Characterization of soluble vs membrane-bound human placental 5'-nucleotidase.

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.

Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (CD73).

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.

NMR studies identify four intermediate states of ATPase and the ion transport cycle of sarcoplasmic reticulum Ca2+-ATPase.

Water proton nuclear relaxation measurements are used to detect and characterize four distinct intermediate states for Gd3+ bound to Ca2+ sites of sarcoplasmic reticulum Ca2+-ATPase in complexes with ATP analogues. In the absence of nucleotides, Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the nonhydrolyzable ATP analogue, Co(NH3)4AMPPCP, a new state for bound Gd3+ (still occluded and with fewer waters of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected in the NMR studies. The first of these probably represents an intermediate state for bound Gd3+ during ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and is probably analogous to the highly occluded E1-P state observed with CrATP [(1987) Biochim. Biophys. Acta 898, 313-322].

NMR relaxation measurements detect four intermediate states of ATPase and transport cycle of sarcoplasmic reticulum Ca2+-ATPase.

At least four of the intermediate states of Ca2+-ATPase (and presumably ion transport) can be trapped and characterized using water proton relaxation measurements. Gd3+ binds to two occluded Ca2+ transport sites on Ca2+-ATPase which have a low accessibility to solvent water. In the presence of the MgATP analogue Co(NH3)4AMPPCP, a new state for bound Gd3+ with one less water of hydration) is observed. In the presence of Co(NH3)4ATP or ATP, two additional states for bound Gd3+ are detected by NMR, the first of which probably represents an intermediate state of ATP hydrolysis. The latter is the most occluded Gd3+ site yet observed in these studies and corresponds to the highly occluded E1-P state observed with CrATP (Vilsen and Andersen, Biochim. Biophys. Acta 898, 313 (1987).

Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase.

Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these sites exhibits an unusually long electron spin relaxation time, consistent with occlusion of these sites and reduced contact with solvent H2O. In this report, the nature of the Gd3+ sites was examined in preparations of the enzyme solubilized with the detergent C12E8. The frequency dependence of water proton relaxation in solutions containing the solubilized Ca2+-ATPase yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of 1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound at site 2. The correlation time itself is frequency dependent below 30 MHz, indicating that the correlation time is dominated by the electron spin relaxation time of bound Gd3+. The long values of the correlation time found in the present study are consistent with a poor accessibility of these Gd3+ sites (particularly site 2) to solvent water molecules. Analytical ultracentrifugation and molecular sieve high performance liquid chromatography indicated that the active fraction of the soluble Ca2+-ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is largely dependent on the tertiary structure of the monomeric ATPase and does not appear to depend on multimeric membrane structures.