Biomedical subjects

C A Broomfield

Publications and source records attributed to C A Broomfield.

At least 19 recordsLinked to original sources

PubMed2004

TNF-alpha expression patterns as potential molecular biomarker for human skin cells exposed to vesicant chemical warfare agents: sulfur mustard (HD) and Lewisite (L).

Studies were conducted to examine the effect of two vesicant chemical warfare agents (VCWA), one of them an arsenical, on cytokine gene expression in normal human epidermal keratinocyte (NHEK) cells. We tested 2,2'-dichlorethylsulfide (sulfur mustard, military designation HD) and 2,chlorovinyldichloroarsine (Lewisite, military designation L), which have significant differences in their chemical, physical, and toxicological properties. Human tumor necrosis factor-alpha (hTNF-alpha) cytokine was detected by using the enzyme-linked immunosorbent assay, a protein multiplex immunoassay, Luminex100, and reverse transcription-polymerase chain reaction (RT-PCR). The messenger RNA expression of hTNF-alpha was determined to provide a semi-quantitative analysis. HD-stimulated NHEK induced secretion of hTNF-alpha in a dose-dependent manner. Dose response effect of Lewisite decreased hTNF-alpha levels. Time-response data indicated that the maximum response for HD occurred at 24 h with an associated cytotoxic concentration of 10(-4) mol/L. NHEK cells stimulated with 10(-4) mol/L HD for 24 h at 37 degrees C increased detectable levels of hTNF-alpha from 5 to 28 ng/ml at an index of cell viability between 85 to 93% as detected by Luminex100. Our results indicated that the increased levels of hTNF-alpha by HD are dependent on the primary cultures, cell densities, and chemical properties of the stimulation. Lewisite under the same conditions as HD caused a reduction of hTNF-alpha from control levels of 1.5 ng/ml to 0.3 ng/ml after stimulation (10(-4) mol/L), with an index of cell viability of reverse similar 34%. We analyzed the transcriptional of hTNF-alpha gene and found that HD (10(-6) to 10(-4) mol/L) activates hTNF-alpha gene in cultured NHEK and that L at 10(-6) to 10(-4) mol/L markedly reduces hTNF-alpha gene. We conclude that the pro-inflammatory mediator, hTNF-alpha, could be a potential biomarker for differentiating between exposure of HD or L.

Adult↗

PubMed2002

Suppression of sulfur mustard-increased IL-8 in human keratinocyte cell cultures by serine protease inhibitors: implications for toxicity and medical countermeasures.

The toxicity of the chemical warfare blistering agent sulfur mustard (2,2'-dichlorodiethyl sulfide; SM) has been investigated for nearly a century; however, the toxicological mechanisms of SM remain obscure and no antidote exists. The similarity of dermal-epidermal separation caused by SM exposure, proteolysis, and certain bullous diseases has fostered the hypothesis that SM vesication involves proteolysis and/or inflammation. Compound screening conducted by the US Army Medical Research Institute of Chemical Defense established that topical application of three tested serine protease inhibitors could reduce SM toxicity in the mouse ear vesicant model. Although most of the drugs with efficacy for SM toxicity in rodent models are anti-inflammatory compounds, no in vitro assay is in current use for screening of potential anti-inflammatory SM antidotes. IL-8 is a potent neutrophil chemotactic cytokine that is increased in human epidermal keratinocyte (HEK) cell cultures following exposure to SM and has been proposed as a marker for SM-induced inflammation. This study was conducted to establish in vitro screening of IL-8 in SM-exposed HEK as a possible model for evaluating candidate compounds prior to in vivo testing. We chose two protease inhibitors, one from those shown as successful in the MEVM (ethyl p-guanidinobenzoate hydrochloride, ICD 1579) and a prototypic inhibitor of trypsin, N-tosyl-L-lysine chloromethyl ketone (TLCK). TLCK (62.5 to 1000 micromol/L) or ICD 1579 (31.25 to 1000 micromol/L) was added to HEK cell cultures 1 h after SM exposure (200 micromol/L) and dose-dependently suppressed SM-increased IL-8. The suppression of SM-increased IL-8 by a class of drug candidate compounds such as protease inhibitors may provide a mechanistic marker that helps predict future medical countermeasures for SM toxicity and reduces the need for testing in animal models.

Animal Testing Alternatives↗

PubMed2001

Progress on the road to new nerve agent treatments.

In the 50 years since nerve agents were developed a great deal has been learned about their acute toxicity, treatment and prophylactic strategies. However, the currently fielded treatments are not significantly different from those available at the end of World War II. Reasons for this lack of progress and strategies to circumvent those intrinsic problems that have impeded progress are discussed, with emphasis on the development of scavengers to be introduced as prophylactics that will significantly reduce the effective dose and thus protect against multiple times the normal LD50.

