Showing papers on "Esterase published in 1973"

A method for the detection of atypical forms of human serum cholinesterase. determination of dibucaine numbers

Abstract: Cases with atypical esterase activity were found by determining esterase inhibition in numerous sera. A suitable inhibitor was the local anaesthetic dibucaine (cinchocaine, TN Nupercaine, Perkain). A good discrimination between typical and atypical sera was obtained under the following conditions: The esterase activity of human serum diluted 1:100 was measured with a recording spectrophotometer at 240 mμ. The substrate was 5 × 10−5 M benzoylcholine dissolved in M/15 phosphate buffer, pH 7.4. The concentration of the inhibitor was 10−5 M. With the experimental temperature around 25 °C, the average inhibition of the typical enzyme was 78.8 ± 0.3%. The inhibition of the atypical esterases was less; in rare cases the inhibition was only 16%. For each person, the inhibition characteristics were constant over a period of several months, and independent of the esterase level. The degree of inhibition measured under these conditions and expressed in per cent has been termed "Dibucaine Number".

A sequence of biochemical events in the antigen-induced release of chemical mediators from sensitized human lung tissue

Abstract: Five sequential steps interspaced between the antigen activation of human lung fragments sensitized with IgE and the release of the chemical mediators, histamine and slow-reacting substance of anaphylaxis (SRS-A), have been delineated. The experimental design that permits this analysis is based upon the capacity to maintain the serine esterase essential to mediator release in its diisopropylphosphofluoridate (DFP)-resistant precursor state despite antigen challenge and upon the ability to arrest reversibly the reaction sequence by various manipulations. When sensitized lung fragments are challenged with antigen in the presence of DFP, a serine esterase is converted to its active DFP-inhibitable state; this conversion is prevented if antigen challenge in the presence of DFP occurs in calcium-free buffer indicating that immunologic activation of the esterase requires extracellular calcium. The fact that calcium depletion alone does not impair antigen-induced histamine release implies that prevention of esterase activation depends upon both the absence of extracellular calcium and the inactivation of any active esterase by DFP to prevent an autocatalytic feedback activation. Arresting the antigen-induced activation of the serine esterase by the combination of DFP in calcium-free buffer precludes the sequence from reaching the labile, 2-deoxyglucose (2-DG)-inhibitable, energy-requiring step, indicating that proesterase activation precedes this energy-requiring stage. The 2-DG-inhibitable step precedes a second calcium-requiring, EDTA-inhibitable stage, as EDTA prevents glucose reversal of 2-DG inhibition of antigen-challenged tissue, while the presence of 2-DG does not prevent calcium reversal of EDTA inhibition. The finding that isoproterenol prevents calcium reversal of EDTA inhibition of mediator release suggests that the inhibitory site of action of increased concentrations of cyclic AMP is coincident with or subsequent to the second calcium-requiring, EDTA-inhibitable step. Therefore, the sequence of biochemical events initiated by the interaction of antigen with tissue-fixed IgE antibodies appears to proceed from the calcium-requiring activation of a DFP-sensitive serine esterase; the further autocatalytic activation of the esterase; a 2-DG-inhibitable energy requirement; a second calcium-requiring, EDTA-inhibitable stage; and a cyclic AMP-inhibitable step to the release of histamine and SRS-A.

Pyrethroid insecticides: esterase cleavage in relation to selective toxicity.

Abstract: The ester group of primary alcohol chrysanthemates is cleaved by mouse hepatic microsomal esterases, more rapidly for the (+)-trans than for the (+)-cis isomers. Substrate-specificity and inhibition studies in vivo establish that these pyrethroid-hydrolyzing esterases probably contribute to the low mammalian toxicity of bioresmethrin and other (+)-trans chrysanthemate insecticide chemicals derived from primary alcohols.

