Human Monocyte Carboxylesterase PURIFICATION AND KINETICS*

Human peripheral blood monocytes were isolated by density gradient centrifugation and purified by counterflow centrifugation elutriation. Membrane-localized carboxylesterase (CBE) was extracted with nonionic detergent (Triton X-100) and purified by ion exchange (DEAE-cellulose), gel filtration (Sephacryl S-300), hydroxylapatite column, and high performance liquid chromatography. The purified enzyme migrated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single protein band with a molecular weight of 60,000. Under nondenaturing conditions, monocyte CBE formed a trimer and eluted from a gel filtration column as a protein with an approximate molecular weight of 200,000. Electrophoretic patterns of the enzyme on polyacrylamide gels run a neutral pH did not vary during enzyme purification. At least four major isoenzymes of human monocyte CBE were observed with isoelectric points between 7.5 and 7.8. Pure human monocyte CBE hydrolyzed short chain alpha-naphthyl, o-nitrophenyl, and p-nitrophenyl esters. Amide esters and thioesters were not hydrolyzed by the enzyme. Short chain alcohols activated the enzyme and organophosphorus compounds, diphenyl carbonate, sodium fluoride, and phenylmethylsulfonyl fluoride inhibited the enzyme. EDTA and sulfhydryl reagents had no effect on enzyme activity. The amino acid content of the enzyme was consistent with other CBEs. Inhibitors reacted either with the active or effector site of the enzyme. Purified enzyme now permits the characterization of CBE structure and regulation.

Human peripheral blood monocytes were isolated by density gradient centrifugation and purified by counterflow centrifugation elutriation.
The purified enzyme migrated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single protein band with a molecular weight of 60,000. Under nondenaturing conditions, monocyte CBE formed a trimer and eluted from a gel filtration column as a protein with an approximate molecular weight of 200,000. Electrophoretie patterns of the enzyme on polyacrylamide gels run at a neutral pH did not vary during enzyme purification.
At least four major isoenzymes of human monocyte CBE were observed with isoelectric points between 7.5 and 7.8.
Pure human monocyte CBE hydrolyzed short chain cY-naphthyl, o-nitrophenyl, and p-nitrophenyl esters. Amide esters and thioesters were not hydrolyzed by the enzyme. Short chain alcohols activated the enzyme and organophosphorus compounds, diphenyl carbonate, sodium fluoride, and phenylmethylsulfonyl fluoride inhibited the enzyme. EDTA and sulfhydryl reagents had no effect on enzyme activity. The amino acid content of the enzyme was consistent with other CBEs. Inhibitors reacted either with the active or effector site of the enzyme. Purified enzyme now permits the characterization of CBE structure and regulation.
undertaken to elucidate its role both in physiological processes and xenobiotic metabolism.
Monocyte carboxylesterase (CBE)' represents a membranebound ectoenzyme of a peripheral blood cell whose primary biological functions are performed, to a large degree, by membrane-associated proteins (16,17). Monocytes are migratory cells that differentiate into tissue histiocytes including Kuppfer cells of the liver. The carboxylesterase, as a monocyte ectoenzyme, is ideally situated to interact with the extracellular environment as shown by the inhibition of its activity by organophosphorus compounds and by the use of this property as a biomarker of organophosphorus exposure (18). Further, the absence of human monocyte carboxylesterase, an enzyme scavenger used for the detoxification of toxic organophosphorus esters and perhaps other toxic organic esters, has led to speculation that such human hosts are at increased risk for cancer (19,20). Variation in the expression of carboxylesterase isoenzymes also functions as a marker for monocyte maturation arrest in leukemias (21,22). Finally, monocyte/ macrophage cell lines represent easily obtainable sources of this enzyme for the investigation of carboxylesterase regulation if such enzymes can be shown to be similar to the native human enzyme.
To clarify the biochemical properties of human monocyte carboxylesterase and its role in xenobiotic metabolism, we have purified to homogeneity peripheral blood monocyte carboxylesterase.
Our studies show that the purified human monocyte carboxylesterase has a preference for certain ester substrates, is inhibited by organophosphorus agents, is activated by alcohols, and has an amino acid content comparable to other enzymes in the e&erase family.

