Table Of ContentEcto-A TPases
Recent Progress on Structure and Function
Ecto-A TPases
Recent Progress on Structure and Function
Edited by
Liselotte Plesner
University ofA arhus
Aarhus C, Denmark
Terence L. Kirley
University of Cincinnati
Cincinnati, Ohio
and
Aileen F. Knowles
University of California
Las Angeles, California
Springer Science+Business Media, LLC
EctO-ATPuos reoent progress an structure and funct,an I edlt6~ by
l,sela,," PI.snor. Terenee l . Klrley. ,nd /1' leen F. t(nowles.
p. C ••
-Praee8~lngs Of t~" Flrst lnternlt,on.' Warkshap en Eete-AlPI.es.
held /lugust 26-]0, 1996, ,n M.r de Plau, Argen, ln.---l.p. versa.
Inelude5 bl~l'egr,ph,e.1 ref"renoe. and Ind.".
ISBN 978-1-4613-7729-0 ISBN 978-1-4615-5955-9 (eBook)
DOI 10.1007/978-1-4615-5955-9
1. Adenoslne trlphaspl,.tase--Cangruse •. 2. EXlr.c.llul.r
cn,y~"s--Congr.sse.. 1 Plesn., . llselotl" li. Klrley. Terenee
l. III. Knewles. AII."n F. IV. Internatlen.l Werkshep an Eela
-AlP".s 11st 1996 M.r d. Plata, Argentina)
[DNLM. 1. /ldenoslnelrlp~asph.t.se--phy.IDlagy--can9res.e.. OU
136 E19 19971
0P609.A3E28 1997
572 .'I75--do21
ONLM/OLC
for l lbr.ry of Congres. 97-3575
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Frol1l c{}\'er: Lighl microscopy based cytoehemical detection of ecto-A TPase activity for
LLC-MK,. HeLa S3. and GI\100637F and 293 cclls: ("Ils ,,"cre Irypsinized, sccded on chambcr
slides and grown for at least 1 Sl lOUrs. Subconfluent cell monolayers were fixed for I hour afler
washing with Tris-buffcred saline. Fixing solution contained 2% glularaldehyde. 5% sucrose.
and O. I M cacodylate buffer (pH 7,4), Subsequcntly, c"lIs wcre washed for 2-3 hours with 0.1
M cacodylalc buffcr (pH 7.4) containing 5% sucrosc. White incubating the cells wi,h a reaction
medium to delect ccto~A TPase activity. 0.2 mM 141CeJ· was used as phosphate capturing and
labei ing agent. [Nislochem. J. 27. 555-564 (1995)J
Proccedings of the Fim Imemational Workshop on Ec!O-A TPases.
held August 26 - 30. 1996. in Mar de Plata. Argentina
Ecto·ATPase Horne Page Address: hUp:/lwww.svf.aau.dk/cctol
ISBN 978-1-4613-7729-0
o 1997 Springer Sciencc+BU$i~ Media New Yor!<
Originally published by Plonum Pres •. New York in 1992
Soflcover "'print of tlle hardcover ISI edilion 19<)2
hllp:flwww.plcnum.com
AII tights reserl'ed
1098765432 1
No pari of this book may bc rcproduccd. slorcd in a re!Ticval systcm. or transmilled in any
fonn or by any means. electronic. mcchanical. photocopying. microfilming. re<:ording. or
olherwise. Wilholll wrinen pennission from the Publishcr
PREFACE
It has been known for almost 50 years that many cells carry enzymes that hydro
lyze extracellular ATP, and the term "ecto-ATPase" was used first by Engelhardt 40 years
ago. But until the end of the 1970's, the idea of an ATPase with its ATP hydrolyzing site
on the outside of the cell membrane was met with substantial skepticism since it was
thought that ATP was strictly intracellular. Nevertheless, ecto-ATPase activity was dem
onstrated using a variety of intact cells. Most ecto-ATPase(s) exhibited three common
characteristics: 1) activation by either Ca2+ or Mg2+, 2) insensitivity to the commonly
used inhibitors ofF-type, P-type, and V-type ATPases, and 3) ability to hydrolyze nucleo
side triphosphates and often nucleoside diphosphates as well. At the same time, the
dominant ATPase activity in many plasma membrane preparations was shown to be dis
tinct from the ion-pump ATPases, but had similar enzymatic properties as the ecto-AT
Pase(s). Thus the term "E-type ATPase activity" has been proposed for ATPase activity
exhibiting these characteristics, and it is assumed that all ecto-ATPases are E-type AT
Pases.
