Potent inactivator of alpha-chymotrypsin: 2,2-dimethyl-3-(N-4-cyanobenzoyl)amino-5-phenyl pentanoic anhydride

We synthesized a novel potent alpha-chymotrypsin inactivator, 2,2-dimethyl-3-(N-4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, which fulfilled the criteria of a mechanism-based inactivator: first-order kinetics, irreversibility, saturation kinetics and substrate protection. The inactivation rate constant (kinact) and the enzyme-inhibitor dissociation constant (KI) were calculated to be 0.017s-1 and 0.071 microM, respectively (kinact/KI = 242,000 M-1s-1). These kinetic parameters indicate that this compound is one of the most powerful alpha-chymotrypsin inactivators ever reported. The average number of alpha-chymotrypsin turnovers per inactivation (partition ratio) was calculated to be 1, which indicates that it is a stoichiometrically ideal inactivator of alpha-chymotrypsin. We compared the IC50 values of this compound with those of several chymotrypsin-like serine proteinases (bovine alpha-chymotrypsin, recombinant human chymase and human neutrophil cathepsin G) and a metallo proteinase, rabbit angiotensin converting enzyme (ACE). Our compound, 2,2-dimethyl-3-(N-4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, inhibited bovine alpha-chymotrypsin potently (IC50 = 1.0 (+/- 0.2) x 10(-9) M) as well as other chymotrypsin-like serine proteinase; recombinant human chymase (IC50 = 7.0 (+/- 1.0) x 10(-8) M) and human neutrophil cathepsin G (IC50 = 1.8 (+/- 0.2) x 10(-7) M). However, rabbit ACE was not inhibited by this compound (IC50 > 1 x 10(-4) M).     

Characterization of the reciprocal binding sites on human alpha-thrombin and factor XIII A-chain

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-alpha-thrombin interactions. Alpha-thrombin activated Factor XIII more efficiently (Km = 0.83 +/- 0.08 x 10(-7) M; V/K = 14.90 +/- 3.20 x 10(-3) min(-1)) than beta-thrombin (Km = 6.14 +/- 1.26 x 10(-7) M; V/K = 3.30 +/- 1.00 x 10(-3) min(-1)) or gamma-thrombin (Km = 6.25 +/- 1.15 x 10(-7) M; V/K = 3.00 +/- 0.80 x 10(-3) min(-1)). Immobilized FPR-alpha-thrombin bound plasma Factor XIII (Kd = 0.17 +/- 0.04 x 10(-7) M) > Factor XIIIa (Kd = 0.69 +/- 0.18 x 10(-7) M) > liver transglutaminase (Kd = 4.73 +/- 1.01 x 10(-7) M) > Factor XIII A-chain (Kd = 49.00 +/- 9.40 x 10(-7) M). FPR-alpha-thrombin and alpha-thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 x 10(-7) M and 13.6 x 10(-7) M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR-alpha-thrombin: IC50 = 1.0 x 10(-7) M, 3.0 x 10(-6) M and 1.52 x 10(-4) M, respectively. Transglutaminase also inhibited Factor XIII binding to alpha-thrombin (IC50 = 2.0 x 10(-6) M). Thrombin-binding site was localized to G38-M731 fragment of Factor XIII A-chain, probably within homologous regions (N72-A493) of transglutaminase. R320-E579 of alpha-thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in alpha-thrombin were essential for binding but not catalytic H363 or residues R382-N394 and R443-G475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.     

Specific substrates for spectrophotometric determination of penicillin acylase activity

Penicillin acylase substrates suitable for colorimetric determination of the enzyme activity have been tested in this study. The kinetic parameters (Km and kcat) have been elucidated for the following nine substrates: six phenylacetic acid derivatives (p-nitroanilide, p-nitrophenyl ester, p-nitro-m-carboxyanilide, p-nitro-o-carboxyanilide, p-nitro-o-hydroxyanilide, m-nitro-p-carboxyanilide), two D-phenylglycine derivatives (p-nitroanilide, p-nitro-m-carboxyanilide), and also p-nitrophenyl ester of acetic acid (p-nitrophenyl acetate). With the exception of p-nitrophenyl acetate, all the compounds studied are highly specific chromogenic substrates for penicillin acylase, but their reactivity is very variable and kcat/Km values are in a range from 0.8.10(4) to 5.10(6) M(-1).sec(-1).     