RESULTS
Purification-Human monocytes were purified by counterflow centrifugation elutriation (23), and their membrane esterases solubilized by a nonionic detergent (Triton X-100). The solubilized membranes were then applied to a column of DEAE-cellulose (DE52) and eluted with a linear gradient of sodium chloride (Fig. 1). This column resolved the solubilized monocyte membranes into multiple protein peaks, but the majority (more than 95%) of the enzyme activity eluted as a single peak to the left of the first protein peak. Less than 5% of the enzyme did not bind to the column and eluted with the detergent (data not shown). The enzyme peak from the ionexchange column was concentrated and then applied to a gel filtration column (Sephacryl S-300) which had been calibrated with standard molecular weight proteins (Fig. 2). The majority (95%) of the enzyme eluted with P-amylase (MI 200,000) and only 5% of the enzyme eluted with bovine serum albumin (Mr 66,000). Thus, the majority of the enzyme was eluted in its high molecular weight form. Higher molecular weight forms of the enzyme were not eluted from this column. Electrophoretie gel patterns of the enzyme from this column separation showed that the enzyme was only 50% pure (Fig. 4A, lane 4), and additional steps were required for the purification of the enzyme. Enzyme peak I from the gel filtration column was applied to a column of hydroxylapatite and eluted with a linear gradient of potassium phosphate (Fig. 3). The enzyme eluted as a single peak from this column with a salt concentration of 0.2 M potassium phosphate. steps by SDS-PAGE (Fig. 4A) and polyacrylamide gel electrophoresis at a neutral pH to determine whether the protein bands exhibited e&erase activity (Fig. 4B). The results of these experiments show that the enzyme was successfully purified to homogeneity by three chromatographic steps (Fig.   4A). The pure enzyme (Fig. 4A, lane 5) had a M, of -60,000 as determined from SDS-PAGE. Further, the enzyme migrated as a single band of high molecular weight, possibly a trimer, in a polyacrylamide gel run at a neutral pH. The migration distance of samples from the different purification steps remained constant (Fig. 4B). In the early steps of the purification, there were larger aggregates of the enzyme than the trimeric form (Fig. 4B, lanes I and 2).
In order to determine whether the single protein band on SDS-PAGE (Fig. 4A, lane 5) consisted of a single or multiple isomers, the purified enzyme was analyzed on isoelectric focusing gels. The enzyme was shown to consist of at least four major isoenzymes (Fig. 4C). Gel slices from the isoelectric focusing gel were eluted with double-distilled water to measure the pH, and the isoelectric points determined for these four isoenzymes. The p1 values for these isoenzymes were between pH 7.5 and 7.8.
Approximately 800 pg of pure enzyme was recovered from 4 x 10' human monocytes. This represented 0.22% of the total protein of the detergent-extracted monocyte homogenate and a 146-fold purification based on the specific activity of the enzyme as measured with a-naphthyl butyrate as substrate (Table I).
Amino Acid Composition-The results of the amino acid analysis of human monocyte carboxylesterase expressed as residues (%) per 60,000 g of enzyme are shown in Table II. As can be seen in that table, the amino acid composition of human monocyte carboxylesterase is similar to published data on the amino acid composition of other human and nonhuman esterases.
Enzyme Stability-The addition of 10% glycerol to the enzyme during purification is used as a stabilizing factor. The enzyme stored in glycerol at -85 "C is stable for months. The gel pattern of samples from different stages of enzyme purification analyzed by 12% SDS-PAGE and treated with silver stain is shown in A. Migration was from top to the bottom (anode) of the gel. The gel pattern for the molecular weight standard proteins is shown in lane 1 of A. Protein bands from the top to the bottom were: bovine serum albumin, iVf, 66,000; ovalbumin, M, 45,000; pepsin, M, 34,700; and trypsinogen, M, 24,000, respectively. Lanes 2-5 in A show the gel pattern of lo-100 rg of monocyte detergent-extracted homogenate and esterase peaks of Figs. 1, 2, and 3, respectively. The gel pattern of samples from different stages of purification analyzed by 6% polyacrylamide gel at a neutral pH after staining with ANA for esterase activity is shown in B. The migration is from top to the bottom (anode). Approximately 5-10 milliunits of the enzyme from each sample was applied to each lane. The electrophoretic gel pattern of monocyte detergent-extracted homogenates, esterase peaks of Figs. 1, 2, and 3 is shown in lanes l-4, respectively. The isoelectric focusing gel pattern of the pure enzyme stained with silver stain is shown in C. Approximately 10 pg of protein was applied to the gel. Migration was from top to the bottom (cathode).
