Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase (PTP ) with high affinity. In this study, we examined the binding of midkine to PTP by solidphase binding assay. Midkine and pleiotrophin binding to PTP were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTP is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the Nand C-terminal halves, and the latter exhibited full activity for PTP binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg 78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys 83 and Lys 84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTP exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg 78 . These results suggested that Arg 78 in midkine plays an essential role in high affinity binding to PTP by interacting with the chondroitin sulfate portion of this receptor.PTP /RPTP␤ 1 is a receptor-like protein-tyrosine phosphatase, which is abundantly expressed in the central nervous system as a chondroitin sulfate proteoglycan (1-4). PTP is composed of an N-terminal carbonic anhydrase-like domain, a fibronectin type III domain, a serine, glycine-rich domain that is thought to be chondroitin sulfate attachment region, a transmembrane segment, and two tyrosine phosphatase domains (1, 2). There are three splice variants of this molecule: (a) the full-length PTP (PTP -A); (b) the short form of PTP , in which most of the serine, glycine-rich region is deleted (PTP -B); and (c) the secreted form (PTP -S), which corresponds to the extracellular region of PTP -A and is also known as 6B4 proteoglycan/phosphacan (3, 5). All these splice variants are expressed as chondroitin sulfate proteoglycans in the brain (6), suggesting that chondroitin sulfate plays an essential role in receptor function.Several proteins such as contactin, tenascin, L1, NCAM, and TAG1 have been reported to bind PTP (7-9). Contactin is thought to be a neuronal receptor of PTP expressed on glial cells (7). Recently, we found that PTP binds with pleiotrophin/ heparin-binding growth-associated molecule (10), in that a chondroitin sulfate portion of PTP constitutes a part of the pleiotrophin binding site and regulates the affinity of PTPpleiotrophin binding (10). We further demonstrated that pleiotrophin-induced neurite outgrowth and neuronal migration were suppressed by chondroitin sulfate, polyclonal antibodies against the extracellular domain of PTP , and sodium vanadate, a protein-tyrosine phosphatase inhibitor. These findings suggested that P...

Prolonged tissue damage or injury often leads to chronic pain states such that noxious stimuli evoke hyperalgesia and innocuous tactile stimuli evoke pain (allodynia). The neuropeptide nociceptin, also known as orphanin FQ, is an endogenous ligand for the orphan opioid-like receptor which induces both hyperalgesia and allodynia when administered by injection through the theca of the spinal cord into the subarachnoid space (that is, intrathecally). Here we show that the nociceptin precursor contains another biologically active peptide which we call nocistatin. Nocistatin blocks nociceptin-induced allodynia and hyperalgesia, and attenuates pain evoked by prostaglandin E2. It is the carboxy-terminal hexapeptide of nocistatin (Glu-Gln-Lys-Gln-Leu-Gln), which is conserved in bovine, human and murine species, that possesses allodynia-blocking activity. We have also isolated endogenous nocistatin from bovine brain. Furthermore, intrathecal pretreatment with anti-nocistatin antibody decreases the threshold for nociceptin-induced allodynia. Although nocistatin does not bind to the nociceptin receptor, it binds to the membrane of mouse brain and of spinal cord with high affinity. Our results show that nocistatin is a new biologically active peptide produced from the same precursor as nociceptin and indicate that these two peptides may play opposite roles in pain transmission.

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.

Seventy-four peptide amides of 7-amino-4-methylcoumarine (Mec) of the type Boc-Xaa-Yaa-Arg-NH-Mec were newly synthesized and tested to find specific substrates for blood-clotting proteases and trypsin. The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively. Among these peptides, the followings were found to be most sensitive substrates for individual enzymes: Boc-Asp(OBz1)-Pro-Arg-NH-Mec (k,,, = 160 s-', K, = 11 pM, k,,,/K, = 15000000 M-' s-') for human a-thrombin, Z-< Glu-Gly-Arg-NH-Mec (k,,, = 19 s-', K, = 59 pM, k,,,/K, = 320000 M-' s-') for bovine factor Xa, BocGln-Gly-Arg-NH-Mec (k,,, = 5.8 s-', K, = 140 pM, k,,,/K, = 42000) for bovine factor XIIa, Boc-Asp(OBz1)-Ala-Arg-NH-Mec (k,,, = 9.2 s-', K,,, = 120 pM, k,,,/K, = 77000 M-' s -l ) for bovine activated protein C, and Boc-Gly-Phe-Arg-NH-Mec (k,,, = 29 s-', K, = 230 pM, kcat/Km = 130000 M-' s-') for bovine plasma kallikrein. Moreover, Boc-Glu(OBz1)-Ala-Arg-NH-Mec (k,,, = 46 s-', K, = 370 pM, kcat/Km = 120000 M-' s-'> was newly found as a good substrate for human factor XIa. Bovine trypsin effectively hydrolyzed peptide-NH-Mec substrates containing Ala and Pro at the P2 site. The most reactive substrate was Boc-Gln-Ala-Arg-NH-Mec (k,,, = 120 sK1, K, = 6.0 pM, k,,,/K, = 20000000 M-' s-').Peptide amides of 7-amino-4-methylcoumarine (Mec) originally developed for the sensitive assay for a-chymotrypsin [l] [7], and horseshoe crab clotting factors [12]. Most of these substrates were designed based on the information from the COOH-terminal sequences of the 'activation peptides', which are liberated during the conversions of plasma serine protease zymogens to their active enzymes. The utility of these fluorogenic peptide substrates has been established for the assay of trace amounts of enzymes because of their high sensitivities. Moreover, the specific substrates for a-thrombin, factor Xa, plasma kallikrein and urokinase so far developed by our group [7] are now commercially available.

Ligand-receptor internalization has been traditionally regarded as part of the cellular desensitization system. Low-density lipoprotein receptor-related protein (LRP) is a large endocytosis receptor with a diverse array of ligands. We recently showed that LRP binds heparin-binding growth factor midkine. Here we demonstrate that LRP mediates nuclear targeting by midkine and that the nuclear targeting is biologically important. Exogenous midkine reached the nucleus, where intact midkine was detected, within 20 min. Midkine was not internalized in LRP-deficient cells, whereas transfection of an LRP expression vector restored midkine internalization and subsequent nuclear translocation. Internalized midkine in the cytoplasm bound to nucleolin, a nucleocytoplasmic shuttle protein. The midkine-binding sites were mapped to acidic stretches in the N-terminal domain of nucleolin. When the nuclear localization signal located next to the acidic stretches was deleted, we found that the mutant nucleolin not only accumulated in the cytoplasm but also suppressed the nuclear translocation of midkine. By using cells that overexpressed the mutant nucleolin, we further demonstrated that the nuclear targeting was necessary for the full activity of midkine in the promotion of cell survival. This study therefore reveals a novel role of LRP in intracellular signaling by its ligand and the importance of nucleolin in this process.

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Neurotoxic peptides from venoms of scorpions and honey bees exhibit a consensus pattern in the two disulfide bridgings related to the sequence portions Cys-X-Cys and Cys-X-X-X-Cys. A revised three-dimensional structure of charybdotoxin, as determined by two-dimensional nmr spectroscopy, confirms that the consensus cystine dislocation generates in all these toxins a common structural element, i.e., the cystine-stabilized alpha-helical (CSH) motif, which may be correlated with their common ion channel blocking activity.

Midkine (MK),Tissue type II transglutaminase (R-glutaminylpeptide: amine ␥-glutamyltransferase, EC 2.3.2.13) is a member of the transglutaminase family that catalyzes Ca 2ϩ