The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.

Phosphofructokinases (PFK; EC 2.7.1.11) are tetrameric enzymes that have a key role in the regulation of glycolysis; as such, they are subject to allosteric activation and inhibition by various metabolites. Eukaryotic PFKs are about twice the size of prokaryotic enzymes and are regulated by a wider repertoire of effectors: for example, the subunit molecular weights of rabbit muscle (RM) PFK and Bacillus stearothermophilus (Bs) PFK are 82,000 and 36,000, respectively. Both enzymes are activated by ADP (or AMP), but RM-PFK is also activated by fructose bisphosphates (FBP) and inhibited by ATP and citrate. This, together with other evidence, has led to speculation that mammalian PFKs have evolved by duplication of a prokaryotic gene, although previous peptide analysis failed to reveal internal homology in RM-PFK. Here we demonstrate clear homology among the N- and C-halves of RM-PFK and Bs-PFK, thus establishing an evolutionary relationship by series gene duplication and divergence. Furthermore, detailed knowledge of the Bs-PFK structure provides the basis for inferences concerning the structural organization of RM-PFK and the evolution of new effector sites in the enzyme tetramer.

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Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurusflavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of a-helix (4"70), but much greater 0-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine. Keywords: amino acid sequence; secondary structure; snake venom fibrolase Fibrinolytic activity has been found in the venom of snakes from the families Crotalidae, Viperidae, and Elapidae, with venom from members of the Crotalidae having the highest levels of fibrinolytic activity (for a recent review, see Markland, 1988). Fibrolase is a direct-acting fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom that does not require bloodborne cofactors for activity (Guan et al., 1991). As previously described (Markland, 1983;Markland et al., 1988; Reprint requests to: Anne Randolph, Chiron Research Laboratories, Retzios & Markland, 1988), the enzyme is a nonhemorrhagic, zinc metalloproteinase which cleaves primarily the a-chain of human fibrinogen and fibrin. Specific cleavage sites have been determined for several venom metalloproteinases using natural or synthetic substrates (Tu et al., 1981;Hagihara et al., 1985;Fox et al., 1986;Mori et al., 1987).