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ATPase associated with microtubules in Tetrahymena, helix, carcinus and Rattus
Comp. Biochem. Physiol. Vol. 67B, pp. 485 to 492 Pergamon Press Ltd 1980. Printed in Great Britain
ATPASE ASSOCIATED WITH MICROTUBULES IN
TETRAHYMENA, HELIX, CARCINUS A N D RATTUS G. A. KERKUT,G. A. SHARP and J. T. R. FITZSlMONS School of Biochemical and Physiological Studies, University of Southampton, Southampton S09 3TU, England Abstract--1. Nerves from the snail, crab and rat were fixed in glutaraldehyde and processed for cytoche-
mical localization of ATPase. 2. 1% glutaraldehyde allowed a lead phosphate precipitate associated with the microtubules in nerve. This was not present in tissue fixed in 2 or 3% glutaraldehyde. 3. There was no enzyme activity associated with the mierotubules if the substrate ATP was missing, or if inorganic phosphate or UTP were given as substrate. GTP will act as a substrate but is not as good as ATP. 4. The microtubular associated ATPase is not inhibited by NaF or cysteine but is inhibited if magnesium ions are missing. 5. The protozoan Tetrahymena vorax was cytologicallyexamined for ATPase activity. ATPase activity was found associated with the outer doublet and central microtubules of the cilia; the basal, post ciliary, longitudinal and transverase microtubules within the cortex. 6. In Tetrahymena, ATP and UTP were equally good substrates for the enzyme but GTP and CTP were much less effective.
INTRODUCTION It is a great privilege to contribute an article to honour Professor Marcel Florkin's contribution to Science. From his important studies and texts on Comparative Biochemistry, Marcel Florkin played a seminal role in interesting students in the subject and encouraging them both by example and direct encouragement. Marcel Florkin by his incisive mind, together with its powerful and gifted application, was able to encompass a wide range of topics and made contributions which will be discussed more fully elsewhere in this volume. The present article is based on a comparative study that we have made on the localization of ATPase in nervous tissue in Carcinus, Helix and Rattus, and in the tubular systems of the protozoan Tetrahymena, Sharp et al. (1978, 1979, 1980). A general picture has emerged that ATPases are associated with microtubular structures in nerve; cells, associated with cilia and flagellar movement and changes in body shape; in pigment cells associated movements of the pigment granules; in secretory cells associated with the movement of the vesicles out of the cell. It would appear that there is either some evolutionary conservatism in the development of motile systems, or else that there are only a limited number of solutions to a given problem and that this solution (microtubules, ATP, actin and myosin) have been developed independently in different systems. It would seem more probable that this motile system is basic within all cells and that it has been conservatively developed to allow for cellular specialization in nerves, secretory cells and pigment cells. MATERIALS AND METHODS
Nervous tissue was removed from the specific animal and dissected from the surrounding muscle under 25% bovine serum albumin. Small pieces of nerve were fixed in 1% glutaraldehyde buffered with 0.1 M cacodylate HCI pH
7.6. Blocks were washed in three changes of cacodylate buffer for 60 rain in each solution. Tissue blocks were then incubated for 5-60 min at 18°C in Wachstein-Meisel (1975) incubation medium containing 0.1 magnesium nitrate, 0.2 M dimethyl glutarate pH 7.2, 4 mM ATP and 3.6 mM lead nitrate. Controls were carried out by incubation in medium (a) without the substrate ATP, (b) with full substrate and 0.1 M sodium fluoride, an inhibitor of acid phosphatase (c) full substrate and cysteine an inhibitor of alkaline phosphatase, (d) medium without magnesium ions, (e) medium with substrate replaced by other nucleotide, i.e. UTP, CTP, GTP, (f) medium with substrate replaced by glucose 6 phosphate, or inorganic phosphate solution. Tetrahymena vorax from a culture were centrifuged down and the pellet fixed in 1% glutaraldehyde solution and treated as above. More practical details are given in the papers (Sharp et al., 1978, 1979, 1980. RESULTS Nerve tissue Our experiments using different concentrations of glutaraldehyde fixative showed that if the concentration was higher than 1% glutaraldehyde, we obtained a good lead deposit on the basement membranes of endothelial cells lining capillaries, and on the axon membrane. Reaction products also filled portion of the tubular smooth endoplasmic reticulum within the dendrites. However no precipitate was found associated with the microtubules in the nerve. With 1% glutaraldehyde fixation there was a consistent deposit of lead on the mierotubules as well as the abovementioned structures. Figure 1 shows a section through the rat cerebral cortex where the tissue had been fixed in 1% glutaraldehyde and incubated in a Wachstein-Meisel medium. In Fig. 1A the medium also contained sodium fluoride (an inhibitor of acid phosphatase) whilst Fig. IB shows the result of the tissue being incubated in the medium with cysteine (an inhibitor of alkaline phosphatase). In both cases there was a deposit of lead phosphate on the microtubules indicating that the reaction was not due to an
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G.A. KERKUT, G. A. SHARP and J. T. R. FITZSIMONS
Fig. 1A, Electron micrograph of a dendrite in the rat cerebral cortex. The tissue was incubated in a Wachstein-Meisel medium containing sodium fluoride. Lead phosphate precipitation can be seen in association with the microtubules (mt) and the tubular endoplasmic reticumlum (ter), Fig. lB. Electron micrograph of part of a dendrite of the rat cerebral cortex incubated for ATPase localization in a medium containing cysteine, Lead phosphate precipitation can be seen in association with microtubnles (mt), tubular endoplasmic reticulum (ter), and the axolemma (a) but not the mitochondria (m).
