Hyaluronidase isoforms from developing embryos of the camel tick Hyalomma dromedarii

Changes in hyaluronidase activity in the camel tick Hyalomma dromedarii were followed throughout embryogenesis. Peak activity of the enzyme on days 21 and 24 during development was accompanied with a complete organization of larvae before hatching on day 27. During purification of hyaluronidase to homogeneity, ion exchange chromatography lead to four forms (HAase1, 2, 3 and 4). HAase2 and HAase4 with highest purity and specific activities after chromatography on Sephacryl S-200. The apparent molecular masses of HAase2 and HAase4 were 25 and 40 kDa, respectively. HAase2 and HAase4 had the same pH optimum of 3.6 and Km values of 0.3 and 0.34 mg/mL hyaluronic acid, respectively. Cleaving activities of HAase2 and HAase4 were demonstrated in the order: hyaluronic acid>chondroitin sulphate A>chondroitin sulphate C>chondroitin sulphate mixed>chondroitin sulphate B>heparin, low M.Wt>heparin. HAase2 and HAase4 had the same temperature optimum (40 degrees C) with heat stability up to 40 degrees C. H. dromedarii HAase2 and HAase4 had broad plateau of NaCl requirement with optimum activity recorded at 0.15 and 0.3 M NaCl, respectively. HAase2 and HAase4 were inhibited by Ca2+, Fe3+, Co2+ and Hg2+ and enhanced by Mg2+ and Mn2+.     

alpha-Amylase from developing embryos of the camel tick Hyalomma dromedarii

alpha-Amylase activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of alpha-amylase III to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). alpha-Amylase III with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of alpha-amylase III was 106 kDa for the native enzyme, composed of two subunits of 43 and 66 kDa, respectively. alpha-Amylase had a value of 10 mg starch/ml. Varying alpha-amylase activity was detected when supplied with various substrates. alpha-Amylase III had a temperature optimum at 40 degrees C with heat stability up to 50 degrees C, and a pH optimum of 7.0. The enzyme activity was activated by CaCl2, MgCl2 and NaNO3, but not activated by NaCl, p-CMB, N-ethylmaleimide and iodoacetamide. EDTA and beta-mercaptoethanol strongly inhibited activity.     

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.     

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed tributyrin hydrolysis. Effect of trypsin treatment

Hepatic triacylglycerol lipase (EC 3.1.1.3) hydrolyzes water-insoluble fatty acid esters, e.g., trioleoylglycerol (lipase activity) and water-soluble fatty acid esters, e.g., tributyrin (esterase activity). Esterase activity of hepatic triacylglycerol lipase is enhanced by triolein emulsion and phospholipid vesicles [1]. The catalytic mechanism and structure of human hepatic triacylglycerol lipase isolated from human post-heparin plasma and the effect of trypsin treatment on the lipase and esterase activities of the enzyme were examined. Treatment of hepatic triacylglycerol lipase with trypsin resulted in loss of its lipase activity, but had no effect on its esterase activity. Chromatography of hepatic triacylglycerol lipase on Bio-Gel A5m showed that hepatic triacylglycerol lipase binds to dipalmitoylphosphatidylcholine vesicles. However, on chromatography of the trypsin-treated enzyme after incubation with dipalmitoylphosphatidylcholine vesicles, a part of hepatic triacylglycerol lipase that retained esterase activity was eluted separately from the dipalmitoylphosphatidylcholine vesicles. Addition of vesicles of dipalmitoylphosphatidylcholine to the trypsin-treated enzyme did not enhance its esterase activity. These results are consistent with the hypothesis that hepatic triacylglycerol lipase has a catalytic site that hydrolyzes tributyrin and a lipid interface recognition site, and that these sites are different: trypsin modified the lipid interface recognition site of the hepatic triacylglycerol lipase but not the catalytic site.     

A new esterase, belonging to hormone-sensitive lipase family, cloned from Rheinheimera sp. isolated from industrial effluent

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to class IV family. The purified enzyme worked optimally at 50 degrees C and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate (C?), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.     

Lipase and esterase formation by psychrophilic and mesophilic Acinetobacter species.