Measuring two-dimensional receptor-ligand binding kinetics by micropipette

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.     

Laser light scattering evidence for a common wormlike growth structure of mixed micelles in bile salt- and straight-chain detergent-phosphatidylcholine aqueous systems: relevance to the micellar structure of bile

We employed quasielastic and static light scattering to measure apparent values of the mean hydrodynamic radii (Rh)app, molecular weights (Mapp), and radii of gyration (Rg)app in solutions containing mixed micelles composed of bile salts (cholate and taurochenodeoxycholate, both cholanoyl derivatives) and the glycoacyl chain detergent, octyl glucoside, with egg yolk phosphatidylcholine (EYPC) as functions of total lipid concentration (0.1-10 g/dL), EYPC/detergent molar ratio (0-1.2), and ionic strength (0.15-0.4 M NaCl) at 20 degreesC and 1 atm. As the mixed micellar phase boundaries were approached by dilution, (Rh)app, Mapp, and (Rg)app values increased markedly by up to 20-fold. For each micellar system, the scaling ratios (Rh)app/Mapp1/2 and (Rg)app/(Rh)app remained essentially constant at 0.018 nm/(g/mol)1/2 and 1.5 (dimensionless), respectively, despite large variations in total lipid concentration, detergent molecular species, and ionic strength. Refined data analysis is inconsistent with a flat "mixed-disc" model for bile salt-EYPC micelles [Mazer, N. A., Benedek, G. B., and Carey, M. C. (1980) Biochemistry 19, 601] and octyl glucoside-EYPC micelles principally because the numerical value of (Rh)app/Mapp1/2 corresponds to a hypothetical disk thickness of approximately 1 nm, which is 4-fold smaller than the bimolecular width of EYPC molecules, and for a disk, (Rg)app/(Rh)app ratios should be close to 1 at low total lipid concentrations. Assuming disc-shaped micelles, we show that intermicellar excluded volume interactions would have only a minor effect on Mapp and cannot account for the unrealistic disk thickness. Instead, locally cylindrical, semiflexible wormlike micelles of diameter d = 4 nm and persistence length xip = 17 nm in solution are compatible with the observed (Rh)app/Mapp1/2 and (Rg)app/(Rh)app values when intermicellar excluded-volume interactions are considered. With EYPC/taurochenodeoxycholate = 0.6 and EYPC/cholate = 1.0 in 0.15 M NaCl, independent micelles grow upon dilution and use of the second virial coefficient [Egelhaaf, S. U., and Schurtenberger, P. (1994) J. Phys. Chem. 98, 8560] is adequate for estimating micellar weights. The systems EYPC/cholate = 1.0 in 0.4 M NaCl, EYPC/cholate = 1.2 in 0.15 M NaCl, and EYPC/octyl glucoside = 0.13 in 0.15 M NaCl all form highly overlapping, semidilute polymer solutions, which mimic the observed scaling ratios. In such semidilute systems, use of the second virial coefficient alone to account for intermicellar interactions is inadequate for estimating micellar weights. The results of the present study, in combination with locations of known phase boundaries of the ternary bile salt-EYPC-water phase diagram at high dilution, suggest that elongation, as well as entanglement of wormlike mixed micelles may occur at concentrations approaching the micellar phase limit.     

Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 x 10(-4) M) had effects similar to those of oxytocin pretreatment (1 x 10(-5) M). PKC depletion with phorbol 12-myristate 13-acetate (1 x 10(-6) M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 x 10(-5) M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 microg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.     

Properties of methemoglobin reductase and kinetic study of methemoglobin reduction