Microtubules in Tetrahymena, Helix, Careinus and Rattus
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500
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.~_ E
Dots counted rnicrotubules Off rnicrotubules
5
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-- 200 76 e0 0
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exp W - M
-ATP
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• GTP
~- UTP
exp U - P
+ phospate
Fig. 2. Counts of the number of lead phosphate dots found on the microtubules, and off microtubules, in EM micrographs of sections of rat cerebral cortex incubated in various media, exp W-M is full Wachstein-Meisel medium; -ATP, is Wachstein-Meisel medium without the substrate ATP; + NaF, +cysteine, is Wachstein Meisel medium with NaF or cysteine added; +GTP, +UTP, +phosphate, has these substances added instead of the ATP substrate to the Wachstein Meisel medium; exp U-P, is incubation in the full Uusitalo-Palkama medium with ATP. There is a greater deposit of the preciptate on the microtubules provided that the substrate (ATP or GTP) is present. It is not inhibited by NaF or cysteine. acid or alkaline phosphatase. A similar result was obtained from snail nerve and crab nerve. In both cases there was a clear deposit associated with the microtubules in the axons and dendrites. A more quantitative approach was made by counting the number of lead phosphate dots in the micrographs. Figure 2 is a graph showing the extent of the lead phosphate precipitate on and off the microtubules in rat brain tissue incubated in various experimental media. There was a very much greater deposit of lead phosphate on the microtubules when the incubation medium was complete, but that there was no difference between that on the microtubules and background axoplasm when ATP was missing, or UTP or inorganic phosphate was substituted for ATP. Similar results were obtained for snail and crab nerve. The results indicate that there is an ATPase associated with microtubules and that the reaction is best with ATP as a substrate, less good with GTP as a substrate and minimal with UTP or inorganic phosphate as a substrate. Tetrahymena vorax Sections of Tetrahymena incubated in the full Wachstein-Meisel medium showed a lead phosphate precipitate on the following microtubular structures; the outer doublet and central microtubules of the cilia, the basal, post ciliary, longitudinal and transverse microtubules within the cortex. Figure 3 shows
an unstained section through Tetrahymena incubated in medium containing UTP. The transverse (tt) longitudinal (It) tubules and the kinetosome showed a good deposit in this medium (as well as if ATP was the substrate). GTP or CTP produced much less precipitate and were not as good as ATP or UTP as substrates. Figure 4 is a photograph through an unstained tangential section through a cilium. Specific lead phosphate deposit indicating ATPase activity is seen on the transverse microtubules (tt) and also on one of each pair of outer doublet (od) microtubules. The inner doublet was unstained indicating that the ATPase was only associated with the outer doublet. Magnesium ions were required to be in the incubation medium otherwise there was no precipitate. DISCUSSION
Nerve In view of the past controversy over the interpretation of cytochemical demonstrations for ATPase localization, (Moses & Rosenthal 1967; Novikoff 1967; 1970; Rosenthal et al., 1970), the results reported have to be interpreted with caution. The reaction product was sharply localized within neurones, but this in itself does not suggest that enzymes have been responsible. No reaction product was observed in tissues heated to 70°C before incuba-
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G.A. KERKUT,G. A. SHARPand J. T. R. FITZSIMONS
Fig. 3. Electron micrograph of an unstained section through the cortex of Tetrahymena vorax fixed in 1~ glutaraldehyde for 30 min and incubated in a UTP medium for 1 hr. The longitudinal (It) transverse (tt) and kinetosome (K) microtubules stain specificallywith lead phosphate indicating a nucleoside phosphatase activity. tion, indicating a heat sensitive substance accounted for the successful deposition of reaction product. It has been argued that boiling destroys some physical or chemical structure that normally attracts lead precipitates. However, another factor in favour of enzyme activity is that the reaction product does not form when the substrate is absent or replaced by a non-hydrolysable phosphate. Axonal membrane. Reaction product was consistently observed in association with the axolemma of crab, snail and rat neurones. This localization suggests the presence of a membrane ATPase. This enzyme has been intensively studied by many workers.
Skou (1957, 1974) biochemically demonstrated that the energy for active transport of sodium and potassium ions across the axolemma, comes from the hydrolysis of ATP. He referred to this enzyme as transport ATPase. Since then there have been many cytochemical demonstrations of the transport ATPase. It has been shown to be inhibited by cardiac glycosides and to require magnesium, sodium and potassium ions for maximum activity. Torack & Barrnett (1963) demonstrated ATPase activity in the membranes of neurones and glia of the rat cerebrum. Novikoff et al. (1966) compared the localization of transport ATPase to the distribution of acetylcholine esterase in the dor-
Mierotubules in Tetrahymena, Helix, Carcinus and Rattus
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Fig. 4. Electron micrograph of an unstained tangential section through a cilium of Tetrahymena vorax. There is a specific lead deposit indicating ATPase activity on the transverse mierotubules (tt) and on one of each pair of outer doublet (od) microtubules. sal root ganglia and peripheral nerve of rat. Sabatini et al. (1968) used the squid nerve, the tissue originally
used in the biochemical demonstration of transport ATPase, to cytochemically demonstrate the enzyme localization. Schlaepfer et al. (1969) cytochemically demonstrated the transport ATPase at the axonalschwann cell interface of myelinated as well as unmyelinated fibres. Such localizations of the enzyme ATPase were taken to indicate that the membrane had an important transport function. Recently, Sumner (1978) observed not the sought after cardiac glycoside-sensitive transport ATPase in association with hypoglossal nucleii membranes, but a membrane ATPase activated by calcium or magnesium ions. The ATPase was insensitive to ouabain (a cardiac glycoside) and to the absence of sodium and potassium ions. This enzyme resembles an actomyosin ATPase (Bray, 1977) and Sumner suggested that this enzyme could be involved in a mechanism for membrane movement. There was an increase in the magnesium or calcium ATPase in hypoglossal nucleii, following axotomy, indicating it may be associated with the increased membrane movement necessary for retraction and growth. From these many cytochemical studies a sodiumpotassium-magnesium ion activated transport ATPase and a magnesium or calcium ion activated
ATPase have been localized in association with the axonal membrane. Our cytochemical staining technique has indicated the presence of both these enzymes in the axonal membranes of crab, snail and rat neurones. Inhibition with sodium fluoride does not completely abolish staining of the axonal membrane. Sodium fluoride as well as inhibiting acid phosphatase, inhibits the sodium-potassium ATPase, but leaves the magnesium ATPase unaffected. The residual staining of the axolemma, after sodium fluoride inhibition of the sodium potassium transport ATPase, is probably the product of the magnesium ATPase activity. Mitochondria. Within the crab and snail neurones fixed with 1, 2 and 3Vo glutaraldehyde, we were able to demonstrate ATPase activity associated with the mitochondrial inner and outer membranes. The ATPase associated with the inner membrane of the mitochondria is concerned with energy capture. Energy supplied by respiration is used to pump protons across the membrane and establish a proton gradient. Using the same Wachstein and Meisel incubation medium, we were unable to localize mitochondrial ATPase within the rat cerebral cortex, fixed in either 1, 2 or 3 ~ glutaraldehyde. Agafonor (1977) was also unable to localize the rat brain mitochondrial
