Atypical Esterase Research Articles

Enzymatic and Molecular Identification of Meloidogyne Species in Tomato Orchards in Paraguay

Tomato is a major crop in Paraguay, where it provides a source of employment and income for households. Tomato production can be affected by root-knot nematodes, especially Meloidogyne spp. The unequivocal identification of Meloidogyne spp. in Paraguay has not been conducted yet. This study aims to identify Meloidogyne species in eight tomato production districts of this country by biochemical and molecular techniques. Females of Meloidogyne spp. were extracted from tomato roots and characterized using esterase isozyme phenotypes. In addition, DNA was extracted from nematode eggs, and species-specific SCARs (sequence-characterized amplified regions) were used to confirm the diagnosis. Nematodes were detected in 100% of studied samples (prevalence), of which M. incognita (Est: I2, Rm: 1.1;1.2) and M. javanica (Est: J3, Rm: 1.0, 1.20, 1.35) were present in 39.13% and 26.08% of samples, respectively. One population (8.69%) of Meloidogyne sp. presenting an atypical esterase profile (Rm: 1.0 and 1.3) was only detected in Julián Augusto Saldívar District. Mixed populations, mostly M. incognita and M. javanica, were observed in 26.08% of samples. The SCAR primers incK14F/incK14R amplified specific fragments for M. incognita (399 bp) and M. javanica (670 bp), confirming the enzymatic results. Here, we present the first study of root-knot nematode identification at the species level in Paraguay.

Structural annotation of the conserved carbohydrate esterase vb_24B_21 from Shiga toxin-encoding bacteriophage Φ24B

Shiga toxin-encoding bacteriophages transfer Shiga toxin genes to Escherichia coli and are responsible for the emergence of pathogenic bacterial strains that cause severe foodborne human diseases. Gene vb_24B_21 is the most highly conserved gene across sequenced Shiga bacteriophages. Protein vb_24B_21 (also termed 933Wp42 and NanS-p) is a carbohydrate esterase with homology to the E. coli chromosomally encoded NanS that deacetylates sialic acid in the intestinal mucus. To assist the functional characterization of vb_24B_21, we have studied its molecular structure by homology modelling its esterase domain and by elucidating the crystal structure of its uncharacterized C-terminal domain at the atomic resolution of 0.97 Å. Our modelling confirms that NanS from the E. coli host is the closest structurally characterized homolog to the esterase domain of vb_24B_21. Like NanS, vb_24B_21 has an atypical active site, comprising a simple catalytic dyad Ser-His and a divergent oxyanion hole. The crystal structure of the C-terminal domain reveals a lectin-like, jelly-roll β-sandwich fold. The domain displays a prominent cleft that bioinformatics analysis predicts to be a carbohydrate binding site without catalytic properties. In summary, our study indicates that vb_24B_21 is a NanS-like atypical esterase that is assisted by a carbohydrate-binding module of yet undetermined binding specificity.

Enhancing the atypical esterase promiscuity of the γ-lactamase Sspg from Sulfolobus solfataricus by substrate screening.

Promiscuous enzymes can be modified by protein engineering, which enables the catalysis of non-native substrates. γ-lactamase Sspg from Sulfolobus solfataricus is an enzyme with high activity, high stability, and pronounced tolerance of high concentrations of the γ-lactam substrate. These characteristics suggest Sspg as a robust enzymatic catalyst for the preparation of optically pure γ-lactam. This study investigated the modification of this enzyme to expand its application toward resolving chiral esters. γ-Lactamase-esterase conversion was performed by employing a three-step method: initial sequence alignment, followed by substrate screening, and protein engineering based on the obtained substrate-enzyme docking results. This process of fine-tuning of chemical groups on substrates has been termed "substrate screening." Steric hindrance and chemical reactivity of the substrate are major concerns during this step, since both are determining factors for the enzyme-substrate interaction. By employing this three-step method, γ-lactamase Sspg was successfully converted into an esterase with high enantioselectivity towards phenylglycidate substrates (E value > 300). However, since both wild-type Sspg and Sspg mutants did not hydrolyze para-nitrophenyl substrates (pNPs), this esterase activity was termed "atypical esterase activity." The γ-lactamase activity and stability of the Sspg mutants were not severely compromised. The proposed method can be applied to find novel multi-functional enzyme catalysts within existing enzyme pools.

