Kinetics of inhibition of platelet calpain II by human kininogens.

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.     

Diversity of plant cell wall esterases in thermophilic and thermotolerant fungi

Fourteen thermophilic and thermotolerant fungal strains isolated from composting soils produced plant cell wall-acting esterases in a medium containing corn cobs and oat spelt xylan. The concentrated and dialyzed protein extracts of these fungi were fractionated using isoelectric-focusing, gels sliced and eluted protein in each slice was assayed for esterase activity against p-nitrophenyl acetate. A total of 84 esterases detected on the basis of pI were found to show distinct preferential substrate specificities towards p-nitrophenyl acetate, p-nitrophenyl ferulate and p-nitrophenyl butyrate, and were putatively classified as acetyl esterases and esterases types I and II. None of the esterases were active against p-nitrophenyl myristate. In addition, these esterases were characterized as acid, neutral or alkaline active.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

BIOCHEMICAL CHANGES DURING SEXUAL DEVELOPMENT IN MARCHANTIA POLYMORPHA: I. ESTERASES

Extracts of uninduced thalli, induced thalli, stalks, antheridiophore and archegoniophore disks of Marchantia polymorpha were subjected to starch-gel zone electrophoresis. Developed gels were treated with appropriate reaction mixtures to detect sites of activity for 12 enzyme systems; only phosphatases, esterases, and peroxidases were observed. Although common sites of phosphatase, peroxidase, and esterase activity were detected in all tested extracts, additional sites of peroxidase and esterase activity were found in extracts from antheridiophore disks. The antheridia provided the additional esterases as determined by the electrophoresis of antheridial extracts and by a histochemical test for esterases in sections of antheridiophores.     

Biochemical properties,homology, and genetic variation of Drosophila “nonspecific” esterases

The biochemical properties and tissue distribution of two major, soluble "nonspecific" esterases have been studied in Drosophila melanogaster, D. pseudoobscura, and related species. The "alpha-like" activity is due to a monomer enzyme (MW congruent to 60 kd) having a nonspecific tissue distribution, which was inhibited by p-hydroxymercuribenzoate (5 X 10(-4)M) plus eserine (1 X 10(-5)M) and was relatively unstable during polyacrylamide gel electrophoresis. Electrophoretograms of this enzyme could be enhanced by treating gels with beta-mercaptoethanol before staining. This procedure allowed the identification of a new alpha-esterase (Est-4) in D. pseudoobscura. The "beta-like" esterase activity (EC 3.1.1.1) is due to a dimer (MW congruent to 120 kd) in most Drosophila species. D. melanogaster and its siblings (D. simulans and D. mauritiana) were exceptions in which this enzyme had an unusual tissue distribution (increased activity in the male reproductive system) and was a monomer (MW congruent to 60 kd). Differences in the genetic variability of these esterases are discussed and interpreted by a population expansion model rather than by differences in biochemical properties of enzyme forms.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Concanavalin A binds of purified prolyl hydroxylase and partially inhibits its enzymic activity.

Prolyl hydroxylase [(EC 1.14.11.2; prolyl-glycyl peptide, 2-oxoglutarate dioxygenase (4-hydroxylating)] was electrophoresed on polyacrylamide gels and the enzyme in the gels was shown to bind [acetyl-³H]concanavalin A. The enzyme-lectin complex was dissociated by treating the gel with methyl α-D-mannopyranoside, a sugar known to inhibit binding of concanavalin A to glycoproteins. Furthermore, prolyl hydroxylase activity was partially inhibited by concanavalin A when the enzyme was assayed in the absence of bovine serum albumin, a protein which enhances enzymic activity. The inhibition of enzyme activity was prevented by sugars known to react with concanavalin A.     

Isolation of Two Acetyl Esterases from Extracts of Bacillus subtilis

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.     

Variations des zymogrammes du tube digestif et du muscle chez Cyclonassa neritea en fonction de la température d''acclimatation

Soluble proteins, esterases 2C, acid phosphatases of the digestive gland and foot muscle of Cyclonassa neritea, were compared using polyacrylamide gradient gels. α-Glucosidases, alkaline phosphatases, l-leucine aminopeptidase and peptidase were studied from digestive gland extracts. Molecular weights of isoenzymes were evaluated with 5000 d accuracy. Variation in activity of the most important isoenzymes of each enzyme under the influence of acclimation temperature was measured. In both muscle and digestive gland, the concentration of soluble proteins is stable. Through the whole acclimation temperature range, esterase activity per mg protein decreased with increased temperature. l-Leucine aminopeptidase activity decreases steadily from 10 to 25°, even though the two alkaline phosphatase isoenzyme activities increase. The other enzymes have their maximum activities at 20°.     