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G. A. KE~KUT,G. A. SHARPand J. T. R. FITZSIMONS
ATPase. It is possible that the mitochondrial ATPase of rat cortex is tightly coupled with a protein for the synthesis of ATP, and unless uncoupled, is unable to catalyze the degradation of ATP. Tubular endoplasmic reticulum. The tubular elements of the smooth endoplasmic reticulum, within dendrites of the rat cerebral cortex, were often partially or completely filled with lead phosphate. This deposition suggests a discrete localization of ATPase activity. Borderson et al. (1978) recently reported a similar distribution of reaction product in dendrites of the rat cerebral cortex. Biochemically ATPase activity has been localized in cell fractions thought to contain fragments of smooth endoplasmic reticulum, (Bradford et al., 1964; Mcllwain 1966). The localization of ATPase in association with the agranular endoplasmic reticulum supports the suggestion that the agranular endoplasmic reticulum is a fast transport system actively involved in the anterograde and retrograde transport of materials, (Droz et al., 1975). Neurofilaments. Using 1, 2 and 3% glutaraldehyde fixation and the Wachstein and Meisel cytochemical staining technique, no lead phosphate deposition was observed in association with neurofilaments. Neurofilaments in tissue slices and tissue blocks of snail, crab and rat neurones reamined free of lead phosphate deposition. Schlaepfer et aL (1969) reported that under certain conditions lead phosphate accumulated along the neurofilaments of myelinated and unmyelinated nerve fibres. These neurofilament deposits were noted after formalin and hydroxyadipaldehyde fixation and occasionally following glutaraldehyde fixation. The deposits were prominent with increasing acidity and its was suggested that they could be related to spurious acid phosphatase deposits. Other authors have suggested that the lead deposits on neurofilaments develop in a similar manner to silver impregnation stains. Silver nucleii form on reducing sites along the neurofilaments, (Peters, 1955), such reducing sites could also attract lead deposits. Wolff & Wolff (1973) demonstrated lead sulphide precipitation in association with neurofilaments of peripheral nerves, when using a modification of the Glick & Fischer (1945) and Mallach et al. (1965) methods, to stain nerves for ATPase localization. Again they suggested that the precipitate on neurofilaments resembled copper binding, (Krammer & Lischka, 1973) and indicated that further investigations were necessary, before an ATPase was described in association with neurofilaments. Microtubules. Lead phosphate deposition, indicating ATPase activity, was only observed in association with microtubules when the nervous tissue had been fixed in 1% glutaraldehyde. 2 and 3% glutaraldehyde completely inhibited this enzyme localization. The precipitation in association with microtubules in I% glutaraldehyde fixed tissues, was inhibited by omission of magnesium ions or ATP from the incubation medium. Such results reported in this thesis, indicate that a magnesium ATPase is present in association with neuronal microtubules. Other cytochemical demonstrations of ATPase within neurones have not reported precipitation in association with microtubules. Usually the workers
have used fixation procedures ranging from 2% glutaraldehyde fixation for 1 hr to 6.5% glutaraldehyde fixation for 24hr. Such harsh fixation conditions could destroy the microtubule-associated ATPase activity, reported in this thesis. Recently accumulating biochemical data indicates an ATPase in association with neuronal microtubules. Khan & Ochs (1974) biochemically measured the magnesium or calcium activated ATPase within the sciatic nerve of the cat. The enzyme was demonstrated by subcellular fractionation to be present mostly in the particulate fraction (61.5%) and to a lesser extent in the mitochondrial fraction (21.4%). The high speed supernatant contained 7.6% of the ATPase activity. Using ligature studies on cat sciatic nerves, Khan & Ochs (1974) demonstrated that the magnesium or calcium ATPase was carried down the nerve fibres by slow axoplasmic transport. They suggested that the magnesium or calcium ATPase was in association with the cross-bridges or side arms seen over the length of microtubules. Burns & Pollard (1974) reported an association between brain microtubules and polypeptides. The mobilities of the polypeptides, on dissociating gelelectrophoresis, were similar to those of the ATPase dynein. Gaskin et al. (1974) demonstrated a low level of ATPase in association with dynein-like polypeptides, separated from brain microtubules. Banks (1976) demonstrated and characterized an ATPase in association with microtubules, reassembled in vitro from bovine splenic nerve. Such biochemical data and the cytochemical demonstration reported in this paper, indicate that a magnesium ATPase could be associated with neuronal microtubules, This enzyme is possibly not localized in the tubulin but is associated to some polypeptide, which purifies and co-polymerizes with tubulin. Tetrahymena Attempts have been made since the 1950s to localize ATPase in cilia and flagella. Workers were aware that these organelles contained ATPase from biochemical studies, (Nelson, 1954). It was also known that these organelles show active movement and that they contain orderly arrays of microtubules. Using cell fractionation and electrophoresis techniques, Gibbons & Rowe (1965) demonstrated that ATPase activity was associated with the side arms of the cilia microtubules. Gibbons called the proteins of Tetrahymena cilia possessing ATPase activity, dynein. Other dyneins have been described since 1965 in association with other cilia and flagella. The isolated flagella from Euglena gracilis have been shown to exhibit ATPase activity (Piccini & Albergoni, 1973) and the activity has been localized by electron micrographical studies to the microtubules and the paraflagellar rod (Piccinni et al., 1975). Cytochemical localization of ATPase in association with the outer doublet microtubules in this study, agrees with the cytochemical observations of Gordon & Barrnett (1967) and Burnasheva et al. (1969). Our findings showed a discrete localization of ATPase activity on one fibre of each outer doublet microtubule. Such a discrete localization of ATPase within cilia
