Proteinase activities of the golden hamster eggs and cells of the cumulus oophorus

Proteinase activities of eggs and cells of the cumulus oophorous of the golden hamster were investigated with highly sensitive fluorogenic amide substrates. Eggs contain a neutral endopeptidase which hydrolyzed Suc-Ala-Ala-Phe-7-amino-4-methylcoumarin amide between the Ala and the Phe residues. Endopeptidase action on this substrate resulted in the accumulation of Phe-7-amino-4-methylcoumarin amide which was monitored by tlc identification. Hamster eggs also contained aminopeptidase and elastase-like activities but no detectable trypsin-like activity. Aminopeptidase, endopeptidase, trypsin-like, and elastase-like activities were detected in cumulus cells.     

New chromogenic and fluorogenic substrates for pyrrolidonyl peptidase.

L-Pyroglutamyl derivatives of p-nitroaniline and 7-amino-4-methylcoumarin were synthesized as new sensitive substrates for pyrrolidonyl peptidase (pyrrolidonecarboxylyl peptidase) from Bacillus amyloliquefaciens. Their hydrolyses could be followed by conventional colorimetric and fluorometric procedures; i.e., in terms of the increase in absorbance at 410 nm caused by the liberation of p-nitroaniline and the emission at 440 nm after excitation at 370 nm depending on the liberation of 7-amino-4-methylcoumarin. Values of Km were estimated to be 0.69 mM for anilide substrate and 0.33 mM for methylcoumarin substrate in the pyrrolidonyl peptidase reaction at pH 8.0. The methylcoumarin compound was about one thousand fold more sensitive than the anilide substrate.     

New fluorogenic substrates for renin

A simple and sensitive fluorometric assay was developed to test renin activity within several hours. Two new fluorogenic peptides, Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (octapeptide-MCA) and a succinyl derivative of the octapeptide-MCA were synthesized and used as a renin substrate. Renin cleaved the substrates at the Leu-Leu bond, releasing Leu-Val-Tyr-MCA. Three amino acids of this product were then successively split off by the auxiliary enzyme, leucine aminopeptidase, to liberate free 7-amino-4-methylcoumarin (AMC). The generation of the fluorescent 7-amino-4-methylcoumarin was proportional to renin concentrations up to 100 mGoldblatt U/tube. The optimal pH of renin reaction for both substrates was 6.5 to 7.0. As low as 5 mGoldblatt U of renin could be detected by this method. This method was applied to the assay of renin during its purification.     

Aprotinin derivatives with chromophoric leaving groups can be used as highly selective active-site titrants for serine proteinases and permit the determination of kinetic constants of enzyme-inhibitor complexes

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.     

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.     

Probing the cruzain S2 recognition subsite: a kinetic and binding energy calculation study

Cysteine proteases are relevant to several aspects of the parasite life cycle and the parasite-host relationship. Moreover, they appear as promising targets for antiparasite chemotherapy. Here, a quantitative investigation on the catalytic properties of cruzain, the papain-like cysteine protease from epimastigotes of Trypanosoma cruzi, is reported. The results indicate that kinetics for the cruzain catalyzed hydrolysis of N-alpha-benzyloxycarbonyl-l-arginyl-l-alanine-(7-amino-4-methylcoumarin), N-alpha-benzyloxycarbonyl-l-phenylalanyl-l-alanine-(7-amino-4-methylcoumarin), and N-alpha-benzyloxycarbonyl-l-tyrosyl-l-alanine-(7-amino-4-methylcoumarin) can be consistently fitted to the minimum three-step mechanism of cysteine proteases involving the acyl.enzyme intermediate E.P; the deacylation step is rate-limiting in enzyme catalysis. Remarkably, these substrates show identical catalytic parameters. This reflects the ability of the cruzain Glu205 residue, located at the bottom of the S(2) subsite, to neutralize the substrate/inhibitor polar P(2) residues (e.g., Arg or Tyr) and to be solvent-exposed when substrate/inhibitor nonpolar P(2) residues (e.g., Phe) fit the S(2) subsite. More complex catalytic mechanisms are also discussed. Binding free-energy calculation provides a quantitative framework for the interpretation of these results; in particular, direct evidence for the compensatory effect between Coulomb interaction(s) and solvation effect(s) is reported. These results appear of general significance for a deeper understanding of (macro)molecular recognition and for the rational design of novel inhibitors of parasitic cysteine proteases.     

New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.     

Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain

A soluble tripeptidylaminopeptidase has been isolated from human post-mortem cerebral cortex by anion exchange, hydrophobic interaction and size-exclusion chromatography. From gel filtration studies the active enzyme can exist in both high molecular weight (Mr>10⁶) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amino-4-methylcoumarin with a pH optimum of around 7.5 and K_m of 148 M. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amino-methylcoumarins and was not inhibited by bestatin. The enzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroisocoumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide showing that its activity is serine and cysteine dependent. The purified enzyme released tripeptides from several naturally occurring neuropeptides with quite broad specificity. Cholecystokinin octapeptide, angiotensin III and neurokinin A were the most rapidly hydrolysed. Peptides with Pro residues arount the point of cleavage were not hydrolysed.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K(m), but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.     

Protease activities present in wheat germ and rabbit reticulocyte lysates

Rabbit reticulocyte lysates and wheat germ lysates were found to contain significant neutral protease activity when assayed against the highly sensitive 7-amino-4-methylcoumarin (AMC) peptide substrates Phe-AMC, succinyl-Ala-Ala-Phe-AMC and t-boc-Ala-Ala-Pro-Ala-AMC (substrates for aminopeptidase, chymotrypsin and elastase-like enzymes, respectively). Additionally, wheat germ lysates contain a trypsin-like activity when assayed against CBZ-Gly-Gly-Arg-AMC and a post-proline cleaving activity which hydrolyzed the Pro-Ala bond of t-boc-Ala-Ala-Pro-Ala-AMC.     

δ-(D-α-Aminoadipoyl)-Cleaving Amidase of Ochrobactrum anthropi

Ochrobactrum anthropi cleaved the delta-(D-alpha-aminoadipoyl)-side chain from delta-(D-alpha-aminoadipoyl)-7-amino-4-methylcoumarin, a beta-lactamase-resistant cephalosporin C analogue. In whole cell conversions up to 1 nkat g(-1) dry cell wt were achieved. O. anthropi possesses also gamma-D-glutamyltranspeptidase activity, 8 nkat g(-1) dry cell wt, the likely cause of delta-(D-alpha-aminoadipoyl)-cleavage.     

A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy

The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). There is no simple method available for measuring the activity of this highly toxic drug. We incubated L-asparaginase from Erwinia chrysanthemi with L-aspartic acid beta-(7-amido-4-methylcoumarin) and measured the release of 7-amino-4-methylcoumarin fluorometrically for 30-300 min. The rate of the hydrolysis of the substrate was linear over a 50-fold range of the concentration of the enzyme. With increasing substrate concentration, the enzyme showed a saturable kinetic pattern with V(max) of 0.547 (SD 0.059) microM/min/mg of enzyme (n = 3) and Km of 0.302 (SD 0.095) mM (n = 3). This assay enables rapid analysis of L-asparaginase activity in biological samples and it can be used, for example, for monitoring of L-asparaginase activity in serum of ALL patients during their L-asparaginase therapy.     

A preliminary study of the use of Colilert for water quality monitoring

Substrate specificities of proteases produced by two putrefactive marine bacteria, Shewanella putrefaciens and Alteromonas haloplanktis , were surveyed by using peptidyl-7-amino-4-methylcoumarin (MCA-substrates). Shewanella putrefaciens produced trypsin-like enzyme(s) showing broad spectrum specificity and chymotrypsin-like enzyme specifically hydrolysing Glt-Gly-Gly-Phe-MCA. Alteromonas haloplanktis produced high activity of ammopeptidase and trypsin-like enzyme(s) preferring Z-Phe-Arg-MCA, Bz-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA. The two organisms would be able to utilize different proteins for their growth.     

Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.     

Rationalization of aminopeptidase activities in human skeletal muscle soluble extract

