Original method for the histochemical demonstration of tripeptidyl aminopeptidase I.

The original histochemical method for the visualization of tripeptidyl aminopeptidase I (TPP I, EC 3.4.14.9) is developed. The method is based on the new synthetic substrates Gly-L-Pro-L-Met(or L-Ala)-5-chloro-1-anthraquinonyl hydrazide (Gly-Pro-Met[Ala]-CAH). The final reaction product is represented as tripeptidyl-5-chloro-1 anthraquinonyl hydrazides (TPP-CAH). Upon the enzyme action the practically in aqueous media insoluble brown-reddish dye 5-chloro-1-anthraquinonyl hydrazine (CAH) is released, which reacts simultaneously with aromatic aldehydes as e.g. 4-anisaldehyde (p-AA) or 4-nitrobenzaldehyde (p-NBA), resulting in microcrystalline or amorphous deeply colored hydrazones. The last compounds mark accurately the locations of enzymatic activity. The biochemically suggested lysosomal localization of this collagen-degrading exopeptidase is thus confirmed on tissue sections. More information about the distribution of TPP I in different rat organs is presented as well.     

Enhanced in situ detection of beta-glucuronidase activity in murine tissue.

We outline here a protocol for high-resolution in situ localization of beta-glucuronidase in murine tissues processed in glycol methacrylate (GMA). Murine tissues were first stained with 5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid (x-gluc), followed by histological processing in GMA. Retention of the blue indigo reaction product after overnight incubations in x-gluc allowed high-resolution localization of beta-glucuronidase activity by brightfield microscopy. When illuminated under darkfield, the x-gluc signal was enhanced, permitting detection even in cells with low-level enzyme activity. This technique offers for the first time a more sensitive enzyme histochemical method of detecting beta-glucuronidase activity in animal tissues and also the opportunity to examine expression at high magni-fication.     

Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Ring-fission, lactonizing and delactonizing enzymes

1. A cell-free system, prepared from Pseudomonas N.C.I.B. 9340 grown on 4-chloro-2-methylphenoxyacetate (MCPA) was shown to catalyse the reaction sequence: 5-chloro-3-methylcatechol --> cis-cis-gamma-chloro-alpha-methylmuconate --> gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide --> gamma-hydroxy-alpha-methylmuconate. 2. The activity of the three enzymes involved in these reactions was completely resolved and the lactonizing and delactonizing enzymes were separated. 3. This part of the metabolic pathway of 4-chloro-2-methylphenoxyacetate is thus confirmed for this bacterium. 4. The ring-fission oxygenase required Fe(2+) or Fe(3+) and reduced glutathione for activity; the lactonizing enzyme is stimulated by Mn(2+), Mg(2+), Co(2+) and Fe(2+); no cofactor requirement could be demonstrated for the delactonizing enzyme. 5. cis-cis-gamma-Chloro-alpha-methylmuconic acid was isolated and found to be somewhat unstable, readily lactonizing to gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide. 6. Enzymically the lactonization appears to be a single-step dehydrochlorinase reaction.     

2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.     

Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.     

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV (DPP IV), based on a newly synthesized substrate Gly-L-Pro-1-hydroxy-4-naphthylamide is proposed. Upon the enzyme hydrolysis of the substrate a strong reducing agent, i.e. 4-amino-1-naphthol is released, which reduces tetrazolium salts to water-insoluble, deeply colored formazans, that precipitate on the sites of enzyme activity, marking them accurately. No auxiliary electron acceptor is needed for the redox reaction. The incubation is performed at the optimal pH of the enzyme. Precise enzyme localization is achieved in all organs studied. Thus, the new method avoids most of the disadvantages of the methods in use and might open new possibilities in peptidases histochemistry.     

Optical biosensor consisting of glutathione-S-transferase for detection of captan

The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 μg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

Halogenated protocatechuates as substrates for protocatechuate dioxygenase from Pseudomonas cepacia

