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.     

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.     

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.     

Labeling of Chlamydomonas 18 S dynein polypeptides by 8-azidoadenosine 5'-triphosphate, a photoaffinity analog of ATP

The 18 S dynein from the outer arm of Chlamydomonas flagella is composed of an alpha subunit containing an alpha heavy chain (Mr = approximately 340,000) and an Mr = 16,000 light chain, and a beta subunit containing a beta heavy chain (Mr = approximately 340,000), two intermediate chains (Mr = 78,000 and 69,000), and seven light chains (Mr = 8,000-20,000). Both subunits contain ATPase activity. We have used 8-azidoadenosine 5'-triphosphate (8-N3 ATP), a photoaffinity analog of ATP, to investigate the ATP-binding sites of intact 18 S dynein. 8-N3ATP is a competitive inhibitor of 18 S dynein's ATPase activity and is itself hydrolyzed by 18 S dynein; moreover, 18 S dynein's hydrolysis of ATP and 8-N3ATP is inhibited by vanadate to the same extent. 8-N3ATP therefore appears to interact with at least one of 18 S dynein's ATP hydrolytic sites in the same way as does ATP. When [alpha- or gamma-32P]8-N3ATP is incubated with 18 S dynein in the presence of UV irradiation, label is incorporated primarily into the alpha, beta, and Mr = 78,000 chains; a much smaller amount is incorporated into the Mr = 69,000 chain. The light chains are not labeled. The incorporation is UV-dependent, ATP-sensitive, and blocked by preincubation of the enzyme with vanadate plus low concentrations of ATP or ADP. These results suggest that the alpha heavy chain contains the site of ATP binding and hydrolysis in the alpha subunit. In the beta subunit, the beta heavy chain and one or both intermediate chains may contain ATP-binding sites.     

Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.     

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.     

Iron(III)-mediated photolysis of outer arm dynein ATPase from sea urchin sperm flagella

Irradiation of outer arm dynein ATPase from sea urchin sperm tail flagella at 365-410 nm in the presence of Fe(III)-gluconate complex and ATP produces photolytic cleavage at two distinct sites on the beta heavy chain, located approximately 250 and approximately 230 kDa from its amino terminus. The former cut is close to or identical with the V1 site of the vanadate-mediated photocleavage (Gibbons, I.R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J.Y., and Gibbons, B.H. (1987) J. Biol. Chem. 262, 2780-2786. The rate of photolysis shows a hyperbolic dependence on Fe(III)-gluconate concentration with half-maximal rate occurring at 23 microM at pH 6.3. In the presence of 0.1-0.5 mM Fe(III)-gluconate-ATP, approximately 58% of the beta chain becomes cleaved with a half-time of about 34 s; the remainder of the beta chain and almost all of the alpha chain are resistant to cleavage. This photolytic cleavage of the beta chain is accompanied by an approximately parallel loss of the dynein latent ATPase activity, whereas the Triton-activated ATPase is lost to a somewhat greater extent. Mg2+ concentrations above approximately 3 mM inhibit photolysis. Substitution of ADP for ATP changes the pattern of cleavage so that both the alpha and beta heavy chain undergo scission but at the 250-kDa site only. AMP, adenyl-5'-yl imidodiphosphate and Fe(II) do not support cleavage at either site. Trivalent rhodium-ATP complexes, as models of MgATP, can also catalyze photolysis of the beta chain at the 250-kDa site. These results suggest that photolysis results from the activation of an Fe(III)-ATP complex bound to the hydrolytic ATP binding site of the beta chain and that both Fe(III) cleavage sites are located close to the nucleotide binding site in the tertiary folding of the beta heavy chain. The cleavage reaction possibly involves initial photoreduction of Fe(III) bound at the Mg2+ binding site in the dynein.Fe.ATP complex, followed by covalent modification of an amino acid side chain that leads to eventual peptide scission.     

