Two heavy chains of 21S dynein from sea urchin sperm flagella

The biochemical properties of 21S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21S dynein preparation contains two distinct heavy chains. These two heavy chains, termed A alpha and A beta, had apparently the same molecular weight of 500,000 but showed different mobilities on SDS-urea gels. The isoelectric points of A alpha and A beta heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar. Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21S dynein into two fractions: the one with larger sedimentation coefficient contained the A alpha heavy chain, and the other with smaller sedimentation coefficient contained the A beta heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.     

Photosensitized cleavage of dynein heavy chains. Cleavage at the V2 site by irradiation at 365 NM in the presence of oligovanadate

Irradiation of the outer-arm dynein ATPase from sea urchin sperm flagella at 365 nm in the presence of 50-200 microM vanadate (Vi) and 1 mM manganese acetate, in the absence of ATP, cleaves the alpha and beta heavy chains at a specific site, termed the V2 site, to form discrete peptides of Mr approximately 260,000 and 170,000 from the alpha chain and of Mr approximately 255,000 and 175,000 from the beta chain, with a yield of 80%. This cleavage at the V2 site is not correlated with any direct effect on the dynein ATPase activity. In the presence of 100 microM Vi, the half-times for cleavage of the alpha and beta chains are about 12 and 50 min, respectively. The rate of heavy chain cleavage shows a sigmoidal dependence upon Vi concentration, with half-maximal rate occurring at 58 +/- 7 microM, consistent with the chromophore responsible for cleavage being tri-vanadate. Addition of 10 microM ATP or ADP, or of 100 microM CTP or UTP, to the irradiation medium inhibits cleavage at the V2 site, and results in a slow cleavage occurring at the V1 site described previously. The peptides produced by sequential cleavage at the V2 and then the V1 sites indicate that the sites are separated by about 100,000 Da along the length of each heavy chain. Photoaffinity labeling with [alpha-32P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) gives specific incorporation of 32P into both the Mr 255,000 and 175,000 peptides of the beta chain but into only the Mr 260,000 peptide of the alpha chain. These results suggest that V2 cleavage occurs on a loop of the heavy chain that forms part of the ATP-binding site, close to the locus of 8-N3ATP attachment.     

Specific cleavage of dynein heavy chains by ultraviolet irradiation in the presence of ATP and vanadate

Irradiation of soluble dynein 1 from sea urchin sperm flagella at 254 nm in the presence of 50 microM ATP and 100 microM inorganic vanadate (Vi) cleaves the alpha and beta heavy chains into approximately equal quantities of two polypeptides of Mr 228,000 and 200,000, with a conversion efficiency of about 63%. A similar cleavage occurs in the presence of Vi and either ADP or 8-azidoadenosine 5'-triphosphate (8-N3ATP); in the latter case, 8-N3ATP becomes covalently bound principally to the Mr 228,000 polypeptide. No detectable amount of these fragments is formed if either the Vi or the nucleotide is omitted or in the presence of Vi and 50 microM AMP. These results emphasize the basic similarity of the two ATPases associated with the alpha and beta heavy chain subunits of dynein 1 and give a mean Mr of 428,000 for the intact heavy chains.     

Photosensitized cleavage of dynein heavy chains. Cleavage at the "V1 site" by irradiation at 365 nm in the presence of ATP and vanadate