Microtubules in Tetrahymena, Helix, Carcinus and Rattus was biochemically demonstrated by Gibbons (1966). He showed that a structural protein, called dynein, formed the side-arms projecting from the A tubules of the outer doublet microtubules. Dynein has high ATPase activity. The ATPase protein associated with microtubules is thought to play an important functional role in the processes underlying motility, since the energy for motility can be provided by the dephosphorylation of ATP. The ATPase has been shown to participate in the mechanochemical activity that is responsible for the linear displacement of microtubules. Microtubules are non-contracile. Addition of ATP induces them to slide in relation to one another, (Satir, 1968; Summers & Gibbons, 1971 ; Warner & Satir, 1974). The longitudinal and transverse microtubules stained for ATPase. They could utilize the energy obtained from the hydrolysis of ATP to alter and maintain cell shape. Kennedy & Zimmerman, (1970) indicate the importance of the longitudinal microtubules in maintaining cell shape by exposing Tetrahymena pyriformis to high hydrostatic pressures of 7500 and 10,000 psi. High hydrostatic pressure caused the depolymerization of the longitudinal microtubules, beginning at the posterior end of the cell and progressing anteriorly. Associated with the disruption of the microtubules was a rounding of the cell in the posterior, suggesting that the longitudinal microtubules have a role in cellular support and form stabilization. The basal microtubules are located at the proximal end of each kinetosome, running parallel with a kinety. They stained with lead phosphate indicating ATPase activity. Their location suggests they may function as a communication line between different cilia of a kinety, (Allen, 1967), The localization of ATPase on the basal microtubules suggests they could actively conduct impulses to co-ordinate the waves of silary beating. Finally the post-ciliary microtubules were seen to be rich in ATPase activity. They may be involved in supporting the cilium and actively involved in preventing rotational or horizontal movements of the cilium, (Allen, 1967). Cytochemical evidence in this study indicates nucleoside phosphatase activity associated with microtubular structures of Tetrahymena vorax. The enzyme, which shows a preference for ATP as substrate, may control and coordinate ciliary motion and cell form. Acknowledgements--We are grateful to the Medical Research Council for a Research Training grant to G. A. Sharp, and to Mr N. Orsin for his technical assistance. REFERENCES AGAFONORV. (1977) Electron cytochemical demonstration of ATPase activity in brain tissue of white rat. Tsitologia 18, 1479-1483. ALLEN R. n. (1967) Fine structure, reconstruction and possible function of components of the cortex of Tetrahymena pyriformis. J. Protozool. 14, 553-565. BANKS P. (1976) ATP hydrolase activity associated with microtubules reassembled from Bovine splenic nerve--a cautionary tale. J. Neurochem. 27, 1465-1471. BRADFORDH. F., GAMMACKD. B. & SWANSONP. D. (1964)
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Constituents of a microsomal fraction from the mammalian brain: their solubilization, especially by detergents. Biochem. J. 92, 247-254. BRAY D. (1977) Actin and myosin in neurones: a first review. Biochimie $9, 1-6. BRODERSON S. H., PATTOND. L. & STAre. W. L. (1978) Fine structural localization of K+-stimulated p-nitrophenylphosphatase activity in dendrites of the cerebral cortex. J. Cell Biol. R13-17. BURNASHEVAS. A., LUBIMOVAM. N. & YURIZINAG. A. (1969) Ultrastructure of flagella in Strigomonas oncopelti and cilia of Tetrahymena pyriformis and locationation of ATPase. Tsitologiia 11, 695-699. BURNSR. G. & POLLARDT. D. (1974) A dynein-like protein from brain. FEBS Lett. 40, 274-280. DROZ B., RAMBOURG A. & KOENIG H. L. (1975) The smooth endoplasmic reticulum: structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport. Brain Res. 93, 1-13. GASKIN F., KRAMERS. B., CANTORC. R. ADELSTEINR. & SHELANSKIM. L. (1974) A dynein-like protein associated with neurotubules. FEBS Lett. 40, 281-286. GIBBONSI. R. (1966) Studies on the ATPase activity of I4S and 30S dynein from cilia of Tetrahymena. d. biol. Chem. 241, 23, 5590-5596. GmBONSL R. & ROWEA. J. (1965) Dynein: a protein with adenosine triphosphatase activity from cilia. Science 149, 424--426. GLICKD. & FISCHERD. D. (1945) The controversy of cholinesterases. Science 102, 100-101. GORDONM. & BARRNETTR. J. (1967) Fine structural localization of phosphatase activities of rat and guinea pig. Expl Cell Res. 48, 395-412. KENNEDY J. R. & ZIMMERMANA. M. (1970) The effects of high hydrostatic pressure on the microtubules of Tetrahymena pyriformis. J. Cell Biol. 47, 568-576. KHAN M. A. & OCHS S. (1974) Magnesium or calcium activated ATPase in mammalian nerve. Brain Res. 81, 413-426. KRAMMERE. B. & LISCHgAM. F. (1973) Schwermetallaffine strukturen des peripheren nerven. Histochemie 36, }69-282. MALLACHH. J., MERGERH. J. & WOLFFJ. (1965) Electronmikroskopische Untersuchungen uner die strukter der forellenmuskulartur und die lokalisation der sauren ATPase im Verlaufe der Tofenstarre. Z. Zellforsch. 66, 794-810. MOSESH. L. & ROSENTHALA. S. (1967) On the significance of lead catalysed hydrolysis of nucleoside phosphates in histochemical systems. J. Histochert Cytochert 15, 354-355. MCILWAINH. (1966) Biochemistry of the Central Nervous System. 3rd edn. Churchill, London. NELSONL. (1954) Enzyme distribution in bull spermatozoa, 1, adenyl pyrophosphatase. Biochim. biophys. Acta 14, 213-320. NOVIKOFFA. B., QUINTANAN., V1LLAVERDEH., FORSCHIRM R. (1966) Nucleoside phosphatase and cholinesterase activities in dorsal root ganglia and peripheral nerve. J. Cell Biol. 29, 525-772. NOXaKOFF A. B. (1967) Enzyme localization with the Wachstein-Meisel procedures: real or artifact. J. Histochert Cytochert 15, 1, 353-354. PETERSA. (1955) Experiments on the mechanism of silver staining. Q. J. Microsc. Sci. 96, 84-102. PICClNNI E. & ALBERGONIV. (1973) ATPase activity in isolated flagella from Euglena gracilis. J. Protozool. 20, 456~58. PICCINNI E., AtaERGONI A. & COPPELLOT'n O. (1975) ATPase activity in flagella from Euglena gracilis. Localization of the enzyme and effects of detergents. J. Protozool. 22, 331-335. ROSENTHALA. S., MOSES H. L. & GANOTEC. E. (1970)
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Interpretation of phosphatase activity cytochemical data. J. Histochem. Cytochem. 18, 915. SABA'rINI M. T., DIPOLO R. & VILLEGAS R. (1968) ATPase activity in the membranes of squid nerve fibres. J. Cell Biol. 38, 176-183. SATIR P. (1965) Studies on cilia II examination of the distal region of the ciliary shaft and the role of the filaments in motility. J. Cell Biol. 26, 805-834. SCHLAEPFER W. W., LAVALLE M. C. & TORACK R. M. (1969) Cytochemical demonstration of nucleoside phosphatase activity in myelinated nerve fibres of the rat. Histochemie 18, 281-292. SHARP G. A., FITZSIMONSJ. T. R. 86 KERKUT G. A. (1978) Microtubular ATPase in Carcinus and HELIX nerve. Comp. Biochem. Physiol. 61A, 29%301. SHARP G. A., FITZSIMONS J. T. R. & KERKUT G. A. (1979) EM localization of ATPase on microtubules of Tetrahymena vorax. Comp. Biochem. Physiol. 63A, 253-260. SHARP G. A., FITZSIMONSJ. T. R. & KERKUT G. A. (1980) Electron microscopic localization of an ATPase associated with microtubules in the cerebral cortex of the rat. Comp. Biochem. Physiol. 66A, 000-000. SKOU J. C. (1957) The influence of some cations on ATPase from peripheral nerve. Biochim. biophys. Acta 23, 394-401.
SKOU J. C. (1974) The N a ÷ - K ÷ activated enzyme system and its relationship to transport of sodium and potassium. Q. Rev. Biophys. 7, 401434. SUMMERS K. E. & GIBBONS I. R. (1971) ATPase induced sliding of tubules in trypsin treated flagella of sea urchin sperm. Proc. natn. Acad. Sci. U.S.A. 68, 3092-3096. SUMNER B. E. H. (1978) Membrane associated ATPase in normal and injured hypoglossal nuclei. Expl Brain Res. 33, 213-225. TORACK R. M. & BARRNETT R. J. (1963) Nucleoside phosphatase activity in membranous fine structures of neurones and glia. J. Histochem. Cytochem. 11,763-772. UUSlrALO R. & PALKAMA A. (1970) Localization of Na ÷-K ÷ stimulated ATPas¢ activity in the rabbit ciliary body using light and electron microscopy. Ann. Med. Exp. Fenn. 48, 84-88. WACHSTEIN M. & MEISELE. (1957) Histochemistry of hepatic phosphatases at a physiologic pH: with special reference to the demonstration of bile canaliculi. Am. J. din. Path. 27, 13-27. WARNER F. D. & SATIn P. (1974) The structural basis of ciliary bend formation. J. Cell Biol. 63, 35-63. WOLFF J. R. & WOLFf A. (1973) Is an ATPase the drive of the axonal flow? 7th Inst. Cong. Neuropath. 2, 305-308.
