Flagellar Movement and Adenosine Triphosphatase Activity in Sea Urchin Sperm Extracted With Triton X-100

The relaxation (straightening) of flagellar rigor waves, which is known to be induced by micromolar ATP concentrations, was investigated with respect to its dependence on the binding and hydrolysis of ATP. Flagellar rigor waves were formed by the dilution of demembranated, reactivated sea urchin ( Lytechinus pictus) spermatozoa into ATP-free buffer . Relaxation in response to nucleotide was quantitated by measuring G, the mean flagellar bend angle per sperm; this novel assay permitted determination of the rate of relaxation . It was found that (a) the rate of flagellar relaxation induced by 4 x 10 -6 M ATP was inhibited 80% by vanadate concentrations of 3 x 10 -5 M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and E-ATP), were also capable of effecting relaxation ; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein arm in the course of flagellar relaxation ; and (d) the ratio of the rate of relaxation to the rate of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP concentration was raised from 2 x 10 -6 to 9 x 10 -6 M. It is concluded that (a) flagellar relaxation depends on ATP hydrolysis ; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein ; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon . A general model of relaxation is proposed based on active microtubule sliding.The relaxation (straightening) of rigor-wave sea urchin sperm flagella is currently favored as a model system for investigating the mechanochemical cycle of flagellar motility (18,19,23,24). This is due to the view that relaxation results from the release of dynein cross-bridges in the flagellar axoneme in response to ATP binding . A number of observations have contributed to this view . Dynein arms were shown to be in the cross-bridged configuration in rigor-wave flagella (11), and this configuration correlated with mechanical indices of flagellar rigidity (10, 11) and stiffness (18). Addition of micromolar concentrations of ATP induced rigor-wave flagella to relax (10). Straightening was accompanied by a 15-fold decrease in flagellar stiffness (18); images of dynein cross-bridges were not reported in relaxed flagella (11). The nonhydrolyzable ATP analogue, adenyl-5'-yl imidodiphosphate (AMP-PNP), was reported to act as a competitive inhibitor of the beat frequency of reactivated sea urchin sperm (19,23) and to effect the relaxation of flagellar rigor waves (23). This and other reports that AMP-PNP also caused the dissociation of dynein arms from isolated outer-doublet microtubules (26), and that relaxation was not blocked (24) (12, 16), were reasonably accommodated by a model that interpreted flagellar relaxation as corresponding to ...

Section: Assays For Nucleotide Hydrolysismentioning

confidence: 99%

Section: Assays For Nucleotide Hydrolysismentioning

confidence: 99%

Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella.

Penningroth¹,

Cheung²,

Olehnik³

et al. 1982

“…9 is not adequate to explain one important feature of flagellar oscillation: although the MgATP2-concentration required for half-maximal beat frequency is -0 .2 mM (6,11,36), much lower MgATP2-concentrations are adequate for relaxation, for dissociation of solubilized dynein arms from Btubule binding sites of the outer doublet microtubules (53) and for half-maximal ATPase activity of uncoupled dynein (2,8,20,32). This discrepancy probably indicates that the control of beat frequency by ATP concentration is not brought about simply by determining the concentration of the enzyme-MgATP2-complex (state 2) .…”

Section: A Kinetic Modelmentioning

confidence: 99%

Inhibition and relaxation of sea urchin sperm flagella by vanadate.

