N-succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide as a sensitive substrate for assaying activity of cysteine proteinases

N-Succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide has been found to be a very sensitive chromogenic substrate for the assay of cysteine proteinase papain, ficin and bromelain. N-Succinyl-alanyl-S-benzylcysteine p-nitroanilide and N-succinyl-alanyl-alanyl-S-benzylcysteine p-nitroanilide are also suitable for this purpose. These substrates were hydrolyzed only very slightly or not hydrolyzed at all by trypsin.     

Studies on Sulfur-containing Carbonates and Their Pesticidal Activities

Several S-substituted-benzyl O-substituted-benzyl dithiocarbonates were synthesized and their acaricidal activities with Tetranychus urticae Koch were examined. Some dithiocarbonates, such as S-benzyl O-4-chlorobenzyl dithiocarbonate, S-benzyl O-4-bromobenzyl dithiocarbonate, S-benzyl O-3-methoxylbenzyl dithiocarbonate, S-3-methylbenzyl O-4-chlorobenzyl dithiocarbonate and S-3-methoxybenzyl O-4-chlorobenzyl dithiocarbonate, showed high ovicidal activities.

Various S-benzyl O-substituted-phenyl dithiocarbonates together with some related compounds were prepared and their biological activities were evaluated. Among them, S-benzyl O-phenyl-, S-benzyl O-2-acetylphenyl- and S-benzyl O-4-methylthiophenyl-dithiocarbonates showed high ovicidal activity to the two spotted spider mite, Tetranychus urticae Koch. S-Benzyl O-2-alkylphenyl-, S-benzyl O-2-allylphenyl- and S-benzyl O-2-dimethyl-aminomethylphenyl-dithiocarbonates showed herbicidal activities to barnyardgrass and radish. S-Benzyl O-4-alkylphenyl dithiocarbonate was herbicidal only to radish. Some structure-activity relationships are discussed.

Several thionocarbonates were synthesized and their antifugal activities were examined. O-Benzyl O-4-nitrophenyl thionocarbonate, O-benzyl O-3-nitrophenyl thionocarbonate and O-benzyl O-4-acetylphenyl thionocarbonate showed excellent activities to Pellicularia sasakii. O-Benzyl O-2- and 4-acetylphenyl thionocarbonate showed the highest activities to Pyricularia oryzae.     

Purification and characterization of aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Alpha-glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

Allosteric activation of chymotrypsin-catalyzed hydrolysis of specific substrates

The rates of hydrolysis of three specific substrates of chymotrypsin, glutaryl-L-phenylalanine p-nitroanilide, acetyl-DL-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide were enhanced by 2,2′-bis[α-(benzyldimethylammonium)methyl] azobenzene dibromide and less so by related compounds. Detailed studies with glutaryl-L-phenylalanine p-nitroanilide showed a 42-fold increase in k_cat with no change in Km. No acceleration (or inhibition) was noted with esters, hydroxamides or proteins as substrates. Tryptic hydrolysis of benzoyl-DL-arginine p-nitroanilide was unaffected. It was concluded that certain quaternary compounds can act as allosteric effectors of chymotrypsin.     

Procedure for obtaining and catalytic properties of trypsin immobilized in cryogels of polyvinyl alcohol

Commercial preparations of trypsin, varying in activity, were immobilized in a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In an organic solvent environment, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-argininine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.     

Substrate effects on the enzymatic activity of alpha-chymotrypsin in reverse micelles

Six different substrates have been used for measuring the activity of alpha-chymotrypsin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilide, succinyl-Phe p-nitroanilide, acetyl-Phe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (kcat and Km) on the water content of the system, on wo (= [H2O]/[AOT]), is different for the different substrates. This indicates that activity-wo profiles for alpha-chymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower kcat (and the higher Km) in aqueous solution, the higher kcat as well as Km in reverse micelles. "Superactivity" of alpha-chymotrypsin could only be found with the ester substrate and with relatively "poor" p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequisite for alpha-chymotrypsin to show "superactivity".     

Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

N-anthraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties

By simple substitution of an N-acyl group for the anthraniloyl(o-aminobenzoyl) group, chromogenic p-nitroanilide substrates are converted into highly sensitive fluorogenic substrates of proteases. The fluorescence of the anthraniloyl group is completely quenched by the p-nitroanilide moiety in the intact substrates and is released during their enzymatic hydrolysis. The approach is exemplified by the synthesis of anthraniloyl-Phe p-nitroanilide, anthraniloyl-Lys p-nitroanilide, and anthraniloyl-Gly-Gly-Phe p-nitroanilide as substrates for chymotrypsin, trypsin, and alkaline mesentericopeptidase, respectively. The kinetic parameters of these substrates are slightly better than those of similar derivatives bearing other acyl groups, suggesting that the enhanced sensitivity is completely due to the method of measurement. Since the conversion does not affect the chromogenic properties of the substrates, the same compounds can be used as usual p-nitroanilide substrates as well.     

