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We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.

Apoptosis is an evolutionarily conserved process that is critical for tissue homeostasis and development including sex determination in essentially all multicellular organisms. Here, we report the cloning of an ankyrin repeat-containing protein, termed F1Aalpha, in a yeast two-hybrid screen using the cytoplasmic domain of Fas (CD95/APO-1) as bait. Amino acid sequence analysis indicates that F1Aalpha has extensive homology to the sex-determining protein FEM-1 of the Caenorhabditis elegans, which is required for the development of all aspects of the male phenotype. F1Aalpha associates with the cytoplasmic domains of Fas and tumor necrosis factor receptor 1, two prototype members of the "death receptor" family. The F1Aalpha protein also oligomerizes. Overexpression of F1Aalpha induces apoptosis in mammalian cells, and co-expression of Bcl-XL or the dominant negative mutants of either FADD or caspase-9 blocks this effect. Deletion analysis revealed the center region of F1Aalpha, including a cluster of five ankyrin repeats to be necessary and sufficient for maximum apoptotic activity, and the N-terminal region appears to regulate negatively this activity. Furthermore, F1Aalpha is cleaved by a caspase-3-like protease at Asp(342), and the cleavage-resistant mutant is unable to induce apoptosis upon overexpression. F1Aalpha is therefore a member of a growing family of death receptor-associated proteins that mediates apoptosis.

Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1-10 microM) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including gamma-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents.

The treatment of purified rat muscle AMP deaminase with pryidoxal-5'-P produces a decrease of the enzyme activity which is time- and pyridoxal-5'-P concentration-dependent. The reaction is reversible and can be changed to an irreversible reaction by reduction with NaBH4. The reduced pyridoxal-5'-P-AMP deaminase complex shows an absorption spectrum which is typical for pyridoxamine derivatives. Chromatographic analyses confirm the formation of a Schiff base between pyridoxal-5'-P and the e-amino group of lysine residues in the enzyme. The loss of activity is not a simple inactivation process due to modification of the catalytic site of the enzyme. GTP and ATP, which are allosteric inhibitors of AMP deaminase, show a protective effect against pyridoxal-5'-P inactivation whereas KCl at high concentration exerts a little protection and Pi and P-creatine are ineffective. The reduced pyridoxal-5'-P-AMP deaminase derivative with 30 to 35% residual activity and 6 to 7 moles of pyridoxal-5'-P bound per mole of protein has different kinetic and regulatory properties from the native enzyme: Vmax is the same but the Km value is increased; the enzyme shows a homotropic cooperativity also in the presence of activators, K+ and ADP, and is no longer sensitive to GTP, ATP, and high salt concentration inhibitions. Pi and P-creatine still exert an inhibitory effect. These kinetic properties are similar to those of the native enzyme in the presence of nucleoside triphosphates. The reduced pyridoxal-5'-P-AMP deaminase derivative prepared in the presence of GTP is still sensitive to inhibition by nucleoside triphosphates. It is likely that pyridoxal-5'-P binds at or near the binding sites for these compounds.

This paper presents a kinetic study of the transamidinase reaction. The following results have been obtained. 1. The kinetic data are in agreement with a reaction mechanism involving two half-reactions with exclusively binary complexes between substrate and enzyme. 2. The true amidine acceptor is the form of the substrate bearing the unprotonated amino group. This has been demonstrated for hydroxylamine and glycine. 3. k1 and k5 vary as a function of the pH. This variation is related to the dissociation of a group or groups of the enzyme with a pK of approximately pH 8.5.