A soluble erythrocyte cytochrome b5 was purified as the substrate of methemoglobin reductase and an electron carrier to methemoglobin. The isoelectric point of this protein was at pH 4.3, and E0' was -0.010 at pH 7.0.. The Km value of the enzyme for this protein was 1 x 10(-4) M, and the turnover number (k5) was 3.4 x 10(4) min-1, with NADH as an electron donor at pH 7.0. The optimum pH of the enzyme was pH 4.6 for ferricyanide and pH 5.5 for cytochrome b5, with a shoulder of activity at pH 7 to 9 for both substrates. The rate equation which represents the reduction of either methemoglobin or cytochrome c was obtained as a function of methemoglobin or cytochrome c, methemoglobin reductase, and cytochrome b5 by considering the E . S complex for both reductase and cytochrome b5, and the rate constants involved were determined. The rate constants between methemoglobin and reduced cytochrome b5 (k1, M-1 min-1) were 1.6 x 10(4), 3.1 x 10(6), and 4.1 x 10(6) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constants between the reduced enzyme and oxidized cytochrome b5 (k'3, M-1 min-1) were 4.3 x 10(8), 12 x 10(8), and 9.3 x 10(8) at pH 7.0, pH 5.2, and pH 5.0, respectively. The rate constant between reduced hemoglobin and oxidized cytochrome b5 (k2) was 35 M-1 min-1 at pH 7.0. The theoretical Km for methemoglobin was 2.1 M at an infinite enzyme concentration at pH 7.0     

Ricin A-chain: kinetics, mechanism, and RNA stem-loop inhibitors

Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a kcat/Km of 4.5 x 10(5) M-1 s-1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower kcat values, but all have Km values of approximately 5 microM. Both the 10- and 18-base substrates have kcat/Km near 10(4) M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem-loop DNA (10 bases) of the same sequence is also a substrate with a kcat/Km of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O-riboside at the depurination site is not a substrate, but binds tightly to the enzyme (Ki = 0.34 microM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pKa of the adenine analogue at N7. Binding of this analogue (Ki = 9.4 microM) is weaker than substrate which indicates that the altered pKa at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (Ki = 0.18 microM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.     

Quantitation of beta 1 triiodothyronine receptor mRNA in human tissues by competitive reverse transcription polymerase chain reaction

Kinetics of photomodification of 26-meric deoxyribonucleotide pTTGCCTTGAATGGGAA-GAGGGTCATT with derivatives of the complementary oligonucleotides pTCTTCCCATTC, pTCTTCCCA, and pTTCCCA bearing a residue of (p-azidotetrafluorobenzoyl)aminopropylamine(-ArN3) attached to the terminal phosphate (reagents I, II, and III, respectively) was studied at 37 degrees C. It was established that during irradiation the reagents are inactivated, loosing their affinity to the target. A kinetic equation describing the modification was suggested. From the dependence of the time-limited modification level on the reagent concentration, the association constants of the reagents with the target were determined: [Kx = (9.9 +/- 0.4) x 10(4), (1.1 +/- 0.1) x 10(5), and (8.4 +/- 2.1) x 10(6) M-1 for reagents I, II, and III, respectively] and the efficiency of the modification in the complex gamma ef (ca. 0.3 for all the reagents) were determined. From the dependence of the modification level [PZ]/p0 on time for reagent II, the rate constant was determined for the rate-determining step of the photomodification k0 = (7.9 +/- 0.9) x 10(-3) s-1, which is close to the rate constant for the photolysis of p-azidotetrafluorobenzoic acid kp = (5.5 +/- 0.3) x 10(-3) s-1.     

Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occurring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the p450 proteins and the corresponding C. maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (Ks= 26 micron) and n-octadecane (Ks = 27 microM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks = 127 microM) and myristic acid (Ks = 134 microM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined for n-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax = 27 microM x min-1, Km = 54 microM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax = 30 microM x min-1, Km = 61 microM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids.     

Conjugation of classic Bowman-Birk soy inhibitor with a copolymer of ethylene oxide and propylene oxide

A classical soybean inhibitor of the Bowman-Birk type (BBI) with a copolymer of ethylene oxide and propylene oxide (PE) has been synthesized. The BBI-PE conjugate contain five covalently bound polymeric chains per one protein molecule and retains its capacity to inhibit trypsin (Ki = 10(-10) M), alpha-chymotrypsin (Ki = 7 x 10(-8) M) and human granulocyte elastase (Ki = 3 x 10(-8) M). The preservation of the antiproteinase activity in the antichymotrypsin center creates a prerequisite for the manifestation of the anticarcinogenic effect of the inhibitor.     

Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P. Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively. In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively. The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.     

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).     

