scholarly journals The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm.

1976 ◽  
Vol 71 (3) ◽  
pp. 823-831 ◽  
Author(s):  
B H Gibbons ◽  
K Ogawa ◽  
I R Gibbons
Keyword(s):  
Atpase Activity ◽  
Beat Frequency ◽  
Rabbit Antiserum ◽  
Motile Sperm ◽  
Tryptic Fragment ◽  
Percentage Decrease ◽  
Bending Waves ◽  
Low Concentrations ◽  
Bend Angles ◽  
Sea Urchin Sperm

Rabbit antiserum prepared against an ATPase-containing tryptic fragment of dynein by Ogawa and Mohri (J. Biol. Chem. 250: 6476-6483) specifically inhibited the ATPase activity of dynein 1 and not that of dynein 2. Varying amounts of this antidynein 1 serum were added to demembranated sperm while they were swimming in reactivating solution containing 1 mM ATP. The sperm continued to form regularly propagated flagellar bending waves, but the beat frequency decreased gradually with time, the greater part of the change occurring in the first 15 min. The beat frequency after 1 h was a function of the amount of antiserum used, and could be as low as 1 Hz. The waveforms of the treated sperm resembled those of normal reactivated sperm except that the bend angles of both the principal and reverse bends were larger in the proximal portion of flagellum. The ATPase activity and corresponding beat frequency of sperm which had been pretreated with varying amounts of antidynein 1 serum for 15 min at 0 degrees C and then diluted were both decreased as a function of the amount of antiserum added, the ATPase activity of homogenized, nonmotile sperm also decreased upon pretreatment with antiserum, but the percentage decrease was less than for motile sperm. For moderate to low concentrations of antiserum, the rates of reaction with motile and with rigor sperm were almost identical. The overall results suggest that antidynein 1 inhibits the functioning of the dynein arms, probably by blocking the ATPase sites of the dynein 1.

scholarly journals Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.

Modification of flagellar waveform and adenosine triphosphatase activity in reactivated sea-urchin sperm treated with N-ethylmaleimide

1983 ◽  
Vol 60 (1) ◽  
pp. 231-249
Author(s):  
M.P. Cosson ◽  
W.J. Tang ◽  
I.R. Gibbons
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Atp Hydrolysis ◽  
Beat Frequency ◽  
Prolonged Treatment ◽  
Bend Angle ◽  
The Asymmetry ◽  
Bend Angles ◽  
The Waves ◽  
Sea Urchin Sperm

Treatment of demembranated sea-urchin sperm for 1–2 min with 10 microM-N-ethylmaleimide (Mal-NEt) at pH 8.0 prior to reactivation with 1 mM-ATP causes the asymmetry of the flagellar waveform to become desensitized to the presence or absence of Ca2+ in the reactivating medium. In such sperm, changes in concentration of free Ca2+ between 10(−7) M and 10(−3) M have no effect on the asymmetry of the waveforms as measured by the turning rate of the sperm in radians per beat cycle, while the beat frequency and the propulsive efficiency of the waves remain unchanged from the values observed in control preparations not treated with MalNEt. A somewhat more prolonged treatment with MalNEt causes a progressive decrease in the bend angles of the flagellar waves, while the beat frequency and the wavelength still remain largely unchanged. Further extension of the treatment with MalNEt causes complete loss of motility. Little ATP-induced sliding of the doublet tubules is observed upon treatment with trypsin of sperm flagella that have been rendered non-motile with MalNEt. However, the preparations of solubilized dynein 1 obtained by 0.6 M-NaCl extraction of axonemes treated with MalNEt appear almost identical to those obtained from untreated axonemes, both in terms of the amount solubilized and in the specific ATPase activities of their latent and Triton-activated forms. These preparations also appear capable of restoring much of the beat frequency of dynein-1-depleted flagella. These results suggest that the observed desensitization to Ca2+ and decrease in bend angle result from the reaction of MalNEt with axonemal polypeptides that are not part of the dynein 1 particle extracted with 0.6 M-NaCl. The rate of ATP hydrolysis by demembranated sperm rendered non-motile with MalNEt remains relatively high, and it decreases about 50% when the flagella are broken by brief homogenization. This ‘homogenizer-sensitive’ ATPase activity appears to be derived from some flagellar regulatory mechanism, which controls the ATPase activity of intact non-motile axonemes.