Aryldialkylphosphatase↗

PubMed2001

Serum paraoxonase (PON1) isozymes: the quantitative analysis of isozymes affecting individual sensitivity to environmental chemicals.

In a recent study on Gulf War veterans who developed delayed neurotoxicity symptoms, we found their levels of serum paraoxonase (PON1) isozyme type Q to be significantly lower than in the control, unaffected veteran group. These results were obtained in 25 ill veterans and 20 well control subjects, of which 10 were deployed and 10 were nondeployed battalion members who remained in the United States during the Gulf War. The blood samples were also assayed for serum butyrylcholinesterase in our laboratory, and more recently in Dr. C. Broomfield's laboratory for somanase and sarinase activities. The cholinesterase activities showed no significant correlation with the PON1 isozyme levels or the severity of the clinical symptoms, but the somanase and sarinase levels ran parallel to the PON1 type Q isozyme concentrations. Although there is no direct evidence that these Gulf War veterans were directly exposed to or encountered either of these nerve gases, they may have been exposed to some environmental or chemical toxin with a similar preference for hydrolysis by the PON1 type Q isozyme. The number of subjects is relatively small, but the results should encourage other investigators to examine both the individual phenotypes and the levels of PON1 isozymes in other groups exhibiting neurological symptoms.

Aryldialkylphosphatase↗

PubMed2001

Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to VX lethality.

The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC 3.1.1.7) was examined in AChE knockout mice. Mice (34-55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 microg/kg in AChE-/-, 17 microg/kg in AChE+/-, and 24 microg/kg in AChE+/+ mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE.

Acetylcholinesterase↗

PubMed2000

Exposure of human epidermal keratinocyte cell cultures to sulfur mustard promotes binding of complement C1q: implications for toxicity and medical countermeasures.

Sulfur mustard (HD)-increased proteolytic activity, HD-enhanced expression of Fc receptor (FcR) on human epidermal keratinocytes (HEK) and associated inflammatory responses may contribute to HD pathology. Like the FcR, the first component of the classical complement (C') cascade, C1q, binds to the Fc region of antibody to mediate inflammatory responses. Complement C1q binds specifically to the C1q receptor (C1qR) on the blebs of apoptotic human keratinocytes and is proposed as a cell surface marker for apoptosis. Assays by fluorescent antibodies demonstrated significantly enhanced binding of C1q to HEK cell cultures exposed to HD. The cell populations of HEK that showed enhanced C1q binding also demonstrated an intermediate uptake of propidium iodide that was greater than in viable unexposed cells but less than in dead cells. The HD-enhanced C1q binding was concentration-dependent, negative by flow cytometry or weakly positive by digital scanning microscopy at 100 microM and positive by both methods at 300 microM. Binding of C1q was also time-dependent, weakly positive at 8 h, and positive at 16 and 24 h after HD exposure. The HD-increased C1qR that binds C1q to the surface of HEK might be a contributing mechanism or a marker for the inflammation and vesication associated with HD exposure.

Antigens, Surface↗

PubMed2000

Response of normal human keratinocytes to sulfur mustard: cytokine release.

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (2,2'-dichlorodiethyl sulfide, HD). This study describes responses of normal human epidermal keratinocytes (NHEK) to HD, defined by interleukin-1beta (IL-1beta), IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) release. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased the release of cytokines. The amounts of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and 4-fold, respectively, above control levels when NHEK were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and at other times it decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM HD and significantly increased levels of IL-6 were observed. Interleukin-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that the cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Biomarkers↗

PubMed2000

Expression of biologically active human butyrylcholinesterase in the cabbage looper (Trichoplusia ni).

This investigation examined the utility of three recombinant protein-expression systems (COS cells, insect cells and insect larvae) to cost-effectively produce biologically active human butyrylcholinesterase (BuChE). It was determined that baculovirus-infected insect cells (Sf9 and High 5) expressed 3.5- and 8.2-fold, respectively, more active enzyme than COS-7 cells. Baculovirus-infected cabbage looper (Trichoplusia ni) insect larvae produced over 26 times more than High 5 cells; in fact, one baculovirus-infected insect larva provided more active protein than 100 ml of insect cell culture. Analysis of the larvally expressed proteins revealed that the vast majority of BuChE expressed was inactive due to extensive degradation that occurred in vivo. However, the active form of BuChE does have enzyme kinetics similar to those of its human serum counterpart. Cabbage looper larvae were also examined for their ability to serve as an in vivo animal model to study protection against anti-cholinesterase toxicity. This was unsuccessful due to their high tolerance to the very toxic organophosphorus compounds tested. This tolerance may be attributed at least in part to a novel endogenous organophosphorus acid anhydride hydrolase activity that is capable of hydrolysing the chemical-warfare nerve agents sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate). These results show that cabbage looper larvae can serve as an inexpensive recombinant protein-expression system for human BuChE.

