Distribution of peptidases in cultured cells from middle ear mucosa: prolyl endopeptidase is localized in fibroblasts and dipeptidyl peptidase IV and II in epithelial cells.

To examine the distribution of prolyl endopeptidase (PEP), dipeptidyl peptidase IV (DPP IV), and dipeptidyl peptidase II (DPP II) in specific cell types, fibroblasts and epithelial cells were selectively cultured from middle ear mucosal tissues of guinea pigs. In fibroblasts, PEP had the highest activity, 12.28 +/- 4.00 nmole/min/mg protein (mean +/- SD), 45-fold higher than corresponding DPP II levels. In epithelial cells, DPP IV activity was the highest, 6.48 +/- 0.90 nmole/min/mg protein. This communication describes, for the first time, the distribution of the enzyme activities of PEP, DPP IV, and DPP II in fibroblasts and epithelial cells, and the occurrence of PEP in fibroblasts.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Purification and characterization of glyoxalase II from Hansenula mrakii

Glyoxalase II [S-(2-hydroxyacyl)glutathione hydrolase], one of the components of the glyoxalase system, catalyzes the hydrolysis of S-lactoylglutathione to glutathione and d-lactic acid. The enzyme was partially purified from the yeast Hansenula mrakii IFO 0895 by successive column chromatographies and polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 22,000 daltons by gel-filtration of Sephadex G-150 column chromatography and 24,000 daltons by SDS-polyacrylamide gel electrophoresis. The enzyme was specific to S-lactoyglutathione and S-acetylglutathione. The activity of the enzyme was strongly inhibited by Cu²⁺, p-chloromercuribenzoate and HgCl₂. The enzyme activity was also inhibited by hemimercaptal, a non-enzymatic condensation product between glutathione and methylglyoxal.     

The Serine Hydroxymethyltransferase of Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH₄)₂SO₄ fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0–3.3 m (NH₄)₂SO₄, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8–7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The K_m was 4.3 × 10^?3m for l -serine and 2.5 × 10^?4m for tetrahydrofolate.     

Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel–nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS–PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH₂-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues −180 to −175, NH₂-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH₂-terminal sequence, LDXNQLY, corresponding to residues −73 to −67 of the proregion peptide and thus were generated by removal of 126 residues from the NH₂-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37°C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k_cat/K_m) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM⁻1 s⁻¹, respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing 2 × 10⁹ High Five cells.     

Dehydrocyclopeptine epoxidase from Penicillium cyclopium

Dehydrocyclopeptine epoxidase (DE) activity was determined in cell free preparations of Penicillium cyclopium. The enzyme transforms dehydrocyclopeptine into cyclopenin by a mixed function oxygenation. It needs molecular oxygen and uses NAD(P)H, ascorbate or d,l-6-methyl-5,6,7,8-tetrahydropteridine as cosubstrates. DE is inhibited by CN^?, SCN^?, 1,10-phenanthroline, EDTA, 2,2′-bipyridine, sodium diethyldithiocarbamate, dicoumarol, p-chloromercuribenzoate and ions of different heavy metals, but not by CO and the lead salt of diethyldithiocarbamate. These properties indicate a specific importance of Fe²⁺-ions, SH-groups and flavins. DE activity is increased by Fe²⁺ and FAD. The enzyme may be therefore a Fe²⁺ activated FAD containing flavoprotein. DE was enriched 268-fold by (NH₄)₂SO₄ precipitation and chromatography on Sephadex G-200. Its MW estimated by Sephadex chromatography, exceeds 480 000.     

Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4–5 hours at 37°C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.     

Purification and characterization of cathepsin B from goat brain

Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH₄)₂SO₄ fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withK _i value of 12.5 × l0⁻⁹M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.     

