Studies on the action mechanism of the antihemostatic effect of lodopeptides.

A synthetic iodopeptide having a glutamic acid-diiodotyrosine molar ratio of 1:1 has been shown to be an effective anticoagulant both in vivo and in vitro. Contrasted with heparin the following general conclusions may be made regarding its action. The iodopeptide does not act through the inactivation of thrombin in plasma. Iodopeptide does interact with fibrinogen to form a complex which, in vitro, is not soluble in buffered saline at physiological pH. At pH 8, iodopeptide interacts with fibrinogen to form a soluble complex in the presence of 0.9% NaCl that is not coaguable either by thrombin or Crotalus venom enzymes. All the available evidence indicates that the fibrinogen to fibrin conversion is not inhibited under these conditions, but that fibrin, once formed, is not able to polymerize due to interference by iodopeptide. Similar results were obtained with heparin in vitro with thrombin-fibrinogen mixtures in the absence of NaCl. Studies with Russell's viper venom in native PRP strongly suggest that the iodopeptide also interferes with processes in the early coagulation pathway associated with prothrombin activation.

Studies on the procoagulant activity of human amniotic fluid. I. Stability and coagulation factor requirements.

A fraction of human amniotic fluid possessing procoagulant activity was purified 35-70 fold by gel filtration on Sepharose 4B. The activity eluted in the void volume indicating a particle size in excess of 5 X 10(6) daltons. The amniotic fluid factor (AFF) activity is stabilized on storage in the presence of Ca++ ions which could not be replaced by Mg++. Addition of phospholipids resulted in accelerated loss of activity. Steps taken to remove factor VII did not affect the activity, but factor X and V are required.

Studies on the procoagulant activity of human amniotic fluid. II. The role of factor VII.

Partially purified Amniotic Fluid Factor (AFF) is found to directly activate human factor X, with a Km for factor X of 0.37 +/- 0.06 M. Added phospholipid has only a slight effect on the activation at low concentrations, and inhibits the reaction at higher concentrations. Both DIPF and PMSF inhibit AFF factor X activation. Although added phospholipid is not required for AFF-activity, phospholipase C rapidly destroys it, indicating the presence of intrinsic phospholipid. Phospholipase C treated AFF releases factor VII activity, which leads to the conclusion that AFF is in fact a thromboplastin: factor VII complex. Both AFF and a human brain thromboplastin factor VII complex prepared in vitro were inhibited by Zn++ ion, while human brain thromboplastin alone is not. AFF is markedly larger than the human brain thromboplastin-factor VII complex as judged by gel filtration.

Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis.

The purification and crystallization of T17, a toxin from Ptychodiscus brevis, is reported. The toxicity of this compound and a second toxin known as T34 are compared by i.v., i.p. and oral administration in mice. Both toxins produce symptoms characteristic of muscarnic stimulants; hypersalivation, rhinorrhea and excessive urination and defecation being the most commonly observed. T17, which is orally toxic, is believed to be the agent responsible for Neurotoxic Shellfish Poisoning.

Bronchoconstriction caused by Florida red tide toxins.

T17, a toxin purified from laboratory cultures of Florida's redtide organism, Ptychodiscus brevis (formerly Gymnodinium breve), produces bronchoconstriction in anesthetized artificially-ventilated guinea pigs. Bronchoconstriction, measured as a resistance to mechanical pulmonary inflation, was antagonized by atropine, but not by interruption of vagal nerve stimulation or diaphragm dissection.

Specific antibodies directed against toxins of Ptychodiscus brevis (Florida's red tide dinoflagellate).

Specific antibodies directed against Ptychodiscus brevis 'brevetoxins' have been produced in a goat. The haptenic toxin T34 was chemically reduced to toxin T17, covalently-linked to succinic acid via anhydride coupling, and coupled to bovine serum albumin using standard carbodiimide condensation procedures. The hapten coupling efficiency ranged from 10.4 to 13.5 moles of toxin bound per mole of protein. Antibody titers were directly related to the frequency of immunization, and weekly intervals appeared optimum for maintaining adequate titers. [3H]Brevetoxin T17 is displaced in a competitive manner from the antibody-antigen complexes by unlabeled toxin, but the antibodies do not distinguish between T17 and T34. The sensitivity achieved, using purified brevetoxins as competitive inhibitors of [3H]T17 binding, was 600 picograms. The assay linearity ranged from 1.5 to 48 ng.