Dialysis of the enzyme using nitrocellulose membranes caused a significant loss of enzyme activity, probably due to the binding of the enzyme to the dialysis membranes.
For this reason, dialysis could not be used to determine whether the enzyme was reactivated after incubation with inhibitors and subsequent dialysis to remove inhibitor.
Monocytes frozen at -85 "C in Dulbecco's phosphate buffered saline without calcium and magnesium chloride and containing 20% glycerol were used for purification after 2-3 months with recovery of activities comparable to preparations made from freshly isolated monocytes.
Molecular Weight-Gel filtration chromatography indicated that the majority of the enzyme (95%) eluted with pamylase and had an apparent molecular weight of 200,000. Approximately 5% of the enzyme activity eluted with bovine serum albumin with a molecular weight of 66,000. When the enzyme peak recovered from the hydroxylapatite column was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major protein band was observed that migrated with an estimated molecular weight of 60,000 (Fig. 4A, lone 5). The data from gel chromatography and electrophoresis suggested that the enzyme was a trimer of high molecular weight consisting of three monomeric units with a molecular weight of 60,000 each. Carbohydrate Content-The enzyme was characterized as a glycoprotein since it contained an N-linked high mannose type carbohydrate chain. Two glucosamine and eight mannose molecules per mol of enzyme were found on carbohydrate analysis of the purified enzyme.
pH Optimum-The rate of ester hydrolysis with a-naphthyl butyrate at saturating substrate concentrations was greatest at pH 8 with little loss of activity at pH 6.5 and 9. Less than 50% of the maximal rate of ester hydrolysis was observed at pH values less than 6 or more than 12 (data not shown).
Substrate Specificity-Only short carbon chain length organic esters were determined to be hydrolyzed by monocyte CBE. Long chain fatty acid esters which represent possible physiological substrates for the enzyme were not hydrolyzed by monocyte CBE either in aqueous solutions or in phospholipid micelles as described by the methods of Shirai and his colleagues (39). Apparent K,,, and V,,,,, values were determined for a-naphthyl acetate and butyrate, p-nitrophenyl valerate, and o-nitrophenyl butyrate (Table III). Such values are all in the same range. Other short chain a-naphthyl esters (anaphthyl caproate, a-naphthyl propionate), were also hydrolyzed by this monocyte enzyme but these substrates have less affinity for the enzyme than ANB. The enzyme did not hydrolyze a-naphthyl caprylate, a-naphthyl laurate, or esters with longer carbon chains. The enzyme also hydrolyzes orthonitrophenyl and para-nitrophenyl esters (Fig. 5). With these ester substrates, enzyme activity is only observed with short chain carbon compounds. The ortho derivatives show activity with acetate, propionate, butyrate, valerate, and caproate esters. Ester substrates of higher carbon chain lengths were  Carboxylesterase not hydrolyzed by the enzyme. For the para-nitrophenyl esters, activity is determined with acetate, propionate, butyrate, valerate, caproate, and caprylate esters. No activity was observed with higher carbon chain substrates in this class. Enzyme activity was slightly higher with the o-nitrophenyl and a-naphthyl esters than with p-nitrophenyl esters. The highest activity (V,,,,,) was seen with butyrate esters. Since liver carboxylesterases also hydrolyze thioesters and amide esters, the classical substrates (acetanilide, acetyl-coenzyme A, and butyl-coenzyme A) for these enzyme reactions were evaluated as substrates for the purified monocyte esterase (32-34). Purified enzyme showed no activity with these esters as substrates. Further, the carcinogens, 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene, were not hydrolyzed by monocyte esterase.