Mannitol Biosynthesis in Pyrenochaeta terrestris I. D-Maunitol-1-Phosphate: NAD Oxidoreductase

Cell-free extracts of mycelial mats of Pgrenochaeta terrestris grown in stationary culture on synthetic glucose or sucrose - salts liquid media contained D-mannitol-1-Phosphate:NAD oxidoreductase (EC 1.1.1.17) activity. Greatest activity occurred early in the growth period. The optimum pH for the reduction of NAD⁺ in the presence of Fru-6-P was 7.4–7.5 while the optimum pH for the oxidation of NADH in the presence of Mtl-1-P was 8.1–8.2. The enzyme was stabilized to some extent in Tris-maleate buffer, pH 7.5, and by the addition of 10% (NH₄)₂SO₄, to this buffer. A 10- to 16-fold purification was attained by a combination of (NH₄)₂SO₄ fractionation and gel filtration on Sephadex G-100. The enzyme was relatively specific in its substrate and coenzyme requirements. The Km values were determined as: Fru-6-P - 3 × 10^?4 M, Mtl-1-P - 1 × 10^?4 M, and NAD⁺ and NADH - 3 × 10^?5 M.     

Alkaline phosphatase from pig kidney. Method of purification and molecular properties

Alkaline phosphatase (EC 3.1.3.1) from pig kidney brush-border membranes was solubilized from membrane precipitates by butan-1-ol at a critical pH of 7.0. The 12000-fold purification procedure included (NH₄)₂SO₄ precipitation, DEAE-and TEAE-cellulose chromatography, Sephadex G-200 gel filtration and neuraminidase digestion followed by DEAE-cellulose chromatography. The purified protein contained 20% (w/w) carbohydrate and had mol.wt. 150000–156000 as estimated by Sephadex filtration and ultracentrifuge analysis. It was a tetrameric glycoprotein consisting of identical subunits, and it had a molecular activity at 25°C of 2600s⁻¹ per tetramer. Its concentration in kidney was estimated to be 8.5–8.8mg/kg.     

Purification and properties of mannitol dehydrogenase from Agaricus bisporus sporocarps

Mannitol dehydrogenase (mannitol: NADP⁺ 2-oxidoreductase: EC 1.1.1.138) was isolated from Agaricus bisporus by fractionation with protamine sulphate and (NH₄)₂SO₄, followed by chromatography on DEAE-Sephadex, then by affinity and gel chromatography. The products of enzyme reaction were identified by GLC and TLC. K_m, optimum pH, MW and pI of the enzyme as well as the influence of temperature, ions and inhibitors on enzymic activity were determined. In the sugar reducing reaction, the enzyme was specific for fructose but, in the reverse direction, some structurally related polyols could substitute for mannitol. The enzyme was very sensitive to alterations in the NADP⁺/NADPH ratio. The results are discussed in relation to the possible role of mannitol dehydrogenase in fungal metabolism.     

Soluble dipeptidyl peptidase IV from terminal differentiated rat epidermal cells: purification and its activity on synthetic and natural peptides

In terminally differentiated epidermal cells dipeptidyl peptidase IV (EC 3.4.14.5) (DPP IV) is present mainly in a soluble form. We purified the enzyme from 2-day-old rat cornified cells to homogeneity by Sephadex G-200 and Mono-Q column chromatography and finally HPLC gel filtration on G3000SW. The enzyme was estimated to be Mr 190,000 by HPLC gel filtration and Mr 90,000 by sodium dodecyl sulfate-electrophoresis. The enzyme showed general properties reported for detergent-solubilized DPP IV from other tissues. It was Con A binding and almost completely inhibited by 1 mM diisopropyl fluorophosphate and Diprotin A. The pI was 5.6 and the pH optimum was 7.5. The specific activity for Gly-Pro-p-nitroanilide was 31.9 units/mg. HPLC analysis demonstrated the release of dipeptides of the N-terminal of substance P, beta-casomorphin, and their related peptides. A stoichiometric reaction of the enzyme on substance P was observed. The epidermal DPP IV had a Km of 0.3 mM and a kcat of 50.3 s-1 for substance P and the Km value decreased by shortening the peptide from the carboxyl-terminal amino acids. The enzyme hydrolyzed human and bovine beta-casomorphin with Km values of 0.025 and 0.05 mM, respectively. Shortening the bovine beta-casomorphin peptide chain did not affect enzyme affinity.     

