(Received for publication, June 7, 1995)
From the
Multiple forms of carboxylesterase have been identified in rat liver, and five carboxylesterases (designated hydrolases A, B, C, S, and egasyn) have been cloned. Hydrolases A, B, C and egasyn all have a C-terminal consensus sequence (HXEL) for retaining proteins in the endoplasmic reticulum, and these carboxylesterases are found in rat liver microsomes. In contrast, hydrolase S lacks this C-terminal consensus sequence and is presumed to be secreted. In order to test this hypothesis, a polyclonal antibody was raised against recombinant hydrolase S from cDNA-directed expression in Escherichia coli. In addition to hydrolases A, B, and C (57-59 kDa), this antibody recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum. The 71-kDa protein detected in rat serum was also detected in the extracellular medium from primary cultures of rat hepatocytes. Non-denaturing gel electrophoresis with staining for esterase activity showed that a serum carboxylesterase comigrated with the 71-kDa protein. Immunoprecipitation of the 71-kDa enzyme from rat serum decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate. The 71-kDa protein immunoprecipitated from rat serum had an N-terminal amino acid sequence identical to that predicted from the cDNA encoding hydrolase S, providing further evidence that hydrolase S is synthesized in and secreted by the liver. The levels of the 67-kDa protein in rat liver microsomes and the levels of the 71-kDa protein in rat serum were co-regulated. Deglycosylation of microsomes and serum converted the 67- and 71-kDa proteins to a 58-kDa peptide, which matches the molecular mass calculated from the cDNA for hydrolase S. These results suggest that the 67-kDa protein in liver microsomes is a precursor form of hydrolase S that undergoes further glycosylation before being secreted into serum. In rats, liver appears to be the only source of hydrolase S because no mRNA encoding hydrolase S could be detected in several extrahepatic tissues. Serum carboxylesterases have been found to play an important role in lipid metabolism and detoxication of organophosphates, therefore, the secretion of hydrolase S and the modulation of its expression by xenobiotics may have physiological as well as toxicological significance.
Carboxylesterases play an important role in the metabolism of
endogenous lipids and foreign compounds, such as drugs and
pesticides(1, 2, 3, 4, 5, 6) .
In addition to hydrolyzing numerous chemicals, carboxylesterases can
catalyze transesterification reactions, which accounts for the
conversion of cocaine (a methyl ester) to ethylcocaine (the
corresponding ethyl ester) in the presence of an alcohol(7) .
Carboxylesterases are abundant cellular components, and they appear to
have certain structural roles, which may be important in cellular
trafficking. For example, egasyn, a liver microsomal carboxylesterase
identified in both rat and mouse, binds to -glucuronidase via its
active site, which results in sequestration of the protein within the
endoplasmic reticulum(8, 9) . In rabbits, microsomal
carboxylesterases have recently been shown to interact with and
regulate the secretion of acute-phase-response proteins, such as
C-reactive protein(10) .
Multiple forms of carboxylesterase
have been identified in rat liver microsomes by isoelectrofocusing or
non-denaturing gel electrophoresis followed by staining for esterase
activity(11, 12) . Previous studies from this
laboratory led to the purification and characterization of two
carboxylesterases in rat liver microsomes(12, 13) .
These two carboxylesterases, designated hydrolases A and B, appear to
be the only abundant enzymes in rat liver microsomes that rapidly
hydrolyze para-nitrophenylacetate. High levels of hydrolase A
are present in testis, whereas high levels of hydrolase B are present
in kidney, as determined by enzyme kinetics and Western immunoblotting (12, 13) . We have recently isolated three full-length
cDNAs from libraries prepared from rat testis, kidney, and liver. These
cDNAs apparently encode hydrolase A, B, and a third microsomal
carboxylesterase designated hydrolase
C(14, 15, 16) . Our initial attempts to clone
hydrolases A and B by immunoscreening a rat liver cDNA library with
antibodies against the purified proteins invariably resulted in the
isolation of a cDNA (partial sequence) encoding a carboxylesterase that
is similar but not identical to those present in rat liver microsomes.
Other investigators have isolated the same cDNA when screening rat
liver cDNA libraries with antibodies against carboxylesterases purified
from rat liver microsomes. Consequently, the cDNA isolated by Takagi et al.(17) with antibody against carboxylesterase E1
does not encode this microsomal carboxylesterase, just as the cDNA
isolated by Long et al.(18) does not encode the
microsomal carboxylesterase they identified as a target for alkylation
by halothane. ()These cDNAs encode the same protein, which
is hereafter designated hydrolase S.
