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PubMedDate not supplied

Synthesis and renin inhibitory properties of a new soluble pepstatin derivative.

A new pepstatin derivative, pepstatinyl arginine methyl ester hydrochloride (pepstatinyl-Arg-O-Me), was synthesized with the aim of increasing water solubility without altering capacity to inhibit the renin-angiotensinogen reaction. Pepstatinyl-Arg-O-Me was shown to inhibit in vitro the reaction between either rat or purified hog renin and the natural rat renin substrate. In a fluorimetric assay, this derivative competitively inhibited the reaction between purified hog renin and a synthetic N-acetyl-tetradecapeptide renin substrate with a Ki of the same order of magnitude as that of pepstatin. In urethaneanesthetized ganglion-blocked rats, binephrectomized 24 hr before the experiments, both the plasma reinin concentration and the rise in blood pressure following intravenous injection of hog renin were inhibited by pepstatinyl-Arg-O-Me disappeared within 60 min. Pepstatinyl-Arg-O-Me inhibited the 1.4 mumoles/kg. The in vivo inhibitory effect of 1.4 mumoles/kg of pepstatinyl-Arg-O-Me disappeared within 60 min. Pepstatinyl-Arg-Op-Me inhibited the reaction of endogenous and exogenous renin with plasma substrate both in vitro and in vivo.

Angiotensin II↗
PubMed1982

Pepstatin inhibition of proteinase-free human renin.

The kinetics of inhibition of proteinase-free human renin and of pepsin by the carboxyl proteinase inhibitor pepstatin have been examined. Inhibition was of the tight-binding type with both enzymes. Inhibition of crude human renin was of the classical, freely reversible type, but most of the renin-like activity of the preparation was due to contaminating proteinases which could be separated chromatographically from the renin. These results clarify previous kinetic studies of pepstatin inhibition of human renin.

Animals↗
PubMed1998

Nucleotide sequence of the gene encoding the precursor protein of pepstatin insensitive acid protease B, scytalidopepsin B, from Scytalidium lignicolum.

A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.

Amino Acid Sequence↗
PubMed1984

An improved procedure for the histochemical demonstration of cathepsin D by the mercury-labeled pepstatin method.

The desirable fixation conditions for the histochemical demonstration of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor were examined biochemically and histochemically. Four well known fixatives, namely, glutaraldehyde (GA), paraformaldehyde (PFA), glutaraldehyde with paraformaldehyde (GA-PFA) and periodate-lysine-paraformaldehyde (PLP), were applied to the prefixation of tissues prior to the reaction of the labeled inhibitor to the enzyme-active site. The effects of fixatives on cathepsin D were biochemically examined using subcellular fractionated lysosomes. Cathepsin D from rat liver lysosomes was rapidly inactivated by the fixatives containing glutaraldehyde, i.e., GA and GA-PFA, whereas the activity of cathepsin D was sufficiently maintained after fixing the enzyme in the PFA or PLP preparations. Effects of the PLP fixative on lysosomal cathepsin D in liver tissues using the mercury-labeled pepstatin method were also studied histochemically. The best result for the visualization of lysosomal cathepsin D in liver tissues was obtained using the PLP fixative with the prefixation time of three hours or more.

Animals↗
PubMedDate not supplied

Pepstatin inhibition of rat myocardial acid protease.

The protease inhibitor, pepstatin, inhibited acid protease in rat heart homogenates with a Ki of 7 x 10(-11) M by a noncompetitive mechanism, but it had no effect on myocardial acid protease activity when given in high doses to intact animals. Exposure of heart slices to 10(-7) M pepstatin for two hours also failed to produce demonstrable acid protease inhibition, suggesting that myocardial acid protease activity is unaffected by this potent inhibitor unless the integrity of the cell is disturbed.

Animals↗
PubMed1990

Crystallographic analysis of a complex between human immunodeficiency virus type 1 protease and acetyl-pepstatin at 2.0-A resolution.