Acinetobacter O16, a psychrophilic species, produced extracellular lipase (measured by hydrolysis of olive oil, tributyrin, or beta-naphthyl laurate) when grown on a complex medium (peptone plus yeast extract). Most lipase was produced during the logarithmic phase of growth. Very little cell-bound lipase was formed. These cells also produced an esterase (measured by the hydrolysis of beta-naphthyl acetate). At first, all esterase was cell bound; significant amounts appeared in the external medium late in growth. Breaking the cells did not increase cell-bound lipase activity. After breaking of the cells, most of the cell-bound lipase and esterase activity was solubilized, even after very high speed centrifugation. No appreciable amounts of these enzymes were released by osmotic shock. Lipase formation was greatly affected by nutrient conditions. Lowering either the yeast extract of the peptone content of the normal complex medium lowered or abolished lipase formation. Esterase activity was lowered to a lesser extent. Cells growing in synthetic amino acid plus vitamin medium or in acid-hydrolyzed casein produced substantial amounts of esterase but no cell-free or cell-bound lipase. However, if sodium taurocholate was added to these media, lipase was produced. Greatest production occurred if a mixture of di- and poly-peptides was also present. Taurocholate also stimulated lipase production in the normal complex medium. Adding Tween 80 or ethanol to the normal complex medium inhibited lipase production. Sodium acetate, oleic acid, olive oil, or Tween 20 added to synthetic media did not affect lipase production. The psychrophile grew more quickly at 30 degrees C than at 15 or 20 degrees C but produced more lipase at the lower temperatures. Esterase production was about the same at 20 and 30 degrees C. A mesophilic Acinetobacter species produced the same amount of lipase and esterase at 20 and 30 degrees C. The best production of lipase by the psychrophile occurred in standing cultures.     

Hydrolysis of dolichyl esters by oviduct membranes and characterization of endogenous dolichyl esters

The chick oviduct system has been employed to study whether dolichol esters might serve as a storage form of dolichol to be converted to dolichyl phosphate (Dol-P) during periods when Dol-P levels increase. Chicken oviduct membranes catalyze the hydrolysis of dolichyl-[14C]oleate; the reaction is dependent on detergent (0.04% NP-40 is optimal), is unaffected by divalent cations and EDTA, and exhibits a pH optimum of 6.0. Oviduct membranes also hydrolyze cholesteryl-[14C]oleate, which exhibits similar properties except the pH optimum is 5.0-5.5. Neither Dol-[14C]palmitate nor Chol-[14C]palmitate is hydrolyzed by membranes. Chol-ester hydrolysis is more sensitive to heat-denaturation than is Dol-ester hydrolysis. Esterase activity was assayed in membranes prepared from immature chicks, chicks treated with diethylstilbestrol, chicks withdrawn from diethylstilbestrol, and mature hens. The highest esterase specific activity was observed in membranes obtained from chicks withdrawn from hormone. In order to characterize the fatty acid composition of Dol-esters they were purified from mature hen oviducts by chromatography on DEAE-cellulose and Fractogel ORPVA-6000, reverse-phase HPLC, and TLC. About 15-25% of oviduct dolichol is in the esterified form. Fatty acid analysis revealed that approximately 85% of the dolichol was esterified to oleic acid. The fact that the highest esterase activity is found in membranes from chicks withdrawn from hormone and that only 20% of the dolichol is esterified argues against a role for Dol-esters as a reservoir of dolichol for conversion to Dol-P.     

Purification and characterization of a non-kallikrein arginine esterase from dog urine

A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.     

Biochemical characterisation of esterases active in hydrolysing xenobiotics in wheat and competing weeds