Carboxylesterases are widely
distributed throughout the body and are present in several subcellular
organelles, but the highest concentration of carboxylesterases is found
in liver microsomes(12) , where they are loosely bound to the
luminal surface of the endoplasmic reticulum. The retention of soluble
proteins in the lumen of the endoplasmic reticulum of yeast and animal
cells is specified by the COOH-terminal consensus sequence,
HXEL or KXEL(19, 20) . Hydrolases A, B, and C all have
the COOH-terminal HXEL consensus sequence and are found in rat liver
microsomes(15) . In contrast, hydrolase S lacks this
COOH-terminal consensus sequence and is presumed to be secreted. It is
believed that HXEL-containing proteins bind ERD2 (for endoplasmic
reticulum retention) or related proteins in the cis-Golgi
apparatus, which leads to retrograde transport of HXEL-containing
proteins to the endoplasm reticulum(21, 22) . Recent
studies have shown that retention or secretion of certain animal
proteins is cell typedependent. For example, protein
disulfide-isomerase is exclusively localized within the lumen of the
endoplasmic reticulum and nuclear envelope in rat hepatocytes, whereas
this protein is secreted by rat pancreatic
cells(23, 24) . Similarly, cDNA directed expression of
thioredoxin and interleukin-1 in COS-7 cells results in the
secretion of thioredoxin but not of interleukin-1
, although both
proteins are secreted by monocytes (25) .
The aim of the present study was to examine the cellular trafficking of hydrolase S and to test the hypothesis that hydrolase S is a serum carboxylesterase synthesized in and secreted by rat liver. We describe the preparation of a high-affinity antibody against recombinant hydrolase S and demonstrate the presence of hydrolase S in rat serum (in vivo) and the secretion of hydrolase S by primary cultures of rat hepatocytes (in vitro). We also present evidence that hydrolase S exists in two forms: a 67-kDa protein, which is the nascent form in liver microsomes, and a 71-kDa protein, which is the mature form in serum. Both proteins are extensively glycosylated. In rats, the liver appears to be the only source of hydrolase S, inasmuch as no mRNA coding for hydrolase S or the 67-kDa protein could be detected in several extrahepatic tissues.
The PCR-amplified cDNA was digested with EcoRI and XbaI and separated electrophoretically on a 1.2% agarose gel. The cDNA was recovered with a Qiaex agarose gel extraction kit (Qiagen Inc., Chatsworth, CA), inserted into EcoRI-XbaI-digested pUC18 plasmid, and sequenced with a Sequenase 2.0 kit (U. S. Biochemical Corp.) as described previously(14) .
Hydrolases A and B were purified from rat liver microsomes, as described by Morgan et al.(12) . Polyclonal antibodies against the purified proteins were raised in rabbits and were subjected to immunoabsorption chromatography to remove cross-reacting antibodies, as described by Morgan et al.(13) . After immunoabsorption, the antibody against hydrolase A was specific for hydrolase A, whereas the antibody against hydrolase B recognized both hydrolase B and C, which are 96% identical in amino acid sequence(16) . Antibody against maltose-binding protein was purchased from New England BioLabs.
Hydrolase S was immunoprecipitated from the partially purified
preparation with antibody against the recombinant protein coupled to
Protein G-agarose. Protein G-agarose (50 µl) was washed three times
with buffer B (10 mM sodium phosphate, pH 7.2, containing 150
mM NaCI) by centrifugation at 500 g for 2
min/wash. After the last wash, the Protein G-agarose pellet was
resuspended in 0.5 ml of buffer B containing either no antibody
(negative control) or 0.15 mg/ml of antibody against hydrolase S or rat
serum albumin (i.e. an irrelevant antibody). After a 4-h
incubation at 4 °C with gentle agitation, the antibody-Protein G
complexes were washed three times as described above. Following the
last wash, 100 µl of partially purified hydrolase S (about 500
µg of protein) and 400 µl of buffer B were added to each tube.
After an overnight incubation at 4 °C with gentle agitation, the
samples were centrifuged at 3000
g for 5 min at 4
°C. The supernatant fractions were analyzed by Western
immunoblotting with antibody against hydrolase S and assayed for
enzymatic activity toward para-nitrophenylacetate and
1-naphthylacetate as described by Morgan et al.(12) .