ATPASE ASSOCIATED WITH MICROTUBULES IN
TETRAHYMENA, HELIX, CARCINUS A N D RATTUS G. A. KERKUT,G. A. SHARP and J. T. R. FITZSlMONS School of Biochemical and Physiological Studies, University of Southampton, Southampton S09 3TU, England Abstract--1. Nerves from the snail, crab and rat were fixed in glutaraldehyde and processed for cytoche-
mical localization of ATPase. 2. 1% glutaraldehyde allowed a lead phosphate precipitate associated with the microtubules in nerve. This was not present in tissue fixed in 2 or 3% glutaraldehyde. 3. There was no enzyme activity associated with the mierotubules if the substrate ATP was missing, or if inorganic phosphate or UTP were given as substrate. GTP will act as a substrate but is not as good as ATP. 4. The microtubular associated ATPase is not inhibited by NaF or cysteine but is inhibited if magnesium ions are missing. 5. The protozoan Tetrahymena vorax was cytologicallyexamined for ATPase activity. ATPase activity was found associated with the outer doublet and central microtubules of the cilia; the basal, post ciliary, longitudinal and transverase microtubules within the cortex. 6. In Tetrahymena, ATP and UTP were equally good substrates for the enzyme but GTP and CTP were much less effective.
INTRODUCTION It is a great privilege to contribute an article to honour Professor Marcel Florkin's contribution to Science. From his important studies and texts on Comparative Biochemistry, Marcel Florkin played a seminal role in interesting students in the subject and encouraging them both by example and direct encouragement. Marcel Florkin by his incisive mind, together with its powerful and gifted application, was able to encompass a wide range of topics and made contributions which will be discussed more fully elsewhere in this volume. The present article is based on a comparative study that we have made on the localization of ATPase in nervous tissue in Carcinus, Helix and Rattus, and in the tubular systems of the protozoan Tetrahymena, Sharp et al. (1978, 1979, 1980). A general picture has emerged that ATPases are associated with microtubular structures in nerve; cells, associated with cilia and flagellar movement and changes in body shape; in pigment cells associated movements of the pigment granules; in secretory cells associated with the movement of the vesicles out of the cell. It would appear that there is either some evolutionary conservatism in the development of motile systems, or else that there are only a limited number of solutions to a given problem and that this solution (microtubules, ATP, actin and myosin) have been developed independently in different systems. It would seem more probable that this motile system is basic within all cells and that it has been conservatively developed to allow for cellular specialization in nerves, secretory cells and pigment cells. MATERIALS AND METHODS
Nervous tissue was removed from the specific animal and dissected from the surrounding muscle under 25% bovine serum albumin. Small pieces of nerve were fixed in 1% glutaraldehyde buffered with 0.1 M cacodylate HCI pH
7.6. Blocks were washed in three changes of cacodylate buffer for 60 rain in each solution. Tissue blocks were then incubated for 5-60 min at 18°C in Wachstein-Meisel (1975) incubation medium containing 0.1 magnesium nitrate, 0.2 M dimethyl glutarate pH 7.2, 4 mM ATP and 3.6 mM lead nitrate. Controls were carried out by incubation in medium (a) without the substrate ATP, (b) with full substrate and 0.1 M sodium fluoride, an inhibitor of acid phosphatase (c) full substrate and cysteine an inhibitor of alkaline phosphatase, (d) medium without magnesium ions, (e) medium with substrate replaced by other nucleotide, i.e. UTP, CTP, GTP, (f) medium with substrate replaced by glucose 6 phosphate, or inorganic phosphate solution. Tetrahymena vorax from a culture were centrifuged down and the pellet fixed in 1% glutaraldehyde solution and treated as above. More practical details are given in the papers (Sharp et al., 1978, 1979, 1980. RESULTS Nerve tissue Our experiments using different concentrations of glutaraldehyde fixative showed that if the concentration was higher than 1% glutaraldehyde, we obtained a good lead deposit on the basement membranes of endothelial cells lining capillaries, and on the axon membrane. Reaction products also filled portion of the tubular smooth endoplasmic reticulum within the dendrites. However no precipitate was found associated with the microtubules in the nerve. With 1% glutaraldehyde fixation there was a consistent deposit of lead on the mierotubules as well as the abovementioned structures. Figure 1 shows a section through the rat cerebral cortex where the tissue had been fixed in 1% glutaraldehyde and incubated in a Wachstein-Meisel medium. In Fig. 1A the medium also contained sodium fluoride (an inhibitor of acid phosphatase) whilst Fig. IB shows the result of the tissue being incubated in the medium with cysteine (an inhibitor of alkaline phosphatase). In both cases there was a deposit of lead phosphate on the microtubules indicating that the reaction was not due to an
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G.A. KERKUT, G. A. SHARP and J. T. R. FITZSIMONS
Fig. 1A, Electron micrograph of a dendrite in the rat cerebral cortex. The tissue was incubated in a Wachstein-Meisel medium containing sodium fluoride. Lead phosphate precipitation can be seen in association with the microtubules (mt) and the tubular endoplasmic reticumlum (ter), Fig. lB. Electron micrograph of part of a dendrite of the rat cerebral cortex incubated for ATPase localization in a medium containing cysteine, Lead phosphate precipitation can be seen in association with microtubnles (mt), tubular endoplasmic reticulum (ter), and the axolemma (a) but not the mitochondria (m).
