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PubMed1994

Use of steady state kinetic methods to elucidate the kinetic and chemical mechanisms of retroviral proteases.

Despite the current plethora of structural data of HIV-1 protease and the availability of potent inhibitors, whose structures are based in part on the presumed mechanism of action of this enzyme, our actual understanding of its chemical mechanism has been until now based largely on the precedents of the mammalian and fungal aspartic proteases and static three-dimensional data. The available steady state kinetic data of the protease, as reviewed here, constitute a first step in a detailed description of the mechanism of the enzyme to complement the structural data.

Amino Acid Sequence↗
PubMed1988

Measurement of IgG blocking antibody in human serum: comparison of ELISA with monoclonal antibody and fluorogenic substrate and Staphylococcus protein A solid-phase RIA.

We compared ELISA with mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate with the Staphylococcus protein A solid-phase radioimmunoassay (SPRIA) in the measurement of specific IgG antibody to short ragweed pollen. Sera from 51 ragweed-allergic patients undergoing allergen immunotherapy were evaluated for ragweed-specific IgG antibodies with the same ragweed extract in the two assay systems. With optimal conditions, the ELISA and SPRIA displayed comparable positive thresholds (approximately 1 ng/ml of ragweed-specific IgG). Both assays also demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation [CV] less than 8.8% for ELISA and less than 8.6% for SPRIA). Reproducibility was determined by constructing precision profiles for intra- and interassay variations over the working ranges of each assay (ELISA, 0.8 to 100 ng/ml; SPRIA, 1 to 250 ng/ml). ELISA intra-assay CVs ranged from 13% near threshold to less than 5% at higher antibody concentrations; SPRIA intra-assay CVs ranged from 4.3% to 2.8%. Interassay reproducibility was somewhat better for SPRIA (4.6% to 9.6%) than for ELISA (10% to 18%). In direct comparison, 41 (80%) of the 51 sera were concordant in the two assays (r = 0.91; p less than 0.001). Although each assay result was reproducible, 10 (20%) of the sera elicited consistently discrepant results in the two assays. In eight of the 10 discordant sera, the SPRIA results were higher than ELISA, suggesting the possibility that some ragweed allergen may be better represented on the short ragweed-pollen extract agarose than on ELISA plate wells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1980

Modification of the 4-(p-nitrobenzyl) pyridine assay for measurement of melphalan and mechlorethamine in plasma.

A sensitive, reproducible spectrophotometric assay for mechlorethamine and melphalan was developed through modifications in the use of 4-(p-nitrobenzyl) pyridine as the chromogenic reagent. Key factors that led to enhanced color development and consistency of results were: (1) protein precipitation with 4 degrees C perchloric acid; (2) control of the pH of the reaction with acetate buffer; (3) precise timing of color development in alkali; and (4) sample reading at the appropriate wavelength. The modifications allowed measurement of mechlorethamine and melphalan levels in perfusate samples obtained from isolated rat liver perfusions. Half-lives calculated from perfusate decay curves after addition of mechlorethamine at 50 microgram/ml and 10 microgram/ml were 15.9 min and 15.0 min respectively. The perfusate half-life after addition of melphalan at 5 microgram/ml was 44.2 min. Plasma levels of mechlorethamine were not detectable by this assay after intrapleural administration of 15 mg to one patient but were detectable after intravenous administration of 20 mg to a second patient.

Animals↗
PubMed1991

Evaluation of a microassay for human kininogens as cysteine protease inhibitors.

Several methods have been described for the identification and quantification of kininogens based on both immunochemical and functional characteristics. This article presents a rapid, cheap and simple microplate assay of kininogens based on their ability to inhibit cysteine proteases. The target enzyme papain is activated by cysteine HCl and the activated enzyme will be inhibited by added kininogens. The residual enzyme activity that is not inhibited in this reaction subsequently hydrolyzes the added substrate, S-2302, generating a yellow color that is read in a microplate reader at 405 nm. This method is very sensitive, the smallest amount of kininogen that causes significant inhibition of papain is established to be 0.01 micrograms. As a quantitative method, the assay performs accurately when approximately 0.1 micrograms of low molecular weight kininogen or high molecular weight kininogen is added to the test system. The within-run coefficient of variation (%) of the method was 1.7% when the inhibition of papain was in the range 45-70% and the day to day variation as low as 2.3% when performed with a papain inhibition of 80%. Applications of the method are presented, studying chromatographic separated kininogens in plasma, ascites, and urine.

Ascites↗
PubMed1988

New spectrophotometric method for continuous recording of the spleen exonuclease activity.