The Effect of Stress Induced by Ether Anaesthesia on Cholesterol Content and Cholesteryl‐Esterase Activity in Rat‐Adrenal Cortex

Abstract: The cholesterol content and the activities of cholesteryl esterase and cyclic-AMP-dependent protein kinase have been studied in the adrenal glands of rats. These studies have been performed on the adrenal glands of rats injected with a protein-synthesis inhibitor, cycloheximide, and in the adrenal glands of rats exposed to ether anaesthesia to increase the blood concentration of adrenocorticotrophic hormone. The analysis of the adrenal steroids revealed that acute stress induced by ether anaesthesia stimulated cholesterol ester depletion in adrenal lipid droplets. Cycloheximide injection into rats did not significantly alter the concentrations of cholesterol and cholesterol esters in the lipid droplets. Injection of cycloheximide followed by ether anaesthesia resulted in an increase in free cholesterol with a concomitant decrease in cholesterol ester concentration within the lipid droplets. Cholesteryl esterase and protein kinase activities in rat adrenal 105000 × g supernatant fractions were significantly higher in animals subjected to stress by ether anaesthesia. Injection of cycloheximide did not prevent the stress-induced enhancement of the activities of either enzyme. Cholesteryl esterase activity was significantly enhanced by the addition of cyclic AMP, ATP and theophylline in the presence of cyclic-AMP-dependent protein kinase. The combination of all three activators proved to be more effective than any one or combinations of any two factors. The degree to which the activators stimulated cholesteryl esterase was higher in untreated animals than in animals subjected to ether anaesthesia. It is postulated that the enhancement of cholesteryl esterase activity induced by ether anaesthesia is due to the effect of adrenocorticotropic hormone on the adrenal gland, and that in vitro activation of cholesteryl esterase by cyclic AMP and ATP involves a protein-kinase-dependent phosphorylation reaction. Subcellular distribution of cholesteryl esterase activity in rat adrenal gland and some properties of the enzyme have been studied.

Studies on the coagulant enzyme from Agkistrodon rhodostoma venom. Isolation and some properties of the enzyme.

Abstract: 1. Arvin, a commercial preparation of the coagulant activity from the venom of Agkistrodon rhodostoma , is shown to contain a non-coagulant caseinolytic fraction. 2. A method is described for the purification of the coagulant enzyme free from any detectable contaminating protein. 3. The coagulant enzyme is identified as a glycoprotein which probably consists of a single polypeptide chain containing approx. 29% by weight of carbohydrate. Amino acid and carbohydrate analyses are reported and the N - and C -terminal amino acid residues identified. 4. Electrophoresis on polyacrylamide gel reveals the polymorphic nature of the glycoprotein. Five forms of the enzyme are observed. 5. The coagulant action is correlated with an arginine esterase activity and kinetic properties are studied with both arginine and lysine esters as substrates. The inhibitory nature of guanidine and arginine toward the esterase activity is reported.

An esterase zymogram of Escherichia coli.

Abstract: SUMMARY: The intracellular esterases of 25 strains of Escherichia coli, growing exponentially on a minimal medium, were analysed by the acrylamide-agarose zymogram technique. Five kinds of esterase bands were defined: three major bands (A, B and C) and two minor ones. The A and B esterase bands hydrolysed α-naphthyl, β-naphythl and indoxyl acetates; they were inhibited by di-iso-fluoropropyl phosphate (DFP). Esterase band B also hydrolysed the α- and β-naphthyl butyrates and was stable at 60°C. Esterase band C hydrolysed only β-naphthyl acetate and it resisted DFP. The A, B and C esterase bands showed variations in electrophoretic mobility which seemed to indicate an intraspecific differentiation of molecular structures of the esterase that could have arisen during microbial evolution.

Biochemical genetics of rat esterases: polymorphism, tissue expression and linkage of four loci

Abstract: Two alleles at each of four esterase loci in Rattus norvegicus are described with regard to tissue expression, electrophoretic characterization, and genetic linkage. A previously described dominant gene for prealbumin serum esterase is demonstrated to exist as two codominant alleles in the genetically determined absence of the characteristic albumin esterase. The allelic composition of 16 inbred strains for four esterase genes is provided, and the heretofore ambiguous nomenclature of rat esterase genetics is standardized. Linkage of Es-1, Es-2, and Es-3 is demonstrated. Es-2 and Es-3 are tightly linked in that no recombination has been observed in 55 offspring. The same offspring demonstrated 9% recombination between Es-1 and the other two loci.

Purification and molecular properties of an unspecific carboxylesterase (E1) from rat-liver microsomes.

