Use of a monoclonal antibody to detect DNA damage caused by the anticancer drug cis-diamminedichloroplatinum (II) in vivo and in vitro.

A monoclonal antibody, MAb62-5, was prepared and used to detect DNA damage due to the anticancer drug cis-diamminedichloroplatinum (II) (or cisplatin). ELISA competition indicated that the binding of MAb62-5 to cisplatin-DNA was competitively inhibited (50% control) by 210 nM of cisplatin bound to DNA, cisplatin/nucleotide (D/N) = 0.2. Using a DNA mobility shift assay, MAb62-5 binding activity was inhibited by 50% by approximately 50-fold molar excess of cisplatin-DNA adducts (D/N = 0.08), whereas there was less than 5% inhibition by UV-DNA adducts or mock-treated DNA. In addition, MAb62-5 showed a similar affinity to the cisplatin-DNA adducts as compared to an endogenous cisplatin-damaged DNA recognition protein. Using ELISA with this antibody, we have demonstrated a 2-fold enhancement in excision repair of cisplatin-DNA adducts in resistant HeLa cells. This is supported by the measurement of repair-associated DNA strand breaks using alkaline elution and host cell reactivation of transfected plasmid DNA carrying cisplatin damage. These findings also provide explanation for the complexicity of immunoassay in cells.

Non-specific activity of (+/-)-CP-96,345 in models of pain and inflammation.

The non-peptide NK1 receptor antagonist, CP-96,345, and its 2R,3R enantiomer CP-96,344, which is not an NK1 receptor antagonist (IC50 > 10 microM), were evaluated for antinociceptive and anti-inflammatory activities in several classical models of pain and inflammation in the rat. Both CP-96,345 and CP-96,344 reduced carrageenin-induced paw oedema and hyperalgesia, and attenuated the second phase of formalin-induced paw licking with equal potency. These results indicate that NK1 antagonism is not responsible for the activity of (+/-)-CP-96,345 in the above animal models.

Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom.

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.

Interactions of alpha-chymotrypsin and Carlsberg subtilisin with methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates.

Methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for alpha-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2,3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1,2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by alpha-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl N alpha-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of alpha-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with Ki values which are generally smaller than the Km values for N alpha-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility necessary for catalysis.

Amino acid sequence of a coagulant enzyme, flavoxobin, from Trimeresurus flavoviridis venom.

The amino acid sequence of a coagulant enzyme, named flavoxobin, isolated from the venom of Trimeresurus flavoviridis (the habu snake) was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides generated by chemical (cyanogen bromide and hydroxylamine) and enzymatic (clostripain, Staphylococcus aureus V8 protease, Achromobacter protease I, and elastase) cleavages. Hydrazinolysis was also employed to determine the C-terminal amino acid. The enzyme consisted of 236 amino acids and had a calculated molecular weight of 25,744. Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from the venom of Bothrops atrox, and 27, 39, and 31% homologous to bovine thrombin, bovine trypsin, and human kallikrein, respectively. The sequence around the active site serine residue deduced from the homology relationship was Phe-Asp-Ser-Gly-Thr, which is different from the common sequence, Gly-Asp-Ser-Gly-Gly, for most serine proteases. Flavoxobin appears to be similar in secondary structure composition to batroxobin.

Purification and characterization of a coagulant enzyme, okinaxobin I, from the venom of Trimeresurus okinavensis (Himehabu snake) which releases fibrinopeptide B.

A coagulant enzyme, named okinaxobin I, has been purified to homogeneity from the venom of Trimeresurus okinavensis (Himehabu) by chromatographies on Sephadex G-100 and CM-Toyopearl 650M columns. The enzyme was a monomer with a molecular weight of 37,000 and its isoelectric point was 5.4. The enzyme acted on fibrinogen to form fibrin clots with a specific activity of 77 NIH units/mg. Fibrinopeptide B was released at a rate much faster than fibrinopeptide A. The enzyme exhibited 2 to 3 times higher activity toward tosyl-L-arginine methyl ester and benzoyl-L-arginine p-nitroanilide than bovine thrombin. The esterase activity was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and to a lesser extent by tosyl-L-lysine chloromethyl ketone, indicating that the enzyme is a serine protease like thrombin. The N-terminal sequence was highly homologous to those of coagulant enzymes from T. flavoviridis and Bothrops atrox, moojeni venoms which preferentially release fibrinopeptide A. In order to remove most, if not all, of the bonded carbohydrates, the enzyme was treated with anhydrous hydrogen fluoride (HF), thereby reducing the molecular weight to 30,000. The protein contained approximately 260 amino acid residues when computation was based on this value. The HF-treated enzyme retained about 50% of the clotting and esterolytic (TAME) activities and preferentially released fibrinopeptide B from fibrinogen. The carbohydrate moiety is not crucial for enzyme activity but might be necessary for eliciting full activity.

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion.

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.

Tryptophan residue essential for activity of Naja naja atra phospholipase A2.

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.

Purification and characterization of phospholipase A2 from Trimeresurus gramineus venom.

Phospholipase A2 was purified to homogeneity from the venom of Trimeresurus gramineus (the Green Habu snake) via three steps consisting of Sephadex G-75, DEAE-cellulose, and DEAE-Toyopearl 650M column chromatographies. Molecular weight determinations showed that the enzyme consists of a single polypeptide chain with a molecular weight of approximately 14,000. The isoelectric point was 4.5. The enzyme is characterized by high contents of acidic amino acids, glycine, and half-cystine. Calcium ion was essential for eliciting activity. The enzyme was inactivated by alkylation of a single histidine residue with p-bromophenacyl bromide following pseudo first-order kinetics, and the rate of the inactivation was depressed in the presence of Ca2+. The N-terminal sequence of this enzyme determined to the 40th residue was found to be highly homologous to that of Trimeresurus okinavensis phospholipase A2 but not to that of Trimeresurus flavoviridis phospholipase A2. The phenylalanine residue at the 27th position of T. gramineus phospholipase A2 is noteworthy because all other phospholipases A2, with only two exceptions, contain a tyrosine residue at this position.