Identification of preussin as a selective inhibitor for cell growth of the fission yeast ts mutants defective in Cdc2-regulatory genes

In order to identify physiological activators of proteinase-activated receptor-2 (PAR-2), a peptide chloromethane inhibitor (biotinyl-Ser-Lys-Gly-Arg-CH2Cl) based on the cleavage site for activation of PAR-2 was synthesised and tested with 12 trypsin-like serine proteinases. The second-order rate constant (ki/Ki) for the formation of the covalent proteinase-inhibitor complex varied by 2 x 10(5)-fold between the proteinases. Biotinyl-Ser-Lys-Gly-Arg-CH2Cl reacted very rapidly with trypsin, acrosin from sperm and tryptase from mast cells: the ki/Ki values with these proteinases were greater than 10(5) M(-1) x s(-1). Thus, the specificity of these proteinases matched the sequence of the activation site of PAR-2 and it can be concluded that these proteinases are potential physiological activators of PAR-2.     

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.     

A novel technique for long-term vascular access in the unrestrained rat

125I-labelled recombinant human interferon alpha 2 (rHuIFN-alpha 2) capable of high-affinity binding (Kd = 2.46 +/- 0.18 x 10(-10) M) with receptors expressed on mouse thymocytes was obtained. Prothymosin alpha (proTM-alpha) but not cholera toxin was found to compete with radiolabelled IFN-alpha 2 for binding to the same receptor (Ki = 3.68 +/- 0.21 x 10(-11) M). The synthetic peptide covering the sequence 130-137 of IFN-alpha 2 (authors' definition: alpha-peptoferon) was shown to have the capacity to displace the labelled IFN-alpha 2 from the IFN-alpha 2/receptor complex (Ki = 7.19 +/- 0.12 x 10(-11) M). It was shown that receptors of this type are localized in plasmatic membrane fraction. Using [125I]-alpha-peptoferon, specific and saturable binding was detected on human fibroblasts and the data fitted a single binding site. Scatchard analysis yielded a Kd of 9.63 +/- 0.17 x 10(-8) M. The binding was competitively inhibited by IFN-alpha 2 (the Ki value in competition assays was 1.37 +/- 0.12 x 10(-8) M), proTM-alpha(Ki = 2.2 +/- 0.2 x 10(-7) M) and cholera toxin B subunit (Ki = 5.5 +/- 0.2 x 10(-7)). The present study has demonstrated that the sequence 130-137 of HuIFN-alpha 2 is involved in the competition of HuIFN-alpha 2, proTM-alpha and cholera toxin B subunit for common receptors on human fibroblasts.     

The interactions between a low molecular weight protease inhibitor of bronchial mucus and chymotrypsin-like cationic proteins of granulocytes

The proteolytic activity of the chymotrypsin-like cationic proteins of human granulocytes is inhibited by complex formation with the acid-stable low molecular weight protease inhibitor of human bronchial mucus (Ki = 1.0 x 10(7)M). The molar combining ratio is 1:1.     

Oxyanion-mediated inhibition of serine proteases

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].     

Perinatal exposure to morphine: reactive changes in the brain after 6-hydroxydopamine

The decapeptide H2N-Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-COOH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of the human immunoglobulin G1 Eu heavy chain and displaying a 43% identity with the antigenic determinant of beta-endorphin was synthesized. Immunorphin was found to compete with 125I-beta-endorphin for high-affinity receptors on murine peritoneal macrophages (K = 2.5 +/- 0.9 x 10(-9) M) and with 3H-morphin for receptors on murine thymocytes (Ki = 2.7 +/- 0.6 x 10(-9) M) and murine macrophages (Ki = 5.9 +/- 0.7 x 10(-9) M). In particular two types of receptors to 125I-beta-endorphin with Kd1 = 6.1 +/- 0.6 x 10(-9) M and Kd2 = 3.1 +/- 0.2 x 10(-8) M were revealed on macrophages. The second type of receptors interacted with 125I-beta-endorphin, 3H-Met-enkephalin, 3H-Leu-enkephalin and 3H-morphin; the first displayed reactivity with 125I-beta-endorphin, 3H-morphin and immunorphin. The first type receptors are not present on murine brain cells nor are inhibited by naloxone. A minimum fragment of immunorphin practically completely retaining its inhibitory activity in the competition tests with 125I-beta-endorphin for common receptors on thymocytes was found to correspond to the tetrapeptide H2N-Lys-Gly-Phe-Tyr-COOH (Ki = 5.6 +/- 0.5 x 10(-9) M).     