Okuno¹

1980

Direct measurements of the stiffness (elastic bending resistance) of demembranated sea urchin sperm flagella were made in the presence of MgATP 2-and vanadate. Under these conditions, the flagellum is in a relaxed state, with a stiffness of -0 .9 x 10-2 ' N m l, which is -5% of the stiffness obtained in the rigor state in the absence of MgATP2-. MgADP -'does not substitute for MgATP 2 " in producing the relaxed state . A progressive inhibition of movement is observed after addition of MgATP 2-to flagella preincubated with vanadate, in which new bend generation, propagation, and relaxation by straightening are distinguished, depending on the ratio of MgATP 2-and vanadate. At appropriate concentrations of vanadate, increase of the velocity of bend propagation is observed at a very low concentration of MgATP 2-that is not enough to induce spontaneous beating . Vanadate enhances competitive inhibition of beat frequency by MgADP -but not by ADP3-, ATP -, or Pi. These observations, and the uncompetitive inhibition of beat frequency by vanadate, indicate that vanadate can only bind to dyneinnucleotide complexes induced by MgATP 2-and MgADP -. The state accessible by MgATP2-binding must be a state in which the cross-bridges are detached and the flagellum is relaxed . The state accessible by MgADP -binding must be a crossbridged state . Bound vanadate prevents the transition between these two states . Inhibition and relaxation by vanadate in the presence of MgATP2-results from the specific affinity of vanadate for a state in which nucleotide is bound, rather than a specific affinity for the detached state .The sliding microtubule hypothesis for flagellar and ciliary movement has been firmly established by a series of experimental (42, 49, 52) and theoretical works (4,5,31, 40), since the first important observations by P. Satir (43,44) . This hypothesis predicts that the dynein arm plays a vital role in generating the bending wave of flagella, because the dynein arm is involved in the transduction of the chemical energy provided by ATP dephosphorylation into mechanical energy (15, 16) . The conformational change of the dynein arm is assumed to involve the cyclic change of its orientation, including association and dissociation with the adjacent microtubule, as in the actomyosin system (26). This mechanochemical behavior of the dynein arm is thought to be tightly coupled with the cyclic dephosphorylation of ATP, because the rate of ATP hydrolysis is tightly coupled with flagellar movement and beat frequency (7, 10, 11), and because dynein ATPase not only has a latency (I, 21, 32, 47, 48) but also can be increased in activity by microtubular protein (l, 39) and tubulin (25).The dynein microtubule coupling mediated with ATP has also been suggested by some physiologi-J . CELL . BIOLOGY

“…Bien que la conservation de la Iongueur des fibres p~riph6riques d6montr6e par les travaux de Satir (49,51), permette de retenir la th6orie du glissement des fibres les unes sur les autres et non celle du raccourcissement des fibres, l'origine de la force de glissement n'est pas encore clairement pr6cis6e. Cette force de glissement pourrait btre engendr6e par l'interaction: (a) soit des fibres p6riph6riques et de la matrice qui les entoure (51); (b) soit des fibres p6riph6riques et des fibres centrales par l'interm6diaire de liens radiaux (68,70); (c) soit des doublets p6riph6riques adjacents (19)(20)(21)(22)10).…”

unclassified

[A functional flagella with a 6 + 0 pattern]

Schrével¹,

Besse²

1975

The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies.The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2 s.The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 ~; the space between the adjacent doublets is about 160 ~. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 ~. The basal body consists of a cylinder of dense material 2,500 ~ long and 1,300-1,400 ~ in diameter; a microtubule 200 ~ in diameter is present in the axis.This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets.The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures.L'armature fibrillaire ou axon~me des cils et des flagelles pr6sente le caract~re structural universel d'etre construit sur la base enn6an~me (9 doublets l~riph~riques + 2 fibres axiales), aussi la diff6r-ence entre ces deux organites concerne les mouvements. La flagelle a un mouvement ondulant. Le battement du cil est de type pendulaire, avec un coup actif et un coup de retour ou de repli.La constance du module 9 + 2 a ~:t6 6tablie par de nombreux travaux (35, 17, 1, 23)et maintes fois rappel6e dans les articles de revues (16,31, 3,59,61,9). Toutefois des exceptions au sch6ma 9 + 2 ont 6t6 signal6es; elles portent, soit sur le nombre de doublets p6riph6riques (48,2,12,39,47,43,8,65), soit sur le nombre de fibres axiales (57,2,58,13,36,11,26,40,28), soit sur la presence de microtubules ~ l'ext6rieur des doublets p6riph6-riques (2,39,47,40,18,7).Quand une exception au sch6ma 9 + 2 est signal6e, la question la plus importante concerne la motilit6 ou la non motilit6 du flagelle ou du cil. Bien que la conservation de la Iongueur des fibres p~riph6riques d6montr6e par les travaux de Satir (49, 51), permette de retenir la th6orie du glissement des fibres les unes sur les autres et non celle du raccourcissement des fibres, l'origine de la force de glissement n'est pas encore clairement pr6cis6e. Cette force de glissement pourrait btre engendr6e 492