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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 The Xaa and Yaa residues of these substrates have been replaced by 12 and 15 different amino acids, respectively.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.

The properties of anhydrous hydrogen fluoride (HF) as a reagent for the acidolysis of various protective groups have been studied. Amino acids with various protective groups were each treated with HF at 0 or 20°C in the presence of anisole, and the reaction products were tested. Thus, HF was found to be much better than the other known reagents, such as hydrogen bromide and trifluoroacetic acid. In the present study, the following groups were shown, for the first time, to be removed safely: the nitro group in nitroarginine, the diphenylmethyl group in diphenylmethylamide, the t-butyl group in S-t-butylcysteine, the isopropyloxycarbonyl group, and the isopropyl ester group. A new and convenient apparatus was designed for the safe handling of HF for peptide synthesis.

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma. on receptor binding and CAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using ["51]rAM revealed the presence of a single class of high-affinity (& 1.3 x lo-* M) binding sites for rAM m VSMC. The apparent K, of rat calcitonin gene-related peptide (rCGRP) was 3 x lo-' M. Affinity labeling of VSMC membranes with ["'I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated CAMP formation with an approximate EC,, of lo-* M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced CAMP response was unaffected by propranalol. indomethacin, or quinacrine, but inhibited by a CGRP receptor antagonist, human CGRP[8-371. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.

A specific and sensitive radioimmunoassay for adrenomedullin has been developed. Half-maximal inhibition of binding of radioiodinated adrenomedullin was observed at 4 fmol/tube. The radioimmunoassay recognized the entire adrenomedullin molecule and has little crossreactivity with adrenomedullin fragment peptides. Adrenomedullin-like immunoreactivity was found to circulate in human plasma at considerable concentration (3.3 + 0.39 fmol/ml). The immunoreactivity of adrenomedullin was eluted at almost the same position as synthetic adrenomedullin on gel-filtration chromatography and reverse-phase high-performance liquid chromatography, suggesting that circulating adrenomedullin recognized by the present radioimmunoassay is identical or very similar to authentic adrenomedullin. Plasma immunoreactive adrenomedullin signiticantly increased in patients with hypertension, with a progressive rise proportionate to disease severity.

A major problem in polypeptide synthesis is the formation of products with a wide range of molecular weights. As a step toward the total synthesis of natural protein, an attempt was made to synthesize a sequential polypeptide with a defined molecular weight. Thus, poly-(L-prolyl-L-prolylglycyl) with ten or twenty repeating units was synthesized by the step-by-step addition of AOC-L-prolyl-L-prolylglycine (I)1) using the solid-phase procedure.2) The synthesis was started from glycine-anchored polystyrene (100-200 mesh, copolymerized with 2% divinylbenzene; 14g, 0.1mmol/g), to which AOC-L-prolyl-L-proline (1.4g)1) was coupled in methylene chloride with dicyclohexylcarbodiimide. The AOCgroup was then removed with 2N hydrogen chloride in acetic acid, and the tripeptide-cycle was extended with I (1.7g per cycle) and dicyclohexylcarbodiimide (0.9g per cycle); each coupling reaction was carried out in methylene chloride for 3hr at room temperature. After nine reaction cycles, when ten repeating units were expected to have been formed, a part of the resin was dried (wt 7g) and placed in an HFreaction cylinder;3) anisole (7ml) was added, and then anhydrous hydrogen fluoride (HF, was introduced over the resin by means of the HFexcess HF was removed, and the peptide liberated was extracted with water. The water extract was treated with IR-45 (OH-form), and then dialyzed against distilled water for 3 days. The final solution was lyophilized (II, wt 0.

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