Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding

We recently demonstrated that addition of the divalent cation Mg++ to demembranated cilia causes the dynein arms to attach uniformly to the B subfibers. We have now studied the dose-dependent relationship between Mg++ or Ca++ and dynein bridging frequencies and microtubule sliding in cilia isolated from Tetrahymena. Both cations promote efficient dynein bridging. Mg++-induced bridges become saturated at 3 mM while Ca++-induced bridges become saturated at 2 mM. Double reciprocal plots of percent bridging vs. the cation concentration (0.05-10 mM) suggest that bridging occurs in simple equilibrium with the cation concentration. When microtubule sliding (spontaneous disintegration in 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.1 mM ATP at pH 7.4) is assayed (A350 nm) relative to the Mg++ or Ca++ concentration, important differential effects are observed. 100% Disintegration occurs in 0.5-2 mM Mg++ and the addition of 10 mM Mg++ does not inhibit the response. The addition of 0.05-10 mM Ca++ to cilia reactivated with 0.1 mM ATP causes a substantial reduction in disintegration at low Ca++ concentrations and complete inhibition at concentrations greater than 3 mM. When Ca++ is added to cilia reactivated with 2 mM Mg++ and 0.1 mM ATP, the percent disintegration decreases progressively with the increasing Ca++ concentration. The addition of variable concentrations of Co++ to Mg++-activated cilia causes a similar but more effective inhibition of the disintegration response. These observations, when coupled with the relatively high concentrations of Ca++ or Co++ needed to inhibit disintegration, suggest that inhibition results from simple competition for the relevant cation-binding sites and thus may not be physiologically significant. The data do not yet reveal an interpretable relationship between percent disintegration, percent dynein bridging, and percent ATPase activity of both isolated dynein and whole cilia. However, they do illustrate that considerable (sliding) disintegration (60%) can occur under conditions that reveal only 10-15% attached dynein cross bridges.     

Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella

The effect of vanadate on the ATP-induced disruption of trypsin-treated axonemes and the ATP-induced straightening of rigor wave preparations of sea urchin sperm was investigated. Addition of ATP to a suspension of trypsin-treated axonemes results in a rapid decrease in turbidity (optical density measured at 350 nm) concomitant with the disruption of the axonemes by sliding between microtubules to form tangles of connected doublet microtubules (Summers and Gibbons, 1971; Sale and Satir, 1977). For axonemes digested to approximately 93 percent of their initial turbidity, 5 {muM} vanadate completely inhibits the ATP-induced decrease in turbidity and the axonemes maintain their structural integrity. However, with axonemes digested to approximately 80 percent of their initial turbidity, vanadate fails to inhibit the ATP-induced decrease in turbidity and the ATP-induced structural disruption of axonemes, even when the vanadate concentration is raised as high as 100 μm. For such axonemes digested to 80 percent of their initial turbidity, the form of ATP-induced structural changes, in the presence of 25 μM vanadate, was observed by dark-field light microscopy and revealed that the axonemes become disrupted into curved, isolated doublet microtubules, small groups of doublet microtubules, and “banana peel” structures in which tubules have peeled back from the axoneme. Addition of 5 μM ATP to rigor wave sperm, which were prepared by abrupt removal of ATP from reactivated sperm, causes straightening of the rigor waves within 1 min, and addition of more than 10 μM ATP causes resumption of flagellar beating. Addition of 40 μM vanadate to the rigor wave sperm does not inhibit straightening of the rigor waves of 2 μM-1 mM ATP, although oscillatory beating is completely inhibited. These results suggest that vanadate inhibits the mechanochemical cycle of dyein at a step subsequent to the MgATP(2-)-induced release of the bridged dynein arms.     

Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella.

To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

ATP causes a structural change in retinal rod outer segments: Disc swelling Is not involved

Mg2+-ATP was found to produce a 15 to 30% drop in the turbidity of suspensions of broken retinal rod outer segments from the toad Bufo marinus, prepared by washing or flotation in sucrose. This in vitro process has a half-time of about two minutes and appears to be irreversible. It is not affected by the bleaching of rhodopsin. Direct measurements show that the drop in turbidity is not due to swelling of the disc internal space measured in outer segments recovered by centrifugation. Instead, the total packed volume of the outer segments increases following incubation in Mg2+-ATP. Under the specific conditions of these experiments, the total pellet volume increase was 26 +/- 22% (13 experiments) when corrected for the percent of rhodopsin recovered in the centrifugal pellet. The magnitude of the ATP effect on turbidity suggests that the majority of the discs are involved in some kind of structural change. Vanadium in the +5 oxidation state (vanadate) is an inhibitor of the Mg2+-ATP effect on turbidity at a half-maximal concentration of 0.2 to 0.4 microM, and inhibition is rapidly reversed by norepinephrine, which complexes vanadate. A Mg2+-ATPase activity in extensively washed outer segment membranes, previously shown to be activated as much as twofold following light exposure of the membranes, is not sensitive to vanadate at the concentrations which block the ATP-dependent change of turbidity.     

