Esterase

About: Esterase is a research topic. Over the lifetime, 7622 publications have been published within this topic receiving 168270 citations.

Microplate adaptation of Gomori's assay for quantitative determination of general esterase activity in single insects.

Abstract: Esterase activity is monitored in mosquitoes and other arthropod species because high levels of these enzymes can be associated with pesticide resistance. In the 1950s, G. Gomori devised a colorimetric method to detect esterase activity based on their capacity to hydrolyze aryl-esters. We modified this method for use in microtiter plates. Mosquito homogenates ( Culex quinquefasciatus Say and C. pipiens L.) from strains susceptible and resistant to insecticides were allowed to hydrolyze a-napthyl acetate in the presence of Triton X-100 and a specific acetylcholinesterase inhibitor. The a-naphthol product was detected colorimetrically by a diazo-coupling reaction with Fast Garnet GBC salt. Triton X-I00 improved the extraction of esterases and maintained the azo compound in solution. The linear range of the method was 2-20 nmoles of a-naphthol; this high sensitivity permitted accurate determinations in 1/30 portions of single adult mosquitoes from the strain with the lowest esterase activity. To avoid variations due to changes in temperature and duration of assay, results were normalized to equivalent enzyme activity units obtained in a spectrophotometer at 25°C. Depending on the number of homogenate dilutions required, performance of the assay in microplates allowed the simultaneous analysis of 20-80 samples. Female mosquitoes showed higher enzyme activity than males when expressed in nmoles/min per mosquito, but differences were reduced when results were expressed as specific activity (nmoles/min per mg protein). A mosquito strain resistant to organophosphates due to the presence of high levels of esterases showed about 200 times more esterase activity than a susceptible strain or a strain resistant due to insensitive acetylcholinesterase.

Prolactin Induction of Enzymes Controlling Luteal Cholesterol Ester Turnover

Abstract: An enzyme in extracts of luteinized rat ovaries was found which catalyzed the synthesis of cholesterol esters from palmitic acid-l-14C and cholesterol and required ATP, Mg++ and Coenzyme A for expression of activity. This enzyme, sterol acyl transferase, was associated predominantly with the 100,000 ×g pellet fraction obtained from 10,000 ×g supernatant fractions of homogenized ovaries. The enzyme which hydrolyzes cholesterol esters, sterol esterase, was assayed simultaneously in 100,000 ×g supernatant fractions. Transferase and esterase levels were both markedly reduced (p <.01) when assayed 3 days after hypophysectomy (90 and 70%, respectively). Administration of rat prolactin (2 IU; sc; b.i.d.) was completely effective in preventing the decline of both enzymes after hypophysectomy, whereas LH (NIH-LH-S14; 50 μg daily in sesame oil+5% beeswax) was without effect. In hypophysectomized animals, LH and prolactin combined were less effective in maintaining transferase activity than prolactin treatment alone...

Biodegradation of polycarbonate-based polyurethanes by the human monocyte-derived macrophage and U937 cell systems

Abstract: The prominent cell type found on implanted medical devices during the chronic inflammatory response is the monocyte-derived macrophage (MDM). Using an activated in vitro cell system, it was possible to show that MDMs possess esterolytic activities that may contribute to the degradation of polyurethanes. In the present study, the U937 cell line was paralleled to the MDM cell system in order to validate the use of a cell line that could expedite studies on biomaterial biocompatibility and biostability. Using 12-o-tetradecanoylphorbol 13-acetate (PMA), the optimum differentiation time for the U937 cells was 72 h based on biodegradation, degradative potential, and (35)S-methionine uptake. After activation of the cells by resuspending from tissue culture polystyrene plates and reseeding onto a (14)C-labeled polycarbonate-based polyurethane(PCNU), both U937 cells and the MDMs elicited comparable radiolabel release (measure of polymer breakdown) and esterase activity (measure of degradative potential) at 48 h. There was no difference in the effect on radiolabel release and esterase activity elicited by both cell types with inhibitors of protein synthesis, esterase activity, and phospholipase A(2). This established that both cell types likely used similar hydrolytic activities and signaling pathways to cause degradation of the PCNU. Immunoblotting demonstrated that both cell systems secreted monocyte-specific esterase and cholesterol esterase enzymes previously shown to degrade PCNUs. The U937 cell system is more convenient and reproducible than MDMs for pursuing possible biological pathways elucidating the mechanism of polyurethane biodegradation. Once established with U937s, the pathways can then be validated with the more physiologically relevant human MDM cell system.

Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum

Abstract: A hypothetical protein FoFaeC-12213 of Fusarium oxysporum was found to have high amino acid sequence identity with known type C feruloyl esterases (FAEs) containing a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase. The putative FAE from the genomic DNA was successfully cloned in frame with the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in Pichia pastoris X-33 to confirm that the enzyme exhibits FAE activity. The molecular weight (62 kDa) and pI (6.8) were in agreement with the theoretical calculated values indicating the correct processing of the secretion signal in P. pastoris. The recombinant FAE was purified to its homogeneity and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme is a type C FAE showing broad hydrolytic activity against the four methyl esters of hydroxycinnamic acids and strong preference for the hydrolysis of n-propyl ferulate. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from Trichoderma longibrachiatum (a maximum of 67% total FA released after 1-h incubation). The esterase showed broad pH stability making it an important candidate for alkaline applications such as pulp treatment in the paper industry.