Substrates containing electron-withdrawing groups were reacted with protocatechuate 3,4-dioxygenase and oxygen. Haloprotocatechuates (5-fluoro-, 5-chloro-, 5-bromo-, 2-chloro-, and 6-chloroprotocatechuates) are oxygenated by the enzyme at rates 28- to 3000-fold lower than that with the native substrate. These lower rates are due to both deactivation of substrate to O2 attack, and to the formation of abortive enzyme-substrate (ES) complexes. Such ES complexes with haloprotocatechuates are spectrally distinct from the normal ES complex. 6-Chloroprotocatechuate produces changes more like those due to protocatechuate. The abortive ES complexes, when rapidly mixed with oxygen, decay to free enzyme and product monophasically, and the dependence of the rates on O2 concentration shows that a rate-limiting step precedes reaction with O2. Thus these complexes are rather unreactive toward O2, and the rate-limiting step in oxygenation is their conversion to active complexes. In contrast, the reaction of O2 with the enzyme and 6-chloroprotocatechuate is biphasic, the first phase being dependent on O2 concentration (2 X 10(4) M-1 S-1) and the second not (7 S-1). The intermediate formed after the first phase strongly resembles the second intermediate seen in the reaction of enzyme with protocatechuate and O2 (Bull, C., Ballou, D. P., and Otsuka, S., (1981) J. Biol. Chem. 256, 12681-12686), implying that the electron-withdrawing effect of the chlorine slows the O2 addition step considerably while the conversion to the second intermediate is hardly affected. When the enzyme cycles through several turnovers with 6-chloroprotocatechuate, an enzyme species is formed that resembles the unreactive ES complexes seen with the other haloprotocatechuates, indicating that a small amount of the unreactive complex is in equilibrium with the reactive complex and that during successive turnovers the enzyme is slowly converted into the unreactive form. The formation of this form correlates with the observation that in assays the rate of product formation gradually decreases with time.     

Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.     

Studies on the histochemical “Leucine aminopeptidase” reaction

Summary This paper summarizes the basic characteristics of the reactants involved in the usual LAP (naphthylamidase) reaction and the tetrazonium dye DBB coupling. In the subsequent experimental part the adsorption characteristics of the reactants, their diffusion rates and their behaviour towards lipids are studied in different model systems. Attention is called to the microcrystalline and insoluble nature of the final reaction product (FRP), and further to the very marked diffusion hindrance to this product in tissue sections. The quantitative aspects of the reaction pattern are considered.The findings lead to the suggestion of extended control experiments, in which the adsorption characteristics of DBB, the 2-Namine and the FRP are separately checked. These recommendations should be applied in cases where there is reason to doubt the correct localization of the FRP.The rate of coupling seems rapid enough to ensure a correct subcellular enzyme localization provided the PRP—the 2-Namine—is liberated and trapped in a compartment containing excess of DBB, i.e. in activated lysosomes and autophagosomes.The following Abbreviations will be used in the text LAP the so-called leucine Amino-peptidase reaction - DBB Diazo Blue B, Fast Blue B, tetrazotized diorthoanisidine (C.I. No. 37235); the amine content not stated (Sigma Comp.) - 1-NA 1-Naphthol - 2-NA 2-Naphthylamine - LNA Leucyl-2-naphthylamide - Arg-NA Arginine-2-naphthylamide - BANA Benzoyl-DL-arginine-2-naphthylamide - PRP The primary reaction product - FRP The final reaction product     

Synthesis of Chlorodideoxynucleosides Using Tris(2,4,6-tribromophenoxy)-dichlorophosphorane (BDCP)

Abstract

5′-Chloro-5′-deoxy-N,3′-O-dibenzoylthymidine (3a), 5′-chloro-5′-deoxy-N⁴, 3′-O-dibenzoyldeoxycytidine(3b), 5′-chloro-5′-deoxy-N⁶,3′-O-dibenzoyldeoxyadenosine(3c), N-benzoyl-1-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)thymine (5a) and N⁶-benzoyl-9-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)adenine (5b) have been synthesized in very high yields using a new efficient reagent, tris(2,4,6-tribrom-ophenoxy)dichlorophosphorane (BDCP). The reaction time was greatly reduced to 5–8 min. NOE data suggested an inversion of configuration at C₃-position and thus an SN2 mechanism has been proposed for the chlorination reaction.

     

Mesentery vascular metabolism of substance P

Dipeptidylpeptidase IV (EC 3.4.14.5), an enzyme which metabolizes substance P, is present in crude homogenates of hog mesenteric artery and aorta. Its subcellular localization is closely correlated with the plasma membrane marker enzyme 5'-nucleotidase (EC 3.1.3.5) in addition to the kinin and angiotensin metabolizing enzymes angiotensin I converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). The highest level of dipeptidylpeptidase IV is found on the surface membrane-enriched fraction and is immunologically identical to the kidney brush border-bound enzyme. Vascular dipeptidylpeptidase IV sequentially removes the N-terminal Arg1-Pro2 and Lys3-Pro4 dipeptides of substance P and exposes the biologically active C-terminal heptapeptide product to rapid degradation by vascular aminopeptidases.     