Abstract: 1 The isolation of an unspecific carboxylesterase from rat liver microsomes is reported. The purification is 66-fold at a yield of about 20%. The enzyme is one of 5 esterase/amidase variants recently separated and corresponds to the fraction designated E1. 2 The enzyme is homogeneous in disc electrophoresis, dodecylsulfate-polyacrylamide gel electrophoresis and in analytical ultracentrifugation, but exhibits slight heterogeneity in isoelectric focusing. 3 Its amino acid composition is presented. 4 The molecular weight of rat liver esterase E1 is 177000 (equilibrium sedimentation), the subunit weight 61 500 (dodecylsulfate disc electrophoresis), and the equivalent weight 66000 (titration with bis[p-nitrophenyl]phosphate). After cross-linking of the enzyme by dimethyl suberimidate and dodecylsulfate-polyacrylamide gel electrophoresis three principal bands are observed. Therefore, a trimeric structure in which each subunit possesses one active site is proposed.

Serum esterase genetics in rats: Two new alleles at Es-2, a new esterase regulated by hormonal factors, and linkage of these loci to the Ag-C blood group locus

Abstract: Two new alleles at the Es-2 locus are described which determine electrophoretic variants of serum esterases of rats. A new esterase protein is described which is detectable in sera of sexually mature females of the appropriate genotype. Evidence is presented for genetic linkage between the Ag-C blood group locus and Es-1, Es-2, and the locus controlling the sex-influenced protein. Since the Ea-1 blood group locus of mice is linked to four esterase loci, it is suggested that Ag-C is the rat homologue of the mouse Ea-1 locus.

Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens

Abstract: An obligately anaerobic ruminal bacterial isolate was selected from 18 tributyrin-degrading isolates and identified as Butyrivibrio fibrisolvens strain 53. The culture in late exponential phase contained enzymes which could be released by sonic disruption. These enzymes degraded substrates at a rate in the order 1-naphthyl acetate (NA) > 1-naphthyl butyrate > 1-naphthyl propionate but did not degrade 1-naphthyl palmitate or 1-naphthyl phosphate. The enzymes on NA were neither stimulated nor inhibited by CoCl(2), MgCl(2), and MnCl (each varied from 10(-6) to 10(-4) M). CaCl at 10(-3) M stimulated esterase activity by 16%. Aliphatic substrates were hydrolyzed at a rate in the order triacetin > tributyrin > tripropionin, and ethyl acetate > ethyl formate. Similarly, aromatic fluorescein diesters were degraded at a rate in the order acetyl > propionyl > caproyl > butyryl > capryl > lauryl. Polyacrylamide gel electrophoretic zymograms indicated that the enzyme composite contained cathodally migrating bands. By column chromatography, these enzymes were separated into six NA-degrading fractions. Fraction V contained an esterase which had an optimal temperature of 39 C, a K(m) of 7.6 x 10(-4) on NA, and a molecular weight of about 66,000. This enzyme was inhibited by paraoxon (41%, 10(-4) M), eserine (17%, 10(-2) M), NaF (17%, 10(-2) M), and diisopropyl fluorophosphate (62%, 10(-4) M) but not by 1-naphthyl N-methyl carbamate at 8.4 x 10(-4) M.

Current problems on the structure and classification of mammalian liver carboxylesterases (EC 3.1.1.1).

Abstract: 1. By isoelectric focusing pig liver esterase (EC 3.1.1.1) is separated into 4 main, but still inhomogeneous peaks. A study of the specificity of these main variants, using acetanilide, methyl butyrate, and o-nitrophenyl acetate as substrates, showed that all 3 compounds were hydrolysed by all enzyme forms. The ratios of the activities, however, differed considerably. 2. Butyryl choline is hydrolysed by highly purified liver esterase, whereas liver esterases from ox and rat are inactive toward this substrate. The activity of pig liver esterase toward butyryl choline and methyl butyrate is completely inhibited by 10 µM physostigmine. 3. The present state of knowledge on the main molecular properties of pig liver esterase is reviewed. The available evidence strongly suggests that the enzyme is a trimer. 4. A comparison is made of the structure of 5 carboxylesterase preparations of different origin. 5. Some current problems on the nomenclature and classification of carboxylesterases are discussed in the light of recent findings from our laboratory.