Microtubules in Tetrahymena, Helix, Careinus and Rattus
487
500
""" 4oo
.~_ E
Dots counted rnicrotubules Off rnicrotubules
5
"O
ID
-- 200 76 e0 0
IO0
exp W - M
-ATP
+ NaF
+Cysteine
• GTP
~- UTP
exp U - P
+ phospate
Fig. 2. Counts of the number of lead phosphate dots found on the microtubules, and off microtubules, in EM micrographs of sections of rat cerebral cortex incubated in various media, exp W-M is full Wachstein-Meisel medium; -ATP, is Wachstein-Meisel medium without the substrate ATP; + NaF, +cysteine, is Wachstein Meisel medium with NaF or cysteine added; +GTP, +UTP, +phosphate, has these substances added instead of the ATP substrate to the Wachstein Meisel medium; exp U-P, is incubation in the full Uusitalo-Palkama medium with ATP. There is a greater deposit of the preciptate on the microtubules provided that the substrate (ATP or GTP) is present. It is not inhibited by NaF or cysteine. acid or alkaline phosphatase. A similar result was obtained from snail nerve and crab nerve. In both cases there was a clear deposit associated with the microtubules in the axons and dendrites. A more quantitative approach was made by counting the number of lead phosphate dots in the micrographs. Figure 2 is a graph showing the extent of the lead phosphate precipitate on and off the microtubules in rat brain tissue incubated in various experimental media. There was a very much greater deposit of lead phosphate on the microtubules when the incubation medium was complete, but that there was no difference between that on the microtubules and background axoplasm when ATP was missing, or UTP or inorganic phosphate was substituted for ATP. Similar results were obtained for snail and crab nerve. The results indicate that there is an ATPase associated with microtubules and that the reaction is best with ATP as a substrate, less good with GTP as a substrate and minimal with UTP or inorganic phosphate as a substrate. Tetrahymena vorax Sections of Tetrahymena incubated in the full Wachstein-Meisel medium showed a lead phosphate precipitate on the following microtubular structures; the outer doublet and central microtubules of the cilia, the basal, post ciliary, longitudinal and transverse microtubules within the cortex. Figure 3 shows
an unstained section through Tetrahymena incubated in medium containing UTP. The transverse (tt) longitudinal (It) tubules and the kinetosome showed a good deposit in this medium (as well as if ATP was the substrate). GTP or CTP produced much less precipitate and were not as good as ATP or UTP as substrates. Figure 4 is a photograph through an unstained tangential section through a cilium. Specific lead phosphate deposit indicating ATPase activity is seen on the transverse microtubules (tt) and also on one of each pair of outer doublet (od) microtubules. The inner doublet was unstained indicating that the ATPase was only associated with the outer doublet. Magnesium ions were required to be in the incubation medium otherwise there was no precipitate. DISCUSSION
Nerve In view of the past controversy over the interpretation of cytochemical demonstrations for ATPase localization, (Moses & Rosenthal 1967; Novikoff 1967; 1970; Rosenthal et al., 1970), the results reported have to be interpreted with caution. The reaction product was sharply localized within neurones, but this in itself does not suggest that enzymes have been responsible. No reaction product was observed in tissues heated to 70°C before incuba-
488
G.A. KERKUT,G. A. SHARPand J. T. R. FITZSIMONS
Fig. 3. Electron micrograph of an unstained section through the cortex of Tetrahymena vorax fixed in 1~ glutaraldehyde for 30 min and incubated in a UTP medium for 1 hr. The longitudinal (It) transverse (tt) and kinetosome (K) microtubules stain specificallywith lead phosphate indicating a nucleoside phosphatase activity. tion, indicating a heat sensitive substance accounted for the successful deposition of reaction product. It has been argued that boiling destroys some physical or chemical structure that normally attracts lead precipitates. However, another factor in favour of enzyme activity is that the reaction product does not form when the substrate is absent or replaced by a non-hydrolysable phosphate. Axonal membrane. Reaction product was consistently observed in association with the axolemma of crab, snail and rat neurones. This localization suggests the presence of a membrane ATPase. This enzyme has been intensively studied by many workers.
Skou (1957, 1974) biochemically demonstrated that the energy for active transport of sodium and potassium ions across the axolemma, comes from the hydrolysis of ATP. He referred to this enzyme as transport ATPase. Since then there have been many cytochemical demonstrations of the transport ATPase. It has been shown to be inhibited by cardiac glycosides and to require magnesium, sodium and potassium ions for maximum activity. Torack & Barrnett (1963) demonstrated ATPase activity in the membranes of neurones and glia of the rat cerebrum. Novikoff et al. (1966) compared the localization of transport ATPase to the distribution of acetylcholine esterase in the dor-
Mierotubules in Tetrahymena, Helix, Carcinus and Rattus
489
Fig. 4. Electron micrograph of an unstained tangential section through a cilium of Tetrahymena vorax. There is a specific lead deposit indicating ATPase activity on the transverse mierotubules (tt) and on one of each pair of outer doublet (od) microtubules. sal root ganglia and peripheral nerve of rat. Sabatini et al. (1968) used the squid nerve, the tissue originally
used in the biochemical demonstration of transport ATPase, to cytochemically demonstrate the enzyme localization. Schlaepfer et al. (1969) cytochemically demonstrated the transport ATPase at the axonalschwann cell interface of myelinated as well as unmyelinated fibres. Such localizations of the enzyme ATPase were taken to indicate that the membrane had an important transport function. Recently, Sumner (1978) observed not the sought after cardiac glycoside-sensitive transport ATPase in association with hypoglossal nucleii membranes, but a membrane ATPase activated by calcium or magnesium ions. The ATPase was insensitive to ouabain (a cardiac glycoside) and to the absence of sodium and potassium ions. This enzyme resembles an actomyosin ATPase (Bray, 1977) and Sumner suggested that this enzyme could be involved in a mechanism for membrane movement. There was an increase in the magnesium or calcium ATPase in hypoglossal nucleii, following axotomy, indicating it may be associated with the increased membrane movement necessary for retraction and growth. From these many cytochemical studies a sodiumpotassium-magnesium ion activated transport ATPase and a magnesium or calcium ion activated
ATPase have been localized in association with the axonal membrane. Our cytochemical staining technique has indicated the presence of both these enzymes in the axonal membranes of crab, snail and rat neurones. Inhibition with sodium fluoride does not completely abolish staining of the axonal membrane. Sodium fluoride as well as inhibiting acid phosphatase, inhibits the sodium-potassium ATPase, but leaves the magnesium ATPase unaffected. The residual staining of the axolemma, after sodium fluoride inhibition of the sodium potassium transport ATPase, is probably the product of the magnesium ATPase activity. Mitochondria. Within the crab and snail neurones fixed with 1, 2 and 3Vo glutaraldehyde, we were able to demonstrate ATPase activity associated with the mitochondrial inner and outer membranes. The ATPase associated with the inner membrane of the mitochondria is concerned with energy capture. Energy supplied by respiration is used to pump protons across the membrane and establish a proton gradient. Using the same Wachstein and Meisel incubation medium, we were unable to localize mitochondrial ATPase within the rat cerebral cortex, fixed in either 1, 2 or 3 ~ glutaraldehyde. Agafonor (1977) was also unable to localize the rat brain mitochondrial