The mode of binding of acetyl-pepstatin to the protease from the human immunodeficiency virus type 1 (HIV-1) has been determined by x-ray diffraction analysis. Crystals of an acetyl-pepstatin-HIV-1 protease complex were obtained in space group P2(1)2(1)2 (unit cell dimensions a = 58.39 A, b = 86.70 A, c = 46.27 A) by precipitation with sodium chloride. The structure was phased by molecular replacement methods, and a model for the structure was refined using diffraction data to 2.0 A resolution (R = 0.176 for 12901 reflections with I greater than sigma (I); deviation of bond distances from ideal values = 0.018 A; 172 solvent molecules included). The structure of the protein in the complex has been compared with the structure of the enzyme without the ligand. A core of 44 amino acids in each monomer, including residues in the active site and residues at the dimer interface, remains unchanged on binding of the inhibitor (root mean square deviation of alpha carbon positions = 0.39 A). The remaining 55 residues in each monomer undergo substantial rearrangement, with the most dramatic changes occurring at residues 44-57 (these residues comprise the so-called flaps of the enzyme). The flaps interact with one another and with the inhibitor so as to largely preserve the 2-fold symmetry of the protein. The inhibitor is bound in two approximately symmetric orientations. In both orientations the peptidyl backbone of the inhibitor is extended; a network of hydrogen bonds is formed between the inhibitor and the main body of the protein as well as between the inhibitor and the flaps. Hydrophobic side chains of residues in the body of the protein form partial binding sites for the side chains of the inhibitor; hydrophobic side chains of residues in the flaps complete these binding sites.

Amino Acid Sequence↗
PubMedDate not supplied

[Isolation of cathepsin D from the aorta using pepstatin sepharose].

Affinity chromatography on pepstatin-Sepharose was used for isolation of cathepsin D from pig aorta. The enzyme was purified 800-fold. The proteolytic activity of cathepsin D was completely inhibited by pepstatin, suggesting that there were no other proteinases in the enzyme preparation obtained.

Animals↗
PubMed1981

A rapid and large-scale isolation of renin from mouse submaxillary gland by pepstatin-aminohexyl-agarose affinity chromatography.

A pressor enzyme, renin, was purified about 60-fold by chromatography on an affinity column including pepstatin-aminohexyl-agarose with a high yield of 83% from the homogenate of adult mouse submaxillary glands. The renin obtained by the one-step purification was electrophoretically homogenous on SDS-polyacrylamide gel and was as active as an absolutely pure renin. The renin purified by the affinity column could be separated into five active components by chromatography on CM-cellulose. Each of these renins gave a symmetrical elution profile on the CM-cellulose column and a discrete protein band on polyacrylamide gel electrophoresis at pH 8.6. Administration of nanogram quantities of each of the two major renin fractions to nephrectomized rats caused a sustained rise of blood pressure and decrease in sensitivity of the animal to angiotensin II. This rapid and large-scale purification method using pepstatin-aminohexyl-agarose eliminates all four fractionation steps reported previously for the isolation of mouse submaxillary gland renin.

Animals↗
PubMed1982

Inhibition of cysteine proteinase activity by Z-Phe-Phe-diazomethane and of aspartic proteinase activity by pepstatin in different organs from some animals and isolated cells from rat liver.

Two methods have been developed to discriminate simultaneously between the main part of cysteine proteinase activity (cathepsin L) and all aspartic proteinase activity (mainly cathepsin D) in rat organs, using Z-Phe-Phe-CHN2 which at 5 mumol/l completely inhibits cathepsin L from rat liver and, on the other hand, pepstatin which at 0.5 mumol/l completely inhibits cathepsin D. Substrates are double-labeled cytosol proteins from rat liver at pH 3.0 or azocasein in 3 mol/l urea at pH 5.0. Several organs from rat, pigeon, frog and carp have been investigated using these methods. Especially kidneys from rat, frog and carp contain a high Z-Phe-Phe-CHN2 inhibited activity. Investigating the different liver cell types we could confirm earlier findings that Kupffer cells and endothelial cells contain more pepstatin inhibited activity than parenchymal cells.

Animals↗
PubMed1993

Purification and characterization of kumamolysin, a novel thermostable pepstatin-insensitive carboxyl proteinase from Bacillus novosp. MN-32.