Immunophenotypic and enzymatic studies do not support the concept of mixed monocytic-granulocytic differentiation in acute promyelocytic leukaemia (M3): a study of 44 cases.

Leukaemic promyelocytes from 30 cases of hypergranular and 14 cases of hypogranular acute promyelocytic leukaemia (M3) were analysed for the presence of monocyte-associated characteristics to determine whether there was any evidence of mixed (hybrid) granulocytic-monocytic differentiation. Cytochemically, a high proportion of hypergranular cases showed significant alpha-naphthyl acetate esterase (ANAE) staining and simultaneous chloroacetate esterase, and ANAE expression by single cells was commonly seen. These atypical staining patterns were, however, not a feature of hypogranular cases. Immunophenotypic studies revealed that most hypergranular M3 cases were HLA-DR- and that monocyte-associated membrane CD14 expression was low in all cases tested. In addition, serum lysozyme concentrations (20 cases) were generally within the normal range and thus inconsistent with monocytic involvement in the leukaemic process. The significance of atypical ANAE staining of leukaemic promyelocytes was further examined by analysing ANAE isoenzyme components (defined by isoelectric focusing) in 11 cases. The patterns obtained (G1 and G2) were identical to those found in normal granulocytes and did not show any evidence of monocyte-associated esterase isoenzyme expression. On the basis of these findings, it is considered that the differentiation process in acute promyelocytic leukaemia is relatively well conserved and that the atypical esterase cytochemistry of hypergranular promyelocytes does not reflect their mixed lineage nature but is simply a consequence of increased granulation.

Esterase cytochemistry in primary myelodysplastic syndromes and megaloblastic anaemias: demonstration of abnormal staining patterns associated with dysmyelopoiesis

Summary. Acid alpha naphthyl acetate esterase (ANAE) and combined ANAE‐chloroacetate esterase cytochemistry was performed on 121 bone marrow aspirates from primary myelodysplastic syndromes (MDS) and a secondary dysplasia—megaloblastic anaemia (MA). The investigation demonstrated the presence of abnormal ANAE positive granulocyte populations in a significant proportion of cases. These cells, in which the staining patterns were characterized by atypical granular ANAE positivity and double ANAE‐chloroacetate reactions, were shown immunologically to lack the receptor and antigenic characteristics of monocytes and morphologically to be granulocytes. Isoelectric focusing, however, indicated that the atypical esterase cytochemistry of these granulocytes was due to the presence of markedly increased concentrations of ANAE isoenzymes usually found in monocytes. Atypical ANAE‐staining granulocytes were particularly evident in MDS marrows showing sideroblastic erythroid changes, whilst in MA they were mainly seen in cases of intermediate severity. It is suggested that these cells are associated with dysmyelopoietic changes in both malignant and non‐malignant conditions.

Esterase cytochemistry in primary myelodysplastic syndromes and megaloblastic anaemias: demonstration of abnormal staining patterns associated with dysmyelopoiesis

Acid alpha naphthyl acetate esterase (ANAE) and combined ANAE-chloroacetate esterase cytochemistry was performed on 121 bone marrow aspirates from primary myelodysplastic syndromes (MDS) and a secondary dysplasia-megaloblastic anaemia (MA). The investigation demonstrated the presence of abnormal ANAE positive granulocyte populations in a significant proportion of cases. These cells, in which the staining patterns were characterized by atypical granular ANAE positivity and double ANAE-chloroacetate reactions, were shown immunologically to lack the receptor and antigenic characteristics of monocytes and morphologically to be granulocytes. Isoelectric focusing, however, indicated that the atypical esterase cytochemistry of these granulocytes was due to the presence of markedly increased concentrations of ANAE isoenzymes usually found in monocytes. Atypical ANAE-staining granulocytes were particularly evident in MDS marrows showing sideroblastic erythroid changes, whilst in MA they were mainly seen in cases of intermediate severity. It is suggested that these cells are associated with dysmyelopoietic changes in both malignant and non-malignant conditions.