Effects ofAlcohols--The effects of several nucleophilic small molecules on the esterase catalyzed hydrolysis of a-naphthyl butyrate are summarized in Table IV. Short chain alcohols like methanol and ethanol activated the enzyme under the conditions of this assay, whereas more complex alcohols (ethylene glycol monoethyl ether and ethylene glycol) had little effect on enzyme activity except at high concentrations (1 M). n-Butanol increased monocyte carboxylesterase activity at low concentrations (0.1 M), but at high concentrations (1 M) it inhibited enzyme activity. Dimethylsulfoxide and phenol at high concentrations also inhibited the hydrolysis of a-naph-thy1 butyrate. The former compound only inhibited the enzyme at high concentrations (1 M), whereas phenol inhibited the enzyme at one-tenth that concentration.
Neither of these latter agents increased a-naphthyl butyrate hydrolysis.
Inhibitors-Monocyte esterase activity was inhibited by a variety of organophosphorus compounds as well as sodium fluoride in high concentration (lo-* M). Sulfhydryl inhibitors @-chloromercuribenzoic acid and N-ethylmaleimide) and a metal chelator (EDTA) caused no change in enzyme activity over a range of concentrations.
In experiments where electrophoresis was used to examine the type of binding between radioactively labeled inhibitor and pure enzyme (Fig. 6A), radioactivity migrated with the purified esterase when 3H-labeled DFP was incubated with the enzyme and the resultant enzyme-inhibitor mixture elec- Human monocyte carboxylestkase (4 fig) was labeled with 1 x lo5 cnm of either 13HlDFP. I"ClTPP. or I"ClDPC. and incubated at room temperature for 36 min: The'samples'were'then electrophoresed through SDS-PAGE slabs (12% acrylamide). The gel was fixed, dried, and examined by autoradiography. The exposure time was 5 days. Lanes 1, 2, and 3 of A showed the binding activity of ("HIDFP, ["CITPP, and [W]DPC with human monocyte carboxylesterase, respectively. In B, human monocyte carboxylesterase was labeled with VHlDFP by methods identical to those used in A, lane I, except that the enzyme was preincubated for 30 min with either no unlabeled inhibitor (lone I). or 10m3 M TPP (lane 2). or 0.1 M DPC (lane 3). The enzyme ias subsequently labeled with'pH]DFP.
The samples were then electrophoresed through SDS-PAGE (12%), and the gel was examined by autoradiography. The exposure time was 9 days.
trophoresed under denaturing conditions. With the other two radiolabeled inhibitors (TPP and DPC), little or no radioactivity was observed to corn&rate with the enzyme when these enzyme-inhibitor mixtures were subjected to electrophoresis and autoradiography. Such data suggested that DFP formed a covalent bond with the monocyte esterase, whereas DPC and TPP did not form stable covalent bonds. Such mechanisms were further supported by experiments in which the enzyme-inhibitor mixture was subjected to electrophoresis in a nondenaturing gel. Under these conditions, enzyme activity was recovered after electrophoresis of the DPC/enzyme and TPP/enzyme mixtures, whereas no enzyme activity was observed with a DFP/enzyme mixture when it was subjected to electrophoresis under the same conditions (data not shown).
To elucidate further the interactions between these organophosphorus agents and monocyte carboxylesterase, competitive binding experiments were done (Fig. 6B). In these experiments, pure monocyte carboxylesterase was preincubated for 30 min with either no unlabeled inhibitor or TPP or DPC, these mixtures were subsequently incubated with identical quantities of radiolabeled DFP. The latter mixtures were then subjected to electrophoresis as described previously. The results of these experiments show that the carboxylesterase, preincubated with TPP and then treated with radiolabeled DFP, incorporated significantly less radioactivity (Fig. 6B, lane 2) than the carboxylesterase incubated with radiolabeled DFP alone (Fig. 6B, lane I) (no TPP preincubation). These data suggest that DFP and TPP compete with the same site on the enzyme. An identical experiment using DPC in place of TPP showed quite different results. In the case of DPC, there was no significant difference in the quantity of [3H] DFP incorporated into enzyme (Fig. 6B, he 3) as compared to the amount of radiolabel incorporated into the control (Fig.  6B, lane I) (no DPC preincubation). These data suggest that TPP and DFP compete for an identical site on monocyte carboxylesterase, whereas DPC interacts with a site other than the active site of the enzyme.
