We describe the opioid properties of a tridecapeptide, the sequence of which corresponds to the NH2-terminal sequence of dynorphin, a novel porcine pituitary endorphin. It The first pituitary opioid peptide to be discovered (1, 2) had properties quite different from those of /3-endorphin. It had a lower apparent molecular weight, was more basic, and had a more persistent effect in the guinea pig ileum bioassay. Also, its biologic activity (unlike that of f3-endorphin) was resistant to destruction by cyanogen bromide. The final stages of purification presented special problems, which led to erroneous conclusions about composition and the presence of a blocked NH2 terminus (3). Losses due to adsorption were particularly troublesome, and the material proved to be remarkably potent; consequently, the yield of purified product was much less than anticipated. However, by means of microsequencing technology (4, 5), we have now been able to determine the sequence of the first 13 residues.To denote its extraordinary potency, the natural peptide has been named "dynorphin" (dyn-from Greek dynamis = power). Synthetic dynorphin-(1-13) proved to have approximately the same high potency as natural dynorphin. We describe here some properties of this synthetic tridecapeptide. MATERIALS AND METHODSPurification and Partial Sequence Determination of Porcine Dynorphin. Starting material was 100 g of melanotropin concentrate, the second oxycellulose adsorbate in commercial corticotropin production from porcine pituitary glands (6). The initial steps, with 25-g batches, have been described (2): extraction and back-extraction with butanol; separation from ,B-endorphin on Bio-Gel P-6; preparative reversed-phase high-performance liquid chromatography on C18 columns, first with a methanol gradient in trifluoroacetic acid and then with an acetonitrile gradient in Tris buffer at neutrality, followed by elution of the active material with trifluoroacetic acid. Every step was monitored by assay on the guinea pig ileum myenteric plexus longitudinal muscle preparation (7,8).Pooled material from four batches, obtained as above, was loaded on a Bio-Gel column (P-6/P-4, 4:1; 1.5 X 90 cm) in 0.1 M (NH4)2CO0 at pH 8.7. Fractions (1.7 ml, 15 min) were tested for activity in the bioassay. The peak of slow-reversing activity (2) (fractions 65-75) was lyophilized and then further purified on CM-Sephadex (0.9 X 30 cm) equilibrated with 12.5 mM sodium borate at pH 11.0. The material was eluted with a 160-ml linear gradient from the starting buffer to 0.1 M phosphate buffer at pH 12.0. Fractions (2.0 ml; 15 min) were collected and assayed. Slow-reversing activity emerged between 94 and 106 ml. The two peak tubes, containing 50% of the activity, were pooled and desalted on Bio-Gel P-2 (1.5 X 90 cm) in n-butanol/acetic acid/water, 2:1:4 (vol/vol).Finally, the desalted material was subjected to reverse-phase chromatography (high-performance liquid chromatography, C18 column) using a 10-50% acetonitrile gradient in 5 mM trifluoroacetic acid. The pea...

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.

Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Microsomal triglyceride transfer protein (MTP) is MTP,1 expressed specifically in the liver and the small intestine, plays a critical role in the assembly and secretion of very low density lipoproteins (VLDLs) and chylomicrons. MTP exists in the lumen of the endoplasmic reticulum as a heterodimer with protein-disulfide isomerase and is involved in the transfer of triglycerides, cholesterol esters, and phospholipids to newly synthesized apo B (1, 2). If the apo B protein is not properly folded or if the enrichment of lipids is insufficient, the apo B protein is degraded by a ubiquitin-dependent proteasome process instead of proceeding to the formation of lipoprotein particles (3-5). In human patients with abetalipoproteinemia the absence of functional MTP results in a defect in the assembly and secretion of plasma lipoproteins containing apo B (6). A vital role of MTP in the formation and secretion of apo B is further supported by the fact that conditional gene knock-out mice specifically lacking hepatic MTP are unable to form VLDLs in the liver (7). Moreover, specific inhibitors of MTP lipid transfer have been developed and have lowered plasma cholesterol levels successfully (8). These findings clearly indicate that changes in MTP activities under various physiological conditions can modulate lipoprotein production and secretion in the liver and intestine.HNF-4 is a highly conserved member of the nuclear receptor superfamily. It is a liver-enriched transcription factor that, together with other factors, plays a key role in the tissuespecific expression of a large number of genes involved in lipid and glucose metabolism. The active form of HNF-4 is a homodimer, and it does not appear to heterodimerize with other members of the nuclear receptor family. Recent investigations have shown that coenzyme A derivatives of certain fatty acids activate the receptor, and these derivatives thus have been characterized as endogenous ligands for HNF-4 (9, 10). A crucial role for HNF-4 in metabolic homeostasis was demonstrated by the finding that mutations in the HNF-4 gene cause the disorder known as maturity onset diabetes of the young (11). Conditional HNF-4 gene knock-out mice, which were produced using the Cre-loxP method with an albumin-Cre transgene, exhibit a great reduction in serum cholesterol and triglycerides because of the decreased levels of MTP and several apolipoproteins (12). Although this result indicates that MTP gene expression is under the control of HNF-4, little is known about the specific step.It has been shown that the transcriptional activity of HNF-4 is regulated by interaction with small heterodimer partner (SHP), an atypical negative nuclear receptor lacking a DNAbinding domain (13,

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