Some of the synthetic chromophoric substrates of various enzymes cannot be used for direct spectrophotometric recording of the reactions, when a difference between the pH optimum of the enzyme reaction and the pH of maximum absorption of the released chromophore exists. In the present paper we describe a new method for following the time course of the spleen exonuclease-catalyzed reaction with thymidine 3'-monophospho-p-nitrophenyl ester as a substrate, based on the difference obtained in the absorbency of the substrate and its products in the far UV (at 330 nm). This difference, not published before, permits direct spectrophotometric recording of the amount of the hydrolyzed chromophoric substrate in acidic pH, whereas the maximum absorption of the product as accepted in the literature, is in alkaline pH. The molar absorption coefficient of the measurement at pH 5.7 is determined to be epsilon = 522 M-1.mm-1.

Chromogenic Compounds↗
PubMed1988

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.

Chromogenic Compounds↗
PubMed1983

Improved visualization of neurons labeled with horseradish peroxidase: silver-intensification of the pyrocatechol/p-phenylenediamine reaction product.

A silver intensification procedure suitable for use with pyrocatechol/p-phenylenediamine (PC-PPD) product of the horseradish peroxidase (HRP) reaction is described. Qualitative and quantitative results from retrogradely labeled neurons in the cat cortex after thalamic injection of HRP demonstrate an increase of the intensity of labeling and in the number of darkly labeled cells after the intensification procedure. In both the non-intensified and the intensified PC-PPD reacted tissue the sensitivity was comparable to that of TMB-treated material. The ratio of lightly to darkly labeled neurons was very similar in intensified PC-PPD and TMB material, suggesting that the lightly labeled cells may have fewer terminals present at the level of the injected target.

Animals↗
PubMed1985

A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP).

In this paper, an alternative procedure for the histochemical detection of HRP using amonium heptamolybdate (AHM) as a stabilizing agent and tetramethyl benzidine (TMB) as a chromogen is reported. This procedure avoids the two main problems that occur in previous methods using sodium nitroferricyanide (SNF) as the stabilizer, namely, the appearance of needle-shaped crystals at non-specific anatomical sites, and intensive tissue shrinkage. A comparative study of both, the TMB-AHM and TMB-SNF methods, was performed in the analysis of cerebral cortex afferent connections of the lizard Podarcis hispanica. This study demonstrates that the two methods are of similar sensitivity. The TMB-AHM reaction can be carried out at physiological pH (from 6 to 8), thus, avoiding tissue contraction. The reaction product is of an intense blue-green colour and, as with the TMB-SNF method, shows granulation. The appearance of non-specific precipitates is completely avoided when the incubation medium is maintained at a pH in excess of 5.

Animals↗
PubMed1988

Stabilization of tetramethylbenzidine (TMB) reaction product at the electron microscopic level by ammonium molybdate.

The ability to use the tetramethylbenzidine (TMB) method for studying neuronal connections at the electron microscopic level is often difficult because the conditions of osmification and dehydration used in processing the tissue may result in significant loss and/or decreased electron density of the reaction product. In the present study, we report that stabilization of TMB reaction product with 5% ammonium molybdate (AM) prior to osmificating the tissue results in the formation of TMB-AM crystals that are many times more electron dense and resistant to ethanol extraction than non-stabilized TMB crystals. The nature of the chemical interaction that underlies the stabilization of TMB by AM is uncertain, but it may involve the formation of an insoluble salt between molybdic ions and the TMB polymer. The use of this simple procedure increases the sensitivity of the TMB procedure at the electron microscopic level and may be used to label neuronal pathways in the peripheral and central nervous systems with equal success.

Animals↗
PubMed1995

A modification of biotinylated dextran amine histochemistry for labeling the developing mammalian brain.

Biotinylated dextran amine (BDA) has proven to be an excellent anterograde tracer in adult mammalian brains, having some advantages over other anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biocytin. However, results are inferior when BDA is used in neonatal mammals. To improve the sensitivity and quality of BDA labeling in neonatal mammalian brains, the tetramethylbenzidine-sodium tungstate (TMB-ST) method for horseradish peroxidase (HRP) histochemistry was modified and used in BDA histochemistry. After BDA application to the visual cortex of neonatal rat and cat, contralateral and ipsilateral cortical and subcortical regions were examined for BDA-labeled exons and terminals. The modified BDA histochemistry produced corpus callosum (CC) axons in neonatal rat and cat that were heavily and continuously labeled. The distribution, trajectories, branching and termination of individual CC axons, and even possible axon-axon contracts, were clearly identified in exquisite detail, even at low magnification. The quality of BDA labeling in the ipsilateral lateral geniculate nucleus and superior colliculus was similar to that of the CC axonal labeling. These results indicate that the modified BDA histochemistry provides a very sensitive and reliable approach to revealing the detailed distribution and morphology of projecting axons and terminals in the developing mammalian nervous system.

Animals↗
PubMed1996

Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains.

