Using a rapid-freeze, deep-etch replica technique, the effects of adenylylimidodiphosphate (AMPPNP), a non-hydrolyzable ATP analogue, on the structure of the outer dynein arm were analyzed in demembranated cilia from Terra/rymena. In the presence of lmM AMPPNP, the lateral view of all axonemes was characterized by egg-shaped outer dynein arms which showed a slightly baseward tilt with a mean inclination of 11° from the perpendicular to the doublet microtubules. These AMPPNP-incubated axonemes were morphologically indistinguishable from those in a rigor state (without ATP). On the other hand, in the presence of l mM ATP and 100 ,uM vanadate, the outer dynein arms were extended and slender, and showed an increased baseward tilt. It is widely accepted that dynein arms generate sliding displacement between the outer doublet microtubules in flagellar and ciliary movement (2, 9, 10). However, there have been little data to explain the molecular mechanism of transducting the chemical energy of ATP into mechanical energy for the interdoublet sliding, although some experimental results favored the idea that the conformational change of dynein arms might cause the sliding (see reference 2). Recently, the ATP-dependent structural changes of the outer dynein arm in cilia and flagella have been successfully demonstrated by Goodenough and Heuser (3) and by us (13) with the rapid-freeze, deep-etch replica technique. It is now clear that the outer dynein arms take two distinct forms, depending upon the medium conditions. In the absence of ATP, all axonemes are characterized by structurally uniform outer dynein arms attached to A-tubules, showing a slightly baseward tilt with a mean inclination of about 11° from the perpendicular to the doublet. In the presence of 1 mM ATP and 100 /1M vanadate, all arms show a characteristically baseward tilt with a mean inclination of about 32° from the perpendicular. Therefore, we have tentatively designated these types of arms with an almost perpendicular position (ll°) and with a tilted position (320), ‘P-type’ and ‘T-type’ arms, respectively (13). A non-hydrolyzable ATP analogue, adenylylimidodiphosphate (AMPPNP), has been used in several studies of ciliary movement. At first, Penningroth and Witman obtained relaxation of rigor bends with AMPPNP (8). However, conflicting results were obtained by Takahashi and Tonomura (11). They demonstrated that AMPPNP failed to cause detachment of dynein arms from B-tubules. Recently, Okuno and Brokaw (6) have reported observations on rigor bend relaxation and stiffness measurements in the presence of AMPPNP which are in agreement with the results of Takahashi and Tonomura (11). In the present study, we used the rapid-freeze, deep-etch replica technique to determine which type of arm configuration, the P-type or the T-type, occurs in the presence of AMPPNP. Dernembranated axonemes were prepared from Tetrtalzynzeria pyrrf0rmr's, strain W, according to the procedure described in the previous paper (13). The axonemes were centrifuged at 12,000 g for 20 min, and the axonemal pellet was gently resuspended in HEPES solution (25 mM KCI, 2.5 mM MgSO4, 1 mM DTT, 10 mM HEPES, pH 7.5). The resuspended axonemes | I
[1]
H. Ishikawa,et al.
ATP-dependent structural changes of the outer dynein arm in Tetrahymena cilia: a freeze-etch replica study
,
1983,
The Journal of cell biology.
[2]
U. Goodenough,et al.
Substructure of the outer dynein arm
,
1982,
The Journal of cell biology.
[3]
H. Ishikawa,et al.
Myosin filaments in smooth muscle cells of the guinea pig taenia coli: a freeze-substitution study.
,
1982,
European journal of cell biology.
[4]
H. Ishikawa,et al.
The cytoskeleton in myelinated axons: A freeze-etch replica study
,
1982,
Neuroscience.
[5]
T. Arata,et al.
Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers.
,
1981,
Journal of molecular biology.
[6]
T. Yanagida.
Angles of nucleotides bound to cross-bridges in glycerinated muscle fiber at various concentrations of ϵ-ATP, ϵ-ADP and ϵ-AMPPNP detected by polarized fluorescence
,
1981
.
[7]
Y. Tonomura,et al.
Kinetic properties of dynein ATPase from Tetrahymena pyriformis. The initial phosphate burst of dynein ATPase and its interaction with ATP analogs.
,
1979,
Journal of biochemistry.
[8]
M. Hines,et al.
Cilia and Flagella of Eukaryotes
,
2022
.