I) The field is covered by the following reviews:

• Banerjee, R.K. (1981) Ecto-ATPase. Mol.Cell.Biochem. 37, 91-99
• Pearson, J.D. (1985) Ectonucleotidases: Measurement of activities and use of inhibitors. Methods of Pharmacol. 6, 83-107
• Ziganshin, A.U., Hoyle, C.H.V., and Burnstock, G. (1994). Ectoenzymes and metabolism of extracellular ATP. Drug Development Research. 32, 134-146
• Plesner, L. (1995) Ecto-ATPases: Identities and Functions. Internat. Review of Cytology, 158, 141-214
• Sarkis, J.J.F., Battastini, A.M.O., Oliveira, E.M., Frassetto, S.S., and Dias, R.D. (1995), ATP diphosphohydrolases: An Overview. Ciencia e Cultura (Journal of the Brazilian Association for the Advancement of Science). 47, 131-136
• Ferreira, S.T., Vasconcelos, E.G. and Verjovski-Almeida, S. (1995). The ecto-ATPases of endoparasites and of blood cells and vessels. Ciencia e Cultura (Journal of the Brazilian Association for the Advancement of Science). 47, 151-166
• Beaudoin, A.R., Sevigny, J., and Picher, M. (1996) ATP-diphosphohydrolases, apyrases, and nucleotide phosphohydrolases: biochemical properties and functions. In: Biomembranes (Ed. Lee, A.G.) Volume 5, pp 367-399, JAI Press, Greenwich, C.T. USA
• Komoszynski, M. and Wojtczak, A. (1996). Apyrases (ATP diphosphohydrolases, E.C. 3.6.1.5.): Function and relationship to ATPases.Biochim.Biophys.Acta. 1310, 233-241
• Zimmermann H (1992) 5´-Nucleotidase - molecular structure and functional aspects. Biochem J 285:345-365
• Whyte MP (1994) Hypophosphatasia and the role of alkaline phosphatase in skeletal mineralization. Endocrine Rev 15:439-461
• Kennedy C, Westfall TD, Sneddon P (1996) Modulation of purinergic neurotransmission by ecto-ATPase. Semin Neurosci 8:195-199
• Zimmermann H (1996) Extracellular purine metabolism. Drug Develop Res 39:337-352
• Zimmermann H (1996) Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 49:589-618
• Resta R, Thompson LF (1997) T cell signalling through CD73. Cell Signal 9:131-139
• Goding JW, Terkeltaub R, Maurice M, Deterre P, Sali A, Belli SI (1998) Ecto-phosphodiesterase/pyrophosphatase of lymphocytes and non-lymphoid cells: Structure and function of the PC-1 family. Immunol Rev 161:11-26
• Resta R, Yamashita Y, Thompson LF (1998) Ecto-enzyme and signaling functions of lymphocyte CD73. Immunol Rev 161:95-109:95-109
• Ehrlich YH, Kornecki E (1999) Ecto-protein kinases as mediators for the action of secreted ATP in the brain. Prog Brain Res 120:411-426
• Zimmermann H, Pearson J (1998) Extracellular metabolism of nucleotides and adenosine in the cardiovascular system. In: Burnstock G, Dobson JG, Liang BT, Linden J (eds) Cardiovascular biology of purines, Kluwer Academic Publishers, Dordrecht, Boston, London, pp 342-358
• Redegeld FA, Caldwell CC, Sitkovsky MV (1999) Ecto-protein kinases: ectodomain phosphorylation as a novel target for pharmacological manipulation? Trends Pharmacol Sci 20:453-459
• Zimmermann H (1999) Two novel families of ecto-nucleotidases: Molecular structures, catalytic properties, and a search for function. Trends Pharm Sci 20:231-236
• Zimmermann H, Braun N (1999) Ecto-nucleotidases: Molecular structures, catalytic properties, and functional roles in the nervous system. Prog Brain Res 120:371-385
• Goding, J.W. (2000) Ecto-enzymes: physiology meets pathology. J.Leukocyte Biol.67. 285-311
• Zimmermann, H., Beaudoin, A.R., Bollen, M., Goding, J.W., Guidotti, G., Kirley, T.L., Robson, S.C., and Sano, K. (2000) Proposed nomenclature for two novel nucleotide hydrolyzing enzyme families expressed on the cell surface. In: Ecto-ATPases and Related Ectonucleotidases, edited by vanDuffel, L. and Lemmens, R.Maastricht:Shaker Publishing BV, , p. 1-8

