The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

The wild-type -Phe*Pro-bond located at the N-terminus of the mature aspnrtic protcinasc of HIV-l was repluccd by -llc-Pro-or -V&Pro-. By this means, processing at this clcavngc junction was prevented and so. extended or precursor fcxs of HIV-protcinase were gcncmted. Thcsc constructs were expressed in ~xchcricl~iu cofi. purified therefrom, and their specificity. activity ut different pH values and susceptibility to the potent inhibitor, Ro3 l-8959, was assessed, A hitherto unobserved cleavage junction (at -Alu-Phe*Lcu-Gin-)in the frame-shift region of the gag-p01 viral genome was identified and contirmcd by demonstrating clcnvagc of a synthetic pcptide corresponding to this region. The implications for viral replication of self-processing at neutral pH by proteinase whilst still present (in a prccuisur tjrm) as a component of the polyprotcin arc considered; such reactions, however, are still blocked cvcn at pH values us high us 8.0 by Ro3 l-8959.HIV-proteinase precursor; Activity and pH dcpcndcnce; Inhibition by Ro31-8959; Novel polyprotein clcuvagc junction

We have extracted, purified, and sequenced an ANP-like peptide from the killifish. The peptide was extracted from whole brains with acidic acetone, and the aqueous phase remaining after evaporation of the acetone was subjected directly to HPLC. A pure peak was obtained after three successive HPLC steps. A key part of our purification method was the deliberate oxidation of methionyl residues in the peptide between the second and third HPLC steps. The purified peptide was chemically sequenced, and its molecular weight was determined by fast atom bombardment mass spectrometry (FABms). The peptide is 22 amino acids long and has considerable sequence similarity to the known natriuretic peptides, especially within the disulfide bonded "ring"; but unlike these known peptides it ends immediately after the second half cystine. Though it lacks a C-terminal "tail," the killifish peptide is equipotent to rat ANP in our radioimmunoassay, which employs an antiserum to the rat peptide. Furthermore, this brain peptide is equipotent to eel ANP in relaxing toadfish aortic rings, though both fish peptides are slightly less potent than rat ANP.

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