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Title: Characterisation of non-specific esterase isoenzyme forms in normal and leukaemic myeloid cells
Author: Patel, Divyen Harshadbhai
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 1992
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Abstract:
The generic term 'esterases' broadly represents enzymes that hydrolyse aliphatic or aromatic esters. and within this definition is a group of esterases that show a preference for short acyl chain esters such as a-naphthyl acetate, propionate or butyrate. These are ti often collectively referred to as 'non-specific esterases' (NSE), but a more accurate and descriptive designation is according to the substrate used. The demonstration of esterases by azo-dye techniques has found particular applications in haematological cytochemistry, and, with specific reference to normal myeloid cells, a-naphthyl acetate esterase (ANAE) 0 cytochemical reactions of moderate-strong diffuse type are typically associated with cells of monocyte/macrophage lineage. In contrast. granulocytes at all levels of morphological differentiation are cytochemically ANAE negative. Isoelectric focusing (IEF) studies of ANAE isoenzymes have further revealed the existence of two species with apparent differences in lineage affiliation. The first (ComEst) is expressed by both granulocytes and monocytes, and comprises a series of isoenzymes with isoelectric points (p1) ranging from 6.3 to 7.9, whereas a second (MonEst) species that is specifically expressed by haemopoietic cells of monocytic/macrophage origin is seen on IEF zymograms as a series of isoforms within a relatively narrow pI range of 5.5-6.2. These present studies were undertaken in order to extend what is currently known of the cellular, kinetic and molecular features of the two main myeloid esterase species. It was considered that these investigations were necessary to clarify the nature of atypical cytochemical reactions in leukaemic and dyshaemopoietic myeloid cells, to establish whether or not the two species were related or distinct enzymes, to gain further insights into their possible functional role(s), and to provide molecular details of relevance for the longer term aim of cloning MonEst protein in particular. Studies reported here of normal myeloid cells confirmed the lineage affiliation of the two main esterase species, and analysis of a large number of acute myeloid leukaemias also resolved the nature of atypical ANAE cytochemistries. Abnormally increased focal and granular reactions of myeloid blasts was shown to be due to over-expression of ComEst, and the lack of ANAE cytochemical staining in a significant proportion of monocytic leukaemias was shown to result from a failure to synthesise MonEst. As a prelude to the biochemical purification of myeloid esterases, the ComEst and MonEst species were also investigated to determine their chromatographic characteristics. This involved an evaluation of a wide range of column gels including ion-exchange, hydrophobic interaction, affinity, and gel filtration. The purification protocol resulting from these evaluations successfully permitted the purification of ComEst to a highly enriched state and MonEst to homogeneity. Subsequent molecular and kinetic analyses revealed that enzymatically active MonEst exists in its native state as an apparent trimer which, under non-reducing conditions, dissociates to inactive 63 kDa monomers. In contrast, native ComEst was shown to be a 68 kDa monomer which retained enzymatic activity following SDS treatment, and was not dissociated under reducing conditions. Lectin affinity studies confirmed that both esterase species were glycoproteins but differed in that MonEst contained oligomannosidic-type glycan(s) whilst ComEst contained a mixture of fucosylated and non-fucosylated biantennary N-acetyllactosamine-type glycan(s). Neuraminidase, a-mannosidase, a-L-fucosidase, and endoglycosidase H were shown to have no effect on the pI distribution of individual ComEst or MonEst isoforms, but endoglycosidase treatment did reduce the Mr of MonEst from 63 to 60 kDa. Enzyme kinetic studies also revealed that purified ComEst preferentially hydrolysed esters of short acyl chain length (C2 and C3) whilst MonEst hydrolysed esters of higher acyl chain length (butyrate > propionate > acetate). However, MonEst failed to hydrolyse a wide range of natural and synthetic peptidase substrates thus tending to exclude its functional role in peptide processing. Possible differences in reaction mechanisms of the two esterase species were also evaluated by examining the inhibitory effects of representative enzyme inhibitors which demonstrated that serine and histidine residues were required. for MonEst but not ComEst activity. N-terminus amino acid sequencing of purified MonEst indicated almost complete identity with human alveolar macrophage esterase. differing only in a Val-Thr substitution at position 12, and close similarities with rabbit liver carboxylesterase. In summary, substrate and inhibitor studies strongly suggested that the MonEst and ComEst species should be classified as carboxylesterases (EC 3.1.1.1) and acetylesterases (EC 3.1.1.6) respectively and that, together with distinct differences in their molecular and biochemical characteristics, it is concluded that these are unrelated myeloid enzymes which share only the ability to hydrolyse a-naphthyl acetate. Although yet to be established, the kinetic and molecular differences reported here may have fundamental relevance with respect to the biofunctional role(s) of these enzymes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.674976  DOI: Not available
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