A METHOD FOR THE DETECTION OF ATYPICAL FORMS OF HUMAN SERUM CHOLINESTERASE. DETERMINATION OF DIBUCAINE NUMBERS*

Cases with atypical esterase activity were found by determining esterase inhibition in numerous sera. A suitable inhibitor was the local anaesthetic dibucaine (cincho-caine, 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 X 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”.

Genetic regulation of plasma cholinesterase in man

Individual variation in response to succinylcholine has stimulated investigations on the variations and genetic control of serum cholinesterase in man. It has been found that the level of cholinesterase varies from essentially no detectable activity to exceedingly high levels due to a number of different genetic mutations. Qualitative variations in the esterase are also inherited. The most common variant of the latter type is the atypical (dibucaine resistant) cholinesterase which differs from the normal esterase in its lower apparent affinity for choline ester substrates and for a number of inhibitors. On the basis of evidence derived from kinetic experiments, both the anionic site and the esteratic site of the atypical esterase are modified. These changes may be due to a difference in the primary structure of the enzyme at one position which affects both sites of the active center of the enzyme. The modified kinetic properties of the atypical esterase are expressed in both the major component (C 4 ) and the minor components (C 1 , C 2 and C 3 ) of the enzyme which is present in serum in multiple molecular forms. Component C 4 (molecular weight of about 300,000) can be converted to component C 3 by treatment with urea, and the latter transformed to a C 1 -like component by sulfhydryl reagents. Both C 1 components (native and derived) have molecular weights of about 80,000 and are approximately one-fourth as large as C 4 . This information should be useful in further studies on the structure of variant forms of the esterase and how the structural alterations account for altered kinetic properties of the variant cholinesterases.

The substrate specificity of atypical cholinesterase in relation to phenotypes

The activities of plasma esterase and phenotypes were studied in a family of four in which the mother had previously responded to the neuromuscular blocking agent succinylcholine with a prolonged paralysis. The plasma of the mothei contained atypical esterase which hydrolyzed succinylcholine and other esters at reduced rates. The increased response to succinylcholine thus relates to an atypical cholinesterase and a lowered total esterase activity. Three phenotypes were indicated by data obtained from dibucaine and fluoride inhibition studies, but each of the four family members had different substrate activity patterns for plasma esterase, as determined by K m values. The study reported here presents an example of two siblings with the same phenotype but with markedly different plasma esterase activity toward a variety of choline and noncholine esters. The father and the one son who have the least difference in enzyme activity patterns have distinctly different phenotypes, as determined by fluoride and dibucaine inhibition. The study emphasizes the need for additional characterizations of atypical esterase within families so that inheritance in relation to plasma esterase may be further understood.

New genetically determined molecular form of erythrocyte esterase in man.

An altered molecular form of erythrocyte esterase was discovered in hemolysates from two males and one female in three generations of the same family. This indicates that the alternate enzyme form is under the control of a single autosomal gene. Evidence suggests that the atypical esterase is a variant form of erythrocyte carbonic anhydrase.

Multiple forms of esterases from human erythrocytes.

SummaryA system is described in which multiple bands of esterase activity can be resolved from human red cell hemolysates by zone electrophoresis in starch gel. Variations from the normal pattern were observed in certain patients with liver disorders and associated anemias. Another atypical esterase pattern was found in a pair of monozygotic twins and their mother indicating that the observed variation is under genetic control.

THE ACTION OF NORMAL AND ATYPICAL CHOLINESTERASE OF HUMAN SERUM UPON A SERIES OF ESTERS OF CHOLINE

The results of a comparison of the actions of individual human sera support evidence previously obtained for the existence of two different types of pseudocholinesterase in man. Maximum rates of hydrolysis of choline esters by human sera containing the normal type of cholinesterase were two to four times greater than the maximum rates obtained with sera containing atypical esterase. Human sera with the normal type of cholinesterase hydrolyzed butyrylcholine at a considerably faster rate than pentanoylcholine; atypical esterase hydrolyzed pentanoylcholine at approximately the same rate as butyrylcholine. All Michaelis constants for the normal enzyme were lower than those for the atypical enzyme by a factor which varied from 1.4 to 6.4. The data were compatible with the assumption of a linear relationship between the log of the Michaelis constants and the number of acyl carbons in the choline esters, but the slopes differed significantly for the two enzymes. For a homologous series of choline esters, there was no correlation between rates of hydrolysis and Michaelis constants.