Tryptic digestion of dynein 1 in low salt medium. Origin and properties of fragment A

Dynein 1 was extracted from sperm flagella of the sea urchin Tripneustes gratilla with 0.6 M NaCl and dialyzed against 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 5 mM imidazole/HCl buffer, pH 7.0, for 24-48 h. In some cases, fractions containing the alpha heavy chain and the beta/intermediate chain 1 complex (beta/IC1) were separated by density gradient centrifugation in the same solution. Treatment of the samples at a trypsin:protein ratio of 1:10 w/w for 32 min at room temperature yields a crude digest from which Fragment A is purified by density gradient centrifugation. The purified Fragment A consists of two principal peptides (Mr = 195,000 and 130,000) that cosediment with the peak of ATPase activity at 12.5 S, which is slightly faster than the 11 S of the original beta/IC1 complex. When digests of the separated alpha chain and of the beta/IC1 complex are followed as a function of time, the early cleavages of the two heavy chains (Mr = 428,000) resemble each other in that both lead to similarly sized peptides of Mr 316,000 and 296,000, but only in the beta/IC1 fraction does the digestion proceed to form Fragment A. The remainder of the beta chain, termed Fragment B, occurs as an Mr 110,000 peptide sedimenting at 5.7 S with no associated ATPase activity. Fragment A has a specific ATPase activity of 4.3 mumol Pi X min-1 X mg-1, with a Km of 29 microM in 0.1 M NaCl medium, and an apparent Ki for inhibition by vanadate of 1.2 microM in the absence of salt, and 22 microM in 0.6 M NaCl. Photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-triphosphate indicates that the ATP binding site on the beta chain of dynein 1 is located on the Mr 195,000 peptide of Fragment A. The possibility that Fragments A and B of the beta/IC1 complex may correspond to the head and tail regions of the tadpole-shaped particle seen by electron microscopy is discussed.     

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella coupled with ATP hydrolysis

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella in relation to ATP hydrolysis was examined by tryptic digestion. Tryptic digestion of the beta chain in the presence of 2 mM ATP (ADP) and 100 microM vanadate (Vi) or in the presence of 4 mM ATP gamma S produced different polypeptides from in the case of no addition. The difference was similar to the result previously reported for 21S outer-arm dynein heavy chains [Inaba, K. & Mohri, H. (1989) J. Biol. Chem. 264, 8384-8388]. Unlike the tryptic digestion pattern of 21S dynein heavy chains, however, the 135-kDa polypeptide was consistently produced from the beta chain, even in the presence of ATP (ADP) and Vi. The tryptic digestion pattern of the 21S particle reconstituted from the separated a chain, the beta/IC1 complex and the IC2/IC3 complex [Tang, W.-J.Y., Bell, C.W., Sale, W.S., & Gibbons, I.R. (1982) J. Biol. Chem. 257, 508-515] was similar to that of intact 21S dynein; the 135-kDa polypeptide was only slightly produced in the presence of ATP and Vi. The digestion rate constant of the 135-kDa polypeptide from the beta chain in the presence of ATP and Vi was significantly decreased as compared with in the case of 21S dynein or that of the reconstituted 21S particle. These results suggest that the trypsin sensitivity of the 135-kDa region of the beta chain changes with the association of the beta/ICI complex with the alpha chain and the IC2/IC3 complex in the presence of ATP and Vi.     

Dynamic conformational changes of 21 S dynein ATPase coupled with ATP hydrolysis revealed by proteolytic digestion

Conformational changes of 21 S dynein ATPase from sea urchin sperm flagella were examined by tryptic digestion under physiological conditions. In the presence of 2 mM ATP or ADP plus 100 microM inorganic vanadate (Vi), dynein heavy chains were digested by trypsin into quite different polypeptides from those obtained in other cases (no addition, 2 mM ATP, 4 mM adenosine 5'-(beta,gamma-imido)triphosphate, 4 mM adenosine 5'-(beta,gamma-methylene)triphosphate, 2 mM ADP, 100 microM Vi). In the presence of 4 mM adenosine 5'-O-(3-thiotriphosphate), however, the digestion pattern was similar to that in the presence of ATP (ADP) and Vi, to a certain extent. In all conditions other than the presence of ATP (ADP) and Vi, 165- and 135-kDa polypeptides were the main products, whereas in the presence of ATP (ADP) and Vi, 200-, 150/148-, and 105/96-kDa peptides were produced and 320-kDa peptide became rather inaccessible to trypsin. The latter digestion pattern was not observed in the absence of divalent cations. These results suggest that, in the ATP hydrolysis cycle, dynein changes its conformation remarkably in the dynein-ADP-Pi state, which is presumably responsible for force generation.     

