N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester as a stable ester-type inhibitor of chymotrypsin-like serine proteases: structural requirements for potent inhibition of alpha-chymotrypsin

We introduce a new potent inhibitor, N-[2, 2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine alpha-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (kinact) and enzyme-inhibitor dissociation constant (Ki) against alpha-chymotrypsin were 0.0028 s-1 and 0.0045 microM, respectively (kinact/Ki = 630 000 M-1 s-1). These kinetic parameters indicate that this inhibitor is one of the most powerful alpha-chymotrypsin inactivators ever reported. On the basis of structure-activity relationship (SAR) and structure-stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl group, beta-substituent at the beta-amino acid residue, and ester structure, we suggest that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the beta-position and 4-cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.     

Inhibitors of acrosin and granulocyte proteinases from human genital tract secretions

Human seminal plasma contains two acid-stable proteinase inhibitors, HUSI-II (Mr approximately 6500) and HUSI-I, (Mr approximately 11 000) with different inhibition specificities. The inhibitory activity of HUSI-II is strongly limited to trypsin and acrosin; both enzyme-inhibitor complexes are very stable (e.g. bovine trypsin-HUSI-II complex: Ki = 1 x 10(-10)M; human acrosin-HUSI-II complex: Ki = 2.7 x 10(-10)M). The inhibitor from human seminal plasma HUSI-II may therefore be seen as the natural antagonist of the sperm protease acrosin. In addition to pancreatic trypsin and alpha-chymotrypsin, HUSI-I forms strong complexes with neutral proteases of the lysosome-like granules from human granulocytes, for example, the elastase (Ki = 2.5 x 10(-9)M) and cathepsin G, the chymotrypsin like protease (Ki = 7 x 10(-8)M).     

The 2.2 A crystal structure of human chymase in complex with succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural explanation for its dipeptidyl carboxypeptidase specificity

Human chymase (HC) is a chymotrypsin-like serine proteinase expressed by mast cells. The 2.2 A crystal structure of HC complexed to the peptidyl inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethylketone (CMK), was solved and refined to a crystallographic R-factor of 18.4 %. The HC structure exhibits the typical folding pattern of a chymotrypsin-like serine proteinase, and shows particularly similarity to rat chymase 2 (rat mast cell proteinase II) and human cathepsin G. The peptidyl-CMK inhibitor is covalently bound to the active-site residues Ser195 and His57; the peptidyl moiety juxtaposes the S1 entrance frame segment 214-217 by forming a short antiparallel beta-sheet. HC is a highly efficient angiotensin-converting enzyme. Modeling of the chymase-angiotensin I interaction guided by the geometry of the bound chloromethylketone inhibitor indicates that the extended substrate binding site contains features that may generate the dipeptidyl carboxypeptidase-like activity needed for efficient cleavage and activation of the hormone. The C-terminal carboxylate group of angiotensin I docked into the active-site cleft, with the last two residues extending beyond the active site, is perfectly localized to make a favorable hydrogen bond and salt bridge with the amide nitrogen of the Lys40-Phe41 peptide bond and with the epsilon-ammonium group of the Lys40 side-chain. This amide positioning is unique to the chymase-related proteinases, and only chymases from primates possess a Lys residue at position 40. Thus, the structure conveniently explains the preferred conversion of angiotensin I to angiotensin II by human chymase.     

Substituted 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives as novel nonpeptide inhibitors of human heart chymase

A series of 3-(phenylsulfonyl)-1-phenylimidazolidine-2,4-dione derivatives have been synthesized and evaluated for their ability to selectively inhibit human heart chymase. The structure-activity relationship studies on these compounds gave the following results. The 1-phenyl moiety participates in a hydrophobic interaction where an optimum size is required. At this position, 3,4-dimethylphenyl is the best moiety for inhibiting chymase and showed high selectivity compared with chymotrypsin and cathepsin G. A 3-phenylsulfonyl moiety substituted with hydrogen-bond acceptors such as nitrile and methoxycarbonyl enhances its activity. Molecular-modeling studies on the interaction of 3-[(4-chlorophenyl)sulfonyl]-1-(4-chlorophenyl)-imidazolidine-2,4-dione (29) with the active site of human heart chymase suggested that the 1-phenyl moiety interacts with the hydrophobic P1 pocket, the 3-phenylsulfonyl moiety resides in the S1'-S2' subsites, and the 4-carbonyl of the imidazolidine ring and sulfonyl group interact with the oxyanion hole and the His-45 side chain of chymase, respectively. The complex model is consistent with the structure-activity relationships.     