Expression, purification, and inhibitory properties of human proteinase inhibitor

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Resynthesis of reactive site peptide bond and temporary inhibition of Streptomyces metalloproteinase inhibitor

Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (Ki =1.14 x 10(-10) M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native inhibitor was resynthesized from the modified inhibitor (Ki =2.18 x 10(-8) M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25 degrees C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors. Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100 (mol/mol) enzyme-inhibitor ratio at 37 degrees C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the Ki values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower Ki values (approximately 2 pM) than thermolysin. In the limited proteolysis experiments weak inhibitors were degraded by the enzymes. SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases. SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon.     

Physical properties of lipid-protein complexes formed by the interaction of dimyristoylphosphatidylcholine and human high-density apolipoprotein A-II

Apolipoprotein A-II (apoA-II) from human plasma high-density lipoproteins associates with dimyristoylphosphatidylcholine (DMPC) to give complexes whose structure is determined by the temperature at which the reaction is conducted. The temperature dependence is related to the gel leads to liquid crystalline transition temperature, Tc, of DMPC which occurs at 23.9 degrees C. At T less than Tc (20 degrees C), T = Tc, and T greater than Tc (30 degrees C), three different complexes can be isolated. At 20 degrees C, at 75:1 (molar ratio of lipid to protein) complex is formed. This complex has a molecular weight (Mt) of 343 000, a Stokes radius, Rs, of 65 A, and a partial specific volume (v) of 0.914 mL/g. At 24 degrees C, two different complexes may be formed. One is similar to the one formed at 20 degrees C and the other is a complex with a DMPC:apoA-II ratio of 241:1; the corresponding physical constants for the latter complex are Mr = 1580 000, Rs = 120 A, and v = 0.948 mL/g. This complex is asymmetric, having a frictional coefficient f/f0 = 1.20. AT 30 degrees C, a 45:1 complex was formed; for this complex, Mr = 229 000, Rs = 57 A, and v = 0.892 mL/g. Electron microscopy reveals that the negatively stained complexes are arranged in rouleaux having subunits with average dimensions of 175 x 60, 250 x 62, and 50 x 55 A for the 45:1, 75:1, and 240:1 complexes, respectively. The multiple lipid-protein species formed by apoA-II and DMPC suggest the possible existence of more than one macromolecular spices of lipid and apoA-II in the plasma.     

Primary structure and possible functions of a trypsin inhibitor of Bombyx mori

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Two-dimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and Mr 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (Ki = 2.8 x 10-10), but does not alpha-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.     

A binding site for heparin in the apple 3 domain of factor XI

Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Nostrand, W. E., and Walsh, P. N. (1997) J. Biol. Chem. 272, 26139-26144), we examined binding of factor XI (FXI) and FXIa to heparin. FXIa binds to heparin (Kd approximately 0.7 x 10(-9) M) >150-fold more tightly than FXI (Kd approximately 1.1 x 10(-7) M). To localize the heparin-binding site on FXI, rationally designed conformationally constrained synthetic peptides were used to compete with 125I-FXI binding to heparin. A peptide derived from the Apple 3 (A3) domain of FXI (Asn235-Arg266) inhibited FXI binding to heparin (Kd approximately 3.4 x 10(-6) M), whereas peptides from the A1 domain (Phe56-Ser86), A2 domain (Ala134-Ala176), and A4 domain (Ala317-Gly350) had no such effect. The recombinant A3 domain (rA3, Ala181-Val271) inhibited FXI binding to heparin (Ki approximately 1.4 x 10(-7) M) indicating that all the information necessary for FXI binding to heparin is contained entirely within the A3 domain. The A3 domain also contains a platelet-binding site (Asn235-Arg266), consisting of three surface-exposed loop structures, Pro229-Gln233, Thr741-Leu246, and Thr249-Phe260 (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1995) J. Biol. Chem. 270, 6734-6740). Only peptide Thr249-Phe260 (which contains a heparin binding consensus sequence, RIKKSKA) inhibits FXI binding to heparin (Ki = 2.1 x 10(-7) M), whereas peptides Pro229-Gln233 and Thr241- Leu246 had no effect. Fine mapping of the heparin-binding site using prekallikrein analogue amino acid substitutions of the synthetic peptide Thr249-Phe260 and alanine scanning of the recombinant A3 indicated that the amino acids Lys252 and Lys253 are important for heparin binding. Thus, the sequence Thr249-Phe260 which contains most of the binding energy for FXI interaction with platelets also mediates the binding of FXI to heparin.     