Structure of the gamma heavy chain of the outer arm dynein from Chlamydomonas flagella

We describe here the vanadate-dependent photocleavage of the gamma heavy chain from the Chlamydomonas outer arm dynein and the pathways by which this molecule is degraded by endoproteases. UV irradiation in the presence of ATP, Mg2+, and vanadate cleaves the gamma chain at a single site (termed V1) to yield fragments of Mr 235,000 and 180,000. Irradiation in the presence of vanadate and Mn2+ results in cleavage of the gamma chain at two other sites (termed V2a and V2b) to yield fragment pairs of Mr 215,000/200,000 and 250,000/165,000. The mass of the intact chain is therefore estimated to be 415,000 D. We have located the major tryptic and staphylococcal protease cleavage sites in the gamma chain, determined the origins of the resulting fragments, and identified the regions which contain the epitopes recognized by two different monoclonal antibodies. Both antibodies react with the smaller V1 fragment; the epitope recognized by antibody 25-8 is within 9,000-52,000 D of the original gamma-chain terminus contained in that fragment, whereas that recognized by antibody 12 gamma B is within 16,000 D of the V1 site. The data permit the construction of a linear map showing the structural organization of the polypeptide. The substructure of the gamma chain is similar to that of the alpha and beta chains of the outer arm dynein with regard to polarity as defined by the sites of vanadate-dependent photocleavage, and to that of the beta chain with regard to a highly sensitive protease site located approximately 10,000 D from the original terminus contained in the smaller V1 fragment.     

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.     

Homology of egg and flagellar dynein. Comparison of ATP-binding sites and primary structure

Unfertilized sea urchin eggs contain a Mg2+-ATPase which shares physical and enzymatic characteristics with dynein, the enzyme which powers ciliary and flagellar movement. To further investigate the homology of the egg ATPase and axonemal dynein, ATP-binding subunits in preparations of each of the enzymes were identified using a photoaffinity probe of ATP, 8-azido-ATP (8-N3ATP), and three high molecular weight (HMW) polypeptide components of the two enzymes were compared by one-dimensional peptide mapping. Two heavy chains (A and B) of both the flagellar and egg ATPases bound [alpha-32P]8-N3ATP. The labeling of the HMW bands was specifically inhibited by ATP or ADP. Both the cytoplasmic ATPase and flagellar dynein utilized 8-N3ATP as a substrate indicating that the reagent binds to the active site. The two HMW ATP-binding polypeptides and one other HMW component of the egg ATPase were compared to flagellar dynein heavy chains by peptide mapping. Digestion of the egg versus flagellar HMW polypeptides with Staphylococcus V8 protease or alpha-chymotrypsin produced a highly similar group of peptides, and each pair of heavy chains was qualitatively estimated to be over 85% homologous. These data support the identification of the egg ATPase heavy chains as components of a cytoplasmic dynein and suggest that the HMW polypeptides form active enzymatic sites in flagellar and egg dynein which are substantially homologous.     

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

Regulation of monomeric dynein activity by ATP and ADP concentrations.

Axonemal dyneins are force-generating ATPases that produce ciliary and flagellar movement. A dynein has large heavy chain(s) in which there are multiple (4-6) ATP-binding consensus sequences (P-loops) as well as intermediate and light chains, constituting a very large complex. We purified a monomeric form of dynein (dynein-a) that has at least three light chains from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein-a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP (> or = 20 microM). Both ATPase activity and translocational velocities in the presence of ADP (> or = 20 microM) fit the Michaelis-Menten equation well. However, in the absence of ADP (< 0.1 microM), neither of the activities followed the Michaelis-Menten-type kinetics, probably due to the effect of two ATP-binding sites. Our results also indicate that dynein-a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of a dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system.     

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.     

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.     

Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.     

The limited tryptic cleavage of chymotryptic S-1: an approach to the characterization of the actin site in myosin heads.

Limited tryptic proteolysis of S-1 (A₁+A₂) or S-1 (A₁) and S-1 (A₂) converts the heavy chain into 3 fragments of Mr = 27K-50K-20K. As a result the actin-stimulated ATPase activity of the fragmented heads is lost. When the digestion is performed using the complex F-actin-S-1, this ATPase activity is completely preserved and the heavy chain is split into only 2 fragments of Mr = 27K–70K. The specific protection by F-actin of the -COOH terminal region of the heavy chain at the joint 50K-20K against tryptic cleavage and loss of activity suggests that this part of the head can be involved in actin binding site and/or Mg²⁺ ATP hydrolysis by the acto-S-1 complex.