Irradiation of soluble dynein 1 from sea urchin sperm flagella at 365 nm in the presence of MgATP and 0.05-50 microM vanadate (Vi) cleaves the alpha and beta heavy chains (Mr 428,000) at their V1 sites to give peptides of Mr 228,000 and 200,000, without the nonspecific side effects produced by irradiation at 254 nm as described earlier (Lee-Eiford, A., Ow, R. A., and Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342). The decrease in intact heavy chain material is biphasic; in 10 microM Vi, approximately 80% occurs with a half-time of 7 min and the remainder with a half-time of about 90 min, and the yield of cleavage peptides is better than 90%. Loss of dynein ATPase activity appears to be a direct result of the cleavage process and is not significantly affected by the presence of up to 0.1 M cysteamine (CA, 60-23-1) or 2-aminoethyl carbamimidothioic acid dihydrobromide (CA, 56-10-0) as free radical trapping agents. The concentration of Vi required for 50% maximal initial cleavage rate is 4.5 microM, while that for 50% ATPase inhibition is 0.8 microM, both in a 0.6 M NaCl medium. In the presence of 20 microM Vi, CTP and UTP support cleavage at about half the rate of ATP, whereas GTP and ITP support cleavage only if the Vi concentration is raised to about 200 microM. Substitution of any of the transition metal cations Cr2+, Mn2+, Fe2+, or Co2+ for the usual Mg2+ suppresses the photocleavage, presumably by quenching the excited chromophore prior to scission of the heavy chain. The photocleaved dynein 1 binds to dynein-depleted flagella similarly to intact dynein 1, but upon reactivation of the flagella with 1 mM ATP their motility is partially inhibited, rather than being augmented as with intact dynein. These results indicate that Vi acts as a photosensitizing catalyst and suggest that the cleavage proceeds through excitation of Vi bound to dynein at the hydrolytic ATP binding site on each heavy chain, probably in a dynein X MgADP X Vi complex. The exquisite specificity of Vi-sensitized photocleavage will aid the peptide mapping of dynein heavy chains and may be of broader use in studies of protein structure.     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Masses of chains and sites of ultraviolet-induced vanadate-dependent cleavage

We report here on the UV-induced vanadate-dependent cleavage of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Both polypeptides are cleaved at a single site (termed the V1 site) by UV irradiation in the presence of Mg2+, ATP, and vanadate. The alpha chain yields fragments of Mr 290,000 and 190,000. Fragments of Mr 255,000 and 185,000 are obtained from the beta chain. Ultraviolet irradiation of the alpha and beta chains in the presence of vanadate and Mn2+ (but no nucleotide) induces cleavage of both molecules at sites (termed the V2 sites) distinct from the V1 sites. The single V2 site within the beta chain is located 75,000 daltons from the site of V1 cleavage within the Mr 255,000 V1 fragment. The alpha chain contains three distinct sites of V2 cleavage; all are located within the Mr 290,000 V1 fragment, 60,000, 90,000, and 100,000 daltons from the site of V1 cleavage. From these studies, we estimate the masses of the alpha and beta heavy chains to be 480,000 and 440,000 daltons, respectively.     

Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.     

Activation of dynein adenosine triphosphatase and flagellar motility of demembranated spermatozoa by monovalent salts at 40 degrees C in the domestic fowl, Gallus domesticus

1. At 40 degrees C, around the normal avian body temperature, demembranated fowl spermatozoa with no addition of monovalent chlorides were immotile. 2. Demembranated spermatozoa become motile at 40 degrees C when 0.1-0.5 M concentrations of NH4Cl, NaCl and KCl were added to the reactivation medium, with maximum motility occurring at 0.2-0.3 M in all cases. 3. The addition of NH4Cl, NaCl and KCl also stimulated the ATPase activity of crude dynein extract. In contrast, LiCl did not appreciably affect motility and ATPase activity. 4. These results showed that the flagellar dynein ATPase activity of fowl spermatozoa could be stimulated by the addition of certain monovalent chlorides, except LiCl, and demembranated spermatozoa might be motile at 40 degrees C.     

The role of the dynein stalk in cytoplasmic and flagellar motility

We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10–12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility. Received: 13 March 1998 / Revised version: 17 April 1998 / Accepted: 17 April 1998     

Regulation of sperm flagellar motility by calcium and cAMP-dependent phosphorylation

There is substantial evidence that cAMP-dependent phosphorylation is involved in the activation of motility of spermatozoa as they are released from storage in the male reproductive tract. This evidence includes observations that in vivo activation of motility can be inhibited by protein kinase inhibitors, can be reversed by protein phosphatase treatment of demembranated spermatozoa, and is associated with phosphorylation of sperm proteins, and observations that spermatozoa that have not been activated in vivo can be activated in vitro by cAMP-dependent phosphorylation. Activation in vivo can often be triggered by conditions that increase intracellular pH, but the relevance of this to in vivo activation under natural conditions and the steps between pH increase and cAMP increase have not been fully established. The relationships between changes in the protein substrates for cAMP-dependent phosphorylation and changes in axonemal function are still unknown. Sperm chemotaxis to egg secretions is widespread; in the sea urchin Arbacia, the egg jelly peptide resact has been identified as a chemoattractant. Response to chemoattractants involves changes in asymmetry of flagellar bending waves, and similar changes in asymmetry can be produced in vitro by increases in [Ca++]. Temporal changes in resact receptor occupancy might lead to transient changes in intracellular [Ca++] and the asymmetry of flagellar bending, but many links in this hypothetical sequence remain to be established. Both of these signalling systems offer immediate opportunities for investigations of biochemical pathways leading to easily assayable biological responses. However, complications resulting from interactions between these two systems need to be considered.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Nucleotide binding sites