A quantitative cytochemical assay of beta-galactosidase in single cultured human skin fibroblasts

A quantitative cytochemical method for the measurement of beta-galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated beta-galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140. Control persons, one carrier of and two patients with beta-galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay. It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of beta-galactosidase deficiency due to the small number of cells needed in the analysis.     

Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors

The time-dependent inactivation of several serine proteases including human leukocyte elastase, cathepsin G, rat mast cell proteases I and II, and human skin chymase by a number of 3-alkoxy-4-chloroisocoumarins, 3-alkoxy-4-chloro-7-nitroisocoumarins, and 3-alkoxy-7-amino-4-chloroisocoumarins at pH 7.5 and the inactivation of several trypsin-like enzymes including human thrombin and factor XIIa by 7-amino-4-chloro-3-ethoxyisocoumarin and 4-chloro-3-ethoxyisocoumarin are reported. The 3-alkoxy substituent of the isocoumarin is likely interacting with the S1 subsite of the enzyme since the most reactive inhibitor for a particular enzyme had a 3-substituent complementary to the enzyme's primary substrate specificity site (S1). Inactivation of several enzymes including human leukocyte elastase by the 3-alkoxy-7-amino-4-chlorisocoumarins is irreversible, and less than 3% activity is regained upon extensive dialysis of the inactivated enzyme. Addition of hydroxylamine to enzymes inactivated by the 3-alkoxy-7-amino-4-chloroisocoumarins results in a slow (t1/2 greater than 6.7 h) and incomplete (32-57%) regain in enzymatic activity at pH 7.5. Inactivation by the 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-4-chloro-7-nitroisocoumarins on the other hand is transient, and full enzyme activity is regained rapidly either upon standing, after dialysis, or upon the addition of buffered hydroxylamine. The rate of inactivation by the substituted isocoumarins is decreased when substrates or reversible inhibitors are present in the incubation mixture, which indicates active site involvement. The inactivation rates are dependent upon the pH of the reaction mixture, the isocoumarin ring system is opened concurrently with inactivation, and the reaction of 3-alkoxy-7-amino-4-chloroisocoumarins with porcine pancreatic elastase is shown to be stoichiometric. The results are consistent with a scheme where 3-alkoxy-7-amino-4-chloroisocoumarins react with the active site serine of a serine protease to give an acyl enzyme in which a reactive quinone imine methide can be released. Irreversible inactivation could then occur upon alkylation of an active site nucleophile (probably histidine-57) by the acyl quinone imine methide. The finding that hydroxylamine slowly catalyzes partial reactivation indicates that several inactivated enzyme species may exist. The 3-alkoxy-substituted 4-chloroisocoumarins and 4-chloro-7-nitroisocoumarins are simple acylating agents and do not give stable inactivated enzyme structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Importance of different tfd genes for degradation of chloroaromatics by Ralstonia eutropha JMP134

The tfdC(I)D(I)E(I)F(I,) and tfdD(II)C(II)E(II)F(II) gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D). However, activity of tfd(I)-encoded enzymes was usually higher than that of tfd(II)-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module. The tfdD(II)-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R. eutropha strains containing different tfd gene modules. The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far.     

Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme.

Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S-2-chloro-3-(imidazol-5-yl)propionate at pH8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity.     

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.     

Anomalous reaction of 4-chloro-7-nitrobenzofurazan with thiol compounds.

The kinetics of reaction of 4-chloro-7-nitrobenzofurazan with thiol groups at pH values above 5 cannot be accounted for solely on the basis of formation of a single product, the 4-thio derivative. Spectroscopic observations indicate that, in addition to the 4-thio derivative, at least two other products are formed. One of these, referred to as P1, is most likely a reversible complex of thiol compound and 4-chloro-7-nitrobenzofurazan of the Meisenheimer type. The other product, P2, which forms primarily when thiol compound is in a large excess, does not appear to result from direct reaction of thiol group and 4-chloro-7-nitrobenzofurazan, but may be a reaction of product P1 and thiol compound. The coloured product, P2, will react further with proteins, such as bovine serum albumin and Escherichia coli RNA polymerase. This reaction irreversibly destroys the catalytic activity of RNA polymerase. The implications of these observations for utilization of 4-chloro-7-nitrobenzofurazan as a protein-modifying agent are discussed.