The Effect of Partial Extraction of Dynein Arms on the Movement of Reactivated Sea-Urchin Sperm

1973 ◽  
Vol 13 (2) ◽  
pp. 337-357 ◽  
Author(s):  
BARBARA H. GIBBONS ◽  
I. R. GIBBONS
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Beat Frequency ◽  
Wave Form ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Elastic Forces ◽  
Dynein Arms ◽  
Triton X ◽  
Sea Urchin Sperm

Sea-urchin sperm were extracted with o.5 M KCl for 45 s at room temperature in the presence of Triton X-100, and then transferred to reactivating solution containing 1 mM ATP. The flagellar beat frequency of these KCl-extracted sperm (16 beats/s) was only about half that of control Triton-extracted sperm that had not been exposed to 0.5 M KCl (31 beats/s), although the form of their bending waves was not significantly altered. Examination by electron microscopy showed that the extraction with 0.5 M KCl removed the majority of the outer arms from the doublet tubules, leaving the inner arms apparently intact. By varying the duration of the KCl-extraction, it was shown that the rate of decrease in beat frequency paralleled the rate of disappearance of the arms. Prolonging the extraction time beyond 45 s at room temperature, or 4 min at o °C, had little further effect on beat frequency. ATPase measurements suggested that 6o-65% of the dynein in the original axonemes had been solubilized when the extraction with KCl was permitted to go to completion. These results indicate that the generation and propagation of flagellar bending waves of essentially typical form are not prevented by the removal of the outer row of dynein arms from the doublet tubules. In terms of the sliding filament model of flagellar bending, the results suggest that the rate of sliding between tubules under these conditions is proportional to the number of dynein arms present. The lack of significant change in wave form implies that the total amount of sliding that occurs during each bending cycle is not affected by the reduced number of dynein arms, but is regulated independently in some manner by the elastic forces generated by other structures in the bent axoneme.

scholarly journals FLAGELLAR MOVEMENT AND ADENOSINE TRIPHOSPHATASE ACTIVITY IN SEA URCHIN SPERM EXTRACTED WITH TRITON X-100

1972 ◽  
Vol 54 (1) ◽  
pp. 75-97 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Adenosine Triphosphatase ◽  
Beat Frequency ◽  
Average Frequency ◽  
Mitochondrial Atpase ◽  
The Difference ◽  
Live Sperm ◽  
Triton X ◽  
Sea Urchin Sperm

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25°C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 µm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 µm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 µmole Pi/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 µmole Pi/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg++, although some motility was also obtained with Mn++ and Ca++. The coupled ATPase activity had a Michaelis constant (Km) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "Km" of 0.2 mM.

Effects of organic solvents on flagellar asymmetry and quiescence in sea urchin sperm

1982 ◽  
Vol 54 (1) ◽  
pp. 115-135
Author(s):  
B.H. Gibbons
Keyword(s):  
Opposite Effect ◽  
Beat Frequency ◽  
Regulatory Protein ◽  
Initial Exposure ◽  
Subsequent Time ◽  
Flagellar Beat ◽  
Low Concentrations ◽  
The Asymmetry ◽  
Live Sperm ◽  
Sea Urchin Sperm

Low concentrations of methanol, 2-propanol and ethylene glycol increase the asymmetry of the flagellar waveforms ad the turning rate of both live sperm and potentially symmetrical sperm reactivated with 1 mM-MgATP2-, while at the same time causing a decrease in the heat frequency. Similar effects are observed if the solvents are added to preparations of potentially symmetrical sperm reactivated in the presence of 1 mM free Ca2+, or to potentially asymmetrical sperm reactivated without added Ca2+, A second group of solvents, N,N-dimethylformamide, formamide and p-dioxane, also decrease the flagellar beat frequency, but have the opposite effect on symmetry, reducing the asymmetry of the waveforms and the turning rate of potentially symmetrical sperm reactivated in the presence of 1 mM free Ca2+. These effects of solvents are all reversible within about 5 min after initial exposure to solvent. Higher concentrations of methanol and 2-propanol (above approximately 5 and 0.8 mole %, respectively) induce quiescence in potentially asymmetrical sperm reactivated with concentrations of MgATP2- ranging from 10 microM to 1 mM. The quiescent flagella initially assume a bent form very similar to that seen in Ca2+-induced quiescence, and show a subsequent time-dependent distortion of the initial bent from with eventual disintegration and splitting off of bundles of microtubules. Dimethylformamide, formamide and dioxane have almost no effect on the intrinsic asymmetry of potentially asymmetrical sperm reactivated in the absence of added Ca2+, but addition of these solvents to potentially asymmetrical sperm that have been induced to become quiescent by addition of 0.1 mM free Ca2+ causes the sperm to resume swimming with flagellar waveforms that are substantially more symmetrical that those of the starting preparation before the addition of Ca2+. Mild digestion with trypsin of reactivated sperm that have been induced either to beat asymmetrically or to become quiescent by addition of methanol causes a gradual appearance of symmetrical flagellar beating, as in the case of Ca2+-induced quiescence. The flagellar beat frequency, however, remains low, at about 20 Hz. The results suggest that the solvents either mimic or block the action of CA2+ by interaction with a Ca2+-dependent regulatory protein, and may also induce alteration in the rate constants of dynein ATPase.