On the inhibition of cholinesterases by pancuronium (author''s transl)

1. The inhibition by pancuronium of acetylcholinesterase (AChE) and of plasma cholinesterase (ChE) was investigated in vitro regarding a) the sensitivity of both enzymes; b) the mechanism and constants of inhibition; and c) the relationship between the neuromuscular blocking and the anticholinesterase activity of pancuronium. 2. Pancuronium is a reversible inhibitor of both AChE and ChE. The inhibitory potency regarding ChE ([I]50=2.7 X 10(-7) M; Ki=4.2 X 10(-8) M) is highly selective and about 1000-fold higher than compared to AChE ([I]50=2.4 X 10(-4) M; Ki=3.5 X 10(-5) M). 3. The kinetic analysis by means of an Lineweaver-Burk plot and an Arunlakshana-Schild plot displayed a pure competitive mechanism of inhibition. 4. The inhibition of AChE and ChE is thought to be induced by a reversible binding of pancuronium to the anionic subsite of the active center, thus decreasing the formation of the primary enzyme-substrate complex. 5. The clinical administration of pancuronium for muscular relaxation during anaesthesia (0.01-0.08 mg/kg) will result in a concentration of approximately 10(-7)=10(-6) M in the extracellular fluid. Thus, an inhibition of plasma ChE can be expected to occur under clinical conditions, however, probably without practical significance.     

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.     

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.     

Optimizing the binding of fullerene inhibitors of the HIV-1 protease through predicted increases in hydrophobic desolvation

We have developed and applied a computational strategy to increase the affinity of fullerene-based inhibitors of the HIV protease. The result is a approximately 50-fold increase in affinity from previously tested fullerene compounds. The strategy is based on the design of derivatives which may potentially increase hydrophobic desolvation upon complex formation, followed by the docking of the hypothetical derivatives into the HIV protease active site and assessment of the model complexes so formed. The model complexes are generated by the program DOCK and then analyzed for desolvated hydrophobic surface. The amount of hydrophobic surface desolvated was compared with a previously tested compound, and if this amount was significantly greater, it was selected as a target. Using this approach, two targets were identified and synthesized, using two different synthetic approaches: a diphenyl C60 alcohol (5) based on a cyclopropyl derivative of Bingel (Chem.Ber. 1993, 126, 1957-1959) and a diisopropyl cyclohexyl C60 alcohol (4a) as synthesized by Ganapathi et al. (J. Org.Chem. 1995, 60, 2954-2955). Both showed tighter binding than the originally tested compound (diphenethylaminosuccinate methano-C60, Ki = 5 microM) with Ki values of 103 and 150 nM, respectively. In addition to demonstrating the utility of this approach, it shows that simple modification of fullerenes can result in high-affinity ligands of the HIV protease, for which they are highly complementary in structure and chemical nature.     

Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.     

Heat-induced conversion of ovalbumin into a proteinase inhibitor

Ovalbumin is a member of the serine proteinase inhibitor (serpin) family but is unable to inhibit proteinases. Here we show that heating transforms it into inhibitory ovalbumin (I-ovalbumin), a potent reversible competitive inhibitor of human neutrophil elastase (Ki = 5 nM) and cathepsin G (Ki = 60 nM) and bovine chymotrypsin (Ki = 30 nM). I-ovalbumin also inhibits bovine trypsin, porcine elastase and alpha-lytic proteinase with Ki values in the micromolar range. Thus, I-ovalbumin differs from active serpins by its inability to form irreversible complexes with proteinases. I-ovalbumin is unusually thermostable: it does not undergo any structural transition between 45 degrees C and 120 degrees C as tested by differential scanning calorimetry, and it retains full inhibitory capacity after heating at 120 degrees C. It has 8% less alpha-helices and 9% more beta-sheet structures than native ovalbumin, as shown by circular dichroism. Our results show that the primary sequence of ovalbumin contains the information required for enabling the first step of the serpin-proteinase interaction to occur, i.e. the formation of the Michaelis-like reversible complex, but does not contain the information needed for stabilizing this initial complex.     