Isolation and partial characterization of alkaline feruloyl esterases from Aspergillus niger CFR 1105 grown on wheat bran

Feruloyl esterases (FAEs) of a strain of Aspergillus niger (CFR 1105) grown in solid state (ssf) and submerged fermentations (smf) using wheat bran both as carbon source and inducer of the enzyme were studied. The feruloyl esterase activity was maximum after 4 days in solid state as well as in submerged fermentations (32.5 and 31.5 U/g dry weight of wheat bran respectively) and the enzyme titers were comparable. The specific activity was maximum on day 2 in ssf (12.8 U/mg protein) and it decreased thereafter, whereas specific activity was maximum on day 3 (11.7 U/mg protein) in smf and it remained constant up to 5 days. Two isoenzymes of feruloyl esterases were isolated and purified to homogeneity by conventional protein purification methods from the day 5 culture filtrate of A. niger grown in smf. On a DEAE-cellulose column, two enzyme activity peaks designated as FAE-1 and FAE-2 were eluted with 0.3 and 0.35 M NaCl, respectively. They were monomeric glycoproteins with approximate molecular weights of 50 kDa (FAE-1) and 55 kDa (FAE-2), respectively. FAE-1 showed a temperature optimum of 40°C whereas FAE-2 showed a wider temperature optimum of 40–50°C. FAE-1 and FAE-2 exhibited pH optima of 9 and 6, respectively, and both were stable over a pH range of 6–9. The ability of the enzyme to be active in alkaline pH may be advantageous in biotechnological applications.     

Nongradient blue native gel analysis of serum proteins and in-gel detection of serum esterase activities

The objective of the present study was to analyze serum protein complexes and detect serum esterase activities using nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). For analysis of potential protein complexes, serum from rat was used. Results demonstrate that a total of 8 gel bands could be clearly distinguished after Coomassie blue staining, and serum albumin could be isolated nearly as a pure protein. Moreover, proteins in these bands were identified by electrospray mass spectrometry and low-energy collision induced dissociation (CID)-MS/MS peptide sequencing and the existence of serum dihydrolipoamide dehydrogenase (DLDH) was confirmed. For studies of in-gel detection of esterase activities, serum from rat, mouse, and human was used. In-gel staining of esterase activity was achieved by the use of either α-naphthylacetate or β-naphthylacetate in the presence of Fast blue BB salt. There were three bands exhibiting esterase activities in the serum of both rat and mouse. In contrast, there was only one band showing esterase activity staining in the human serum. When serum samples were treated with varying concentrations of urea, esterase activity staining was abolished for all the bands except the one containing esterase 1 (Es1) protein that is known to be a single polypeptide enzyme, indicating that majority of these esterases were protein complexes or multimeric proteins. We also identified the human serum esterase as butyrylcholinesterase following isolation and partial purification using ammonium sulfate fractioning and ion exchange column chromatographies. Where applicable, demonstrations of the gel-based method for measuring serum esterase activities under physiological or pathophysiological conditions were illustrated. Results of the present study demonstrate that nongradient BN-PAGE can serve as a feasible analytical tool for proteomic and enzymatic analysis of serum proteins.     

Cancer-associated serum galactosyltransferase activity. Demonstration in an animal model system.

Two different lines of solid tumors were produced in outbred hamsters by subcutaneous injection of polyoma transformed BHK cells. Growth of the tumors correlated with the appearance in serum of an electrophoretically distinct peak of galactosyltransferase: NeuAc-, Gal-free fetuin acceptor activity on polyacrylamide gels. This slow moving peak of enzyme activity (GT-HH) was detected before solid tumors could be grossly observed and the amount of activity in this peak was also found to be linearly related with growth of the tumor. GT-IIH was not detectable in control animals and separated from a faster migrating major area of serum galactosyltransferase activity (GT-IH) found in sera of both control and tumor-bearing hamsters. These two activities were shown to maintain their respective mobilities on re-electrophoresis. Solubilized enzyme derived from excised tumors demonstrated an electrophoretic mobility on polyacrylamide gels identical to that for GT-IIH present in serum from tumor-bearing animals. In contrast, enzyme activity solubilized from livers of both control or tumor-bearing hamsters showed a mobility similar to that of the faster moving serum galactosyltransferase enzyme activity, i.e. GT-IH. In addition, medium derived from nonconfluent BHKpy cells in tissue culture contained galactosyltransferase activity which co-electrophoresed with the slower migrating characteristics of galactosyltransferase activities derived from serum (control and tumor-bearing), solid tumors, liver and BHKpy cells in tissue culture were compared. All kinetic properties were similar with the exception that the Km UDP-galactose of GT-IIH (1.0 X 10(-5) M) was half that of GT-IH (2.0 X 10(-5) M).     