Two distinct forms of human mast cell chymase--differences in affinity for heparin and in distribution in skin, heart, and other tissues

Chymase, a chymotrypsin-like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin-agarose chromatography of high-salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0-1.2 M NaCl (peak B) and 1.8-2.0 M NaCl (peak C), with peak B containing 75-90% of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S-200. Both had a molecular mass of 28-29 kDa, yielded the N-terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.     

Irreversible inactivation of Caldariomyces fumago chloroperoxidase by hydrogen peroxide. A kinetic study in chloride and bromide system

The rate constants for the H2O2-induced irreversible inactivation (kinact) of chloroperoxidase from Caldariomyces fumago evaluated from the analysis of complete kinetic curves of chlorination or bromination of monochlorodimedon were found to follow the rate law kinact = k[H2O2]/(K + [H2O2]) with k = 0.009 > or = 0.002 and 0.0095 > or = 0.010 s-1 and K = (13 > or = 4) x 10(-3) and (9 > or = 2) x 10(-3) M in the presence of 0.01 M chloride and bromide, respectively, at pH 2.75 and 25 degrees C. The data show that chloroperoxidase investigated is more than by a factor of 10 less resistant toward hydrogen peroxide compared to horseradish peroxidase. The possible reason for it and the biotechnological implications are briefly discussed.     

Bacterial translocation and wasting in stressed mice

The structures of YM-51084 and YM-51085, new protease inhibitors produced by Streptomyces sp. Q21705, were determined by 1H- and 13C-NMR and mass spectrometry. Both were characterized by the basic structures of an acyl-tripeptide. YM-51084 was elucidated to be isovaleryl-tyrosyl-valyl-phenylalaninal and YM-51085 was the reduced phenylalaninol form of YM-51084. These compounds proved to strongly inhibit human kidney cathepsin L; the IC50 values being 9.6 x 10(-9) M and 3.5 x 10(-7) M, respectively.     

Induction of cytochrome P4503A by the antiglucocorticoid mifepristone and a novel hypocholesterolaemic drug

An acidic proteinase was purified from human kidney cortex. The enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtration, and isoelectric points of 5.2 and 6.1. The optimum pH for hydrolysis of bovine hemoglobin was about 3.5. Reverse-phase HPLC analysis of the incubation mixture of the enzyme with human plasma showed the presence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. The specific activities were 2.91 micrograms MLBK equivalent mg-1.min-1 at pH 3.5 and 2.15 micrograms MLBK equivalent mg-1.min-1 at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstatin A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage points. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Leu-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Arg-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred at the Arg-Ser linkage. Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp++ + was hydrolyzed by the renal acidic proteinase and yielded the peptide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinectic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp (K(m) = 0.69 +/- 0.08 microM; Kcat = 0.052 +/- 0.0095 s-1; Kcat/K(m) = 0.075 +/- 0.005 microM-1.s-1) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K(m) = 1.56 +/- 0.16 microM; Kcat = 0.0048 +/- 0.0001 s-1; Kcat/K(m) = 0.003 +/- 0.0003 microM-1.s-1). Human liver cathepsin D had no activity on C-terminal sequences and human pepsin hydrolyzed them at the Ser-Ser bond. The results suggest that the renal acid proteinase is distinct from human pepsin and human liver cathepsin D and releases MLBK from human kininogen.     

Human chymase, an enzyme forming novel bioactive 31-amino acid length endothelins

We report the novel role of human chymase in the production of bioactive 31-amino acid length endothelins (ETs), which may play a role in allergies and vascular diseases. In the bronchi of asthmatic patients, the vascular tissue in atherosclerosis, and the heart muscle in cardiac hypertrophy, both ET-like immunoreactivity and the accumulation of mast cells significantly increase. Chymase from human mast cells selectively cleaves big ET-1, -2 and -3 at their Tyr31-Gly32 bonds, and produces novel bioactive 31-amino acid length ETs, ETs(1-31), without any further degradation products. However, chymases from other species, human cathepsin G, and porcine alpha-chymotrypsin, degrade big ETs. ETs(1-31) at concentrations between 10(-9) M and 10(-7) M exhibited various contractile potencies in rat tracheae and porcine coronary arteries in a dose-dependent manner. Furthermore, ET-1(1-31) at concentrations between 10(-14) M and 10(-10) M caused a significant increase in the intracellular free Ca2+ concentration. The contractile activity of ETs(1-31) may not be the consequence of conversion to the corresponding ETs(1-21) by phosphoramidon-sensitive ET converting enzyme(s) or other chymotrypsin-type proteases and metallo-endopeptidases, because the contractile activity was not significantly inhibited on treatment with inhibitors of these proteases prior to the addition of ET-1(1-31).     

Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.     

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.     

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.     

Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle

A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.     

Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bovine liver cytochrome b5 in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration (in the presence of 1 mM (Ru(NH3)6)3+, pH 7.0 and I=0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b5, respectively. A comparative two-dimensional NMR study of cytochrome b5 and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42 approximately Glu44 segment in cytochrome b5 may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b5 was studied by optical difference spectra of cytochrome c and variants of cytochrome b5. The association constants (KA) for the wild-type, E44A, E56A, and E44/56A mutants of cytochrome b5 with cytochrome c, are 4.70(+/-0. 10)x10(6) M-1, 1.88(+/-0.03)x10(6) M-1, 2.70(+/-0.13)x10(6) M-1, and 1.14(+/-0.05)x10(6) M-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b5 and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b5 and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrome b5 (1.074(+/-0.49)x10(7) M-1 s-1) is higher than those of the mutant protein E44A (8.98(+/-0.20)x10(6) M-1 s-1), E56A (8.76(+/-0. 39)x10(6) M-1 s-1), and E44/56A (8.02(+/-0.38)x10(6) M-1 s-1) at 15 degreesC, pH 7.0, I=0.35 M. The rate constants are strongly dependent on ionic strength and temperature. These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b5 and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.     

Design of mechanism-based carboxypeptidase A inactivators on the basis of the X-ray crystal structure and catalytic reaction pathway

The X-ray crystal structure of the complex of carboxypeptidase A (CPA) and Gly-Tyr, has been documented. The crystal structure reveals that both the amide carbonyl oxygen and the terminal amino nitrogen of Gly-Tyr coordinate to the active site zinc ion of CPA in a bidentate fashion, whereby the zinc-bound water molecule is displaced by the amino group. As to the catalytic mechanism of CPA, it is generally believed that while in the cases of ester substrates the carboxylate of Glu-270 functions as the nucleophile which attacks the scissile carbonyl carbon (anhydride pathway), in the case of peptide substrates the zinc-bound water molecule attacks the scissile peptide bond (general base pathway). In light of the X-ray crystal structure and the proposed catalytic mechanism for the enzyme, it is envisioned that the ester bond of O-(hydroxyacetyl)-L-beta-phenyllactic acid (L-1) would be hydrolyzed by the attack of the carboxylate of Glu-270 to generate an anhydride intermediate. The latter intermediate would then undergo an intramolecular rearrangement initiated by the attack of the hydroxyl to result in to form an ester bond with the Glu-270 carboxylate. This ester formation impairs the catalytic activity of CPA. We have demonstrated using kinetic analysis that L-1 is indeed an inactivator for the enzyme having the Kinact/KI value of 0.057 M-1 s-1. We have also demonstrated that N-(hydroxyacetyl)-L-phenylalanine (L-2) inactivates the enzyme with the kinact/KI value of 0.071 M-1 s-1, suggesting that the carboxylate becomes to attack the peptide carbonyl carbon to generate the same anhydride intermediate as that formed in the inactivation of CPA by L-1. The formation of the anhydride intermediate rather than a tetrahedral transition state that is expected for peptide type substrates was envisioned to occur on the ground that the zinc-bound water molecule is displaced by the hydroxyl of L-2 upon binding to the enzyme.     

