Substrate and inhibitor specificities have been used to classify esterases. The purpose of this work was to determine the substrate and inhibitor specificities of the esterases in an aquous extract of lyophilyzed carrots. Esterase activity was determined manometrically at 37°C. The optimum pH of the carrot esterases with several substrates varied from 6.8 to 7.2, and a pH of 7.2 was used in this study. The carrot extract hydrolyzed acetyl, propionyl and butyryl esters of phenol, 2-naphthol-6-SO₃Na and glycerol. As the acyl chain length was increased, activity decreased. Long-chain naphthyl esters and triolein were not hydrolyzed, which suggested the absence of lipase in the extract. Lack of activity with choline esters indicated the absence of cholinesterases. EDTA did not exhibit appreciable activation of carrot esterases. The effect of parathion, diisopropyl phosphorofluoridate (DFP) and tetraethyl pyrophosphate (TEPP) at various concentrations on the rate of hydrolysis of the acetyl, propionyl and butyryl esters, of phenol, 2-naphthol-6-SO₃Na and glycerol indicated the presence of six esterases. Inhibitor and substrate specificities of five esterases were similar to carboxylesterases (EC 3.1.1.1). The sixth esterase was similar to arylester as e (EC 3.1.1.2). An enzyme hydrolyzing DFP and TEPP was suggested in the carrot extract. / Graduation date: 1968

This investigation was conducted to differentiate the esterases in a water extract of freeze-dried green beans on the basis of inhibitor and substrate specificities. An attempt was made to classify these esterases according to the criteria used for animal esterases. Activity of the esterases was measured manometrically using the Gilson differential respirometer. Aqueous extracts of green beans were found to hydrolyze the acetyl, propionyl, and n-butyryl esters of glycerol, phenol, and 2-naphthol-6-SO₃Na. No hydrolysis of triolein or long-chain 2-naphthol-6-SO₃Na esters was noted, indicating the absence of lipases. A small amount of activity was observed when the choline esters served as substrates, but this was attributed to esterases other than cholinesterases. Optimum activity of the extract on triacetin, tripropion, tributyrin, phenyl acetate, and phenyl propionate occurred at pH 7.2. The effects of organophosphorus inhibitors, diethyl p-nitrophenyl thiophosphate, tetraethyl pyrophosphate, and diisopropylphosphorofluoridate, at concentrations ranging from 10⁻¹ M to 10⁻¹⁰ M on the esterase activity was studied. These data show that the green bean extract contains at least three esterases. One was resistant to certain organophosphorus compounds, suggesting similarity to animal arylesterases (aryl ester hydrolase, EC 3.1.1.2). Various concentrations of organophosphorus compounds inhibited the activity of the two other esterases. These were classified as carboxylesterases (carboxylic ester hydrolase, EC 3.1.1.1). / Graduation date: 1968

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiii, 195 p.; also includes graphics Includes bibliographical references (p. 177-195). Available online via OhioLINK's ETD Center

Resumo: Vários trabalhos na literatura têm discutido a importância dos polimorfismos enzimáticos em relação ao estado de ser adaptado dos organismos. Nesse trabalho foram estudadas algumas propriedades bioquímicas das aloenzimas EST-5S e EST-5F de Drosophila mulleri, que poderiam estar relacionadas às diferenças em valores adaptativos em indivíduos que apresentam essas variantes enzimáticas. Os resultados sugerem que a aloenzima EST-5S apresenta maior resistência à desnaturação térmica e a agentes caotrópicos, bem como maior atividade em altas concentrações salinas. Os resultados do experimento de produtividade a 29º C mostram que os indivíduos homozigotos para o alelo Est-5S deixam mais descendentes que os indivíduos homozigotos para o alelo Est-5F. Quanto as caractetísticas cinéticas, essas aloenzimas não diferem de forma significante. Os valores de Vmax e Km obtidos para as enzimas não purificadas foram, respectivamente, para a EST-5S: 32,54 µM/min e 1,43 mM, e para a EST-5F: 33,34 µM/min e 1,54 mM. A purificação das aloenzimas foi obtida pela separação em SDS-PAGE seguida da remoção dos géis, eluição em agitador magnético para a difusão das enzimas e posterior concentração desse material em membrana filtrante de 50 kDa (Millipore). O concentrado foi então analisado quanto à atividade esterásica e quanto à pureza (SDS-PAGE). Ambas as aleloenzimas apresentaram uma massa molecular entre 66 e 68 kDa para a subunidade, em SDS-PAGE. Os resultados do presente estudo não são concordantes com a hipótese de neutralidade para a manutenção dos polimorfismos, tanto em populações naturais como de laboratório, indicando a atuação da seleção natural sobre as aleloenzimas estudadas. / Abstract: Several works in literature have discussed the importance of the enzymatic polymorphisms in respect to the state of being adapted of the organisms. In this work, some biochemical properties of Drosophila mulleri of the allozymes EST-5S and EST-5F, which could be related to the differences in fitness values in individuals that present those enzymatic variants, were studied. The results suggest that the EST-5S allozyme presents larger resistance on the thermic denaturation and the chaotropics agents, as well as higher activity in elevated salt concentrations. The results of the productivity experiment at 29°C show that the homozygous individuals to the Est-5S allele let more offspring than the homozygous individuals to the Est -5F allele. Regarding the kinetic characteristics, those allozymes do not differ in a significant way. The Km and Vmax values obtained for crude extracts allozymes were, respectively, to the EST-5S: 32,54 µM/min and 1,43 mM, and to the EST-5F: 33,34 µM/min and 1,54 mM. The purification of the allozymes was obtained by the separation on SDS-PAGE followed by their removal from gels, elution in magnetic shaker for the diffusion of the enzymes and concentration of this material in filtrant membranes of 50 kDa (Millipore). The concentrated materials were then analyzed regarding to their enzymatic activity and to the purity (SDS PAGE). Both allozymes presented subunit molecular weights around 66 and 68 kDa on SDS PAGE. The results of the present study do not agree to neutrality hypothesis for polymorphisms maintenance, in natural and laboratory populations, indicating a natural selection action of the on the studied allozymes. / Orientador: Carlos Roberto Ceron / Coorientador: Gustavo Orlando Bonilla Rodriguez / Banca: Hamilton Cabral / Banca: Fátima Pereira de Souza / Mestre