We have found a novel type of thermostable, pepstatin-insensitive carboxyl proteinase in the culture filtrate of Bacillus novosp. MN-32. The carboxyl proteinase, which was named kumamolysin, was purified about 8,300-fold by column chromatography including DEAE-Sepharose CL-6B, Sephadex G-100, and TSKgel DEAE-5PW. The purified kumamolysin gave a single band corresponding to 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of kumamolysin was estimated to be 40 kDa by gel filtration. The isoelectric point of kumamolysin was estimated to be pH 3.5 by isoelectric focusing. Kumamolysin has maximum proteolytic activity at 70 degrees C and at pH 3.0. Kumamolysin specifically hydrolyzed the Leu15-Tyr16 peptide bond in oxidized insulin B-chain (Km = 9.0 x 10(-5) M, Kcat = 71 s-1; at pH 3.0, 30 degrees C), and additional cleavage at Phe25-Tyr26 was detected at a considerably lower rate. Kumamolysin is insensitive to the known carboxyl proteinase inhibitors pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Kumamolysin has no similarity to the thermostable acid protease thermopsin from Sulfolobus acidocaldarius (Lin, X.-L., and Tang, J. (1990) J. Biol. Chem. 265, 1490-1495). Thus, the substrate specificity, the inhibitor sensitivity, the molecular mass, and the thermostability all suggest that kumamolysin is a novel type of carboxyl proteinase.

Amino Acid Sequence↗
PubMed2006

An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium.

In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.

Animals↗
PubMed1977

Structural and functional determinants of Mucor miehei protease VI. Inactivation of the enzyme by diazoacetyl norleucine methyl esters, pepstatin and 1,2-epoxy-30(p-nitro-phenyoxy)propane.

Mucor miehei protease (EC 3.4.21 -- ), an acid protease of fungal origin, was rapidly inhibited at pH 5.0 and 10 degrees C by a 78-fold molar excess of diazoacetyl norleucine methyl ester (N2Ac-Nle-OMe) when simultaneously added with a 78-fold molar excess of Cu(II). Preincubation with Cu(II) before the addition of N2Ac-Nle-OMe reduced the initial rate of activity loss presumably due to a copper-induced structural change as deduced from an examination of CD spectra. Cof norleucine and 1.02 +/- 0.041 mol of copper. The conformation of the N2Ac-Nle-OMe-inhibited enzyme appeared to be somewhat altered since the rate of H-3H exchange determined for the slowest exchanging class of hydrogens was reduced by more than 10-fold although the estimated number of hydrogens in this class remained constant. Mucor miehei protease was also inhibited by pepstatin; complete inactivation required a 6-fold molar excess of inhibitor and was associated with a major conformational change as determined from CD spectra. Loss of activity also occurred in the presence of 1,2-epoxy-3-(p-nitrophenyoxy)propane (EPNP).

Aminocaproates↗
PubMed1980

Binding of Streptomyces pepsin inhibitor (acetyl-pepstatin) with chymosin (Rennin).

Chymosin (Rennin) was effectively purified using an AH-Sepharose 4B column. Binding of Streptomyces pepsin inhibitor (acetul-pepstatin) with chymosin was studied spectroscopically. The binding caused ultraviolet difference and CD spectral changes suggesting microenvironmental changes around tryptophan and/or tyrosine residue(s) in chymosin. The fluorescence intensity of a hydrophobic probe, 2-p-toluidinylnaphthalene-6-sulfonate, increased in the presence of chymosin and was further amplified when Streptomyces pepsin inhibitor was added to the chymosin-2-p-toluidinylnaphthalene-6-sulfonate solution. The binding and dissociation-rate constants between chymosin and the inhibitor were determined using 2-p-toluidinylhnaphthalene-6-sulfonate as a probe. The binding constant was determined from the binding and dissociation-rate constants, to be 3.1 . 10(7) M-1 at 25 degrees C, pH 5.5.

Chymosin↗
PubMed1982

Distribution of pepstatin and statine following oral and intravenous administration in rats. Tissue localisation by whole body autoradiography.