Figure 1: SDS-PAGE analysis of the maltose-binding protein-hydrolase S fusion protein before and after proteolytic cleavage with factor Xa. E. coli were transformed with an expression plasmid harboring a cDNA encoding hydrolase S linked via a factor Xa cleavage site to the gene encoding MBP, as described under ``Materials and Methods.'' The fusion protein was purified by affinity chromatography on an amylose column and cleaved with factor Xa. After cleavage, hydrolase S was separated from MBP by amylose column chromatography. Samples were subjected to SDS-PAGE, and proteins were stained with Coomassie Blue R250. Lane 1 contained 1 µg of purified, intact fusion protein (MBP linked via the factor Xa site to hydrolase S). Lane 2 contained 2 µg of purified fusion protein cleaved with factor Xa. Lane 3 contained 1 µg of purified hydrolase S, which was recovered in the void volume when Factor Xa-cleaved fusion protein was subjected to amylose column chromatography. Lane 4 contained 0.6 µg of purified hydrolase B. Lane 5 contained 0.4 µg of purified MBP from New England BioLabs. The apparent molecular masses of the intact fusion protein, hydrolase S, and MBP were 100, 58, and 42 kDa, respectively.
Figure 2:
Immunoblots of purified hydrolases A and
B, rat liver microsomes and rat serum probed with antibodies against
hydrolase A, hydrolase B, or recombinant hydrolase S. A mixture of
purified hydrolases A and B (0.3 µg each), liver microsomes (20
µg, lane 2), and serum (0.5 µl) was analyzed by
Western immunoblotting, as described under ``Materials and
Methods.'' The liver microsomes and serum were pooled samples from
five Sprague-Dawley male rats (12-week old). Left panel,
immunoblot probed with antibody against hydrolase A that was
immunoabsorbed against hydrolase B. Middle panel, immunoblot
probed with antibody against hydrolase B that was immunoabsorbed
against hydrolase A. Right panel, immunoblot probed with a
polyclonal antibody against purified, recombinant hydrolase S that
cross-reacts with hydrolases A (57 kDa) and B (
59
kDa).
As shown in Fig.3, primary cultures of rat hepatocytes secreted a protein recognized by antibody against hydrolase S, and this protein comigrated on SDS-PAGE with the 71-kDa protein in rat serum. This 71-kDa protein was not present in the culture medium itself and was detected in the medium only when hepatocytes were cultured under conditions that restored near normal cell morphology and the expression of liver-specific genes (results not shown). Inasmuch as the hepatocytes were cultured in serum-free medium, the results in Fig.3establish that, if the 67-kDa protein is a precursor to the 71-kDa protein, this conversion occurs in the liver prior to secretion of the 71-kDa protein. In other words, the 67-kDa protein is not secreted and subsequently converted to the 71-kDa protein in the serum.
Figure 3: Secretion of hydrolase S from primary cultures of rat hepatocytes as determined by Western immunoblotting. Hepatocytes from 7- to 8-week old Sprague-Dawley male rats were isolated and cultured in Chee's medium, as described under ``Materials and Methods.'' The secretion of hydrolase S into the culture medium was analyzed by Western immunoblotting with a polyclonal antibody against purified, recombinant hydrolase S that cross-reacts with hydrolases A and B. Lane 1 contained 20 µg of rat liver microsomes (hydrolase A, hydrolase B, and a 67-kDa protein were detected). Lane 2 contained 30 µl of extracellular medium from primary cultures of rat hepatocytes (a 71-kDa protein was detected). Lane 3 contained 0.5 µl of rat serum (a 71-kDa protein was detected). Lane 4, contained 30 µl of Chee's medium prior to culturing rat hepatocytes. Lane 5 contained 0.3 µg of rat serum albumin.
Figure 4: Immunostaining and esterase activity staining of rat liver microsomes, rat serum, and purified hydrolase A and B after non-denaturing gel electrophoresis. Purified hydrolase A (0.3 µg), purified hydrolase B (0.3 µg), pooled liver microsomes (10 µg), and pooled rat serum (0.5 µl) were subjected to non-denaturing gel electrophoresis as described under ``Materials and Methods.'' Staining for esterase activity was based on the formation of a black, insoluble complex between diazotized Fast Blue RR and 1-naphthol, which was released enzymatically from 1-naphthylacetate (left panel). The identical samples were also subjected to non-denaturing gel electrophoresis and transferred electrophoretically to Immobilon for immunostaining with anti-hydrolase S (right panel).