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Esterase activity of an aqueous extract of the green bean was separated into fourteen bands, while aqueous extracted pea esterases revealed seven bands, by polyacrylamide-gel electrophoresis. The fourteen bands of bean esterase activity formed three groups; slow, intermediate and fast moving. α-Naphthyl acetate, propionate, and n-butyrate and AS naphthol acetate were hydrolyzed at various rates by the bean and pea esterases. No hydrolysis of β-naphthyl laurate was observed, indicating the absence of a lipase in the aqueous extracts of these vegetables. Since all the esterase bands active toward α-naphthyl acetate were inhibited by organophosphorus compounds (diisopropylphosphorofluoridate, diethyl p-nitrophenyl thiophosphate and diethyl p-nitrophenyl phosphate), these esterases were classified as carboxylesterases (carboxylic ester hydrolase, EC 3.1.1.1). To study the carboxylesterases of the green bean in greater detail, a protamine sulfate treated aqueous extract was separated into three fractions (S₁, S₂ and S₃) by chromatography on Sephadex G-100. Subsequent analysis of each fraction by polyacrylamide-gel electrophoresis demonstrated the presence of the slow moving group in fraction S₁, slow and intermediate moving groups in fraction S₂ and the fast moving group in fraction S₃. Hence, these studies suggest that the three groups of esterase activity in beans were dissimilar in molecular size and the relative molecular size was slow > intermediate > fast moving group. Chromatography of fraction S₁ on carboxymethyl (CM) cellulose with sodium chloride linear-gradient elution resulted in three fractions (CM₁, CM₂ and CM₃). Similarly, fraction S₂ yielded three fractions (DE₁, DE₂ and DE₃), while fraction S₃ produced two fractions (DE₄ and DE₅), by chromatography on microgranular diethylaminoethyl (DEAE) cellulose. Polyacrylamide-gel electrophoresis revealed the presence of the first three bands of the slow moving group in CM₁ and only the first two bands in CM₂. DE₁ possessed mainly the first two bands and DE₂ the last two bands of the five bands in the slow moving group. The five bands of the intermediate moving group of esterase activity was found only in fraction DE₃. The first two bands of the four bands of the fast moving group were separated into fraction DE₄, while the last two bands were in DE₅. Nine substrates and various concentrations of three inhibitors were used to characterize some of the fractions obtained from ion-exchange chromatography. Although most of the fractions hydrolyzed the substrates used in this study, each fraction differed to some extent in substrate specificity. Inhibitor studies indicated the presence of a sensitive and a resistant component of esterase activity in each fraction studied. These results suggest that the esterase fractions were composed of two enzymes. To account for the fourteen bands of esterase activity a hypothetical model of polymers consisting of two monomers was proposed. This hypothesized model suggests that the slow moving group contained six pentamers, the intermediate group five tetramers and the fast moving group four trimers. Most characteristics of the carboxylesterases of beans observed in these studies could be explained on the basis of the hypothetical model. / Graduation date: 1969

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