was strongly immobilized even after 1 min of reaction, when ATPase activity increased twofold. The proportion of strongly immobilized label increased with longer times of reaction. Addition of ATP to SLM-labeled axonemes caused a small decrease in the height of the spectral peak corresponding to strongly immobilized label as compared with that of weakly immobilized label, indicating an increase in rotational freedom of some covalently bound label. The results suggest that ATP causes a conformation change affecting a sulfhydryl group(s) involved in the mechanochemical system. It was also shown that beta,gamma-methylene ATP(AMP-PCP) is an inhibitor of dynein ATPase. This analogue of ATP is not hydrolyzed by whole cilia or by the extracted dyneins and does not cause a pellet height response. With Mg2+ as divalent cation, AMP-PCP inhibits 30S dynein more than it inhibits 14S dynein; with Ca2+, the inhibition of 30S dynein is reduced, and there is no inhibition of 14S dynein. Under conditions where AMP-PCP inhibited 30S dynein ATPase it was much less effective than ATP in protecting against the loss of ATPase activity by SLM. Although SLM inhibited Mg2+-activated 14S and 30S dyneins in solution, it did not inhibit ciliary ATPase activity. These results support the view that at least 2 SH groups are involved in ciliary motility and that their reactivity to SH reagents depends on whether the dyneins are in situ or have been extracted.     

Microtubule translocation properties of intact and proteolytically digested dyneins from Tetrahymena cilia

Tetrahymena cilia contain a three-headed 22S (outer arm) dynein and a single-headed 14S dynein. In this study, we have employed an in vitro assay of microtubule translocation along dynein-coated glass surfaces to characterize the motile properties of 14S dynein, 22S dynein, and proteolytic fragments of 22S dynein. Microtubule translocation produced by intact 22S dynein and 14S dynein differ in a number of respects including (a) the maximal velocities of movement; (b) the ability of 22S dynein but not 14S dynein to utilize ATP gamma S to induce movement; (c) the optimal pH and ionic conditions for movement; and (d) the effects of Triton X-100 on the velocity of movement. These results indicate that 22S and 14S dyneins have distinct microtubule translocating properties and suggest that these dyneins may have specialized roles in ciliary beating. We have also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two-headed proteolytic fragments of this three-headed molecule and examining their motile activity in vitro. Unlike the single-headed 14S dynein, the single-headed fragment of 22S dynein did not induce movement, even though it was capable of binding to microtubules. The two-headed fragment, on the other hand, translocated microtubules at velocities similar to those measured for intact 22S dynein (10 microns/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating microtubule movement, which raises the possibility that multiple heads may serve some regulatory function or may be required for maximal force production in the beating cilium.     

Outer-arm dynein from trout spermatozoa: substructural organization.

Outer-arm dynein purified from trout spermatozoa was disrupted by low-ionic-strength dialysis, and the resulting subunits were separated by sucrose density-gradient centrifugation. The intact 19 S dynein, containing the alpha- an beta-heavy chains, intermediate chains (ICs) 1-5 and light chains (LCs) 1-6, yielded several discrete particles: a 17.5 S adenosine triphosphatase (ATPase) composed of the alpha- and beta-chains ICs 3-5 and LC 1; a 9.5 S complex containing ICs 1 and 2 together with LCs 2, 3, 4, and 6; and a single light chain (LC 5), which sedimented at approximately 4 S. In some experiments, ICs 3-5 also separated from the heavy chain complex and were obtained as a distinct subunit. Further dissociation of the 17.5 S particle yielded a 13.1 S ATPase that contained the beta-heavy chain and ICs 3-5. The polypeptide compositions of the complexes provide new information on the intermolecular associations that occur within dynein. Substructural features of the trout dynein polypeptides also were examined. The heavy chains were subjected to vanadate-mediated photolysis at the V1 sites by irradiation at 365 nm in the presence of Mg2+, ATP, and vanadate. Fragment pairs of relative molecular mass (Mr) 245,000/185,000 and 245,000/170,000 were obtained from the alpha- and beta-heavy chains, respectively. Photolysis of these molecules at their V2 sites, by irradiation in the presence of vanadate and Mn2+, yielded fragments of Mr 160,000/270,000 and 165,000/250,000, respectively. These values confirm that the alpha- and beta-heavy chains have masses of 430,000 and 415,000 daltons, respectively. Immunological analysis using monoclonal antibodies revealed that one intermediate chain from trout dynein (IC 2) contains epitopes present in two different intermediate chains from Chlamydomonas dynein. This indicates that specific sequences within the dynein intermediate chains have been highly conserved throughout evolution.     

