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.