Using two different substrate concentrations of a-naphthyl butyrate (0.1 and 0.2 mM), a Dixon plot (38) was constructed to determine the mode of action of the apparent reversible inhibitors, DPC, TPP, and tetraphenylresorcinol diphosphate. The latter inhibitor, like its analog, TPP, did not bind covalently to the enzyme (data not shown). By Dixon plot, TPP and tetraphenylresorcinol diphosphate, were observed to be competitive inhibitors of carboxylesterase, whereas DPC was determined to be a noncompetitive inhibitor. These observations confirm the electrophoretic data characterizing enzyme-inhibitor interactions between TPP and DFP binding site but showing no interaction between DPC and that binding site. The inhibitor constants (Ki) for each reversible inhibitor are shown in Table V. To calculate the affinity of the inhibitors for monocyte carboxylesterase as well as their inactivation constant (&activation), we have assumed the following reactions.
K *ssxiatio" k. l"aCtlWtlO" ' + I (loose binds 'I (tight binds E1 By this kinetic scheme, Kassmistion is equal to the reciprocal of KD. The data derived from these assumptions are also shown in Table V. The Kassmiation values provide a rank order of affinity of the various inhibitors for carboxylesterase which are consistent with the inhibitor constants (KJ.

DISCUSSION
In this report, we have described a relatively simple method for purifying to homogeneity the membrane-associated human monocyte carboxylesterase from normal, resting peripheral blood monocytes using only three different column chromatographic systems for purification and high performance liquid chromatography to remove nonprotein contaminants. Under nondenaturing conditions, the majority of the enzyme (>95%) present in detergent-extracted human monocyte homogenates had a molecular weight of approximately 200,000 (Figs. 2 and 4B,, and the pure enzyme protein migrated as a single band on SDS-PAGE as observed with silver staining (Fig. 4A, lone 5). By the latter electrophoretic method, the molecular weight was determined to be 60,000. Treatment with SDS presumably resulted in the dissociation of the trimeric form of the enzyme to a monomeric species. Although our data were not analyzed by a Ferguson plot to derive a Stokes radius for the enyzme, we believe existing data are sufficient to permit the reasonable inference that human monocyte carboxylesterase exists in monomeric and trimeric forms. Others have shown that covalent cross-linking of pig and ox liver carboxylesterase subunits with dimethyl suberimidate, and subsequent polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gives three bands with molecular weights of 60,000, 120,000, and 180,000 (40). Identical data have been derived for human liver carboxylesterase (3). Further, electron microscopic examination of negatively stained pig liver esterase has shown enzyme particles to be clover-leaf shaped trimers (41). In a review of carboxylesterase molecular weights and subunit structures, these same enzymes have been examined from nine different species including the human by a variety of methods and found to have a trimeric structure (42). Such findings are also compatible with those observed by Yourno (43) who purified an esterase from an acute myeloid leukemia cell line (ML-l). In that report, a monomer of 68 kDa and a trimer with a molecular mass of 205 kDa were described and observed by PAGE run at pH 9.5. In contrast to that report, human monocyte carboxylesterase was observed as a single band in its presumed trimeric form on PAGE run at pH 7. These subunit and undissociated enzyme molecular weights are similar to those observed for human liver carboxylesterase, human intestinal esterase, human brain esterase, and esterases purified from nonhuman species (3,6,15,(44)(45)(46)(47)(48)). An esterase from the human brain with a preference for butyl ester substrates is an exception to these findings since it has been found to have a molecular mass of 340 kDa (46). Whether this enzyme represents an aggregate of lower molecular weight enzymes or a different enzyme cannot be determined from the data. The similarity of the amino acid content of esterases lends additional support to the possibility that the primary structure of these enzymes share common elements between species (Table II). Sequence data from human monocyte carboxylesterase and other esterases would provide a more quantitative estimation of the proposed homogeneity of this enzyme from species to species.
Isoelectric focusing gel electrophoresis of the purified human monocyte carboxylesterase showed at least four major isoenzymes with pIs between 7.5 and 7.8 (Fig. 4C). Multiple esterase isoenzymes have also been identified in a number of human tissues, and investigations by Coates and his colleagues (49) have reported that at least nine structural gene loci control the expression of human esterase isoenzymes. The recent cloning of carboxylesterase from rat liver provides further evidence that a family of carboxylesterase genes exist (15). Like the monocyte CBE isolated by us, rat liver microsomal CBE is a high mannose-type glycoprotein (50, 51). Despite the reported capacity of carboxylesterases to hydrolyze both xenobiotic and endogenous substrates, the true biological function of this family of enzymes remains poorly understood.