To determine whether the 71-kDa protein contributes significantly to the esterase activity of rat serum, esterase activity toward 1-naphthylacetate and para-nitrophenylacetate was measured before and after immunoprecipitation of the 71-kDa protein with antibody against hydrolase S. Immunoprecipitation of the 71-kDa protein directly from rat serum caused only a small decrease in the rate of hydrolysis of 1-naphthylacetate or para-nitrophenylacetate, which suggests that enzymes other than the 71-kDa protein in rat serum contribute more to the hydrolysis of these carboxylic esters (results not shown). The 71-kDa protein was partially purified from rat serum by a combination of ammonium sulfate fractionation and anion-exchange chromatography. The final preparation contained at least five proteins in addition to the 71-kDa protein, as determined by SDS-PAGE (results not shown). Immunoprecipitation of the 71-kDa protein from partially purified hydrolase S with antibody against hydrolase S decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate by 52 and 22%, respectively, as shown in Fig.5.
Figure 5: Effects of immunoprecipitation of hydrolase S on the hydrolysis of para-nitrophenylacetate and 1-naphthylacetate by partially purified hydrolase S from rat sera. Hydrolase S partially purified from rat sera was prepared by ammonium sulfate and ion-exchange chromatography. The partially purified hydrolase S was incubated overnight at 4 °C with the complex of Protein G-agarose and antibody against hydrolase S, the complex of Protein G-agarose and antibody against rat serum albumin, or the same amount of Protein G-agarose alone as described under ``Materials and Methods.'' After the immunoprecipitates were removed by centrifugation, the supernatant fractions were assayed for hydrolytic activities with 1 mMpara-nitrophenylacetate or 1 mM 1-naphthylacetate as described previously(1) . Rates are expressed relative to control (partially purified hydrolase S + Protein G agarose alone). The supernatant fractions were also analyzed by immunoblotting as described under ``Materials and Methods.''
The 71-kDa protein was isolated from the immunoprecipitate by SDS-PAGE, blotted onto a polyvinyldifluoride membrane, and subjected to protein microsequencing. Nine of the first 10 amino acid residues at the N terminus were unambiguously identified, and the sequence matched that deduced from the cDNA encoding hydrolase S beginning with residue 19, indicating that the signal peptide is cleaved from hydrolase S prior to secretion of the 71-kDa protein.
Figure 6:
Effects of treating Sprague-Dawley rats
with various xenobiotics on the expression of microsomal and serum
hydrolase S and its mRNA. Male rats (7 weeks old, five per group) and
female rats (troleandomycin treatment only) were treated with various
xenobiotics at dosages known to induce liver microsomal enzyme
inducers, as described under ``Materials and Methods.'' Liver
microsomes (25 µg of protein) and serum samples (0.5 µl) were
subjected to SDS-PAGE and transferred electrophoretically to Immobilon
for immunostaining with a polyclonal antibody against hydrolase S. For
Northern blotting analysis, total RNA was isolated from the treated or
control rats with a modified acid phenol-chloroform extraction method (14) . Pooled total RNA samples (10 µg) of each group were
subjected to 2.2 M formaldehyde-agarose electrophoresis and
transferred to Nytran nylon membrane by a vacuum blotting system. The
blots were probed with a cDNA encoding hydrolase S, which was
radiolabeled with [P]dCTP by random primer
extension. To normalize the abundance of 28 S rRNA contained in each
sample, the same membrane was stripped by boiling 2
15 min and
reprobed with an oligonucleotide (hybridize with 28 S rRNA)
radiolabeled with [
P]ATP by T4 polynucleotide
kinase. Note: the cross-reaction of anti-hydrolase S
with hydrolases A and B in rat liver microsomes is not shown to
facilitate the comparison between microsomal and serum hydrolase
S.
It should be noted that the antibody against hydrolase S also recognized hydrolases A and B (57 and 59 kDa) in rat liver microsomes. The effect of xenobiotic treatment on the expression of these carboxylesterases was previously reported by Morgan et al.(13) , hence, this portion of the immunoblot was deleted from Fig.6for clarity.