Pharmacological characterization of tachykinin-induced intracellular calcium rise in a human NK2 receptor transfected cell line

We have examined the effect of various natural and synthetic tachykinins on the steady state Ca(++)-rise ([Ca++]i) in transfected chinese hamster ovary cells expressing recombinant human Neurokinin 2 (NK2) receptors. The rank order of potency with natural tachykinins was NeurokininA > Neurokinin B > Eledoisin > Physaelamin > substance P. The selective NK2 agonist, [beta-Ala8]NKA(4-10) was very potent, with an EC50 value of 4.83 x 10(-9) M whereas Senktide, MePhe7NKB and Sar9, (MetO2)11 substance P, selective NK3 and NK1 agonists, respectively, did not have any effect on [Ca++]i in hrNK2CHO cells, suggesting a selective and preferential recognition and activation of NK2 receptors in these cells. (+/-) SR 48968, a selective NK2 antagonist, abolished the beta-AlaNKA-induced [Ca++]i with an IC50 value of 0.7 nM. Two other peptidic NK2 antagonists, MEN 10376 and L-658977, were less active with IC50 values of 49 nM and 5.29 microM, respectively. In contrast, (+/-) CP-96,345 and (+/-)CP-99,994 and RP 67580, all selective NK1 antagonists, did not have any effect on the beta-AlaNKA-induced [Ca++]i in hrNK2CHO cells (+/-) SR 140333, a potent and selective NK1 antagonist, had a 35% inhibition under similar conditions. These data demonstrate a high selectivity and sensitivity to NK2 receptor mediated [Ca++]i in rhNK2R-CHO cells and may be of value as a rapid, selective test of drug action at the human NK2 receptors in vitro.     

Adaptive survival trials

1. An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2-4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonyl-methyl] acrylamide) has been identified. 2. This compound displaced [3H]-BK binding to B2 receptors present in guinea-pig ileum membranes with an IC50 of 5.6 x 10(-10) M and in rat uterus with an IC50 of 1.5 x 10(-9) M. It did not inhibit different specific radio-ligand binding to other receptor sites. 3. In human lung fibroblast IMR-90 cells, FR173657 displaced [3H]-BK binding to B2 receptors with an IC50 of 2.9 x 10(-9) M and a Ki of 3.6 x 10(-10) M, but did not reduce [3H]-des]Arg10-kallidin binding to B1 receptors. 4. In guinea-pig isolated preparations, FR173657 antagonized BK-induced contractions with an IC50 of 7.9 x 10(-9) M, but did not antagonize acetylcholine or histamine-induced contractions even at a concentration of 10(-6) M. FR173657 caused parallel rightward shifts of the concentration-response curves to BK at concentrations of 10(-9) M and 3.2 x 10(-9) M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration-response curve at a concentration of 10(-8) M. Analysis of the data yield a pA2 of 9.2 +/- 0.2 (n = 5) and a slope of 1.5 +/- 0.2 (n = 5). 5. In vivo, the oral administration of FR173657 inhibited BK-induced bronchoconstriction dose-dependently in guinea-pigs with an ED50 of 0.075 mg kg-1, but did not inhibit histamine-induced bronchoconstriction even at 1 mg kg-1. FR173657 also inhibited carrageenin-induced paw oedema with an ED50 of 6.8 mg kg-1 2 h after the carrageenin injection in rats. 6. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.     

The effects of reverse micelles on the reaction mechanism of alpha-chymotrypsin

Reverse micelles were employed to test the accuracy of the widely accepted mechanism for alpha-chymotrypsin in a highly structured aqueous system similar to intracellular conditions. Results yielded from spectrophotometrical assays of the alpha-chymotrypsin catalyzed hydrolysis of both p-nitrophenyl acetate (p-NPA) and p-nitrophenyl trimethylacetate (p-NPTA) were kinetically analyzed to determine constants typical of the proposed mechanistic model. This was accomplished through the establishment of a control, i.e. the well studied buffer system, for comparison between the reverse micellular environment and a bulk aqueous solution. Control group results yielded kinetic constants in favor of the proposed mechanism (Km = 1.55 x 10(-5) +/- 1.40 x 10(-6) M for p-NPA and a Km = 4.97 x 10(-6) +/- 2.29 x 10(-7) M, Km(app) = 4.92 x 10(-6) +/- 2.33 x 10(-8) M, k2 = 4.34 x 10(-3) +/- 1.31 x 10(-3), k(cat) = 1.96 x 10(-3) +/- 2.47 x 10(-4), and Ks = 1.60 x 10(-5) +/- 4.61 x 10(-6) M for p-NPTA). In contrast, similar reactions of the enzyme in a reverse micellular system produced kinetic constants atypical to that representative of the textbook mechanism. (Km = 1.59 x 10(-4) +/- 2.70 x 10(-5) M, Ks = -8.67 x 10(-5) +/- 4.46 x 10(-5) M and Km(app) = -4.80 x 10(-5) +/- 7.05 x 10(-5) M for p-NPA and Km = 1.95 x 10(-4) +/- 9.28 x 10(-5) M, Km(app) = -1.79 x 10(-4) +/- 2.36 x 10(-5) M, and Ks = -3.95 x 10(-4) +/- 1.18 x 10(-4) M for p-NPTA). In addition to negative kinetic constants, alpha-chymotrypsin seemed to display characteristics indicative of super-activity and a hysteretic response. Overall, the widely accepted mechanism for alpha-chymotrypsin appeared to fail within the confines of reverse micelles, due to the direct influence of the system's highly structured form.     