Isolation and characterization of carboxylesterase from vinyl acetate-assimilating bacterium isolated from soil

A bacterium Pseudomonas species which is able to assimilate vinyl acetate and other esters as a sole source of carbon was isolated from soil. The bacterium contained three kinds of esterases (esterases I, II and III) which were separable on DEAE-cellulose column chromatography. Esterase II was purified and characterized. The enzyme is a monomer with the molecular weight of 27,000 and it hydrolyzed various esters most efficiently at pH 8.0. The activity of the enzyme was inhibited by diisopropylfluorophosphate. The purified enzyme was different from other esterases so far found in Pseudomonas sp. with respect to molecular structure and substrate specificity.     

Short-term effect of a high-protein/low-carbohydrate diet on aminopeptidase in adult rat jejunoileum. Site of aminopeptidase response.

The short-term effects of high-protein/low-carbohydrate diet on aminopeptidase N activity were studied in the brush-border membranes of proximal jejunum and proximal ileum of adult rats. The animals were starved overnight and re-fed for 15 h either with a standard diet (20% protein, 55% carbohydrate, in terms of energy content) or with a high-protein/low-carbohydrate diet of equal energy content (70% protein, 5% carbohydrate). All rats consumed similar amounts of diet, and measurements were made 15 h after initiation of re-feeding. In the proximal jejunum a slight increase in aminopeptidase activity was observed after the high-protein intake. In contrast, considerable stimulation (52%) of the enzyme specific activity was obtained in the proximal ileum. This increase in ileal aminopeptidase activity was more prominent in the mature cells of the upper villus. To determine if the increase of aminopeptidase activity was due to an increased amount of enzyme protein, rocket immunoelectrophoresis was performed with detergent-solubilized brush-border protein from ileum on agarose gels containing anti-(rat brush-border) antiserum. When the same amount of enzyme activity was loaded on the gels, the peaks of immunoprecipitate for aminopeptidase were similar for animals fed on a standard or a high-protein diet. When the same amount of protein was loaded, the peak of immunoprecipitate for aminopeptidase was higher (81%) after a high-protein diet. These results showed that the high protein intake evoked an increase in aminopeptidase activity, with a concomitant increase in the amount of immunoreactive protein.     

Histochemical patterns of lipolytic enzymes of the hepatopancreas of Scylla serrata and their possible relation to eyestalk factor(s)

Of the lipolytic enzymes studied histochemically, lipase (Tween 80 specific enzyme) showed highest activity while non-specific esterases (Tween 60, α-naphthyl acetate, β-naphthyl acetate and 5-bromo-indoxyl acetate esterases) and Tween 20 and Tween 40 esterases exhibited medium and lowest activities respectively. The pattern of distribution of these enzymes was found to be variable among the various elements of the hepatopancreas. Lipase. Tween 20 and Tween 40 esterases were localized at the apical parts of the R-cell, F-cell cytoplasm, the B-cell vacuolar contents and the lumina of the hepatopancreatic acini. Non-specific esterases were primarily present in the cytoplasm of the R-cells, brush border of the tubules and connective tissue. A striking reduction in the lipase activity of the R- and B-cells was apparent 4 hours after the bilateral extirpation of eyestalks. Restoration of activity was observed 48 hours after the operation. On the other hand, a rise in the level of non-specific esterases was conspicuous 4 hours after the eyestalk amputation. This increased enzyme activity was restored to normal 24 hours after the operation. Administration of eyestalk extract into normal and de-stalked animals caused an increase in the lipase activity of the R- and B-cells. Surprisingly, the activity of non-specific esterases also enhanced considerably in the R-cell cytoplasm and connective tissue. The enhanced activity of lipase and non-specific was noted 4 hours after the treatment. From the present findings it appears that the eyestalk hormone(s) are directly involved in regulating the activity of the lipolytic enzymes. The physiological role of the F- and B-cells seems to be secretion and extrusion of lipase respectively. The R-cells and connective tissue appear to be associated with storage and mobilization of lipids.     

Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases

Alpha-synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of alpha-synuclein has not yet been known. Here we have shown that alpha-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of alpha-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with alpha-synuclein. Our results indicate that alpha-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.     

The nature of the prealbumin 'esterases' of horse serum

Evidence is presented to suggest that the acidic prealbumin esterases in horse serum represent a protease-inhibitory protein. The esterase activity may arise from residual enzymic activity of the bound protease.