Animals↗

PubMed1999

Characterization of a soluble mouse liver enzyme capable of hydrolyzing diisopropyl phosphorofluoridate.

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (approximately 39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

Amino Acid Sequence↗

PubMed1999

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

Animals↗

PubMed1999

Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure.

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD). This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2'-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) release. A new method for detaching cell to cell adhesion between keratinocytes has been applied. This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention. NHEK (typical average cell density 4.4 x 10(6) cells/mL) were exposed to HD (100 and 300 microM) in keratinocyte growth medium (KGM) for 24 h at 37 C in humidified air. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased release of cytokines. IL-1beta (exposed: 1.41 x 10(-5) pg/ cell+/-1.60 x 10(-6) pg/cell: control 7.10 x 10(-6) pg/ cell+/-1.20 x 10(-6) pg/cell), TNF-alpha (exposed: 1.06 x 10(5) pg/cell+/-7.3 x 10(-7)pg/cell; control: 4.04 x 10(-6)+/-2.80 x 10(-7) pg/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell+/- 3.26 x 10(-6) pg/cell; control: 2.99 x 10(-6) pg/cell+/-8.80 x 10(-7) pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 microM for 24 h (exposed: 1.47 x 10(-6)+/-1.60 x 10(-7) pg/cell; control: 1.28 x 10(-6)+/-8.40 x 10(-8) pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 microM HD (4.67 x 10(-5) pg/cell+/-3.90 x 10(-6) pg/cell; control: 3.99 x 10(-6) pg/cell+/-5.50 x 10(-7) pg/cell). The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM and significantly increased levels of IL-6 were observed. IL-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Cell Adhesion↗

PubMed1998

Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes.

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.

Amino Acid Sequence↗

PubMed1998

Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase.

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

Amino Acid Substitution↗

PubMed1998

Sulfur mustard exposure enhances Fc receptor expression on human epidermal keratinocytes in cell culture: implications for toxicity and medical countermeasures.

Sulfur mustard (HD) is a chemical warfare blister agent. The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists. Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology. Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses. FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes. Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 micromol/L). The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication. HD-increased FcR on the surface of HEK might be a mechanism of vesication.

Cells, Cultured↗

PubMed1997

Identification of a recombinant synaptobrevin-thioredoxin fusion protein by capillary zone electrophoresis using laser-induced fluorescence detection.

Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site. TSB-51 was derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve amino acids as SDKIIHLTDDSF. TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51.

Amino Acid Sequence↗

PubMed1997

Cutaneous exposure to bis-(2-chloroethyl)sulfide results in neutrophil infiltration and increased solubility of 180,000 Mr subepidermal collagens.

Exposure to bis-(2-chloroethyl)sulfide (BCES; "sulfur mustard") causes delayed formation of slowly healing skin blisters. Although the histopathology of BCES injury is well characterized [reviewed in Smith et al., J Am Acad Dermatol 32: 767-776, 1995], little is known of the cutaneous toxicity at the molecular level. To identify biological markers of exposure, epidermal and subepidermal extracts were prepared from 48 individual hairless guinea pigs (HGP) at successive 3-hr intervals following exposure to BCES vapor, and compared using gel electrophoresis, and lectin- and antisera-binding. Inflammation was assessed by measuring edema and myeloperoxidase activity. Edema reached peak levels at 15-18 hr and remained elevated above controls at 24 hr. Recruitment of neutrophils, deduced from increased myeloperoxidase, occurred as early as 3 hr after BCES exposure with maximum infiltration at 6-12 hr. Binding of concanavalin-A lectin revealed increased amounts, relative to contralateral control sites, of two approximately 180,000 Mr polypeptides in subepidermal protein extracts from the BCES-exposed skin obtained > or = 12 hr after exposure. This alteration was not found in epidermal protein extracts prepared from the same animals. Based upon the determined amino acid compositions, both polypeptides had significant collagenous triple helical content (>75%). They could be distinguished immunologically from collagen types I, III, and IV by using polyclonal antisera. We conclude that exposure of HGP skin to BCES results in an early neutrophil infiltration that precedes epidermal-dermal separation and selective alterations of the subepidermal extracellular matrix.

Amino Acids↗

PubMed1997

A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase.

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

Animals↗

PubMed1996

Mustard gas crosslinking of proteins through preferential alkylation of cysteines.

Mustard gas, bis(2-chloroethyl)sulfide, treatment of proteins is shown to generate significant amounts of covalently crosslinked protein dimers. This is due to the preferential alkylation of cysteine residues. Crosslinking does not occur in the model protein staphylococcal nuclease, which has no cysteine residues. Treatment of cysteine-containing mutants of staphylococcal nuclease with this chemical warfare agent did result in crosslinking. However, these dimers are slowly cleaved back to monomers by an unknown mechanism. The alkylation and crosslinking of cysteine-containing proteins by mustard gas may contribute to its toxicity.

Alkylation↗