The distribution in rats of two radiolabelled derivatives of the potent pepsin inhibitor, pepstatin A, has been monitored by whole-body autoradiography. There was a very poor absorption of pepstatinyl-[14C]glycine and N-[3H]acetylstatine across the gastric and intestinal mucosae after oral administration. Both inhibitors were rapidly cleared from the blood by the liver and kidneys after intravenous administration. Neither inhibitor was localised in the gastric mucosa at a concentration that could be expected to be effective in inhibiting intracellular pepsin activity.

Administration, Oral↗
PubMed1988

Kinetics of in vivo inhibition of tissue cathepsin D by pepstatin A.

1. We have investigated the kinetics of inhibition of cathepsin D in heart, liver and skeletal muscle of CD-1 mice following administration of 25, 50, 100 and 200 mg/kg i.p. of pepstatin A, a specific inhibitor of this protease. 2. In the liver, a significant inhibition of cathepsin D occurred up to at least 15 days, whereas, in heart and skeletal muscle, this inhibition lasted for a much shorter period of time. 3. These results show that the recovery of enzyme activity to normal values is dose-dependent and that, at the same dose level, marked differences occur in the recovery of enzyme activity in these organ tissues, the liver being the most sensitive one.

Animals↗
PubMed1992

Bovine brain cathepsin D: inhibition by pepstatin and binding to concanavalin A.

1. Cathepsin D from bovine brain has been purified 1100-fold in 46% recovery. Three isozymes are present with pI (+/- 0.05) = 6.10, 6.30 and 6.40. 2. The isozymes are single polypeptide chains with apparent Mr = 42,000 and are similar with respect to substrate binding and cleavage; the pH-optimum is 3.5 with virtually no activity at neutral pH. 3. Pepstatin inhibits the enzyme and kinetic data are consistent with a "tight binding" mechanism. 4. The dissociation constant for the concanavalin A-enzyme complex is Kd = 19 nM at pH 5.0. 5. Under conditions where 90% of the enzyme is bound to soluble concanavalin A, full enzymatic activity is observed.

Animals↗
PubMed1994

Sorption kinetics and breakthrough curves for pepsin and chymosin using pepstatin A affinity membranes.

Isotherms and kinetic parameters for pepsin and chymosin sorption to immobilized pepstatin A were measured in batch experiments. The measured single-solute parameters were used in an affinity-membrane model which included competitive sorption kinetics, axial diffusion and dead volume mixing. The predictions made using the affinity-membrane model matched the experimental breakthrough curves, whereas predictions made using local-equilibrium theory were a distinct mismatch. The performance of affinity-membrane separations was dominated by slow sorption kinetics.

Adsorption↗
PubMed1992

Substrate specificity and kinetic properties of pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. No. 101.

The substrate specificity of the pepstatin-insensitive carboxyl proteinase isolated from Pseudomonas sp. No. 101 was studied by using a series of synthetic chromogenic substrates with general structure P5-P4-P3-P2-P1 *(NO2)Phe-Arg-Leu (P5, P4, P3, P2, P1: a variety of amino acids, (NO2)Phe is p-nitro-L-phenylalanine). The nature of the residues occupying the P2, P3 and P4 positions as well as P1 position had strong influences on kinetic parameters. Among those tested, Lys-Pro-Ile-Glu-Phe*(NO2)Phe-Arg-Leu was the best substrate (Km = 3 microM, kcat = 6.9 s-1, kcat/Km = 2300 mM-1 s-1). The S2 subsite of the enzyme was found to contain one or more basic amino acids while the S4 subsite probably includes one or more acidic amino acids. The pH-dependence of the hydrolysis of Ser-Pro-Ala-Lys-Phe*(NO2)Phe-Arg-Leu was studied. The pK1 and pK2 values for enzyme-substrate complex were found to be 2.97 and 4.92, respectively. Coupled with other results, it seems likely that two active carboxyl residues are involved in the catalytic action of the enzyme. In addition, it was found that a specific peptide inhibitor of the enzyme, tyrostatin, is a compeptive inhibitor with a ki value of 2.6 nM.

Amino Acid Sequence↗