Esterase activities toward model xenobiotic substrates ( p -nitrophenyl acetate, naphthyl acetate) and pesticide esters (diclofop methyl, bromoxynil octanoate, binapacryl) have been compared in crude extracts from wheat (Triticum aestivum L.) and Triticum progenitors of wheat. Esterase activities were also determined in the weeds, wild oat ( Avena fatua ) and two populations of black-grass ( Alopecurus myosuroides ), one of which (Rothamsted) is susceptible to herbicides, while the other (Peldon) shows cross-resistance to multiple classes of herbicides. Esterase activity toward the model substrates was highest in wheat, while the weeds were more active in hydrolysing the pesticides. Using isoelectric focussing (pH 4–8), 13 proteins with esterase activity toward α -naphthyl acetate could be resolved in hexaploid wheat (genome AABBDD). The pattern of these activities was most similar to that of the diploid progenitor T. tauschii (DD), excepting a major acidic esterase (pI 4.6), which originated from T. urartu (AA). Resolved esterase activities in the weeds were distinct from those observed in the Tritcum species. However, unlike the case with other classes of xenobiotic-metabolising enzymes, the complement of esterases in the Peldon and Rothamsted populations of black-grass appeared to be identical. In all species, the more basic esterases (>pI 5.0) were sensitive to inhibition by organophosphate and carbamate insecticides, suggesting that they were B-class esterases. In contrast, the acidic wheat esterase (pI 4.6) with the greatest activity toward α -naphthyl acetate was insensitive to insecticides. This wheat-specific esterase was purified 7000-fold by a combination of hydrophobic interaction chromatography, gel filtration and anion-exchange chromatography. The purified esterase behaved as a monomeric 45-kDa protein showing high activity toward p -nitrophenyl acetate and α -naphthyl acetate, but limited activity toward the pesticides.     

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.     

Purification and properties of aminopeptidase H from chicken skeletal muscle.

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.     

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.     

厌氧真菌菌系乙酰酯酶的特性研究

利用厌氧培养技术,采用产酶培养基,培养课题组自行构建的一组厌氧真菌菌系,使之产乙酰酯酶。采用硫酸铵分级沉淀、透析袋透析、DEAE-纤维素离子交换柱层析、Sephadex G-75凝胶过滤柱层析,分离纯化所得到乙酰酯酶,研究其酶学性质。酶活力动态分析表明,乙酰酯酶在产酶培养基上,培养至第3天酶活力达到最高。乙酰酯酶最适温度为41℃,最适pH为9．0,Mg^2＋、K^＋、Ca^2＋对酶有一定的激活作用,Fe^3＋对酶有很强的抑制作用。该厌氧真菌菌系所产的乙酰酯酶,对于发酵木质纤维素类物质具有潜在应用价值。     

The extracellular metalloprotease ofSerratia marcescens: I. Purification and characterization

Summary An extracellular protease ofSerratia marcescens produced during growth on skim milk medium was isolated by ethanol precipitation. The protease was purified by salt fractionation, DEAE-cellulose ion exchange chromatography and gel filtration chromatography on Agarose P-100. It has a broad optimum from pH 6.0 to 9.0 and a temperature optimum of 45°C for proteolytic activity on casein. It was classified as a metallo-protease by virtue of its inactivation by metal-ion chelators and reactivation by ferrous ions. Proteolytic activity was not affected by diiso-propylfluorophosphate, p-chloromercuribenzoate and dithiothreitol.     

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.     

High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5

An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.     

Properties of the lipid body lipase of Pinus eduUs and electrophoretic purification of its 64 kDa subunit

Lipase (triacyiglycerol acylhydrolase, EC 3.1.1.3) in oilseeds can be associated with either the lipid body or glyoxysomal membrane and can have various pH optima and substrate specificities. There is conflicting evidence for the subcellular location of lipase in gymnosperms, and little information exists on its activity characteristics. In this report, Pinus edulis (pinyon) was found to have an acid lipase, which was associated with the lipid body fraction and the activity of which increased during germination. Active lipase from the solubilized lipid body membrane was determined by gel permeation chromatography to have a molecular weight of 260 000. Further attempts to purify the active enzyme were unsuccessful. A lipid body membrane protein of 64 kDa which increased in parallel with lipase activity during germination was isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. excised, and polyclonal antibodies were made against it. Using these antibodies, active lipase was immunoprecipitated from solution, thus indicating that the 64 kDa protein is a subunit of the lipase. Pinus edulis lipase had a pH optimum of ca 4.5. and it exhibited little specificity for triacyiglycerol substrates in vitro. The lipase was specific in activity against fluorometric substrates, with the highest activity against methyl-umbelliferyl laurale. Lipase activity was inhibited by high concentrations of non-ionic detergent. This lipid body acid lipase appears to be primarily responsible for lipid hydrolysis during pinyon germination.     

Purification and characterization of a new arginine carboxypeptidase in human serum

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.     

Purification and characterization of the extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius

The extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS-PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0-7.0. Under the conditions tested, the activity is maximal between 45 and 50 degrees C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.