490
G. A. KE~KUT,G. A. SHARPand J. T. R. FITZSIMONS
ATPase. It is possible that the mitochondrial ATPase of rat cortex is tightly coupled with a protein for the synthesis of ATP, and unless uncoupled, is unable to catalyze the degradation of ATP. Tubular endoplasmic reticulum. The tubular elements of the smooth endoplasmic reticulum, within dendrites of the rat cerebral cortex, were often partially or completely filled with lead phosphate. This deposition suggests a discrete localization of ATPase activity. Borderson et al. (1978) recently reported a similar distribution of reaction product in dendrites of the rat cerebral cortex. Biochemically ATPase activity has been localized in cell fractions thought to contain fragments of smooth endoplasmic reticulum, (Bradford et al., 1964; Mcllwain 1966). The localization of ATPase in association with the agranular endoplasmic reticulum supports the suggestion that the agranular endoplasmic reticulum is a fast transport system actively involved in the anterograde and retrograde transport of materials, (Droz et al., 1975). Neurofilaments. Using 1, 2 and 3% glutaraldehyde fixation and the Wachstein and Meisel cytochemical staining technique, no lead phosphate deposition was observed in association with neurofilaments. Neurofilaments in tissue slices and tissue blocks of snail, crab and rat neurones reamined free of lead phosphate deposition. Schlaepfer et aL (1969) reported that under certain conditions lead phosphate accumulated along the neurofilaments of myelinated and unmyelinated nerve fibres. These neurofilament deposits were noted after formalin and hydroxyadipaldehyde fixation and occasionally following glutaraldehyde fixation. The deposits were prominent with increasing acidity and its was suggested that they could be related to spurious acid phosphatase deposits. Other authors have suggested that the lead deposits on neurofilaments develop in a similar manner to silver impregnation stains. Silver nucleii form on reducing sites along the neurofilaments, (Peters, 1955), such reducing sites could also attract lead deposits. Wolff & Wolff (1973) demonstrated lead sulphide precipitation in association with neurofilaments of peripheral nerves, when using a modification of the Glick & Fischer (1945) and Mallach et al. (1965) methods, to stain nerves for ATPase localization. Again they suggested that the precipitate on neurofilaments resembled copper binding, (Krammer & Lischka, 1973) and indicated that further investigations were necessary, before an ATPase was described in association with neurofilaments. Microtubules. Lead phosphate deposition, indicating ATPase activity, was only observed in association with microtubules when the nervous tissue had been fixed in 1% glutaraldehyde. 2 and 3% glutaraldehyde completely inhibited this enzyme localization. The precipitation in association with microtubules in I% glutaraldehyde fixed tissues, was inhibited by omission of magnesium ions or ATP from the incubation medium. Such results reported in this thesis, indicate that a magnesium ATPase is present in association with neuronal microtubules. Other cytochemical demonstrations of ATPase within neurones have not reported precipitation in association with microtubules. Usually the workers
have used fixation procedures ranging from 2% glutaraldehyde fixation for 1 hr to 6.5% glutaraldehyde fixation for 24hr. Such harsh fixation conditions could destroy the microtubule-associated ATPase activity, reported in this thesis. Recently accumulating biochemical data indicates an ATPase in association with neuronal microtubules. Khan & Ochs (1974) biochemically measured the magnesium or calcium activated ATPase within the sciatic nerve of the cat. The enzyme was demonstrated by subcellular fractionation to be present mostly in the particulate fraction (61.5%) and to a lesser extent in the mitochondrial fraction (21.4%). The high speed supernatant contained 7.6% of the ATPase activity. Using ligature studies on cat sciatic nerves, Khan & Ochs (1974) demonstrated that the magnesium or calcium ATPase was carried down the nerve fibres by slow axoplasmic transport. They suggested that the magnesium or calcium ATPase was in association with the cross-bridges or side arms seen over the length of microtubules. Burns & Pollard (1974) reported an association between brain microtubules and polypeptides. The mobilities of the polypeptides, on dissociating gelelectrophoresis, were similar to those of the ATPase dynein. Gaskin et al. (1974) demonstrated a low level of ATPase in association with dynein-like polypeptides, separated from brain microtubules. Banks (1976) demonstrated and characterized an ATPase in association with microtubules, reassembled in vitro from bovine splenic nerve. Such biochemical data and the cytochemical demonstration reported in this paper, indicate that a magnesium ATPase could be associated with neuronal microtubules, This enzyme is possibly not localized in the tubulin but is associated to some polypeptide, which purifies and co-polymerizes with tubulin. Tetrahymena Attempts have been made since the 1950s to localize ATPase in cilia and flagella. Workers were aware that these organelles contained ATPase from biochemical studies, (Nelson, 1954). It was also known that these organelles show active movement and that they contain orderly arrays of microtubules. Using cell fractionation and electrophoresis techniques, Gibbons & Rowe (1965) demonstrated that ATPase activity was associated with the side arms of the cilia microtubules. Gibbons called the proteins of Tetrahymena cilia possessing ATPase activity, dynein. Other dyneins have been described since 1965 in association with other cilia and flagella. The isolated flagella from Euglena gracilis have been shown to exhibit ATPase activity (Piccini & Albergoni, 1973) and the activity has been localized by electron micrographical studies to the microtubules and the paraflagellar rod (Piccinni et al., 1975). Cytochemical localization of ATPase in association with the outer doublet microtubules in this study, agrees with the cytochemical observations of Gordon & Barrnett (1967) and Burnasheva et al. (1969). Our findings showed a discrete localization of ATPase activity on one fibre of each outer doublet microtubule. Such a discrete localization of ATPase within cilia