Dimeric beta-cyclodextrin-based supramolecular ligands and their copper(II) complexes as metalloenzyme models

New supramolecular ligands possessing linear 13- and 15-membered pyridine diamidetriamine chelators between the primary sides of two beta-cyclodextrin cavities were synthesized, and characterized by MALDI-MS, NMR, IR and UV-Visible spectroscopy. Fluorescence and pH-metric titration were carried out in order to ascertain their behavior as bifunctional hosts for fluorescent guests and Cu(II) ion. The pKa value for the Cu(II) promoted deprotonation of amide ligands was determined to be 6.2 from pH-absorbance profile. Above pH 8.0, two deprotonated amides and three amino groups chelated Cu(II) ion, and yielded penta-coordinated Cu(II) complexes. The Cu(II) complexes catalyzed the hydrolysis of p-nitrophenyl acetate, adamantate and amino acids. Especially, the complex containing 13-membered chelator is an artificial metalloesterase with catalytic rate constant kcat = 3.8 x 10(-3) min-1 and Michaelis constant K(m) = 3.5 x 10(-4) M for the hydrolysis of p-nitrophenyl adamantate via metal-hydroxide mechanism.     

Studies on the mechanism of inhibition of tRNA methylation by 3,4-dihydroxyphenylethylamine

A Lineweaver-Burk analysis of a kinetic study of tRNA methylation by a 30-50% (NH4)2SO4 fraction from a weanling rat liver extract showed competitive inhibition with a Km for S-adenosylmethionine = 0.66 - 10(-6) M and a Ki for 3,4-dihydroxyphenylethylamine (dopamine) = 4 - 10(-5) M. The dopamine-inhibited methylation of tRNA appears to be linear with time. Rapid-flow dialysis studies indicated a S-adenosylmethionine binding constant of 0.65 - 10(-6) M. Dopamine appeared to interfere with the binding of S-adenosylmethionine to the weanling rat liver protein preparation but did not affect the binding of S-adenosylmethionine to protein in several systems in which dopamine did not inhibit tRNA methylase activity.     

M3-type muscarinic receptors predominantly mediate neurogenic quick contraction of bovine ciliary muscle

1. The present experiments were designed to investigate which subtypes of muscarinic receptors are involved in the neurogenic quick contraction of bovine ciliary muscle in connection to quick eye focal accommodation. 2. Transmural electrical stimulation (TES) produced a transient contraction, which was abolished in the presence of 3 x 10(-7) M tetrodotoxin and 10(-6) M atropine, but greatly augmented by 3 x 10(-7) M physostigmine. 3. The exogenously applied acetylcholine (ACh: 10(-9) to 3 x 10(-6) M) produced a concentration-dependent contraction, which was competitively antagonized by 10(-6) M atropine and augmented by 3 x 10(-7) M physostigmine, but unaffected by 3 x 10(-7) M tetrodotoxin. 4. The magnitude and time to peak of the maximal contraction produced by TES were significantly greater (1267.5 +/- 86.0 mg, P < 0.005) and shorter (9.0 +/- 0.2 sec, P < 0.005) than corresponding values (97.0 +/- 9.9 mg and 20.3 +/- 2.1 sec, respectively) of the phasic contraction caused by exogenously applied 10(-5) M ACh, at which concentration the agonist caused the maximal contraction. The velocity (140.6 +/- 7.8 mg/sec) of the transient contraction caused by TES was approximately 28-fold greater than that of the phasic contraction caused by ACh (5.1 +/- 0.9 mg/sec). 5. The contractions produced by TES were greatly attenuated by 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) as an M3 antagonist and slightly by pirenzepine as an M1 antagonist (20.2 +/- 7.9% inhibition at the highest concentration), but not by methoctramine (MET) as an M2 antagonist. The IC50 value (-log M) for 4-DAMP was determined to be 7.17 +/- 0.14. 6. Scatchard plot analysis of [3H]-quinuclidinylbenzilate (QNB) binding revealed that the binding sites constituted a single population with a Kd of 31.2 +/- 0.8 pM and a Bmax of 895.5 +/- 93.2 fmol/mg protein. The activity in inhibiting [3H]-QNB binding was most potent with 4-DAMP (-log Ki = 7.98 +/- 0.02), but less potent with pirenzepine (-log Ki = 6.43 +/- 0.04) and MET (-log Ki = 7.32 +/- 0.16). 4-DAMP was approximately 35- and 5-fold more potent than pirenzepine and MET in terms of -log Ki values, respectively, suggesting the predominant localization of M3 receptor subtypes in the bovine ciliary muscle membrane. 7. These results suggest that TES produces a neurogenic quick contraction of the bovine ciliary muscle, which would be mediated mainly by ACh released from the intramural nerve terminals and subsequent excitation of M3 receptor subtypes localized on the ciliary muscle cells, and that neurogenic quick contraction of the ciliary muscle is possibly involved in part in eye focal accommodation.     