The converse is not true, however, since soluble E-type ATPases were shown to ex
sist in plants, microorganisms, and the saliva of blood sucking insects. These enzymes
could be easily purified, and exhibited very high specific activity. In contrast, the mem
brane bound E-type ATPases (the ecto-ATPases) were extremely difficult to isolate, and
studies on partially purified preparations yielded conflicting and often controversial re
sults with respect to enzymology and molecular identity.
Meanwhile, the important multiple physiological effects of extracellular nucleotides
were being established and as a result, interest in ecto-A TPases was considerably in
creased. Purification efforts were intensified in spite of the technical difficulties. In 1992
the first truly homogeneous preparation of ecto-ATPase was obtained from T -tubule mem
branes of rabbit skeletal muscle, which showed an extraordinarily high specific activity. It
led to the realization that probably all ecto-ATPases are low abundance proteins, a some
what discouraging prospect for most investigators. Although molecular cloning of a rat
liver ecto-ATPase was reported earlier, subsequent studies using this cDNA generated
confusion rather than the expected success of the cloning of other ecto-A TPases.
Thus, a sense of uneasy uncertainty prevailed in the field, which was evident when
Liselotte Plesner visited a number of ecto-A TPase researchers in early 1994 in preparation
for writing a review on ecto-ATPases for the International Review of Cytology. It became
clear during these visits that an international meeting would be a most welcome opportu
nity to improve communication and foster collaboration in the field. Dr. Plesner then ap
proached the organizers of the VIU,h International Conference on Na+,K+-ATPase,
scheduled to take place in Argentina in August of 1996, and obtained their sponsorship of
v
vi Preface
the First International Workshop on Ecto-ATPases. Subsequently, Aileen F. Knowles and
Terence L. Kirley agreed to join Liselotte Plesner in forming an organizing committee.
The Organizing Committee was very gratified by the response of the much larger
than expected number of colleagues who expressed interest in attending the workshop.
However, the development which gave the workshop its focus occured only a few months
preceding the meeting. In a report on the molecular cloning of potato apyrase, it was
shown that five E-type A TPases from plants and microorganisms share consensus se
quences which were also found in human and murine CD39. CD39 is a cell surface pro
tein initially shown to be involved in lymphoid cell activation and adhesion.
Subsequently, it was verified by expression in COS cells that CD39 possesses ATPase ac
tivity. Thus, an E-type ATPase gene family, including both soluble and membrane bound
A TPases, was born.
During the meeting in Mar del Plata, 47 participants presented their latest data in 41
talks, 7 posters, and 3 discussion sessions over five days in August of 1996. Eleven coun
tries and four continents were represented in the workshop which included participants
from several related fields. The ubiquitous nature of the ecto-A TPases was demonstrated
by reports of the enzyme(s) from a variety of organisms and tissues, ranging from potato
tubers, protozoa, parasitic worms to vertebrate tissues. For the first time, the interrelation
ship of these seemingly disparate enzymes became apparent. Thus, after a long period of
controversy concerning the molecular identity and structure of the ecto-A TPases, a con
sensus was reached that E-type ATPases from organisms throughout the phylogenetic tree
are related, and that the ecto-A TPase family in vertebrates is characterized by a core pro
tein with a molecular mass of 50--60 kDa, which is glycosylated to varying degrees in dif
ferent cells, and appears to exist and function as homooligomers of 2-3 monomers.
Nevertheless, E-type ATPases may also exist among proteins outside this category, as ex
emplified by the neural cell adhesion molecule (N-CAM) which was shown to have low
level E-type ATPase activity and an extracellular ATP binding site.
In addition to structural information, clear cut evidence was also presented with re
spect to the function of some ecto-A TPases (including vascular CD39) in regulation of
platelet aggregation. These results highlighted the important roles of ecto-ATPases in
thromboregulation, transplant rejection, and parasite survival. The implication that these
ecto-A TPases and relevant reagents may be potential therapeutics for diseases such as
atherosclerosis as well as for diseases prevalent in third world countries such as schis
tosomiasis and malaria is truly exciting. Moreover, results were presented on the possible
role of ecto-A TPases in lymphocyte function and other clinically relevant areas such as
hearing, kidney diseases, epilepsy, and cancer.
A special discussion session was dedicated to assigning an acceptable nomenclature
for the E-type ATPase, the lack of which has been another source of confusion in the lit
erature. The terms often used to refer to these enzymes include apyrase, ecto-ATPase, and
NTPase, all of which fail to adequately describe the E-type A TPases. However, as the
family is sure to expand rapidly in the near future when clear distinction of subfamilies
will be established, the workshop participants decided to postpone the assignment of an all
encompassing name for the E-type A TPases.