Although a wide range of aminoacyl-7-amino-4-methylcoumarin derivatives (which are used to measure aminopeptidase activity) were found to be hydrolysed by human skeletal muscle soluble fraction, fractionation of the latter via anion-exchange and gel-filtration chromatography resolved only five types of separable aminopeptidase (with activity relative to alanyl aminopeptidase in parentheses): alanyl aminopeptidase (alpha-aminoacyl-peptide hydrolase, EC 3.4.11.14, 100%), arginyl aminopeptidase (two isoenzymes, L-arginyl-L-lysyl)-peptide hydrolase, EC 3.4.11.6, 15%); pyroglutamyl aminopeptidase (5-oxoprolyl-peptide hydrolase, EC 3.4.19.3, 3%); leucyl aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1, 1.5%) and alpha-glutamyl aminopeptidase (0.2%). Thus over 80% of the total aminopeptidase activity (expressed in relative terms) in human skeletal muscle soluble fraction can be accounted for by a single enzyme, the major aminopeptidase. A single peak of activity, which co-eluted with the major aminopeptidase after anion-exchange and gel-filtration chromatography, was obtained after assay with the following aminoacyl-7-amino-4-methylcoumarin derivatives: glycyl-, isoleucyl-, lysyl-, methionyl-, ornithyl-, phenylalanyl-, prolyl-, seryl-, tyrosyl- and valyl-. Thus, the hydrolysis of these derivatives by skeletal muscle soluble fraction occurs principally via the major aminopeptidase and not by specific enzymes, as previously suggested (Wada and Aoyagi, 1983). These results illustrate the difficulty in measuring individual aminopeptidase activities in muscle homogenate and soluble fraction, and the danger in ascribing apparent aminopeptidase activity to 'specific' enzymes.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K_m, but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25 °C but concealed at 37 °C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25 °C, but not at 37 °C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.     

Novel thiocoumarins as inhibitors of TNF-alpha induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation

Different coumarin/thiocoumarin derivatives, that is, 7-hydroxy-4-methylcoumarin, 7,8-dihydroxy-4-methylcoumarin, 7-acetoxy-4-methylcoumarin, 7,8-diacetoxy-4-methylcoumarin, 7-hydroxy-4-methylthiocoumarin, 7,8-dihydroxy-4-methylthiocoumarin, 7-acetoxy-4-methylthiocoumarin and 7,8-diacetoxy-4-methylthiocoumarin were synthesized and evaluated for their effects on TNF-alpha induced expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and on NADPH-catalyzed rat liver microsomal lipid peroxidation with a view to identify modulators for expression of cell adhesion molecules and to establish structure-activity relationship. We found that dihydroxy and diacetoxy derivatives of thiocoumarin were more potent in comparison to the corresponding coumarin derivatives in inhibiting TNF-alpha-induced expression of ICAM-1. However, coumarin derivatives were found to be more potent in comparison to the corresponding thiocoumarins in inhibiting microsomal lipid peroxidation. We have also tested the intermediate compounds 7,8-dibenzyloxy-4-methylcoumarin and 7,8-dibenzyloxy-4-methylthiocoumarin for their inhibitory activity on TNF-alpha-induced ICMA-1 expression. We found that dibenzyloxy-4-methylthiocoumarin is better than dibenzyloxy-4-methylcoumarin. The mechanisms underlying the observed activities of coumarins and thiocoumarins have been discussed with reference to their structures. Such structure-function relationship studies may help in developing molecules with better anti-inflammatory and anti-oxidant activities.     

Isoelectric focusing (IEF) and band detection with fluorogenic protease substrates

Summary In a previous paper, combined dye histochemistry and analytical isoelectric focusing (IEF) of supernatants from organ homogenates have been shown to yield good results for the detection of protease isoenzymes. Difficulties arise when the protease to be studied is partially or completely inhibited y diazonium salts and when synthetic peptide substrates different from 4-methoxy-2-naphthylamine (MNA) amino acids and peptides are to be used. In this paper a technique is described in which cellulose acetate foils impregnated with MNA, 7-amino-4-methylcoumarin (AMC) and 7-amino-4-trifluoromethylcoumarin (AFC) substrates are overlaid on electrophoresis strips after IEF. After incubation, the foils are viewed with an UV-lamp and photographed. The MNA, AMC and AFC peptides are equally suitable for fluorescence band detection. Using this technique, occasionally protease isoenzymes are found which are more sensitive towards diazonium salts, e.g. aminopeptidase A and M. Sometimes it is also possible to detect thiolproteases which is not the case when employing dye histochemistry.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.     

Biosynthesis of coumarin glycosides by transgenic hairy roots of Polygonum multiflorum

To investigate the substrate specificity and regio-selectivity of coumarin glycosyltransferases in transgenic hairy roots of Polygonum multiflorum, esculetin (1) and eight hydroxycoumarins (2-9) were employed as substrates. Nine corresponding glycosides (10-18) involving four new compounds, 6-chloro-4-methylcoumarin 7-O-β-D-glucopyranoside (15), 6-chloro-4-phenylcoumarin 7-O-β-D-glucopyranoside (16), 8-hydroxy-4-methylcoumarin 7-O-β-D-glucopyranoside (17), and 8-allyl-4-methylcoumarin 7-O-β-D-glucopyranoside (18), were biosynthesized by the hairy roots.