Microtubules in Tetrahymena, Helix, Carcinus and Rattus was biochemically demonstrated by Gibbons (1966). He showed that a structural protein, called dynein, formed the side-arms projecting from the A tubules of the outer doublet microtubules. Dynein has high ATPase activity. The ATPase protein associated with microtubules is thought to play an important functional role in the processes underlying motility, since the energy for motility can be provided by the dephosphorylation of ATP. The ATPase has been shown to participate in the mechanochemical activity that is responsible for the linear displacement of microtubules. Microtubules are non-contracile. Addition of ATP induces them to slide in relation to one another, (Satir, 1968; Summers & Gibbons, 1971 ; Warner & Satir, 1974). The longitudinal and transverse microtubules stained for ATPase. They could utilize the energy obtained from the hydrolysis of ATP to alter and maintain cell shape. Kennedy & Zimmerman, (1970) indicate the importance of the longitudinal microtubules in maintaining cell shape by exposing Tetrahymena pyriformis to high hydrostatic pressures of 7500 and 10,000 psi. High hydrostatic pressure caused the depolymerization of the longitudinal microtubules, beginning at the posterior end of the cell and progressing anteriorly. Associated with the disruption of the microtubules was a rounding of the cell in the posterior, suggesting that the longitudinal microtubules have a role in cellular support and form stabilization. The basal microtubules are located at the proximal end of each kinetosome, running parallel with a kinety. They stained with lead phosphate indicating ATPase activity. Their location suggests they may function as a communication line between different cilia of a kinety, (Allen, 1967), The localization of ATPase on the basal microtubules suggests they could actively conduct impulses to co-ordinate the waves of silary beating. Finally the post-ciliary microtubules were seen to be rich in ATPase activity. They may be involved in supporting the cilium and actively involved in preventing rotational or horizontal movements of the cilium, (Allen, 1967). Cytochemical evidence in this study indicates nucleoside phosphatase activity associated with microtubular structures of Tetrahymena vorax. The enzyme, which shows a preference for ATP as substrate, may control and coordinate ciliary motion and cell form. Acknowledgements--We are grateful to the Medical Research Council for a Research Training grant to G. A. Sharp, and to Mr N. Orsin for his technical assistance. REFERENCES AGAFONORV. (1977) Electron cytochemical demonstration of ATPase activity in brain tissue of white rat. Tsitologia 18, 1479-1483. ALLEN R. n. (1967) Fine structure, reconstruction and possible function of components of the cortex of Tetrahymena pyriformis. J. Protozool. 14, 553-565. BANKS P. (1976) ATP hydrolase activity associated with microtubules reassembled from Bovine splenic nerve--a cautionary tale. J. Neurochem. 27, 1465-1471. BRADFORDH. F., GAMMACKD. B. & SWANSONP. D. (1964)
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Constituents of a microsomal fraction from the mammalian brain: their solubilization, especially by detergents. Biochem. J. 92, 247-254. BRAY D. (1977) Actin and myosin in neurones: a first review. Biochimie $9, 1-6. BRODERSON S. H., PATTOND. L. & STAre. W. L. (1978) Fine structural localization of K+-stimulated p-nitrophenylphosphatase activity in dendrites of the cerebral cortex. J. Cell Biol. R13-17. BURNASHEVAS. A., LUBIMOVAM. N. & YURIZINAG. A. (1969) Ultrastructure of flagella in Strigomonas oncopelti and cilia of Tetrahymena pyriformis and locationation of ATPase. Tsitologiia 11, 695-699. BURNSR. G. & POLLARDT. D. (1974) A dynein-like protein from brain. FEBS Lett. 40, 274-280. DROZ B., RAMBOURG A. & KOENIG H. L. (1975) The smooth endoplasmic reticulum: structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport. Brain Res. 93, 1-13. GASKIN F., KRAMERS. B., CANTORC. R. ADELSTEINR. & SHELANSKIM. L. (1974) A dynein-like protein associated with neurotubules. FEBS Lett. 40, 281-286. GIBBONSI. R. (1966) Studies on the ATPase activity of I4S and 30S dynein from cilia of Tetrahymena. d. biol. Chem. 241, 23, 5590-5596. GmBONSL R. & ROWEA. J. (1965) Dynein: a protein with adenosine triphosphatase activity from cilia. Science 149, 424--426. GLICKD. & FISCHERD. D. (1945) The controversy of cholinesterases. Science 102, 100-101. GORDONM. & BARRNETTR. J. (1967) Fine structural localization of phosphatase activities of rat and guinea pig. Expl Cell Res. 48, 395-412. KENNEDY J. R. & ZIMMERMANA. M. (1970) The effects of high hydrostatic pressure on the microtubules of Tetrahymena pyriformis. J. Cell Biol. 47, 568-576. KHAN M. A. & OCHS S. (1974) Magnesium or calcium activated ATPase in mammalian nerve. Brain Res. 81, 413-426. KRAMMERE. B. & LISCHgAM. F. (1973) Schwermetallaffine strukturen des peripheren nerven. Histochemie 36, }69-282. MALLACHH. J., MERGERH. J. & WOLFFJ. (1965) Electronmikroskopische Untersuchungen uner die strukter der forellenmuskulartur und die lokalisation der sauren ATPase im Verlaufe der Tofenstarre. Z. Zellforsch. 66, 794-810. MOSESH. L. & ROSENTHALA. S. (1967) On the significance of lead catalysed hydrolysis of nucleoside phosphates in histochemical systems. J. Histochert Cytochert 15, 354-355. MCILWAINH. (1966) Biochemistry of the Central Nervous System. 3rd edn. Churchill, London. NELSONL. (1954) Enzyme distribution in bull spermatozoa, 1, adenyl pyrophosphatase. Biochim. biophys. Acta 14, 213-320. NOVIKOFFA. B., QUINTANAN., V1LLAVERDEH., FORSCHIRM R. (1966) Nucleoside phosphatase and cholinesterase activities in dorsal root ganglia and peripheral nerve. J. Cell Biol. 29, 525-772. NOXaKOFF A. B. (1967) Enzyme localization with the Wachstein-Meisel procedures: real or artifact. J. Histochert Cytochert 15, 1, 353-354. PETERSA. (1955) Experiments on the mechanism of silver staining. Q. J. Microsc. Sci. 96, 84-102. PICClNNI E. & ALBERGONIV. (1973) ATPase activity in isolated flagella from Euglena gracilis. J. Protozool. 20, 456~58. PICCINNI E., AtaERGONI A. & COPPELLOT'n O. (1975) ATPase activity in flagella from Euglena gracilis. Localization of the enzyme and effects of detergents. J. Protozool. 22, 331-335. ROSENTHALA. S., MOSES H. L. & GANOTEC. E. (1970)
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Interpretation of phosphatase activity cytochemical data. J. Histochem. Cytochem. 18, 915. SABA'rINI M. T., DIPOLO R. & VILLEGAS R. (1968) ATPase activity in the membranes of squid nerve fibres. J. Cell Biol. 38, 176-183. SATIR P. (1965) Studies on cilia II examination of the distal region of the ciliary shaft and the role of the filaments in motility. J. Cell Biol. 26, 805-834. SCHLAEPFER W. W., LAVALLE M. C. & TORACK R. M. (1969) Cytochemical demonstration of nucleoside phosphatase activity in myelinated nerve fibres of the rat. Histochemie 18, 281-292. SHARP G. A., FITZSIMONSJ. T. R. 86 KERKUT G. A. (1978) Microtubular ATPase in Carcinus and HELIX nerve. Comp. Biochem. Physiol. 61A, 29%301. SHARP G. A., FITZSIMONS J. T. R. & KERKUT G. A. (1979) EM localization of ATPase on microtubules of Tetrahymena vorax. Comp. Biochem. Physiol. 63A, 253-260. SHARP G. A., FITZSIMONSJ. T. R. & KERKUT G. A. (1980) Electron microscopic localization of an ATPase associated with microtubules in the cerebral cortex of the rat. Comp. Biochem. Physiol. 66A, 000-000. SKOU J. C. (1957) The influence of some cations on ATPase from peripheral nerve. Biochim. biophys. Acta 23, 394-401.
SKOU J. C. (1974) The N a ÷ - K ÷ activated enzyme system and its relationship to transport of sodium and potassium. Q. Rev. Biophys. 7, 401434. SUMMERS K. E. & GIBBONS I. R. (1971) ATPase induced sliding of tubules in trypsin treated flagella of sea urchin sperm. Proc. natn. Acad. Sci. U.S.A. 68, 3092-3096. SUMNER B. E. H. (1978) Membrane associated ATPase in normal and injured hypoglossal nuclei. Expl Brain Res. 33, 213-225. TORACK R. M. & BARRNETT R. J. (1963) Nucleoside phosphatase activity in membranous fine structures of neurones and glia. J. Histochem. Cytochem. 11,763-772. UUSlrALO R. & PALKAMA A. (1970) Localization of Na ÷-K ÷ stimulated ATPas¢ activity in the rabbit ciliary body using light and electron microscopy. Ann. Med. Exp. Fenn. 48, 84-88. WACHSTEIN M. & MEISELE. (1957) Histochemistry of hepatic phosphatases at a physiologic pH: with special reference to the demonstration of bile canaliculi. Am. J. din. Path. 27, 13-27. WARNER F. D. & SATIn P. (1974) The structural basis of ciliary bend formation. J. Cell Biol. 63, 35-63. WOLFF J. R. & WOLFf A. (1973) Is an ATPase the drive of the axonal flow? 7th Inst. Cong. Neuropath. 2, 305-308.