Thus, in this historical meeting of ecto-ATPase research, the primary goal of reach
ing consensus on several issues was successfully accomplished. We believe all of us can
look forward to a new era of rapid progress in the field. As more ecto-ATPases are cloned,
the establishment of subfamilies based on sequence will commence. Correlations can then
be made linking the subfamily classification with enzymology and localization of the vari
ous isozymes, which will aid in the establishment of a consistent and accurate nomencla-
Preface vii
ture for the E-type A TPases, as well as revealing possible connections to physiological
functions. Other areas of future research, such as establishment of definitive functions in
specific systems, elucidation of interplay between purinoceptors and ecto-A TPases, inves
tigation of structure-function relationships, higher order protein structural determination,
development of therapeutic agents (including specific inhibitors of E-type A TPases and
injectable soluble forms of these enzymes), will surely bring this field the attention it de
serves.
Finally, we wish to express our gratitude to the organizers of the VIII1h International
Conference on Na+,K+-ATPase, Professors Patricio J. Garrahan, Luis A. Beauge, David C.
Gadsby and Rolando C. Rossi, for their sponsorship of the workshop and their hospitality.
Their splendid management of the meeting allowed us to enjoy the science to the fullest.
Liselotte Plesner
Terence L. Kirley
Aileen F. Knowles
ACKNOWLEDGMENTS
I would like to personally thank the organizers of the VIII,h International Conference
on Na +,K' -A TPase: Patricio 1. Garrahan, Luis A. Beauge, David C. Gadsby and Rolando
C. Rossi for their foresight, effectiveness, and kind support which were of decisive impor
tance for the implementation of the plan for a first international meeting on ecto-A TPases.
I am grateful to colleagues at the Department of Biophysics, University of Aarhus,
Denmark, for their willingness to absorb in the departmental funds the administrative
costs during the preparation of the workshop, and to the department secretary, Dorthe
Abildskov, for her help when the paperwork surpassed my capacity.
Liselotte Plesner
ix
CONTENTS
I. Ecto-ATPase: Characterization and Localization
1. Ecto-ATPases of the Nervous System
Agnes K. Nagy
2. Evidence for Ectonuc1eotidases in the Guinea-Pig Cochlea: In Vivo and in Vitro
Biochemical Studies ........................................... 15
Srdjan M. Vlajkovic, Peter R. Thome, Gary D. Housley, and
David J. B. Munoz
3. Solubilization and Characterization of an ATP Diphosphohydrolase (EC 3.6.1.5)
from Rat Brain Synaptic Plasma Membranes . . . . . . . . . . . . . . . . . . . . . . . . 21
Ana Maria O. Battastini, Edilamar M. Oliveira, Cleci M. Moreira,
Carla D. Bonan, Joao Jose F. Sarkis, and Renato Dutra Dias
4. The Hydrolysis of Extracellular Adenine Nuc1eotides by Cultured Vascular Cells
and Cardiac Myocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
L. L. Slakey, E. S. Dickinson, S. J. Goldman, E. L. Gordon, P. Meghji, and
J. D. Pearson
5. Distribution of Different ATP-Diphosphohydrolase Isofonns in Mammalian
Organs. . . . . .. . . .. . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 33
Adrien R. Beaudoin, Gilles Grondin, and Jean Sevigny
6. Solubilized E-Type ATPase Is Released from Intact Rat Tissues in the
Simultaneous Presence ofNucleotides and Detergents. . . . . . . . . . . . . . . . 41
L. Plesner
7. Ecto-ATP-Diphosphohydrolase from Normal and Abnormal Placenta. . . . . . . . . 49
A. M. Kettlun, L. C. Collados, L. Garcia, L. Chayet, M. Mancilla,
C. G. Acevedo, 1. Bravo, E. Aranda, A. Traverso-Cori, and
M. A. Valenzuela
xi
xii Contents
8. ATP-Diphosphohydrolase Activity from Mammary Gland: Regulation and
Localization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
M. A. Valenzuela, M. Galleguillos, A. Alvarez, L. Chayet, L. C. Collados,
L. Garcia, A. M. Kettlun, M. Mancilla, A. Traverso-Cori, and D. Miranda
9. Role of Ecto-ATPases, Based on Histochemical Investigations: Evidences and
Doubts.. . . . . .. .. . .. . . . . . . . . .. . . . . .. . . . . . . . . .. . . . .. . . . . . . . . .. 65
Agnes Kittel