Phosphorylation of double-stranded DNAs by T4 polynucleotide kinase.

The phosphorylation by T4 polynucleotide kinase of various double-stranded DNAs containing defined 5'-hydroxyl end group structures has been studied. Particular emphasis was placed on finding conditions that allow complete phosphorylation. The DNAs employed were homodeoxyoligonucleotides annealed on the corresponding homopolymers, DNA duplexes corresponding to parts of the genes for alanine yeast tRNA, and a suppressor tyrosine tRNA from Escherichia coli. The rate of phosphoylation of DNAs with 5'-hydroxyl groups in gaps was approximately ten times slower than for the corresponding single-stranded DNA. At low concentrations of ATP, 1 muM, incomplete phosphorylation was obtained, whereas with higher concentrations of ATP, 30 muM, complete phosphorylation was achieved. In the case of DNAs with 5'-hydroxyl groups at nicks approximately 30% phosphorylation could be detected using 30 muM ATP. A DNA containing protruding 5'-hydroxyl group ends was phosphorylated to completion using the same conditions as for single-stranded DNA, i.e., a ratio between the concentrations of ATP and 5'-hydroxyl groups of 5:1 and a concentration of ATP of approximately 1 muM. For a number of DNAs containing protruding 3'-hydroxyl group ends and one DNA containing even ends incomplete phosphorylation was found under similar conditions. For all these DNAs a plateau level was observed varying from 20 to 45% of complete phosphorylation. At 20 muM and higher ATP concentrations, the phosphorylation was complete also for these DNAs. With low concentrations of ATP a rapid production of inorganic phosphate was noted for all the latter DNAs. The apparent equilibrium constants for the forward and reverse reaction were determined for a number of different DNAs, and these data revealed that the plateau levels of phosphorylation obtained at low concentrations of ATP for DNAs with protruding 3'-hydroxyl group and even ends is not a true equilibrium resulting from the forward and reverse reaction. It is suggested that the plateau levels are due to formation of inactive enzyme-substrate and enzyme-product complexes. For all double-stranded DNAs tested, except DNAs containing protruding 5'-hydroxyl group ends, addition of KCl to the reaction mixture resulted in a drastic decrease in the rate of phosphorylation, as well as in the maximum level phosphorylated. Spermine, on the other hand, had little influence. Both of these agents have previously been shown to activate T4 polynucleotide kinase using single-stranded DNAs as substrates (Lillehaug, J.R., and Kleppe, K. (1975), Biochemistry 14, 1221). The inhibition of phosphorylation of double-stranded DNAs by salt might be the result of stabilization of the 5'-hydroxyl group regions of these DNAs.     

Adenylate kinase in sea urchin embryonic cilia

Sea urchin embryos swim by ciliary movement. Hypertonic shock causes deciliation and loss of motility. Within 2-4 h, cilia regenerate and the embryos swim again. Regeneration of cilia occurs multiple times. The adenylate kinase (AK) activity of isolated cilia was studied. A 130-kDa Sp-AK isozyme, present in sperm flagella, is also present in embryonic cilia. AK activity is responsible for approximately 93% of nonmitochondrial ATP regeneration from ADP in embryonic cilia. This is unlike sea urchin sperm flagella, where approximately 31% of the nonmitochondrial ATP regeneration is from the 130-kDa Sp-AK isozyme and approximately 69% from the flagellar creatine kinase (Sp-CK). Embryos were deciliated 1-3 times and after a 2-h period of regeneration the major ciliary axonemal proteins such as the tubulins appeared constant in amount. However, a moderate decrease in ATPase activity, and a large decrease of total AK activity, were measured. The decrease in AK activity paralleled the decrease in embryo swimming velocity. Embryos were deciliated once and cilia regeneration followed for 4 h. ATPase activity recovered to control levels by 3 h, but AK activity and swimming velocity remained lower than in controls. Detergent solubility data and kinetic experiments indicate that, in addition to the 130-kDa Sp-AK, there is at least one additional AK isozyme in embryonic cilia. Analysis of the S. purpuratus genome indicates five AK isozymes in addition to the 130-kDa Sp-AK isozyme. Decreased swimming velocity of embryos with regenerated cilia suggests that regenerated cilia are not as functionally perfect as naturally grown cilia.     

Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.     

Polarity of dynein-microtubule interactions in vitro: cross-bridging between parallel and antiparallel microtubules

Ciliary doublet microtubules produced by sliding disintegration in 20 muM MgATP2-reassociate in the presence of exogenous 30S dynein and 6 mM MgSO4. The doublets form overlapping arrays, held together by dynein cross-bridges. Dynein arms on both A and B subfibers serve as unambiguous markers of microtubule polarity within the arrays. Doublets reassociate via dynein cross-bridges in both parallel and antiparallel modes, although parallel interactions are favored 2:1. When 20 muM ATP is added to the arrays, the doublets undergo both vanadate-sensitive and insensitive forms of secondary disintegration to reproduce the original population of doublets. The results demonstrate that both parallel and antiparallel doublet cross-bridging is sensitive to dissociation by ATP even though normal ciliary motion depends strictly on dynein interactions between parallel microtubules.     

The effects of adenosine triphosphate and related compounds on some hydrodynamic properties of glycerinated cilia

Cilia were isolated from Tetrahymena pyriformis by a glycerol method. The addition of low concentrations of ATP, but not of other nucleoside triphosphates, caused an increase of up to twofold in the amount of cilia pelleted in a low-speed centrifugation assay and decreased the density of the pellets compared to control pellets. Pellet size and density depend on pH, both in the absence and in the presence of low concentrations of ATP. High concentrations (5 mM and above) of ATP and of other nucleoside triphosphates tend to "dissolve" the cilia. Heating the cilia for 11 min at 40°C abolishes the increase in pellet size and the decrease in pellet density caused by low ATP, but slightly increases the ATPase activity of the cilia. Heat treatment, however, does not prevent the dissolving effect of high ATP. There are, thus, two independent effects of ATP on the hydrodynamic properties of cilia suspensions.     

Manganese (II) and substrate interaction with unadenylylated glutamine synthetase (Escherichia coli w). II. Electron paramagnetic resonance and nuclear magnetic resonance studies of enzyme-bound manganese(II) with substrates and a potential transition-state analogue, methionine sulfoximine.

The enhancement of the longitudinal proton relaxation rate of solvent water protons which occurs when Mn(II) is bound to the "tight" metal ion site of unadenylylated glutamine synthetase (GS) was used to determine the binding constant of L-methionine (SR)-sulfoximine to GS-Mn(II) complexes. The binary enhancement for GS-Mn(II) is 22 at 24 MHz, 25 degrees C. The enhancement is lowered in the presence of the sulfoximine and the computed dissociation constant is 30 muM with epsilont, the enhancement for the ternary complex, equal to 3.0. Titration curves for the sulfoximine were also obtained in the presence of Mg-ADP, Mg-ADP plus Pi, and Mg-ATP. The dissociation constants were 9, 5, and 0.8 muM, respectively. The progressive tightening of the dissociation constants is symptomatic of conformational changes at the active site as the total subsite occupied by ATP is filled. The number of rapidly exchanging water molecules drops from 2 to approximately 0.1 when saturating concentrations of L-methionine (SR)-sulfoximine and nucleotide are present. The kinetically determined KI value of approximately 4 muM for the sulfoximine is about three orders of magnitude tighter than thee Km' value of approximately 3 mM for L-glutamate. The previously mentioned dissociation constants obtained by enhancement titrations are also orders of magnitude tighter than Km'. These data suggest that L-methionine (SR)-sulfoximine is a "transition-state" analogue for the glutamine synthetase reaction. ...     

Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100

Sonicated unilamellar and large multilamellar liposome suspensions have been treated with the non-ionic detergent Triton X-100, and the subsequent changes in turbidity have been studied as a function of time. Sonicated liposome suspensions exhibit an increase in turbidity that takes place in two stages, a fast, low-amplitude one is completed in less than 100 ms, and a slow large-amplitude one occurs in 20-40 s. The first increase in turbidity is associated to detergent incorporation into the bilayer, and the second one, to vesicle fusion. The fast stage may be detected at all detergent concentrations, while the slow one is only seen above the critical micellar concentration of Triton X-100. Both processes may be interpreted in terms of first-order kinetics. Studies of the variation of kexp with lipid and detergent concentration suggest a complex multi-step mechanism. In the case of multilamellar liposomes, a fast increase in turbidity is also seen after detergent addition, which is followed by a slow (20-60 s) decrease in turbidity and a very slow (up to 12 h) large scale decrease in turbidity. These processes do not conform to single-exponential patterns. The fast stage is also thought to reflect surfactant incorporation, while the decrease in turbidity is interpreted as bilayer solubilization starting with the outer bilayer (slow stage) and proceeding through the remaining ones (very slow stage).     