The photoaffinity analogs 2-azidoadenosine 5'-tri(di)-phosphate (2-N3AT(D)P) and 8-azidoadenosine 5'-triphosphate (8-N3ATP) have been used to probe the substructural organization of the nucleotide binding pockets within the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Both 2-N3ATP and 8-N3ATP are competitive inhibitors of dynein ATP hydrolysis, and both analogs are themselves hydrolyzed by the alpha-beta dimer. Following vanadate-dependent photolysis at the V1 site (by UV irradiation in the presence of Mg2+, ATP, and vanadate), both probes exclusively labeled the larger fragment from the alpha chain. In contrast, within the beta chain the predominant insertion sites for the two analogs were located on opposite sides of the V1 site. Therefore, the hydrolytic pockets of these two molecules have different substructures. Vanadate-dependent photolysis of the alpha and beta chains at the V2 sites (by UV irradiation in the presence of vanadate and Mn2+) profoundly affected the predominant modification sites; for example, following photolysis at the V2a site neither fragment of the alpha chain was photolabeled by 2-N3ATP or 8-N3ATP. Based on the photolabeling patterns obtained, the single V2 site within the beta chain is predicted to be analogous to the V2b site within the alpha chain. The results support the hypothesis that the V2 sites occur within the ATP binding pockets, and indicate that these functional domains are composed of portions of the heavy chains which are linearly separated by up to at least 100,000 daltons. Thus, the central region of each dynein heavy chain must be extensively folded so as to bring the widely separated photocleavage and photolabeling sites together within a single catalytic unit.     

The force-velocity relationship for microtubule sliding in demembranated sperm flagella of the sea urchin

We studied the relationship between the force and velocity of microtubule sliding in demembranated sperm flagella of the sea urchin, Hemicentrotus pulcherrimus, under auxotonic conditions following a quick release of the tension between sliding microtubules. The shape of the force-velocity curve was independent of the concentration of Mg-ATP over the range of 3.7 to 350 microM and appeared either linear or was the reverse of the hyperbolic curve seen for muscle. The power, calculated as the product of velocity and force, passed through a peak at c. 0.7 Fmax (the maximal isometric force). Thus, the maximal power is attained at a larger relative load than in muscle. The sliding velocity at 0.1 Fmax showed a hyperbolic dependence on Mg-ATP concentration, with a Km of 210 microM and a Vmax of 19 micron.sec-1. The maximal force did not significantly change over the Mg-ATP concentration range of 3.7 to 350 microM. These results are discussed in terms of a crossbridge model similar to the one originally proposed by Huxley. It is suggested that the dynein crossbridge cycle is characterized by a relatively rapid rate of attachment and a relatively slow rate of detachment.     

Distinct roles of 1alpha and 1beta heavy chains of the inner arm dynein I1 of Chlamydomonas flagella

The Chlamydomonas I1 dynein is a two-headed inner dynein arm important for the regulation of flagellar bending. Here we took advantage of mutant strains lacking either the 1α or 1β motor domain to distinguish the functional role of each motor domain. Single- particle electronic microscopic analysis confirmed that both the I1α and I1β complexes are single headed with similar ringlike, motor domain structures. Despite similarity in structure, however, the I1β complex has severalfold higher ATPase activity and microtubule gliding motility compared to the I1α complex. Moreover, in vivo measurement of microtubule sliding in axonemes revealed that the loss of the 1β motor results in a more severe impairment in motility and failure in regulation of microtubule sliding by the I1 dynein phosphoregulatory mechanism. The data indicate that each I1 motor domain is distinct in function: The I1β motor domain is an effective motor required for wild-type microtubule sliding, whereas the I1α motor domain may be responsible for local restraint of microtubule sliding.     