10. Ecto-ATPase Activity in Goldfish Hepatocytes .............. . . . . . . . . . . . . . 73
Michael E. Frischmann, P. J. Schwarzbaum, G. Krumschnabel, R. C. Rossi,
and W. Wieser
11. Ecto-ATPases of the Kidney. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Tihana Zanic-Grubisic and Lorena Griparic
12. Ecto-ATPase Activity in the Kidney. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Luc Vanduffe1, Raf Lemmens, and Henri Teuchy
13. Inhibition of Porcine Renal Ecto-ATP Diphosphohydrolase by Ca2+ Channel
Blockers and Neuroleptic Drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Jens Rengelshausen, Christa Schweickhardt, and Gerhard Burckhardt
14. Human Tumor Ecto-ATPases 101
Aileen F. Knowles
II. Ecto-ATPase: Purification and Sequencing
15. Purification, Characterization, and Molecular Cloning of the Chicken Gizzard
Smooth Muscle Ecto-ATPase .................................... III
Terence L. Kirley and James G. Stout
16. A Strategy for Monoclonal Antibody Production to Ecto-ATPases . . . . . . . . . . . . 127
Randy S. Strobel and Murray D. Rosenberg
17. Ecto-ATPase of Tetrahymena: Role in Purinergic Responses and Purification of
a Soluble Form ............................................... 135
Tom M. Smith, Mark Y Kim, Terence L. Kirley, and Todd M. Hennessey
18. Purification and Identification by Immunological Techniques of Different
Isoforms of Mammalian ATP Diphosphohydrolases .................. 143
Jean Sevigny, France Dumas, and Adrien R. Beaudoin
19. ATP Diphosphohydrolase from Schistosoma mansoni Belongs to a New Family
of Apyrases .................................................. 153
Sergio Verjovski-Almeida, Eveline G. Vasconcelos, Sergio T. Ferreira,
Ana M. Kett1un, Marta Mancilla, and M. Antonieta Valenzuela
Contents xiii
III. Ecto-ATPases: Function and Pathology
20. Immunochemical Expression of Ecto-ATP-Diphosphohydrolase in Experimental
and Clinical Disease ........................................... 161
W. W. Bakker, P. K. Cheung, and K. Poelstra
21. Control of Platelet Reactivity by an Ecto-ADPase on Human Endothelial Cells 167
A. J. Marcus, M. J. Broekman, J. H. F. Drosopoulos, N. Islam,
T. Alyonycheva, L. B. Safier, K. k Hajjar, D. N. Posnett,
M. A. Schoenborn, K. Schooley, and C. R. Maliszewski
22. Vascular ATP Diphosphohydrolase (CD39/ATPDase) . . . . . . . . . . . . . . . . . . . . . . 171
Elzbieta Kaczmarek, Jonathan B. Siegel, Jean Sevigny, Katarzyna Koziak,
Wayne W. Hancock, Adrien Beaudoin, Fritz H. Bach, and
Simon C. Robson
23. Expression of Glomerular Ecto-ATPase in Idiopathic Nephrotic Syndrome 187
P. K. Cheung, J. F. W. Baller, M. L. C. van der Horst, and W. W. Bakker
24. Role of Ecto-ATPase in Lymphocyte Function ........................... 197
Kenneth E. Dombrowski, Yong Ke, and Judith A. Kapp
25. ATP Diphosphohydrolase: A Possible Relation of This Enzyme with Alzheimer's
Disease and Thrombosis ........................................ 209
J. J. F. Sarkis, C. D. Bonan, S. S. Frassetto, C. Pilla, A. M. O. Battastini, and
R. D. Dias
26. ATP Diphosphohydrolase and Sf-Nucleotidase Activities trom Hippocampal
Synaptosomes after Brain Ischemia ............................... 213
M. R. C. Schetinger, C. D. Bonan, R. Schierholt, k Webber, J. J. F. Sarkis,
R. D. Dias, and C. A. Netto
27. The ATP-Diphosphohydrolase of Schistosoma mansoni: Ecto-Localization and
Possible Roles in Host-Parasite Interactions ........................ 221
Eveline G. Vasconcelos, Christiane R. Torres, Samantha M. Martins,
Sergio Verjovski-Almeida, and Sergio T. Ferreira
28. Potato Apyrase: An Overview: Characteristics and Localization of
ATP-Diphosphohydrolase from Solanum tuberosum Tuber ........... " 227
A. Traverso-Cori, M. Mancilla, k M. Kettlun, and M. A. Valenzuela
IV. Interrelationship of Ecto-ATPase and Purinoceptor Function
29. P2 Nucleotide Receptor Structure and Function .......................... 231
G. A. Weisman, J. T. Turner, L. L. Clarke, F. k Gonzalez, M. Otero,
R. C. Garrad, and L. Erb
30. Ectonucleotidases and Purinoceptors in the Cochlea and Their Putative Role in
Hearing ..................................................... 239
P. R. Thorne, G. D. Housley, S. M. Vlajkovic, and D. J. B. Munoz