Figure 7: Changes in the electrophoretic mobility of the 67-kDa protein in microsomes and the 71-kDa protein in serum following deglycosylation with endoglycosidases. Microsomes (25 µg) or serum (16 or 4 µg) pooled from five rats were incubated with 1.0 milliunits of endoglycosidase H (Endo H) or 3.0 milliunits of endoglycosidase F (Endo F) under SDS-denaturing condition at 37 °C for 20 h, as described under ``Materials and Methods.'' The samples were subjected to SDS-PAGE, transferred electrophoretically to Immobilon, and probed with antibody against hydrolase S. The three bands with control microsomal sample are the 67 kDa form of hydrolase S, hydrolase A (57 kDa), and hydrolase B (59 kDa).
Figure 8:
Tissue
distribution of microsomal hydrolase S as determined by Western
immunoblotting and Northern blotting. Microsomes from various organs
were prepared from 7-week-old Sprague-Dawley rats. The microsomal
samples (25 µg of protein) pooled from four rats were subjected to
SDS-PAGE and transferred electrophoretically to Immobilon for
immunostaining. The blots were probed with a polyclonal antibody
against hydrolase S that recognizes hydrolase A (57 kDa), hydrolase B
(59 kDa), and microsomal hydrolase S (67 kDa) as shown in the top
panel. For Northern blot analysis, total RNA was isolated from the
treated or control rats with a modified acid phenol-chloroform RAN
extraction method(14) . Pooled total RNA samples (10 µg)
from five rats were subjected to 2.2 M formaldehyde-agarose
gel electrophoresis and transferred to Nytran nylon membrane by a
vacuum blotting system. The blot was probed with a cDNA encoding
hydrolase S, which was radiolabeled with [P]dCTP
by random primer extension as described under ``Materials and
Methods.''
The tetrapeptide sequence KXEL or HXEL is found at the
carboxyl terminus of luminal and some transmembrane proteins in the
endoplasmic reticulum. This sequence is necessary to ensure the
retention of proteins in the lumen of the endoplasmic reticulum despite
the high rate of forward vesicular transport to the Golgi apparatus and
subsequent organelles of the secretory
pathway(19, 20, 39, 40) . Hydrolases
A, B, and C all have this COOH-terminal HXEL consensus sequence and are
found in rat liver microsomes(15) . In contrast, hydrolase S
lacks this COOH-terminal consensus sequence and has been postulated to
be secreted into blood. The results of the present study support this
hypothesis inasmuch as Western immunoblotting with antibody against
recombinant hydrolase S detected a 71-kDa protein in both serum and the
medium of cultured hepatocytes. The 71-kDa protein in serum has
esterase activity toward 1-naphthylacetate and para-nitrophenylacetate ( Fig.4and Fig. 6),
consistent with this protein being a carboxylesterase. Furthermore, the
NH-terminal amino acid sequence of the 71-kDa
protein is identical to that deduced from the cDNA encoding hydrolase
S.
The 67-kDa protein in liver microsomes recognized by the antibody
against recombinant hydrolase S is likely the precursor form of
hydrolase S (71 kDa) inasmuch as these two proteins were coregulated in
rats treated with 11 different xenobiotics (Fig.6). The 67-kDa
protein is likely the newly synthesized protein of hydrolase S in the
rough endoplasmic reticulum, rather than a protein that is transported
back from the cis-Golgi apparatus like HXEL-containing
proteins. Several lines of evidence from the present study support this
interpretation. Based on Western immunoblotting, the level of the
67-kDa protein was only 5% of the levels of hydrolases A and B.
Such a low level likely corresponds to the newly synthesized protein.
Furthermore, changes in the level of the 67-kDa protein paralleled
changes of the level of mRNA for hydrolase S, suggesting that the
accumulation of the 67-kDa protein in microsomes depends on the rate of
synthesis of hydrolase S (Fig.6). Deglycosylation studies
indicated that the hydrolase S peptide (58 kDa) is converted to a
67-kDa protein by N-linked glycosylation (Fig.7),
which takes place in the rough endoplasmic reticulum as seen with other
carboxylesterases(41) . During its passage through the Golgi
apparatus and secretory pathway, hydrolase S is apparently glycosylated
further to produce the 71-kDa protein secreted into serum.
Glycosylation of hydrolase S increases its apparent molecular mass
by 20% (from 58 to 71 kDa), whereas glycosylation increases the
size of rat microsomal carboxylesterases (e.g. hydrolases A,
B, and C) by less than 5%(17) . The higher degree of
glycosylation of hydrolase S is consistent with the fact that hydrolase
S contains five putative N-linked glycosylation sites, whereas
the rat liver microsomal carboxylesterases contain no more than
three(14, 15, 16) . Other secretory proteins
such as coagulation factors V and VIII have been shown to contain both N- and O-linked carbohydrate chains(42) .