Hydrolysis of succinyl-trialanine p-nitroanilide by two enzymes associated with human high-density lipoproteins

Succinyl-trialanine p-nitroanilide (Suc-Ala3-pNA), a synthetic substrate for the determination of elastase activity, was hydrolyzed in sequence by two enzymes that were found to be associated with human high-density lipoproteins. The enzymes involved in the sequence of reaction were separated by ion-exchange chromatography from apo-lipoprotein A-I and A-II, major apoproteins of high-density lipoproteins. One, designated as fraction MK, cleaves Suc-Ala3-pNA to succinyl-dialanine and alanine p-nitroanilide (Ala-pNA), and the other, designated as fraction U, cleaves Ala-pNA to alanine and p-nitroaniline. Fraction MK was inhibited by dithiothreitol, EDTA, and 1,10-phenanthroline, whereas fraction U was inhibited by 1,10-phenanthroline and bestatin. In addition to these findings, fraction MK also hydrolyzed 2,4-dinitrophenyl-prolyl-glutaminyl-glycyl-isoleucyl-alanyl-glycyl-glutaminyl- arginine (DNP-octapeptide), a specific substrate for the determination of vertebrate collagenase. Neither native elastin nor native collagen was hydrolyzed by a mixture of the two enzymes. Fraction U was very similar to aminopeptidase M with respect to its enzyme characteristics studied.     

Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.

After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.     

S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II

Three cysteine derivatives, (R)-S-(2-hydroxyethyl)cysteine, together with (R(S)R(C))- and (S(S)R(C))-S-(2-hydroxyethyl)cysteine sulfoxides, have been isolated from the roots of Petiveria alliacea. Furthermore, three additional amino acids, S-methyl-, S-ethyl-, and S-propylcysteine derivatives, were detected. They were present only in trace amounts (<3 microg g(-1) fr. wt), precluding determination of their absolute configurations and oxidation states. In addition, four thiosulfinates, S-(2-hydroxyethyl) (2-hydroxyethane)-, S-(2-hydroxyethyl) phenylmethane-, S-benzyl (2-hydroxyethane)- and S-benzyl phenylmethanethiosulfinates, have been found in a homogenate of the roots. The formation pathways of various benzyl/phenyl-containing compounds previously found in the plant were also discussed.     

Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia.

Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed.     

Synthesis and structure-activity relationships of metal-ligand complexes that potently inhibit cell migration

We screened the National Cancer Institute Diversity Set compound collection for small molecules that affect mammalian cell migration and identified NSC 295642 as an inhibitor of cell motility with nanomolar potency. We found by LC-MS and X-ray crystallography that NSC 295642, a Cu(II) complex of the Schiff base product of condensation of S-benzyl dithiocarbazate and 2-acetylpyridine, has a bridged dimeric Cu2Cl2(L)2 structure with distorted square pyramidal geometry. Each of the two copper atoms is five-coordinated to one of the two tridentate chelating ligands and both bridging chlorine atoms. To define structure-activity relationships,we investigated the bioactivity of related metal-ligand complexes derived from different metal(II) atoms and different ligands. Complexation of the NSC 295642 ligand with Zn(II) or Ni(II), delivered as metal(II) chloride salts under conditions identical to those used for preparation of the original Cu(II) complex, instead results in distorted octahedral bis-chelate structures, where a single metal atom is six-coordinated to two ligands. The Zn(L)2 complex possesses a potency similar to that of the Cu2Cl2(L)2 complex, while the Ni(L)2 has no antimigratory activity at all. We carried out density functional theory calculations to obtain the electronic ground state geometry of the complexes, both in vacuum and implicit water solvent. The X-ray crystal and energy-minimized structures are very similar and exhibit a transoid orientation of the S-benzyl groups relative to the central metal-coordinated rings for both of the bioactive Cu2Cl2(L)2 and Zn(L)2 complexes, despite their different coordination geometries. In contrast, the biologically inactive Ni(L)2 complex adopts a cisoid conformation. Varying the ligand structure, we found that hydrophobic S-alkylaryl groups are required for activity. Complexes with a simple S-methyl group, S-benzyl groups with polar substitutions or a carboxylated pyridine ring exhibit dramatically reduced activity. We tested the most potent metal-ligand complex in a number of cancer cell lines and found cell-type selectivity in its effect on cell motility. Collectively, these results suggest that a two-ligand structure with bulky nonpolar S-substituents in a transoid conformation is important for the antimigratory activity of these metal-ligand complexes.     

MS and NMR investigation of bioactive quinazolones

To prepare effective PARP [poly(ADP)-ribose)polymerase-1] inhibitors, starting from 2-mercapto-4(3H)-quinazolone (1), several S-alkyl derivatives--2-alkylsulfanyl-3H-quinazolin-4-ones (2-5, 7-9)--as well as an S-benzyl derivative (10) were prepared using a simple alkylation method. Some of them are known compounds. Their structure was studied thoroughly by MS and NMR methods.     