The effect of short chain fatty acids on transmural electrical potential across rat small intestine in vivo.

Short chain fatty acids suddenly produce a phasic increase in transmural electrical potential difference (PD) when placed in the lumen of rat small intestine in vivo. With concentrations of propionate ranging from 50 muM to 1000 muM the amplitude of the response in jejunum is about 5.5 mV. The concentration giving half this effect is about 20 muM. With 10 mM propionate the duration of the response is 3-5 min; after this, PD again equals the control value and the gut is refractory to further additions. Removing propionate from the mucosal surface produces no change in PD, but does restore responsiveness to subsequent exposure to short chain fatty acids. This effect is independent of a variety of other alterations in PD such as those caused by sugars, amino acids, bile salts, theophylline, prostaglandins, and ATP. Mechanism and significance of this surprisingly sensitive response remain obscure.     

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3', 5'-AMP-stimulated protein kinase.

The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.     

A mathematical analysis of the substrate effect observed in 3beta-hydroxysteroid dehydrogenase reactions of rat testicular microsomes.

The substrate effect in enzyme reactions has been explained mostly in terms of an additional substrate binding site on the enzyme other than the catalytic site. A rate equation for the reaction is introduced according to the steady state mechanism as follows: v = (Ps3+Qs2+Rs)/(s3+Ls2+Ms+N), were the six parameters, L,M,N,P,Q, and R, can be determined by the least-squares method from the experimental points. The v vs. s curve has an asymptote parallel to the s abscissa, and can be classified into one of four types. The type A curve has an intersection with the asymptote and an apparent maximum velocity; the curve descends toward the asymptote. Type B has no intersection and no stationary point; the curve ascends toward the asymptote. Type C has two intersections and two stationary points, an apparent maximum velocity and a minimum velocity; the curve ascends toward the asymptote. Type D has no intersection and two stationary points; the curve ascends toward the asymptote. The equation was applied to the 3beta-hydroxysteroid dehydrogenase [EC 1.1.1.145] reaction of rat testicular microsomes. The conversion of 3beta-hydroxyandrost-5-ene-17-one was represented by type C, with an apparent maximum velocity of 0.338 nmole/min/mg protein at 0.912 muM of the substrate concentration, minimum velocity of 0.108 nmole/min at 16.6 muM, and saturating velocity of 0.169 nmole/min at infinite concentration of the substrate. The converson of 3beta-hydroxypregn-5-ene-20-one was of type B, having two inflexion points, 0.320 nmole/min at 2.735 muM and 0.814 nmole/min at 12.39 muM, and a saturating velocity of 3.80 nmoles/min at infinite concentration of the substrate.     

Kinetics of tryptophan fluorescence enhancement in myofibrils during ATP hydrolysis

The mechanism of ATP hydrolysis in myofibrils can be studied by following the time course of tryptophan fluorescence. Stoichiometric quantities of ATP produce an enhancement of the tryptophan fluorescence in stirred suspensions of rabbit psoas myofibrils at pCa greater than 7. Approximately 1 mol of ATP/myosin head is required to obtain the maximum fluorescence enhancement of 4-6%. Upon the addition of quantities of ATP greater than 1 mol/mol of myosin head, the fluorescence rapidly increases to a steady state, which lasts for a period that is proportional to the amount of ATP added. The fluorescence then decays to the initial level with a half-time of approximately 40 s at 20 degrees C. Hydrolysis of [gamma-32P]ATP at pCa greater than 7 in myofibrils has an initial burst of approximately 0.7 mol/mol of myosin head that is followed by a constant rate of hydrolysis. The duration of the steady state hydrolysis is identical to the duration of the enhancement of tryptophan fluorescence. A lower limit of 5 X 10(5) M-1 S-1 was obtained for the second order rate constant of the fluorescence enhancement by ATP. At pCa of 4, the duration of the fluorescence enhancement is one-tenth to one-twentieth as long as at pCa greater than 7; this is consistent with the increased steady state rate of ATP hydrolysis at higher calcium concentrations. The time course of the fluorescence enhancement observed in myofibrils during ATP hydrolysis is qualitatively and quantitatively similar to that observed with actomyosin-S1 in solution. These results suggest that the kinetic mechanism of ATP hydrolysis that has been well established by studies of actomyosin-S1 in solution also occurs in myofibrils.     

