Membranes of pig kidney cortex tissue were solubilized in the presence of Triton X-100. Partial purification of ATP diphosphohydrolase (ATPDase) was achieved by successive chromatography on concanavalin A-Sepharose, Q-Sepharose Fast Flow, and 5'-AMP-Sepharose 4B. Monoclonal antibodies against ATPDase were generated. Further purification of the ATPDase was obtained by immunoaffinity chromatography with these monoclonal antibodies. NH(2)-terminal amino acid sequencing of the 78-kDa protein showed a sequence very homologous to mammalian CD39. The protein is highly glycosylated, with a nominal molecular mass of approximately 57 kDa. The purified enzyme hydrolyzed di- and triphosphates of adenosine, guanosine, cytidine, uridine, inosine, and thymidine, but AMP and diadenosine polyphosphates could not serve as substrates. All enzyme activities were dependent on divalent cations and were partially inhibited by 10 mM sodium azide. The distribution of the enzyme in pig kidney cortex was examined immunohistochemically. The enzyme was found to be present in blood vessel walls of glomerular and peritubular capillaries.

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth muscle, spleen and lung.The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence indicated seven putative N-glycosylation sites and one potential intracellular cGMP-and cAMP-dependent protein kinase phosphorylation site. As expected, the protein has a very high homology to other known mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions.Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7 cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by non±covalent interactions.

1. Using the incorporation of [methyl-3H]thymidine as a proliferation marker, the effects of various nucleosides and nucleotides on endothelial LLC-MK2 cells were studied. We found that ATP, ADP, AMP and adenosine in concentrations of 10 microM or higher stimulate the proliferation of these cells. 2. Inhibition of ecto-ATPase (EC 3.6.1.15), 5'-nucleotidase (EC 3.1.3.5) or alkaline phosphatase (EC 3.1.3.1) significantly diminished the stimulatory effect of ATP, indicating that the effect is primarily caused by adenosine and not by adenine nucleotides. Also, the effect depends only on extracellular nucleosides, since inhibition of nucleoside uptake by dipyridamole has no influence on proliferation. 3. Other purine nucleotides and nucleosides (ITP, GTP, inosine and guanosine) also stimulate cell proliferation, while pyrimidine nucleotides and nucleosides (CTP, UTP, cytidine and uridine) inhibit proliferation. Furthermore, the simultaneous presence of adenosine and any of the other purine nucleosides is not entirely additive in its effect on cell proliferation. At the same time any pyrimidine nucleoside, when added together with adenosine, has the same inhibitory effect as the pyrimidine nucleoside alone. 4. Apparently these proliferative effects are neither caused by any pharmacologically known P1-purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP, or D-myo-inositol 1,4,5-trisphosphate as second messenger, nor by extracellular Ca2+. 5. Therefore, we conclude that various purine and pyrimidine nucleosides can influence the proliferation of LLC-MK2 cells by acting on putative purinergic and pyrimidinergic receptors not previously described.

No abstract

Basal‐lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free‐flow electrophoresis. In each type of membrane, azide‐insensitive, low‐affinity Ca2+‐ATPase and Mg2+‐ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPase: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca‐ATP or Mg‐ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 μM of vanadate, N,N′‐dicyclohexylcarbodiimide, diethylstillbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types. The HCO3−‐stimulated Mg2+‐ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3−‐stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.

A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing 141Ce3+ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5'-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce3+ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.

The protein responsible for the (Ca'+ or Mg2+)-ATPase activity in brush-border membranes from pig kidney tubular cells was characterized to distinguish this enzyme from the N-ethylmaleimide-sensitive Mg2+-ATPase, also present in renal brush borders. Both enzymes are clearly different in their pH optimum and their sensitivity to divalent cations, nucleoside 5'-triphosphates and inhibitors.Solubilization of the (Ca'+ or Mg")-ATPase from brush-border membrane vesicles was accomplished with Nonidet P-40 or dodecylmaltoside. However, simultaneous inactivation of the enzyme was inevitable.A tenfold enrichment of the ATPase activity was obtained by chromatofocusing of Nonidet-P-40-solubilized brush borders. A similar degree of purification was achieved by ion-exchange chromatography of dodecylmaltoside-solubilized preparations. From the SDS/polyacrylamide gels of partially purified (Ca2+ or Mg'+)-ATPase, a few protein bands could still be tentatively identified as responsible for the enzyme activity.Labeling of solubilized brush-border preparations with several radioactive ATP analogues also revealed that a protein band of molecular mass 90kDa is the most probable candidate for the catalytic peptide of the (Caz+ or Mg'+)-ATPase.Finally, immunoprecipitation as well as semi-dry blotting with antibodies generated against partially purified enzyme preparations, confirmed that a 90-kDa component is a reasonable candidate for the (Ca'+ or Mg'+)-ATPase in renal brush-border membranes.

No abstract