Brevetoxin binding: molecular pharmacology versus immunoassay.

Brevetoxin PbTx-3 isolated from Florida's red tide dinoflagellate Ptychodiscus brevis has been produced recently in tritiated form by reductive tritiation of brevetoxin PbTx-2. Tritiated PbTx-3 has been used as a specific probe in competitive radioimmunoassays developed to detect brevetoxins in food sources, and this probe has also been utilized to characterize the brevetoxin binding component in rat brain synaptosomes. Brevetoxins PbTx-2 and PbTx-3, possessing the same structural backbone (type-1) as the tritiated probe, and PbTx-1 and PbTx-7, possessing a second structural backbone (type-2), have been compared quantitatively in their individual abilities to competitively displace tritiated PbTx-3 from its specific binding site in each assay. Type-1 toxins displaced labeled probe with ED50 values of 20-22 nM and 12-17 nM in radioimmunoassay and synaptosomes, respectively. Type-2 toxins displaced labeled probe with ED50 values of 92-93 nM and 3.5-4.1 nM in RIA and synaptosomes, respectively. Synaptosome assays reflect potency of each toxin examined, while radioimmunoassay reflects structural similarities to the immunizing toxin PbTx-3.

A heat stable paraoxonase (O,O-diethyl O-p-nitrophenyl phosphate O-p-nitrophenyl hydrolase) from Russell's viper venom.

Fractionation of Russell's viper venom revealed separate phosphohydrolase activities directed against p-nitrophenyl phosphate, bis(p-nitrophenyl) phosphate, p-nitrophenylthymidylic acid, and O,O-diethyl p-nitrophenyl phosphate (paraoxon). On gel fractionation, the first two activities are eluted ahead of the latter. They could be resolved further by phosphocellulose cation exchange chromatography. The hydrolytic activities directed against p-nitrophenylthymidylic acid hydrolyzing component is heat labile, while the paraoxon hydrolyzing component manifests an unusually high degree of heat stability. Gel filtration yields 9600 for the molecular weight of the "paraoxonase". This enzyme, as all known enzymes of this type, requires the presence of a divalent cation. Maximum activity is obtained in the presence of Ca2+. In the presence of Sr2+ the reaction rate is 50% of that of Ca2+; other divalent cations show lower activities. The presence of the enzyme is species specific. Of four species tested, only Russell's viper venom showed significant paraoxonase activity. Enzyme activity is intact following incubation with iodoacetate of p-chloromercuribenzoate. Activity is partially preserved even in the presence of 8 M urea.

A study of the binding of sulfur to rat liver microsomes which occurs concurrently with the metabolism of O, O-diethyl O-p-nitrophenyl phosphorothioate (parathion) to O, O-diethyl O-p-nitrophenyl phosphate (paraoxon).

In order to investigate the nature of the sulfur that is bound to liver microsomes concurrently with the metabolism of parathion to paraoxon, an isolated rat liver microsomal preparation was labeled with [35S] parathion and the purified product was examined by chemical degradation. Characterization of the degradation products by thin-layer chromatography indicated that the sulfur is bound as a polysulfide or hydrodisulfide formed by the combination of a reactive form of sulfur, released from parathion, with cysteine residues of the microsomal protein. Preliminary investigations revealed similar binding occurs when the microsomal preparation is replaced with purified rabbit cytochrome P-450 plus NADPH-cytochrome P-450 reductase and NADPH.

Brevetoxins, unique activators of voltage-sensitive sodium channels, bind to specific sites in rat brain synaptosomes.

The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique site associated with rat brain synaptosomes. Using [3H]brevetoxin PbTx-3 as a specific probe, binding was determined at 4 degrees in rat brain synaptosomes using a rapid centrifugation technique. Rosenthal analysis yields a KD of 2.9 nM and a Bmax of 6.8 pmol of toxin/mg of protein. Labeled probe can be displaced by unlabeled PbTx-3, PbTx-2, or synthetic PbTx-3 (reduced PbTx-2) but not by a nontoxic, synthetic oxidized derivative of PbTx-2. Competition experiments using natural toxin probes specific for sites 1-4 of the voltage-dependent sodium channel have illustrated that PbTx-3 does not bind to any of the previously described sites associated with the channel. A fifth site is proposed. In addition, because of the varied nomenclature associated with the brevetoxins, a new classification system is proposed.

Modification of adenylate cyclase by photoaffinity analogs of forskolin.

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.