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A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykinin or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin x bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8]bradykinin with IC50 values of 36.5 +/- 6 and 118.0 +/- 16.0 pmol/l from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/l. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 pmol/l, low-affinity IC50 = 22.6 nmol/l) in the absence of guanosine 5'-O-(3-thio-trisphosphate) (GTP gamma S); they shifted to a model of one affinity (8.1 nmol/l) in the presence of GTP gamma S. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/l to 1 mumol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/l) from a basal level of 72.8 +/- 16 pmol/mg protein to 433 +/- 35.5 at the highest (1 mumol/l) concentration. We conclude that bradykinin B2 receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.

Because of the importance of bradykinin in improving heart function in some conditions or in enhancing glucose uptake by skeletal muscle, we investigated kininases in these tissues. In P 3 fraction of the heart and skeletal muscles, angiotensin I-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) are the major kininases, as determined first with specific substrates and second with bradykinin. ACE activity was highest in guinea pig heart (2.7 ± 0.07 μmol·h −1 · mg protein −1 ) but decreased in other species in this order: dog atrium, rat heart, dog ventricle, and human atrium. The specific activity of NEP was lower: 0.45 μmol · h −1 · mg protein −1 in cultured neonatal cardiac myocytes and varying between 0.12 and 0.05 μmol · h −1 · mg protein −1 in human, dog, rat, and guinea pig heart. In the skeletal muscle P 3 , ACE was most active in guinea pig and rat (1.2 and 1.1 μmol · h −1 · mg protein −1 , respectively) but less so in dog (0.09 μmol · h −1 · mg protein −1 ). NEP activity was higher in dog P 3 (0.28 μmol · h −1 · mg protein −1 ) but lower in rat and guinea pig (0.19 and 0.1 μmol · h −1 · mg protein −1 , respectively). Continuous density gradient centrifugation enriched NEP activity in dog and rat (from 0.3 to 1.0 and 0.49 μmol · h −1 · mg protein −1 , respectively). Immunoprecipitation with antiserum to purified NEP proved the specificity of the rat enzyme. Bradykinin (0.1 mmol/1) was inactivated in the presence and absence of inhibitors by rat skeletal muscle NEP, as measured by high-performance liquid chromatography. Here, 36% of the activity was caused by NEP and 19% by ACE. In radioimmunoassay (bradykinin 10 nmol/1), 46 and 55% of kininase in rat and dog skeletal muscle P 3 , respectively, was due to ACE; 36 and 28%, respectively, was due to NEP. Aside from these enzymes, an aminopeptidase in rat P 3 also inactivates bradykinin. Thus, in conclusion, heart and skeletal muscle membranes contain kininase II-type enzymes, but their activity depends on the species.

Two arrowhead proteinase inhibitors (inhibitors A and B) were characterized and their primary structures were determined. Both inhibitors A and B are double-headed and multifunctional protease inhibitors. Inhibitor A inhibits an equimolar amount of trypsin and chymotrypsin simultaneously and weakly inhibits kallikrein. Inhibitor B inhibits two molecules of trypsin simultaneously and inhibits kallikrein more strongly than does inhibitor A. The amino acid sequences of inhibitors A and B were determined by sequencing the reduced and S-carboxamidomethylated proteins and their peptides produced by cyanogen bromide or proteolytic lysylendopeptidase or Staphylococcus aureus V8 protease cleavage. Inhibitors A and B consist of 150 amino acid residues with three disulfide bonds (Cys 43-Cys 89, Cys 110-Cys 119, and Cys 112-Cys 115) and share 90% sequence identity, with 13 different residues. Since the primary structures are totally different from those of all other serine protease inhibitors so far known, these inhibitors might be classified into a new protease inhibitor family.

A new peptide thrombin inhibitor was found in the Streptomyces griseus strain 254 isolated from a soil sample from Tongan, Fujian province, China, the inhibitor being a secondary metabolic product. The production of the inhibitor reached a maximum after 3 days culture of bacteria at 28°C in a rotary shaker. The inhibitor excreted in the culture filtrate was purified by absorption on macroporous resin, followed by ion exchange chromatography on DEAE-52, CM-32 cellulose, affinity chromatography on the immobilized thrombin and high performance liquid chromatography. The amino acid composition of the inhibitor was determined to be Val(2), Met(l), Ile(l), Leu(2) and Arg (1), similar to that of the amino acid residues around the reactive site of human antithrombin III, the critical plasma inhibitor of thrombin. The NH2-terminal residue of the inhibitor seems to be blocked by the alkyl group due to the negative reaction to ninhydrin, whereas the COO-terminal residue is most likely to be arginal because of that Arg was not found in the amino acid analysis, unless the peptide was oxidized by performic acid before acid hydrolysis. The chromogen substrates Bz-Phe-Val-Arg-PNA and Bz-Gly-Pro-Lys-PNA were used to determine the thrombin and plasmin activities, respectively. Besides thrombin, the purified inhibitor also exhibits a weak inhibitory activities on trypsin and much weak on plasmin, but not on chymotrypsin and other protein-ases.