Human monocyte CBE did not hydrolyze long chain saturated or unsaturated fatty esters (C&-C& in aqueous solution. Further, the use of lipid micelles containing a variety of pure phospholipids as well as mixtures of phospholipids along with these long chain fatty acid esters as substrates for CBE did not result in ester hydrolysis. Other investigators (52) have reported that monocyte CBE metabolized N-acetylated arylamines to their carcinogenic metabolites using an indirect assay. In our hands, purified human monocyte CBE did not deacetylate the potent acetylated aminofluorene carcinogens using a direct method to measure hydrolytic products. Another xenobiotic, triphenyl phosphate, has been reported to be hydrolyzed to phenol and monophenyl phosphates by mono&e CBE (53). These workers used a whole cell preparation and radiolabeled TPP as an enzyme source and substrate, respectively.
Our results demonstrated TPP to be a relatively potent CBE inhibitor, but we did not observe the generation of any hydrolytic products (phenol and phenyl phosphates) during its incubation with purified monocyte CBE. Since TPP in aqueous solution hydrolyzes slowly to phenol and phenyl phosphates, the hydrolysis observed by others (53) may represent the chemical hydrolysis of TPP or the activity of another organophosphorus-hydrolyzing enzyme (54). We have now examined crude and partially purified hepatic preparations of porcine, rabbit, rat, and human CBE for their capacity to hydrolyze TPP under a variety of experimental conditions and find no evidence of enzymatic hydrolysis of TPP using several different assays to measure the products of hydrolysis, phenol, and phenylphosphates. Neither amidase nor thioesterase activity was demonstrated using classical substrates for these reactions and purified monocyte CBE. In summary, purified monocyte CBE was unable to hydrolyze xenobiotics proposed as substrates for this enzyme and was incapable of metabolizing long chain fatty acid esters which would have characterized a physiological role for this enzyme.
Human monocyte CBE did hydrolyze a-naphthyl esters, pnitrophenyl esters, o-nitrophenyl esters of different carbon chain lengths (Fig. 5), but the role of these short chain esters in physiological processes remains elusive. Relatively low concentrations of alcohols (ethanol or ethylene glycol monoethyl ether) caused a significant increase in the total rate of a-naphthyl butyrate hydrolysis by pure monocyte CBE. These findings are consistent with the existence of an acyl-enzyme intermediate as proposed by other workers (55), who showed that substrate activation occurred via effector sites on the enzyme which were different from catalytic sites. Effector site occupancy may induce a conformational change in the enzyme permitting a more rapid ingress and egress of substrates to the catalytic site. Recent investigations of pig liver esterase have determined that modulators of its catalytic reaction alter inhibition kinetics (56), and the results indicated that an aromatic or hydrophobic structure and a carbonyl group were required for optimal interaction with the effector site. Since the human monocyte CBE inhibitor, diphenyl carbonate, has these properties, our data are consistent with the possibility that DPC interacts with an effector site on the enzyme. On the basis of the inhibitor profile of human monocyte CBE, the enzyme is likely to be a serine esterase without metal or sulfhydryl requirements essential for its activity. Its inhibitor profiles are similar to human hepatic and intestinal esterases which are also inhibited by DFP, paraoxon, and phenylmethylsulfonyl fluoride, but not by EDTA (6, 44). Our data also suggest that there is more than one enzyme site with which inhibitors interact since diphenyl carbonate is a noncompetitive inhibitor whereas the organophosphorus compounds are competitive inhibitors.
We have postulated on the basis of its structure that DPC may interact with an effector site on the enzyme, and studies are underway to test this hypothesis.
We have attempted to sequence the purified human monocyte carboxylesterase directly from the N terminus but the data suggested that the N terminus was blocked. Nonetheless, the purification of this monocyte enzyme to homogeneity provides enzyme protein for sequencing by other methods and the subsequent synthesis of oligonucleotide probes for cloning the monocyte carboxylesterase.
Such information will permit a better understanding of the molecular basis of both the catalytic and effector sites of this enzyme, the structural variations of the human mutant enzymes (20, 57), and the biological functions of this enzyme.
T. Riley of the Howard