毕赤酵母表达的HBV全长Pres蛋白的分离纯化

巴斯德-毕赤酵母工程菌株GS115-PreS经发酵在甲醇诱导下可高效表达分泌型全长PreS蛋白。Western blot证明发酵液中存在着可溶性的分子量为48kD的PreS蛋白和蛋白质颗粒,蛋白质颗粒主要成分为48kD的全长Pres蛋白和28kD的S蛋白,电镜观察发现蛋白质颗粒直径为30nm。发酵液经过脱盐、浓缩处理后,上清液经DEAESFF阴离子交换柱得到纯化的PreS蛋白;超速离心和蔗糖密度梯度离心得到蛋白颗粒。该颗粒的主要组分为全长PreS蛋白（PreS1＋PreS2＋S）,还有少量的主蛋白（S）。ELISA检测证明全长PreS蛋白和蛋白颗粒有着良好的抗原性, P/N显示蛋白颗粒的抗原性比PreS蛋白的抗原性高。     

Bacteriophage T4 Head Morphogenesis III. Some Novel Properties of Gene 13-Defective Heads

The nature of phage precursors in gene 13-defective infected cells was studied by electron microscopy and pulse-chase isotopic labeling experiments. Our results suggest that both stable (20%) and fragile (70%) filled-head precursors accumulated in the absence of gene 13 product. Upon extraction, the fragile heads were found to lose most of their deoxyribonucleic acid and appeared unfilled with an average density of 1.34 g/cm(3) and a sedimentation coefficient of 300S. These unfilled heads differed from empty gene 13-defective heads which did not have any associated deoxyribonucleic acid and banded at an average density of 1.31 g/cm(3). Furthermore, it was found that a tsN38 (temperature-sensitive mutant in gene 13)- infected culture maintained at 41.5 C for increasing times led to a decrease in specific infectivity of 1,000S phagelike particles. Electron microscopy of these particles revealed that the decreased infectivity was due to an improper union of head and tails.     

Immunological relation between 14 S dynein and 30 S dynein from the cilia of Tetrahymena pyriformis.

The immunological relation between 14 S dynein and 30 S dynein obtained from Tetrahymena cilia was investigated by using antisera specific for each dynein subunit or some dynein subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although 14 and 30 S dynein main subunits have different electrophoretic mobilities, our immunodiffusion tests showed that there exists a close immunological relation between them. At least three immunologically different polypeptides designated polypeptides A, B and C are included in the 30 S dynein main band which has been recognized as a single component by electrophoresis, and that the polypeptides designated A',B' and C' are included in the 14 S dynein main bands. Polypeptides A and A',B and B', or C and C' appeared to have a certain common antigenic determinant(s). Polypeptide C of 30 S dynein was shown to possess a certain antigenic determinant(s) specific for 30 S dynein, besides the determinant common with that of polypeptide C' of 14S dynein. The second main component of 30 S dynein proved to be a specific polypeptide of 30 S dynein but not to be a degraded product of the main polypedtide. All antisera reacted with native dynein molecules to some extent, but did not inhibit dynein ATPase (ATP phosphohydrase, EC 3.6.1.3) activity significantly.     

Identification of the human ortholog of the t-complex-encoded protein TCTE3 and evaluation as a candidate gene for primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease that is caused by impaired ciliary and flagellar functions. About 50% of PCD patients show situs inversus, denoted as Kartagener syndrome. In most cases, axonemal defects in cilia and sperm tails can be demonstrated by electron microscopy, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. In order to identify novel PCD genes we have isolated the human ortholog of the murine TCTE3 gene. The human TCTE3 gene encodes a dynein light chain and shares high similarity to dynein light chains of other species. The TCTE3 gene is expressed in tissues containing cilia or flagella, it is composed of four exons and located on chromosome 6q25-->q27. To elucidate the role of TCTE3 as a candidate gene for PCD a mutational analysis of thirty-six PCD patients was performed. We detected five polymorphisms in the coding sequence and in the 5' UTR of the TCTE3 gene. In one patient a heterozygous nucleotide exchange was identified resulting in an arginine to isoleucine substitution at the amino acid level. However, this exchange was also detected in one control DNA. Our results indicate that mutations in the TCTE3 gene are not a main cause of primary ciliary dyskinesia.     

Interrelationships between thermal-, N-ethylmaleimide-, and Ca2+-calmodulin-mediated activation/inactivation of dynein ATPase activities

Interactive effects between calmodulin activation of 30 S dynein ATPase activity and activation by heat or N-ethylmaleimide (NEM) have been studied. Addition of calmodulin during the heat treatment caused a larger increment in ATPase activity (above that caused by heating alone) than did addition of calmodulin after the heat treatment. Similar results were obtained in experiments where activation was caused by NEM treatment. For both the heat and NEM treatments, the synergistic effect of calmodulin when present during the treatment was Ca²⁺ dependent although activation of ATPase activity by either treatment alone was not Ca²⁺ dependent. Heating 14 S dynein inhibited its ATPase activity and reduced the effectiveness of calmodulin as an activator. The activating effect of calmodulin added after heat or NEM treatments was about the same as if the calmodulin was present during the treatment, i.e., interactive effects were minimal. Concentrations of NEM that had little effect on the ATPase activity of 14 S dynein largely eliminated the ability of calmodulin to activate its ATPase activity. Chromatography of the heat-treated 14 S dynein on calmodulin-Sepharose 4B indicated that the loss of sensitivity of 14 S dynein ATPase to calmodulin was not due to loss of ability of the dynein to bind to calmodulin. Retention of calmodulin binding ability was also shown for heat-treated 30 S dynein. These results suggest that calmodulin and heat/NEM activate solubilized 30 S dynein ATPase by separate mechanisms which may include a common process.