We report an improved immunohistochemical protocol for revealing anterograde axonal transport of the subunit B of cholera toxin (CTB) which stains axons and terminals in great detail, so that single axons can be followed over long distances and their arbors reconstructed in their entirety. Our modifications enhance the quality of staining mainly by increasing the penetration of the primary antibody in the tissue. The protocol can be modified to allow combination in alternate sections with tetramethylbenzidine (TMB) histochemical staining of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Using the protocol, we tested the performance of CTB as an anterograde tracer under two experimental paradigms which render other anterograde tracers less sensitive or unreliable: (1) labeling the entire retinofugal projection to the brain after injections into the vitreal chamber of the eye, and (2) labeling developing projections in the cortex and thalamus of early postnatal mammals. Qualitative comparisons were made with other tracers (Phaseolus vulgaris leucoagglutinin, dextran rhodamine, biotinylated dextran, free WGA, or WGA-HRP) that were used to label these same projections. From these observations it is clear that CTB, visualized with our protocol, provides more sensitive anterograde labeling of retinofugal projections as well as of axonal connections in the neonatal forebrain.

Animals↗
PubMed1988

Sensitive fluorogenic enzyme immunoassay on nitrocellulose membranes for quantitation of virus.

A highly sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes nitrocellulose membranes as solid phase support, has been developed for the detection and identification of virus in clinical samples. Reagents were standardized and, using purified Newcastle disease virus (NDV) as a model, the theoretical lower limits of test sensitivity of the FELISA were compared, in both "sandwich" and "indirect" formats, to those of a comparable chromogenic enzyme-linked immunosorbent assay (CELISA). Of the systems evaluated, the "sandwich" FELISA exhibited maximum sensitivity and detected 10 fg of purified virus protein per milliliter of test sample (500 ag per test volume). Specificity of the "sandwich" FELISA was evaluated by challenging the system with heterologous strains of NDV and with other serologically related and unrelated viruses. In a clinical trial in which fecal materials from chickens undergoing vaccination with NDV were assayed directly by FELISA, the virus was detected from the first to approximately the tenth day post-vaccination. The test is simple to perform and results can be obtained in approximately 4 h.

Animals↗
PubMed1996

Kinetic analysis of plasmepsins I and II aspartic proteases of the Plasmodium falciparum digestive vacuole.

Plasmepsins I and II are Plasmodium falciparum aspartic proteases implicated in hemoglobin degradation. Using a synthetic fluorogenic peptide substrate based on the initial hemoglobin cleavage site, we have analyzed kinetic parameters of the two enzymes in native and recombinant forms. Both native plasmepsins cleave the model substrate well. Recombinant plasmepsin II behaves similarly to native enzyme, substantiating its usefulness for inhibition and structural studies. In contrast, recombinant plasmepsin I does not resemble its native homolog kinetically. A hybrid molecule, in which the polyproline loop of plasmepsin I has been replaced by the homologous sequence from plasmepsin II, still maintains the specificity/kinetics of plasmepsin II. This suggests that the polyproline loop, important for substrate recognition in the mammalian aspartic protease renin, does not play a similar role in the plasmepsins.

Amino Acid Sequence↗
PubMed1986

The action of factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions.

Kinetic parameters for the action of bovine Factor Xa (EC 3.4.21.22) on 25 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kc/Km, ranges from 1.5 X 10(1) to 2 X 10(6) M-1 X s-1 for the poorest and the best substates, respectively. The best substrates for Factor Xa were identified as those with arginine in the P1 position, and glycine in the P2 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effect of NaCl and CaCl2 concentrations and temperature on the action of Factor Xa on selected substrates have been assessed. Km values for Factor Xa hydrolysis of most substrates are greater than 100 microM. Solubility of the substrates consequently restricts measurements of reaction velocities to concentrations lower than desirable for optimally determining kc. Comparison of these kinetic parameters for Factor Xa with those of thrombin (Lottenberg, R., Hall, J.A., Blinder, M., Binder, E. and Jackson, C.M. (1983) Biochim. Biophys. Acta 742,539-557) for these same substrates indicates that the greater hydrolytic efficiency of thrombin is due primarily to lower Km values.

Anilides↗
PubMed1987

The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2.

Variation in the kinetic parameters, kcat and Km, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1') residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and the P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO-) of pig pepsin and Thr (OH) or His (ImH+) in P3 of a substrate. Similarly, Lys-220 (NH3+) of chymosin and a Glu (COO-) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO-) of E. parasitica proteinase and a Glu (COO-) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1990

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi.

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.

Amino Acid Sequence↗
PubMed1991

Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.

The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents.

Amino Acid Sequence↗
PubMed1993

Comparison of several new chromogenic galactosides as substrates for various beta-D-galactosidases.

The kinetic characteristics of beta-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli beta-D-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed to interactions not involving the catalytic site. When the product of the maximum observed velocity (Vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The beta-D-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.

Animals↗