Brutons's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N-terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X-ray synchrotron radiation scattering in solution. Low resolution shapes of the full-length protein and several deletion mutants determined ab initio from the scattering data indicated a linear arrangement of domains. This arrangement was further con®rmed by rigid body modelling using known high resolution structures of individual domains. The ®nal model of Btk displays an extended conformation with no, or little, inter-domain interactions. In agreement with these results, deletion of non-catalytic domains failed to enhance the activity of Btk. Taken together, our results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity. Keywords: ab initio models/protein tyrosine kinase/rigid body re®nement/Tec kinases/XLA Introduction Bruton's protein tyrosine kinase (Btk) is a member of the Tec family of non-receptor protein tyrosine kinases (nrPTK) (Neet and Hunter, 1996;Robinson et al., 2000;Smith et al., 2001). In humans its activity is required for the normal maturation of B lymphocytes, which are the source for circulating antibodies. Naturally occurring mutations in the Btk gene result in X-linked agammaglobulinaemia (XLA), a genetic disease characterized by the lack of mature B-cells, low levels of gammaglobulins and high susceptibility to bacterial infections (Smith et al., 1994b;Qiu and Kung, 2000;Cariappa and Pillai, 2002). Similar defects in mice produce a milder immunode®-ciency (Xid) (Rawlings et al., 1993;Thomas et al., 1993). Tec kinases share with Src and Abl/Arg family PTKs a basic cassette composed of SH3, SH2 and kinase domains. The SH3 and SH2 domains have important modular functions targeting signalling molecules to speci®c locations and may also regulate intramolecular interactions (Pawson and Nash, 2003). However, while Src and Abl contain lipid modi®cation signals at the N-terminus, Btk and other Tec family kinases display a Pleckstrin homology (PH) domain, which acts as a membrane targeting unit (Scharenberg et al., 1998;Baraldi et al., 1999). The region between the PH and the SH3 domains is unique to Tec kinases and has been called the Tec homology (TH) region (Smith et al., 1994b;Vihinen et al., 1994). It contains a zinc binding region called the Btk motif (BM), which docks against the PH domain and two proline-rich peptides. In some family members the proline-rich regions have been found to associate intramolecularly with the SH3 domain Hansson et al., 2001).Btk is expressed in all haematopoietic cell lineages except T cells (Smith et al., 1994a). A complete picture of Btk's role in B-cell development does not yet exist, but like other nr...

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Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na(+)K(+)2Cl(-)-cotransporter in both hybridoma and myeloma cells. Further, the Na(+)/H(+) exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH (inf4) (sup+) for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na(+)/H(+) exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.

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