II) As a further introduction to the ecto-ATPases

please find below the preface of the proceedings of the first international meeting within this field of research. The book is in press and planned to be released from Plenum Publishing Corporation in Feb./Mar. of 1997 with the title:

The preface was written by the organizers.

Preface

It has been known for almost 50 years that many cells carry enzymes that hydrolyze 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 demonstrated 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 of F-type, P-type, and V-type ATPases, and 3) ability to hydrolyze nucleoside triphosphates and often nucleoside diphosphates as well. At the same time, the dominant ATPase activity in many plasma membrane preparations was shown to be distinct from the ion-pump ATPases, but had similar enzymatic properties as the ecto-ATPase(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 ATPases.

The converse is not true, however, since soluble E-type ATPases were shown to exsist 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 membrane bound E-type ATPases (the ecto-ATPases) were extremly difficult to isolate, and studies on partially purified preparations yielded conflicting and often controversial results 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-ATPases was considerably increased. 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 membranes of rabbit skeletal muscle, which showed an extraordinary high specific activity. It led to the realization that probably all ecto-ATPases are low abundance proteins, a somewhat 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-ATPases.

Thus, a sense of uneasy uncertainty prevailed in the field, which was evident when Liselotte Plesner visited a number of ecto-ATPase 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 opportunity to improve communication and foster collaboration in the field. Dr. Plesner then approached the organizers of the "8th International Conference on Na,K-ATPase", scheduled to take place in Argentina in August of 1996, and obtained their sponsorship of the First International Workshop on Ecto-ATPases. Subsequently, Aileen F.Knowles and Terence L. Kirley agreed to join Liselotte Plesner 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 ATPases from plants and microorganisms share consensus sequences which were also found in human and murine CD39. CD39 is a cell surface protein initially shown to be involved in lymphoid cell activation and adhesion. Subsequently, it was verified by expression in COS cells that CD39 possess ATPase activity. Thus, an E-type ATPase gene family, including both soluble and membrane bound ATPases, was born.

During the meeting in Mar del Plata, 45 participants presented their latest data in 41 talks, 7 posters, and 3 discussion sessions over 5 days in August of 1996. Eleven countries and four continents were represented in the workshop which included participants from several related fields. The ubiquitous nature of the ecto-ATPases 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 interrelationship of these seemingly disparate enzymes became apparent. Thus, after a long period of controversy concerning the molecular identity and structure of the ecto-ATPases, a consensus was reached that E-type ATPases from organisms throughout the phylogenetic tree are related, and that the ecto-ATPase family in vertebrates is characterized by a core protein with a molecular mass of 50-60 kDa, which is glycosylated to varying degrees in different 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 exemplified 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 respect to the function of some ecto-ATPases (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-ATPases and relevant reagents may be potential therapeutics for diseases such as atherosclerosis as well as for diseases prevalent in third world countries such as schistosomiasis and malaria is truly exciting. Moreover, results were presented on the possible role of ecto-ATPases 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 literature. 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 ATPases. 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 ATPases.

Thus, in this historical meeting of ecto-ATPase research, the primary goal of reaching 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 various isozymes, which will aid in the establishment of a consistent and accurate nomenclature for the E-type ATPases, 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-ATPases, investigation of structure-function relationships, higher order protein structural determination, development of therapeutic agents (including specific inhibitors of E-type ATPases and injectable soluble forms of these enzymes), will surely bring this field the attention it deserves.

Finally, we wish to express our gratitude to the organizers of the VIIIth 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