Characterization of neutral proteinases from human spleen]

Several proteinases hydrolyzing histone and caseine in neutral media were obtained by Sephadex G-100 fractionation of water and salt (1 M KCl) extracts of human spleen. The level of the activity of proteinases in the extracts was very low as a result of the presence of an inhibitor. Neutral proteinases were found in two protein fractions. The "high-molecular-weight-" proteinases were inhibited by DFP and therefore they were attributed to a group of serine proteinases. The "low-molecular-weight" fraction contained neutral SH-dependent proteinase(s) and DFP-inhibited enzymes. In this fraction, the kininogenase activity and the hydrolysis of Boc-1-ananine p-nitrophenyl ester, N-benzoyl-L-tryosine ethyl ester and N-benzoyl-DL-arginine p-nitroanilide were observed.     

Proteases of swine small intestine enterocytes. Kinetics of substrate hydrolysis and inhibition of aminopeptidase N from brush border vesicles

The kinetics of hydrolysis of L-leucine p-nitroanilide and some p-nitrophenylalanine dipeptides by vesicular aminopeptidase N from the porcine small intestine brush border membrane was studied. It was shown that the catalytic properties of the vesicular enzyme are very similar to those known for its solubilized counterpart. Both enzymes are inhibited by o-phenanthroline, ZnCl2 and puromycin with Ki = 10(-5)-10(-6) M. The data obtained offer new possibilities for investigating the role of aminopeptidase N in the amino acid and peptide transport across the enterocyte membrane.     

Protease activity in gut of Daphnia magna: evidence for trypsin and chymotrypsin enzymes

Two major protease activities were present in gut homogenates of the cladoceran crustacean Daphnia magna: (i) a trypsin activity that hydrolysed the synthetic substrate N-benzoyl-dl-arginine p-nitroanilide and was strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF) and not inhibited by chymostatin; and (ii) a chymotrypsin activity that hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, did not hydrolyse N-benzoyl-l-tyrosine p-nitroanilide, and was strongly inhibited by chymostatin and not by TLCK and APMSF. Both activities had alkaline pH optima (pH 7-10), but were shown to be due to distinct types of proteases. These two enzyme activities accounted for 75-83% of the proteolytic activity of gut contents. Substrate SDS-polyacrylamide gel electrophoresis revealed nine different proteases ranging from 15 to 73 kDa.     

Synthesis and fluorescence properties of intramolecularly quenched fluorogenic p-nitroanilides containing coumarin or quinolinone derivatives as fluorophores.

Nine model intramolecularly quenched fluorogenic substrates (IQFS) of the general structure F-Phe-NH-Np, containing coumarin or quinolinone derivatives as fluorophores (F) and the p-nitroanilide group (Np) as quencher, were synthesized. The study of the fluorescence properties of the substrates synthesized and the corresponding fluorophores showed that efficient quenching of fluorescence (>89%) was observed in all cases. The combination of 7-glutarylamido-4-methyl-coumarin (Mec-NH-Glt-OH) or 7-methoxy-4-coumaryl-acetic acid (Mca) with the p-nitroanilide group gave the best results (97.2 and 98.8% quenching, respectively). These fluorophores can be used to convert peptide p-nitroanilides into IQFS, which, retaining their chromogenic properties, may be applied in both fluorometric and colorimetric assays.     

Characterization of proteases formed by Bacteroides fragilis

Bacteroides fragilis NCDO 2217 produced three major proteases, P1, P2 and P3 of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P1 weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease:arylamidase ratios of these enzymes indicated that they were probably endopeptidases. Experiments with protease inhibitors suggested that P1 and P2 had characteristics of serine and metalloproteases respectively and that P3 was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn2+, Ca2+ and Mg2+, but was strongly inhibited (about 95%) by Cu2+ and Zn2+. The temperature optimum for protein hydrolysis was 43 degrees C. Proteolysis was temperature sensitive, however (90% reduction at 60 degrees C) and was maximal at alkaline pH, with two broad peaks at pH 7.9 and pH 8.8. Cell fractionation showed that P1 was located intracellularly and in the periplasm, whereas P2 and P3 were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 M-NaCl suggested that ionic interactions were involved in the association of these enzymes with the membranes.     

Studies on the proteolytic activity of Bacteroides fragilis

The proteolytic activity of the intestinal bacterium Bacteroides fragilis NCDO 2217 was cell-bound during exponential growth, but was progressively released from the cells in stationary phase. Proteins hydrolysed included casein, trypsin, chymotrypsin, azocasein and the proteins in azosoya bean flour. Collagen, azocoll, elastin, gelatin, ovalbumin and bovine serum albumin were either weakly degraded or completely refractory to proteolysis. Arylamidase activity was exhibited against leucine p-nitroanilide (LPNA), leucine beta-naphthylamide, glycyl-proline p-nitroanilide and valyl-alanine p-nitroanilide. The bacterium grew with ammonia, peptone or casein as sole nitrogen source. Azocasein- and LPNA-hydrolysing activities were consistently higher when grown on casein. Cell-bound protease activity increased concomitantly with growth rate in both carbon- and nitrogen-limited continuous culture. Leucine arylamidase activity was also growth-rate-dependent, being 3-fold greater at D = 0.18 h-1 compared to D = 0.03 h-1. Extracellular proteolytic activity was only detected at low growth rates, accounting for about 25% of total protease activity.