A kinetic model for the competitive reactions of ozone with amino acid residues in proteins in reverse micelles

Lysozyme and 10 other proteins are solubilized in reverse micelles formed by 0.1 M sodium di-2-ethyl-hexylsulfosuccinate and 2.0-2.5 M water (pH 7.4) in isooctane solvent. Exposure of the protein-containing reverse micellar solutions to ozone causes oxidative damage to the proteins, as assessed by the oxidation of tryptophan residues. The oxidation product of the protein-bound tryptophan has a molar absorption coefficient of 3275 +/- 81 M-1 cm-1 (mean +/- S.D., n = 6) at 320 nm. The product is suggested to be a Criegee ozonide or a tautomer of the Criegee ozonide and not N-formylkynurenine. Ozonation of lysozyme in reverse micelles results in the formation of hydrogen peroxide in yields of only approximately 0.07 mol/mol of tryptophan residues oxidized. The recovery of hydrogen peroxide added as an internal standard to the lysozyme-containing reverse micellar solutions ranges from 84 to 88%, whether or not the samples are subjected to ozonation. This suggests that hydrogen peroxide is neither destroyed during the process of ozonation nor consumed by the protein to a significant extent in an adventitious reaction. A kinetic model for the overall reaction of ozone with the proteins is developed, taking into account the concentrations and the reactivities of individual amino acid residues toward ozone. The model predicts the fractional reaction of ozone with tryptophan residues in the proteins, despite differences in amino acid composition, molecular weight, and tertiary structures. The lack of influence of protein structure is confirmed further by the observation that the native lysozyme (with and without external S-carboxymethylcysteine) and S-carboxymethylated lysozyme give identical values of the fractional reaction of ozone with tryptophan residues. The kinetic equations for the competitive reactions of ozone with amino acid residues in proteins, with some minor modification, are applicable to ozonations on complex mixtures of lipids, proteins, and antioxidants.     

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.     

Higher frequency of concerted evolutionary events in rodents than in man at the polyubiquitin gene VNTR locus

The polyubiquitin gene is an evolutionarily conserved eukaryotic gene, encoding tandemly repeated multiple ubiquitins, and is considered to be subject to concerted evolution. Here, we present the nucleotide sequences of new alleles of the polyubiquitin gene UbC in humans and CHUB2 in Chinese hamster, which encode a different number of ubiquitin units from those of previously reported genes. And we analyze the concerted evolution of these genes on the basis of their orthologous relationship. That the mean of the synonymous sequence difference Ks which is defined as the number of synonymous substitution relative to the total number of synonymous sites, within the UbC and CHUB2 genes (0.192 +/- 0.096) is significantly less than Ks between these genes (0.602 +/- 0.057) provides direct evidence for concerted evolution. Moreover, it also appears that concerted evolutionary events have been much more frequent in CHUB2 than in UbC, because Ks within CHUB2 (0.022 +/- 0.018) is much less than that within UbC (0.362 +/- 0.192). By a numerical simulation, postulating that the major mechanism of concerted evolution in polyubiquitin genes is unequal crossing over, we estimated the frequency of concerted evolutionary events of CHUB2 at 3.3 x 10(-5) per year and that of UbC at no more than 5.0 x 10(-7) per year.     

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K+ channel (hKv1.5) stably expressed in Ltk- cells and using the whole-cell configuration of the patch-clamp technique. 2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K(D) values of 4.4+/-0.3 microM and 9.2+/-1.6 microM, respectively. 3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17+/-0.55 (n=10) and 0.16+/-0.81 (n=16). 4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9+/-0.9) x 10(6) M(-1) s(-1) and 39.5+/-4.2 s(-1), respectively. For 5-hydroxy-propafenone these values averaged (2.3+/-0.3) x 10(6) M(-1) s(-1) and 21.4+/-3.1 s(-1), respectively. 5. Both drugs reduced the tail current amplitude recorded at -40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a 'crossover' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed. 6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied. 7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.