Shrinkage of growing Escherichia coli cells by osmotic challenge.

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.     

Intracellular Ca2+ regulates the phosphorylation and the dephosphorylation of ciliary proteins via the NO pathway

The phosphorylation profile of ciliary proteins under basal conditions and after stimulation by extracellular ATP was investigated in intact tissue and in isolated cilia from porcine airway epithelium using anti-phosphoserine and anti-phosphothreonine specific antibodies. In intact tissue, several polypeptides were serine phosphorylated in the absence of any treatment (control conditions). After stimulation by extracellular ATP, changes in the phosphorylation pattern were detected on seven ciliary polypeptides. Serine phosphorylation was enhanced for three polypeptides (27, 37, and 44 kD), while serine phosphorylation was reduced for four polypeptides (35, 69, 100, and 130 kD). Raising intracellular Ca2+ with ionomycin induced identical changes in the protein phosphorylation profile. Inhibition of the NO pathway by inhibiting either NO synthase (NOS), guanylyl cyclase (GC), or cGMP-dependent protein kinase (PKG) abolished the changes in phosphorylation induced by ATP. The presence of PKG within the axoneme was demonstrated using a specific antibody. In addition, in isolated permeabilized cilia, submicromolar concentrations of cGMP induced protein phosphorylation. Taken together, these results suggest that the axoneme is an integral part of the intracellular NO pathway. The surprising observation that ciliary activation is accompanied by sustained dephosphorylation of ciliary proteins via NO pathway was not detected in isolated cilia, suggesting that the protein phosphatases were either lost or deactivated during the isolation procedure. This work reveals that any pharmacological manipulation that abolished phosphorylation and dephosphorylation also abolished the enhancement of ciliary beating. Thus, part or all of the phosphorylated polypeptides are likely directly involved in axonemal regulation of ciliary beating.     

On the role of sulfhydryl groups in the ATPase activity and pellet height response of Tetrahymena cilia

The effects of N-ethylmaleimide and p-hydroxymercuribenzoate on the ATPase activity of glycerinated Tetrahymena cilia, of 30 S dynein extracted from the cilia, and on the residual ATPase remaining after extraction were studied and correlated with the effects of these reagents on the pellet height response of these cilia. Simultaneous addition of N-ethylmaleimide and ATP to cilia caused a slight inhibition of ATPase activity. Preincubation of the cilia with low N-ethylmaleimide in the absence of ATP, however, enhanced the ATPase activity; the enhancement decreased with increasing time of preincubation. Preincubation of cilia with high N-ethylmaleimide caused increasing inhibition. p-Hydroxymercuribenzoate was more potent than N-ethylmaleimide, usually causing only an inhibition which increased if the cilia were preincubated with p-hydroxymercuribenzoate in the absence of ATP.The pellet height response of these cilia, which serves as a convenient assay of some events related to ciliary motility, was inhibited about 50% by high concentrations of N-ethylmaleimide in the presence of ATP. Preincubation of the cilia with low concentrations of N-ethylmaleimide led to complete loss of the pellet height response. p-Hydroxymercuribenzoate was a more potent inhibitor of the pellet height response than N-ethylmaleimide; complete inhibition was attained even in the presence of ATP, while preincubation with a low concentration of p-hydroxymercuri-benzoate caused a very rapid loss of pellet height response.The ATPase activity of the crude dynein obtained by extraction of cilia and removal of the axonemes was approximately doubled by preincubation with N-ethylmaleimide. 30 S Dynein, obtained from the crude dynein by sedimentation on a sucrose density gradient, was slightly inhibited by N-ethylmaleimide; p-hydroxy-mercuribenzoate was more potent. The residual ATPase activity remaining on the axonemes after two extractions was also only inhibited by N-ethylmaleimide and by p-hydroxymercuribenzoate.These results demonstrated that SH groups influence both the ATPase activity of dynein and the pellet height response of glycerinated cilia. The possible significance of the similarity in enhancing effect of N-ethylmaleimide on cilia ATPase and on myosin ATPase was discussed.