THE ACTION OF NORMAL AND ATYPICAL CHOLINESTERASE OF HUMAN SERUM UPON A SERIES OF ESTERS OF CHOLINE

The results of a comparison of the actions of individual human sera support evidence previously obtained for the existence of two different types of pseudocholinesterase in man. Maximum rates of hydrolysis of choline esters by human sera containing the normal type of cholinesterase were two to four times greater than the maximum rates obtained with sera containing atypical esterase. Human sera with the normal type of cholinesterase hydrolyzed butyrylcholine at a considerably faster rate than pentanoylcholine; atypical esterase hydrolyzed pentanoylcholine at approximately the same rate as butyrylcholine. All Michaelis constants for the normal enzyme were lower than those for the atypical enzyme by a factor which varied from 1.4 to 6.4. The data were compatible with the assumption of a linear relationship between the log of the Michaelis constants and the number of acyl carbons in the choline esters, but the slopes differed significantly for the two enzymes. For a homologous series of choline esters, there was no correlation between rates of hydrolysis and Michaelis constants.

Some statistical data on atypical cholinesterase of human serum.

SummaryThe occurrence of atypical serum cholinesterase (pseudocholinesterase) was determined in a population which consisted of 2000 healthy persons and 2400 mental patients. The type of esterase was assessed by determining the Dibucaine Number (DN), i.e. the susceptibility of the esterase to inhibition.The distribution of DN in the population was consistent with the assumption of two autosomal allelic genes giving rise to two types of esterase. The frequency of the gene for atypical esterase in the healthy population was estimated to be 0.019 ± 0.002.The great majority of all investigated persons were of European extraction. There was no evidence for a different incidence of atypical esterase among Europeans, or between Europeans and North Americans, or between religious groups.The incidence of heterozygosity did not vary with age, sex, or body weight, although these three factors were correlated with esterase levels.There was no obvious relation between mental illness and incidence of atypical esterase but a connexion between some of the less common mental diagnoses and atypical esterase could not be excluded.

The activity of various esterase inhibitors towards atypical human serum cholinesterase

Guided by genetic studies, human sera were selected and postulated to contain either the usual type of serum cholinesterase (pseudocholinesterase), or an atypical variety of this enzyme, or a mixture of these two enzymes. The shape of concentration-inhibition curves obtained with sera of the last-named category permitted no other interpretation than the presence of a mixture of two enzymes. The concept of the existence of a usual and of an atypical form of human serum cholinesterase was thereby supported. The values for I 50 (concentration of inhibitor causing 50 per cent inhibition) of 16 inhibitors for normal and atypical esterase were compared under standard conditions. Both types of esterase were equally affected by the phosphate inhibitors DFP and TEPP. All other inhibitors blocked the atypical esterase less than the normal one. In relation to their usual blocking capacities, the bisquaternary compounds C 10 and succinylcholine were particularly weak inhibitors of the atypical esterase. For the 12 remaining inhibitors, which included tetramethylammonium, procaine, and eserine, the normal and atypical I 50 could be related by the exponential equation I 50-atypical = (I 50-usual) 0.785; that is, the difference between corresponding I 50-values increased with increasing potency of the inhibitors. Several compounds, e.g., decamethonium and chlorpromazine, were competitive inhibitors of atypical esterase, but they were not completely competitive inhibitors of the usual enzyme. Every fourth molecule of C 10 and of succinylcholine seemed to block two active centres of the normal esterase, but each of these molecules blocked only one active centre of the atypical enzyme.

A METHOD FOR THE DETECTION OF ATYPICAL FORMS OF HUMAN SERUM CHOLINESTERASE. DETERMINATION OF DIBUCAINE NUMBERS

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

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".