However, hydrolase S apparently contains only N-linked
carbohydrate chains because treatment of serum with endoglycosidase F
converted the 71-kDa protein to a 58-kDa peptide. Slight differences in
the degree to which hydrolase S is glycosylated may be responsible for
microheterogeneity of serum hydrolase S, which appeared as a diffusive
band on Western immunoblots ( Fig.2and Fig. 5).
Glycosylation of hydrolase S may be required for the secretion of this
protein, as previously shown for coagulation factors V and
VIII(42) . Extensive glycosylation of rat hydrolase S may be
also required for its enzymatic activity and stability in serum,
inasmuch as the recombinant hydrolase S produced in E. coli was devoid of esterase activity (both before and after
cleavage of the fusion protein), and cleavage of the fusion protein
with factor Xa decreased the stability of hydrolase S (data not shown).
We have recently attempted to express the cDNA encoding hydrolase S in a baculovirus/Sf21 system to study the catalytic properties of hydrolase S. The Sf21 cells infected with recombinant baculovirus, but not the cells infected with wild-type baculovirus, synthesized a truncated form of hydrolase S. Unexpectedly, this truncated form of hydrolase S was retained in the endoplasmic reticulum. Other cell lines have also failed to correctly process hydrolase S. COS cells transfected with plasmid containing a cDNA encoding hydrolase S produced a carboxylesterase that did not comigrate with any of the carboxylesterases present in rat serum, as determined by non-denaturing gel electrophoresis with staining for esterase activity (43) or by Western immunoblotting(44) . It is not unprecedented for cells to differ in their ability to process secretory proteins. For example, endoplasmin (a protein related to glucose-regulated protein) is present in the microsomal fraction of rat hepatocytes, whereas it is secreted by rat pancreatic cells(24) . These findings suggest that primary cultures of rat hepatocytes may be required to establish the trafficking mechanisms that govern whether hydrolase S is retained or secreted by the liver. In this regard, it is significant that we established cell culture conditions that support the secretion of hydrolase S from rat hepatocytes.
The antibody against hydrolase S cross-reacted with hydrolases A and B, which was anticipated based on the structural relatedness of these carboxylesterases (Table1). Although antibodies against hydrolases A and B did not recognize the 67-kDa protein in microsomes or the 71-kDa in serum (Fig.2), they recognized recombinant hydrolase S produced by E. coli (data not shown). We and others have attempted to clone hydrolases A and B from rat liver cDNA libraries with antibodies against these microsomal proteins, but succeeded instead in cloning hydrolase S(17, 18, 45) . The results of the present study provide a likely explanation for this phenomenon. The mRNA encoding hydrolase S is abundant and probably well represented in cDNA libraries prepared from rat liver. When synthesized by E. coli, hydrolase S is not glycosylated and can be recognized by the antibodies against microsomal carboxylesterases. This interpretation is reinforced by our successful cloning of hydrolases A and B from cDNA libraries prepared from rat testis and kidney(14, 15) , respectively, two tissues that contain high levels of microsomal carboxylesterases but do not express hydrolase S.
The susceptibility of animals to organophosphate
toxicity is inversely related to serum carboxylesterase
levels(46) . Previous studies have shown that phenobarbital
treatment protects rats from parathion toxicity(47) , whereas
treatment with -naphthoflavone has the opposite
effect(48) . The results of the present study show that
treatment of rats with phenobarbital increases the serum level of
hydrolase S, whereas treatment with
-naphthoflavone decreases it.
This suggests that hydrolase S may play a role in detoxifying
organophosphates although further studies are required to test this
possibility. Hydrolase S may also have physiological functions. In
rats, fasting followed by refeeding with a fat-free diet significantly
decreased serum esterase activity(35) . In mice, a serum
carboxylesterase (pEs-N) has been shown to rapidly hydrolyze steroid
hormone esters(49) . Therefore, hydrolase S may play an
important role in lipid and steroid hormone metabolism. The mechanisms
by which xenobiotics regulate the levels of serum carboxylesterase are
not known. The results of the present study show that
xenobiotic-induced changes of the 67 and 71 kDa forms of hydrolase S
reflect increase or decrease in mRNA levels. This suggests that
hydrolase S is regulated by xenobiotics at the levels of transcription
and/or mRNA turnover.