40 Biochimica etBiophvsica Acta 847 (1985) 40 47 Elsevier BBA 11567 Identification of ecto-nucleoside triphosphate pyrophosphatase in human articular chondrocytes in monolayer culture Alison M. Caswell and R. Graham G. Russell Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, SIO 2RX (U.K.) (Received April 15th, 1985) Key words: Nucleoside triphosphate pyrophosphatase; ATP; (Chondrocyte) In cultured monolayers of human articular chondrocytes we have observed an enzyme activity which catalyzes the extracellular conversion of ATP to AMP and PPi- The enzyme was active at very low concentrations of ATP (/~M) and exhibited optimal activity at concentrations of ATP of approx. 100/~M. The enzyme was active in intact cells as judged by measurement of the release of the cytoplasmic marker enzyme lactate dehydrogenase. No increase in production of PPi from ATP was observed on mechanically disrupting the cells and no activity was shed into the medium by intact cells. Activity was stable between days 4 and 8 after subculturing the cells and was not affected by the timing of the final medium change prior to assay. Activity was also observed with other nucleoside triphosphates (GTP, CTP and UTP). We suggest that this activity is attributable to ecto-nucleoside triphosphate pyrophosphatase. This observation may be important in relation to the pathogenesis of the human disease of chondrocalcinosis in which crystals of calcium pyrophosphate dihydrate deposit in articular cartilage. Introduction The presence of enzyme activities on the cell surface is an increasingly recognized phenomenon. The term 'ectoenzyme' is used to define those enzymes whose active site is on the outside of the cell and which are an integral part of the plasma membrane [1]. One such activity is nucleoside tri- phosphate pyrophosphatase, which catalyzes the conversion of nucleoside triphosphate (NTP) to nucleoside monophosphate (NMP) and inorganic pyrophosphate (PPi)- This activity was first puri- fied from rat liver plasma membranes by Lieber- man et al. [2]. Subsequent work with rat and Abbreviations: Hepes, 4-(2-hydroxyethyl)-l-piperazineethane- sulphonic acid; Mops, 4-morpholinepropanesulphonic acid; NTP, nucleoside triphosphate; NMP, nucleoside monophos- phate. mouse liver preparations suggested that the en- zyme is active towards a variety of substrates including//,y-substituted derivatives of nucleoside triphosphates, nucleotide sugars, the activated sulphate derivatives and phosphodiester substrates [3-7]. Evidence has accumulated that the rat liver enzyme is an ectoenzyme [8-11] and studies with various substrates have suggested that similar ac- tivities exist in many other tissues of the rat. However, with most rat tissues, the location of this activity at the cell surface and the question of substrate specificity have yet to be adequately defined [6,7]. Work with human tissues has been more limited, but activities towards one or more of the various substrates have been observed in homogenates of several tissues, e.g., skin, kidney and brain [12-15]. Observations of activity against ATP by intact HeLa cells [16] and against UDP galactose by 0167-4889/85/$03.30 ~' 1985 Elsevier Science Publishers B.V. (Biomedical Division)