Abstract
We identified a 60-kDa diisopropylfluorophosphate-(DFP) reactive protein in human bronchoalveolar lavage fluid, at a yield of 50-100 pmol/lavage. The protein is associated with the cell-free lavage fluid sediment, which consists mainly of surfactant. [3H]DFP labeling is inhibited by heating to 56 degrees C, 2 mM phenylmethylsulfonylfluoride and 1 mM bis(4-nitrophenyl)-phosphate. An identical 60-kDa [3H]DFP-reactive protein is present in the insoluble fraction of alveolar macrophage-conditioned culture medium and in total membrane preparations of alveolar macrophages. The [3H]DFP-labeled protein was purified approximately 30-fold from lavage fluid sediment by size-exclusion (Sephacryl S-200) and ion-exchange (Mono-Q) chromatography. Cyanogen bromide treatment of the partially purified protein produced a major labeled peptide of 14 kDa with an NH2-terminal sequence 90% identical to a region of form 1 rabbit liver microsomal carboxylesterase. Esterase activity in unlabeled starting material, detected using p-nitrophenyl valerate as substrate, copurified with the [3H]DFP-labeled enzyme. Degenerate oligonucleotide primers were designed based on the partial amino acid sequence and on a highly conserved region of known liver carboxylesterase sequences. Polymerase chain reaction using these primers and reverse-transcribed human alveolar macrophage mRNA yielded a 354-base pair product which was then used to screen a human alveolar macrophage cDNA library. A complete esterase sequence was obtained from two incomplete, overlapping clones, and is virtually identical to human liver carboxylesterase partial sequences. Northern blot analysis demonstrated a single approximately 1.7-kilobase transcript in human monocytes and alveolar macrophages, with much higher levels in the latter. These data indicate that human alveolar macrophages both contain and release a serine esterase that is apparently identical to liver microsomal carboxylesterase. Its enzymatic profile suggests it is a major component of alveolar macrophage-nonspecific esterase activity. We hypothesize that it acts as a detoxication enzyme in the lung.
Highlights
We identified a 60-kDa diisopropylfluorophosphate- Monocytic cells possess caharacteristic nonspecific esterase (DFP) reactive protein in human bronchoalveolar la- activity which can be detected using standard staining techvage fluid, at a yield of 50-100 pmol/lavage
The pro- niques originally described by Gomori (1)and subsequently tein is associated with the cell-free lavage fluid sedi- modified (2, 3)
Effective inhibitors of this activity include ment, which consists mainly of surfactant. ["HIDFP NaF and organophosphorous compounds such as DFP' and labeling is inhibited by heating to 56 "C, 2 mM phen- BNPP, which indicates that serine esterases areresponsible
Summary
Aliquots from each fraction were used for liquid scintillation counting. Protein D~terminarions-Protein measurements were made using H bicinchoninic acid spectrophotometric assay ( 2 7 )and RSA protein standards supplied by the manufacturer (Micro RCA Protein Assay, Pierce ChemicalCo.). Thismethod was used because it allowed protein measurements to be made in solutions containing up to 1%. ("HIDFP Labeling-Labeling cell-free alveolarlavage fluid with ["HIDFP revealed a single ["HjDFP-binding protein that migratedonSDS-PAGEat 60 kDa under reduced (Fig. 1, lane 2 ) andnonreducedconditions.Ourresults, described below, indicate that this proteinis a member of the family of liver microsomal carboxylesterases. Terase purified withthesedimentablefraction of cell-free degenerate oligonucleotide lavage fluid and remained associated with this fraction after treatment with 1 M NaCl or pH 3 buffer (data not shown)
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