The structure and function of cytosolic Ca 2؉ -binding proteins containing EF-hands are well understood. Recently, the presence of EF-hands in an extracellular protein was for the first time proven by the structure determination of the EC domain of BM-40 (SPARC (for secreted protein acidic and rich in cysteine)/osteonectin) (Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R., Jansonius, J. N., and Engel, J. (1996) Nat. Struct. Biol. 3, 67-73). The structure revealed a pair of EF-hands with two bound Ca 2؉ ions. Two unusual features were noted that distinguish the extracellular EF-hands of BM-40 from their cytosolic counterparts. An insertion of one amino acid into the loop of the first EF-hand causes a variant Ca 2؉ coordination, and a disulfide bond connects the helices of the second EF-hand. Here we show that the extracellular EF-hands in the BM-40 EC domain bind Ca 2؉ cooperatively and with high affinity. The EC domain is thus in the Ca 2؉ -saturated form in the extracellular matrix, and the EF-hands play a structural rather than a regulatory role. Deletion mutants demonstrate a strong interaction between the EC domain and the neighboring FS domain, which contributes about 10 kJ/mol to the free energy of binding and influences cooperativity. This interaction is mainly between the FS domain and the variant EF-hand 1. Certain mutations of Ca 2؉ -coordinating residues changed affinity and cooperativity, but others inhibited folding and secretion of the EC domain in a mammalian cell line. This points to a function of EF-hands in extracellular proteins during biosynthesis and processing in the endoplasmic reticulum or Golgi apparatus.

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Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.

Zusammenfassung:In KCl-stillgestellten Kaninchenherzen und in isch misch stillgestellten Hundeherzen war die Abbaurate der Adeninnucleotide etwa gleich gro . Nach 30 Min. waren 25-30% der Nucleotide zu Nucleosiden und Basen katabolisiert, nach 60 Min. rund 50%. Der Inosingehalt stieg bis auf 70-80% der Summe von Nucleosiden und Basen und erreichte nach l Std. in den Hundeherzen 3,61 μΜοΙ/g. Wie die geringeren Hypoxanthinkonzentrationen zeigten, begrenzt die Purinnucleo-sid-Phosphorylase-Reaktion den weiteren Abbau. Der Adenosingehalt lag in den Kaninchenherzen um 0,1 μΜοΙ/g und in den Hundeherzen um 0,2 μΜοΙ/g. Adenin und Xanthin waren nur in Spuren nachzuweisen. In den In-situ-Versuchen wurde ein Teil der Basen und Nucleoside aus dem Myocard ausgeschwemmt. Glykogen wurde in Kaninchen-und Hundeherzen etwa gleich schnell gespalten. (16-18 μΜο! Glucosylreste/g und Std.), obwohl sich die Ausgangswerte stark unterschieden. Summary: Degradation