Disruption of genes encoding predicted inner arm dynein heavy chains causes motility phenotypes in Tetrahymena

The multi-dynein hypothesis [Asai, 1995: Cell Motil Cytoskeleton 32:129-132] states: (1) there are many different dynein HC isoforms; (2) each isoform is encoded by a different gene; (3) different isoforms have different functions. Many studies provide evidence in support of the first two statements [Piperno et al., 1990: J Cell Biol 110:379-389; Kagami and Kamiya, 1992: J Cell Sci 103:653-664; Gibbons, 1995: Cell Motil Cytoskeleton 32:136-144; Porter et al., 1996: Genetics 144:569-585; Xu et al., 1999: J Eukaryot Microbiol 46:606-611] and there is evidence that outer arms and inner arms play different roles in flagellar beating [Brokaw and Kamiya, 1987: Cell Motil. Cytoskeleton 8:68-75]. However, there are few studies rigorously testing in vivo whether inner arm dyneins, especially the 1-headed inner arm dyneins, play unique roles. This study tested the third tenet of the multi-dynein hypothesis by introducing mutations into three inner arm dynein HC genes (DYH8, 9 and 12) that are thought to encode HCs associated with 1-headed inner arm dyneins. Southern blots, Northern blots, and RT-PCR analyses indicate that all three mutants (KO-8, 9, and 12) are complete knockouts. Each mutant swims slower than the wild-type cells. The beat frequency of KO-8 cells is lower than that of the wild-type cells while the beat frequencies of KO-9 and KO-12 are not different from that of wild-type cells. Our results suggest that each inner arm dynein HC is essential for normal cell motility and cannot be replaced functionally by other dynein HCs and that not all of the 1-headed inner arm dyneins play the same role in ciliary motility. Thus, the results of our study support the multi-dynein hypothesis [Asai, 1995: Cell Motil Cytoskeleton 32:129-132].     

Domains in the 1alpha dynein heavy chain required for inner arm assembly and flagellar motility in Chlamydomonas.

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1alpha Dhc of the I1 inner dynein arm. The 1alpha Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH(2)- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1alpha Dhcs were recovered. The truncated 1alpha Dhc fragments lacked the dynein motor domain, but still assembled with the 1beta Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1alpha motor domain in the I1 structure and further revealed the role of the 1alpha Dhc in flagellar motility and phototactic behavior.     

Conformational changes of dynein: mapping and sequence analysis of ATP/Vanadate-dependent trypsin-sensitive sites on the outer arm dynein b heavy chain from sea urchin sperm flagella

Conformational changes of dynein during ATP hydrolysis are demonstrated by the difference in the tryptic fragments of the dynein heavy chain between in the absence and presence of ATP and vanadate. Here tryptic sites in the presence of ATP and vanadate (Tav sites) have been mapped on the betaheavy chain of outer arm dynein from sea urchin sperm flagella. Tav sites are located not only near the central catalytic domain which includes four P-loops, but also near the carboxyl-terminal coiled-coil region. The Tav2 site is located in the most carboxyl-terminal region, which is nearly 850 amino acid residues apart from the the fourth P-loop (P4 site). The region from the most amino-terminal Tav site (Tav1 site) to the Tav2 site covers approximately 2,100 amino acid residues, which is almost half the whole betaheavy chain. Comparison of the sequences around the tryptic sites of the sea urchin b chain and those of the dynein heavy chains from other organisms reveals that the sequence around the Tav1 site is highly conserved in both cytoplasmic and axonemal dyneins but that around Tav2 sites is only conserved in axonemal dyneins, suggesting functional differences in the Tav2 region between the two subfamilies of dynein.     

Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Location of epitopes and protease-sensitive sites

We describe here the pathways by which the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella are degraded by endoproteases. By probing the digestion products with monoclonal antibodies, we have located a number of protease-sensitive sites within both polypeptides and identified the regions of each molecule from which specific fragments are derived. These data also define the regions within each chain which contain the epitopes recognized by our monoclonal antibodies. The locations of the cleavage sites reveal both differences and similarities between the two molecules. The sites at which the beta chain is initially cleaved by elastase differ depending upon whether the particle is digested in situ or in solution, indicating that the beta chain undergoes a significant conformational change following extraction from the axoneme. Evidence was also obtained that heterogeneity exists among the purified alpha chain molecules. The possible arrangement of the different regions of the heavy chains within the alpha-beta dimer is discussed.