Solution structure, rotational diffusion anisotropy and local backbone dynamics of Rhodobacter capsulatus cytochrome c2

The solution structure, backbone dynamics and rotational diffusion of the Rhodobacter capsulatus cytochrome c2 have been determined using heteronuclear NMR spectroscopy. In all, 1204 NOE-derived distances were used in the structure calculation to give a final ensemble with 0.59(+/-0.08) A rms deviation for the backbone atoms (C, Calpha and N) with respect to the mean coordinates. There is no major difference between the solution structure and the previously solved X-ray crystal structure (1.07(+/-0.07) A rms difference for the backbone atoms), although certain significant local structural differences have been identified. This protein contains five helical regions and a histidine-heme binding domain, connected by a series of structured loops. The orientation of the helices provides an excellent sampling of angular space and thus allows a precise characterization of the anisotropic diffusion tensor. Analysis of the hydrodynamics of the protein has been performed by interpretation of the 15N relaxation data using isotropic, axially asymmetric and fully anisotropic diffusion tensors. The protein can be shown to exhibit significant anisotropic reorientation with a diffusion tensor with principal axes values of 1.405(+/-0.031)x10(7) s-1, 1.566(+/-0.051)x10(7) s-1 and 1.829(+/-0.054)x10(7) s-1. Hydrodynamic calculations performed on the solution structure predict values of 1.399x10(7) s-1, 1.500x10(7) s-1 and 1.863x10(7) s-1 when a solvent shell of 3.5 A is included in the calculation. The optimal orientation of the diffusion tensor has been incorporated into a hybrid Lipari-Szabo type local motion-anisotropic rotational diffusion model to characterize the local mobility in the molecule. The mobility parameters thus extracted show a quantitative improvement with respect to the model-free analysis assuming isotropic reorientation; helical regions exhibit similar dynamic properties and fewer residues require more complex models of internal motion. While the molecule is essentially rigid, a tripeptide loop region (residues 101 to 103) exhibits flexibility in the range of 20 to 30 ps, which appears to be correlated with the order in the NMR solution structure.     

Characterization of electrogenic Na/K pump in rat neostriatal neurons

The electrogenic Na/K pump current (Ip) was studied in the dissociated neostriatal neurons of the rat by using the nystatin-perforated patch recording mode. The Ip was activated by external K+ in a concentration-dependent manner with an EC50 of 0.7 mM at a holding potential (VH) of -40 mV. Other monovalent cations also caused Ip and the order of potency was Tl+>K+, Rb+>NH4+, Cs+>Li+. The Ip decreased with membrane hyperpolarization in an external solution containing 150 mM Na+, while the Ip did not show such voltage dependency without external Na+. Ouabain showed a steady-state inhibition of Ip in a concentration- and temperature-dependent manner at a VH of -40 mV. The IC50 values at 20 and 30 degrees C were 7.1 x 10(-6) and 1.3 x 10(-6) M, respectively. The decay of Ip after adding ouabain well fitted with a single exponential function. At a VH of -40 Mv, the association (k+1) and dissociation (k-1) rate constants estimated from the time constant of the current decay at 20 degrees C were 4.0 x10(2) s-1 M-1 and 6.3 x 10(-3) s-1, respectively. At 30 degrees C, k+1 increased to 2.8 x 10(3) s-1 M-1 while k-1 showed no such change with a value of 1.8 x 10(-3) s-1. A continuous Na+ influx was demonstrated by both the Na+-dependent leakage current and tetrodotoxin-sensitive Na+ current, which resulted in the continuous activation of the Na/K pump. It was thus concluded that the Na/K pump activity was well-maintained in the dissociated rat neostriatal neurons with distinct functional properties and that the activity of the pump was tightly connected with Na+ influxes.