Mechanism of enhanced Na-K-ATPase activity in cortical collecting duct from rats with nephrotic syndrome

The maximal hydrolytic activity of Na-K-ATPase is specifically increased in the cortical collecting duct (CCD) of rats with puromycin-induced nephrotic syndrome (NS). This stimulation is independent of aldosterone and of endogenous ouabain-like substance. To investigate the mechanism responsible for this change, we compared the maximal Na-K-ATPase hydrolytic activity, the ouabain sensitive 86Rb influx, the specific [3H]ouabain binding, and the sensitivity of Na-K-ATPase to ouabain in the CCD of control rats and of rats given an intraperitoneal injection of puromycin 7 d before study. Both Na-K-ATPase activity and ouabain-sensitive 86Rb influx increased two-fold in rats with NS (ATPase activity: 34.1 +/- 2.1 vs. 18.0 +/- 0.7 pmol.mm-1 x min-1 +/- SE, n = 6, P < 0.001; Rb influx: 14.4 +/- 0.7 vs. 7.4 +/- 0.4 peq.min-1 +/- SE, n = 6, P < 0.001) whereas specific [3H]ouabain binding decreased in rats with NS (6.9 +/- 0.7 vs. 9.0 +/- 0.6 fmol.mm-1 +/- SE, n = 6, P < 0.005). Therefore, the maximal turnover rate of Na-K-ATPase increased over twofold in rats with NS (5,053 +/- 361 vs. 2,043 +/- 124 cycles.min-1 +/- SE, n = 6, P < 0.001). Analysis of the curves of inhibition of Na-K-ATPase by ouabain showed the presence of two Na-K-ATPase populations in both control and NS rats: a highly sensitive population (apparent Ki: 1.4 x 10(-6) M and 0.9 x 10(-6) M) and a less sensitive moiety (apparent Ki: 2.6 x 10(-4) M and 1.1 x 10(-4) M). The enhancement of Na-K-ATPase activity observed in the CCD of rats with NS was entirely due to the stimulation of the population of Na-K-ATPase with low ouabain sensitivity. These results suggest that a dysregulation of this subclass of Na-K-ATPase might be the primary cause of sodium retention in this model of nephrotic syndrome.     

Pharmaceutical and pharmacological development of antitumor prostaglandins]

The stoichiometry of the interaction between Erythrina variegata chymotrypsin inhibitor ECI and chymotrypsin was reinvestigated by analysis of their complex with ultracentrifugation and with amino acid analysis of the components separated. The amino acid analysis clearly showed that the stoichiometry of ECI and chymotrypsin was 1:1, though the apparent molecular mass of the complex was estimated to be 60 kDa. To examine the contribution of Leu64 (the P1 residue) to the inhibitory activity of ECI, a complete set of mutated inhibitors in which the amino acid at position 64 was replaced by 19 other amino acid residues was constructed by means of site-directed mutagenesis. Potent inhibitory activities (Ki, 1.3-4.6 x 10(-8) M) exceeding that of the wild-type ECI (Ki, 9.8 x 10(-8) M) were present in the mutant proteins L64F, L64M, L64W, and L64Y. The inhibitory activity of the mutant L64R was practically identical to that of the wild-type ECI. All other mutants exhibited slightly decreased inhibitory activities with Ki values of 1.9-4.6 x 10(-7) M. These results indicate that ECI-chymotrypsin interaction involves not only the P1 site residue but also other residue(s) of ECI. A series of individual alanine mutations was then constructed in residues Gln62 (P3), Phe63 (P2), Ser65 (P1'), Thr66 (P2'), and Phe67 (P3') in order to evaluate the contribution of each residue in the primary binding loop to the inhibitory activity. Replacement of Gln62, Phe63, and Phe67 with Ala residues decreased the inhibitory activity, the Ki values being increased by approximately 3-4-fold; but replacement of Ser65 and Thr66 had relatively little effect. This suggests that the P2, P3, and P3' residues, together with the P1 residue, in the primary binding loop play an important role in the inhibitory activity toward chymotrypsin.     

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.