Sliding Velocity Between Outer Doublet Microtubules of Sea-Urchin Sperm Axonemes

1980 ◽  
Vol 38 ◽  
pp. 600-603 ◽  
Author(s):  
Y. Yano ◽  
T. Miki-Noumura
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Sliding Velocity ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Cylindrical Form ◽  
Activity Form ◽  
Dynein Arms ◽  
The Relationship ◽  
Sea Urchin Sperm

The flagellar axonemes have a cylindrical form, which consists of nine doublet microtubules surrounding a central pair of single microtubules. Each doublet tubule has two parallel rows of projections, called outer and inner arms. Sliding movement between doublet microtubules was first reported by Summers and Gibbons, who observed that doublet tubules were extruded from trypsin-treated axonemes of sea-urchin sperm flagella on addition of ATP. Their observation indicated that the bending movement of flagella results basically from these active sliding movements between the adjacent doublet tubules in the axonemes. Experimental evidence suggests that the dynein arms projecting from the doublet tubules interact with the adjacent tubules and by hydrolysing ATP, produce the mechanical force to slide. According to Gibbons and Gibbons the outer arms were removed from the doublet tubules by extracting the demembranated sea-urchin sperm with 0.5 M KCl or NaCl, while the inner arms and other axonemal structures remained apparently intact. Although the form of their bending waves was not significantly altered, the KCl-extracted sperm had only about half the flagellar beat frequency of the demembranated sperm. The 21S latent ATPase activity form of dynein 1 restored up to 90% of the outer arms on the doublet tubules and increased the beat frequency of KCl-extracted sperm from 14 Hz to 25 Hz. We found that the NaCl-extracted axonemes of sea-urchin sperm had the ability to extrude outer doublet tubules on addition of ATP and trypsin, in a similar manner to that of the intact axonemes. We attempted to compare the sliding velocity of the outer doublet tubules in the arm-depleted axonemes and in the arm-recombined axonemes, with that in the intact axonemes, in order to find the relationship between the sliding velocity and the number of arms in these axonemes.

scholarly journals Effect of beat frequency on the velocity of microtubule sliding in reactivated sea urchin sperm flagella under imposed head vibration.

1995 ◽  
Vol 198 (3) ◽  
pp. 645-653 ◽  
Author(s):  
C Shingyoji ◽  
K Yoshimura ◽  
D Eshel ◽  
K Takahashi ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Vibration Frequency ◽  
Beat Frequency ◽  
Distal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Flagellar Beat ◽  
Tripneustes Gratilla ◽  
Sea Urchin Sperm

The heads of demembranated spermatozoa of the sea urchin Tripneustes gratilla, reactivated at different concentrations of ATP, were held by suction in the tip of a micropipette and vibrated laterally with respect to the head axis. This imposed vibration resulted in a stable rhythmic beating of the reactivated flagella that was synchronized to the frequency of the micropipette. The reactivated flagella, which in the absence of imposed vibration had an average beat frequency of 39 Hz at 2 mmol l-1 ATP, showed stable beating synchronized to the pipette vibration over a range of 20-70 Hz. Vibration frequencies above 70 Hz caused irregular, asymmetrical beating, while those below 20 Hz induced instability of the beat plane. At ATP concentrations of 10-100 mumol l-1, the range of vibration frequency capable of maintaining stable beating was diminished; an increase in ATP concentration above 2 mmol l-1 had no effect on the range of stable beating. In flagella reactivated at ATP concentrations above 100 mumol l-1, the apparent time-averaged sliding velocity of axonemal microtubules decreased when the imposed frequency was below the undriven flagellar beat frequency, but at higher imposed frequencies it remained constant, with the higher frequency being accompanied by a decrease in bend angle. This maximal sliding velocity at 2 mmol l-1 ATP was close to the sliding velocity in the distal region of live spermatozoa, possibly indicating that it represents an inherent limit in the velocity of active sliding.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Effect of Azide on the ATPase Activity of Isolated CF1

1991 ◽  
Vol 46 (7-8) ◽  
pp. 621-628 ◽  
Author(s):  
Ignat B. Minkov ◽  
Heinrich Strotmann
Keyword(s):  
Atpase Activity ◽  
Enzyme Activation ◽  
Applied Method ◽  
Spinach Chloroplasts ◽  
Dependent Atpase ◽  
Low Concentrations ◽  
Activation Heat

Abstract The effect of azide on M g2+-and Ca2+-dependent ATPase of differently activated CF1 isolated from spinach chloroplasts was studied. It is shown that Mg2+-ATPase activity is sensitive towards azide irrespective of the applied method of enzyme activation. Heat-or trypsin-acti-vated Ca2+-ATPase is also inhibited, whereas methanol-and octylglucoside-stimulated or DTT-activated Ca2+-ATPase is not affected by azide. Preincubation of the DTT-activated enzyme with low concentrations of Mg2+ induces azide susceptibility of the Ca2+-dependent ATPase.

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