Frequency distribution and linkage disequilibrium of active and null esterase isozymes in natural populations of drosophila montana

Abstract: The mode of inheritance of the 15 electrophoretically different α-naphthyl acetate-specific esterases found in five population samples of Drosophila montana collected near Gothic, Colorado, was delineated by mating a strain which exhibited no esterase activity to 237 wild-caught flies, and analyzing the progeny. This analysis showed that the α-esterases conform to a four-locus model with a high frequency of a null allele at each locus. Over 90% of the flies had an active esterase produced at only three or four of the possible eight loci; 75% of the chromosomes had two active alleles, presumably the optimum number. Extremely similar frequency distributions of the common allozymes were observed in all populations studied and over 3 years. From this analysis the frequencies of chromosomes of different allozyme types could be calculated. From these chromosome frequencies the probability of an active or a null allele at each locus was calculated. This, in turn, allowed determination of whether the presence of ...

Properties of a stable cell-free esterase from soil.

Abstract: The properties of a stable, extracellular phenyl esterase which hydrolyzes the organophosphorus insecticide, malathion, to its monoacid were investigated following its isolation from soil by 0.2 N alkali extraction and 560-fold purification by MnC12 treatment, protamine sulfate treatment, and QAE Sephadex A-50 chromatography. Phosphonate and phenyl thiophosphate anticholinesterase insecticides were potent competitive inhibitors of soil esterase activity. Inhibi- tion was also observed with mono- and dithiols, but not with diisopropyl fluorophosphate or sulfhydrol compounds. The esterase was not susceptible to enzymatic proteolysis nor A heat-labile component has been isolated from soil which degrades the insecticide, malathion (diethyl mercaptosuc- cinate, S-ester with 0,O-dimethyl phosphorodithioate), to its monoacid, and evidence was presented that the substance is a stable, cell-free enzyme (Getzin and Rosefield, 1968, 1971). It was partially purified by procedures employed for isolating proteins and exhibited typical Michaelis-Menten kinetics. It was electrophoretic, filterable, heat-labile, nondialyzable, and susceptible to denaturation by acid and alkali. Its exis- tence as a stable, cell-free enzyme was postulated from evi- dence based upon the persistence and adsorptive character- istics of the partially purified material in soil. Soil enzymes must possess certain characteristics to ensure their existence in the cell-free state for extended periods. The malathion esterase has some of these properties (Getzin and Rosefield, 1971). It is not easily destroyed by heat, resists microbial attack, loses little or no activity upon prolonged storage, and is resistant to desiccation in soil. Perhaps most significant, it withstands the comparatively drastic alkali treatment necessary for freeing it from soil and thus enables one to examine its properties in vitro. The present investiga- tions were designed to further purify and characterize the enzyme and to examine some of the properties that contribute to its unusual stability in soil. Experimental Section Materials. Chehalis clay loam (pH 5.5-6.0) was collected from a cultivated area in western Washington and used as a source of the enzyme. It contained 8 z organic matter and 30 z clay. (I4C)Malathion (14.0 pCi/mg), labeled at the 2 and 3 positions of the succinyl moiety, was purchased from Amer- sham/Searle Corp ., Chicago, Ill. Nonradioactive malathion (98.5 purity) and malaoxon were obtained from American

The genetics of Cepaea esterases I. Cepaea nemoralis

Abstract: Using Polyacrylamide disk electrophoresis, at least 23 zones of general esterase activity can be distinguished in extracts of Cepaea nemoralis hepatopancreas. Breeding experiments have thrown light on the inheritance of three esterase systems. It has been suggested that the four most cathodally migrating esterase zones may be controlled by four linked loci, forming an esterase supergene. Zone Est. 5 also seems to be part of the same system and might be coded for by another linked locus. At each of these loci three alleles have been postulated, one producing no detectable enzyme, another producing a weak enzyme and the third coding for an active enzyme. Each locus seems to control enzyme activity at one level in the gel. Another esterase system is controlled by three alleles at one locus. The enzyme produced is a dimer, heterozygotes having three zones of activity. Attemps to hybridise “parental” enzymes so as to give a hybrid zone on electrophoresis failed. The third esterase system examined is one of presence and absence of the enzyme. It is controlled by two alleles at one locus with the presence of enzyme being dominant to its absence. There is no linkage detectable between any of the (effective) esterase loci or between the esterase loci and three loci determining the shell colour and banding morphs.

Genetische Untersuchungen bei Picea abies mit Hilfe der Isoenzym-Identifizierung

Abstract: Using techniques of starch gel zone electrophoresis, a considerable variation among esterase and leucine aminopeptidase isoenzyme patterns was found in the endosperm of dormant seeds of Norway spruce (Picea abies). Since the so-called primary endosperm of conifers is only a further developmental stage of the haploid female gametophyte, simple Mendelian segregations can be determined in seeds of individual open-pollinated trees. It was therefore possible to identify some esterase and leucine aminopeptidase loci only with regard to phenotypic frequency distributions without difficult crossing procedures.

Protein electrophoresis as an aid to oat variety identification

Abstract: Ten cultivars of oats (Avena sativa L.) were investigated for their isozymes of esterase (E), leucine aminopeptidase (LAP), and anodal and cathodal peroxidase (APX and CPX).

Studies of the Synthetic Model Enzyme. The Synthesis of Cyclo(His–Glu–Cys- D -Phe–Gly) 2 as the Esterase Model

Abstract: To study the mechanism of enzyme action, cyclo(His–Glu–Cys-D-Phe–Gly)2 was prepared as an esterase model from the corresponding linear decapeptide by the azide methods. The esterase-like activity of this cyclic decapeptide in reaction to p-nitrophenyl acetate was three times greater than that of the linear decapeptide, the curve of the catalytic coefficient versus the different pH is of a bell type, and the optimum pH was recorded at about 7.6. The kinetics of the hydrolysis obeyed Michaelis-Menten’s typical equation.

Acid phosphatase and esterase activity in orchid mycorrhiza.

Abstract: A cytochemical study was made to examine the possibility that acid phosphatase may be specifically involved in the digestion of endophytic hyphae in orchid mycorrhiza. Esterase activity was studied for comparison. Frozen sections of unfixed or glutaraldehyde-fixed protocorms of Dactylorhiza purpurella infected by Thanatephorus cucumeris (Rhizoctonia solani) were reacted for acid naphthol AS BI phosphatase, acid β-glycerophosphatase or naphthol AS D esterase. A marked increase in particulate acid naphthol AS BI phosphatase activity was observed during infection of host, central, parenchyma cells shortly before hyphae lysed; a diffuse reaction of high activity was localised on lysed fungus. Acid β-glycerophosphatase was present at particulate sites only in fungal cytoplasm and as a diffuse reaction on lysed fungus. Naphthol AS D esterase showed highest activity at hyphal apices. Esterase seems to be associated with growth and differentiation of hyphae in orchid cells, rather than lysis of the fungus.

Analysis of Tissue Esterases from Patients with Hodgkin's Disease and Other Types of Advanced Cancer by Isoelectric Focusing in Acrylamide Gel

Abstract: Summary Isoelectric focusing analysis of esterase activity has been carried out in acrylamide gel on a variety of human cancers (Hodgkin9s disease, lymphosarcoma, adenocarcinoma of the colon, adenocarcinoma of the breast) and on nonneoplastic tissues from cancer patients (liver, kidney, spleen, brain, and red blood cells). Eighteen distinct nonspecific esterase bands have been identified and characterized with respect to substrate preference and inhibitor profile. Each of the normal tissues contains a readily recognizable esterase pattern; the tumor extracts present greater heterogeneity. The patterns permit grouping into acetylesterases and butyrylesterases, i.e., carboxylesterases. The former are relatively cationic and resistant to the inhibitory effect of diethyl p-nitrophenyl phosphate and 10 m urea. The butyrylesterases tend to be rather anionic in charge; they are with but a single exception sensitive to inhibition with diethyl p-nitrophenyl phosphate. The cationic acetyl-esterases have substrate specificity and inhibitor profiles similar to those described for lysosomal nonspecific esterase. These acetylesterases are particularly prominent in homogenates of kidney, spleen, and Hodgkin9s disease. The possible relationship between these esterase activities and some of the clinical stigmata of histiocytic neoplasms is considered.