Histochemistry of some proteases in the normal rabbit,pig and ox corneas

Summary The distribution of activities of membrane aminopeptides (aminopeptidases M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), -gluamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4°C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4°C) for the demonstration of lysosomal enzymes.In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity.Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Coconut α-d-galactosidase isoenzymes: isolation,purification and characterization

α-d-Galactosidases (α-d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α-d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α-d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K_m and V_max of α-d-galactosidase A were obtained at 3.46 × 10^?4M and 1.38 × 10^?3 M p-nitrophenyl α-<d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α-d-galactosidase was localized in the 40–70 % (NH₄)₂SO₄ cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K_m was obtained at 6.75 × 10^?4 M p-nitrophenyl α-d-galactoside while the V_max was noted at 5.28 × 10^?3 M p-nitrophenyl α-d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α-d-galactosidase activity in makapuno is discussed.     

Study on dipeptidylpeptidase II (DPP II)

Summary The activity of dipeptidylpeptidase II (DPP II; E.C. 3.4.14.2) was investigated by biochemical and histochemical methods in rat, mouse and guinea-pig organs as well as in human enterobiopsies. Lys-Pro-MNA and Ala-Pro-MNA showed the most favorable kinetic properties (K _m , V _max) and proved to be the most sensitive substrates for biochemical and histochemical studies of DPP II. Lys-Ala-MNA is more specific and is to be preferred due to its relatively low hydrolysis by DPP IV. Lys-Ala-2NA is suitable for the biochemical determination of DPP II activity. Lys-Ala-1NA, Leu-Ala-2NA, Phe-Pro-2NA and Phe-Pro-MNA are inferior. The pH optimum of DPP II amounts to 5.5. Cacodylate, phosphate, citric acid phosphate and succinate buffers deliver similar hydrolysis rates; with citrate and acetate buffers the recorded activities are lower. The reaction can be inhibited by 1 mM DFP, 50 mM Tris and 10 mM puromycin. In the ileum of suckling rats and in human enterobiopsies similar data (K _m , pH optimum, optimal substrate concentration) were obtained by biochemical determination and by quantitative histochemistry (microdensitometry) with Lys-Ala-MNA. For the histochemical demonstration of DPP II freeze-dried celloidin-coated cryostat sections are very suitable. Frozen sections of formaldehyde and glutaraldehyde fixed tissue blocks are inferior due to a higher inhibition of DPP II and less precise localization of the azo-dye. K _m values and optimal pH are identical in fresh and fixed material. Fast Blue B is the best coupling agent for light microscopical localization. DPP II is present in all organs and tissues investigated. Conspicious organ and species differences exist. In adult rats the highest DPP II activity resides in the kidney, epididymis and spleen; in guinea-pigs the epididymis and testis are the most active organs. In the majority of guinea-pig organs the DPP II activity is lower than in rats. The histochemical demonstration of DPP II shows, in addition, cell-dependent differences of DPP II activity. In most cells the enzyme activity is depicted in lysosomes. Highly active are lysosomes of cells of proximal renal tubules, macrophages, thyroid cells, clear and principal cells of the epididymis of adult animals and of enterocytes of suckling rats. Lysosomes of endocrine cells of adenohypophysis, pancreaas, stomach, small intestine and nerve cells display moderate activity. In lysosomes of smooth muscle cells (intestine, myometrium), myocardial cells, and fibers of striated muscle the enzyme is also present. Spermatids and sperms of guinea-pigs are highly active. In some cases secretion granules of endocrine and exocrine gland cells display a positive reaction. Possibly the Golgi apparatus and the endoplasmic reticulum also show a positive staining in the principle cells of the rat and mouse epididymis. Furthermore, DPP II seems to be secreted into the lumen of several organs.Supported by Deutsche Forschungsgemeinschaft (SFB 105)     

A role of cathepsin B1 in polymorphonuclear leukocytes chemotaxis.

Cathepsin B_I¹ was purified from rat liver lysosomal fraction by ammonium sulfate fractionation, followed by chromatography on Sephadex G-200 and DEAE-Sephadex. Formation of chemotactic factor for guinea pig polymorphonuclear (PMN) leukocytes was demonstrated $\text{in vitro}$ when guinea pig serum was incubated with cathepsin B_I. This factor formation was dependent on SH-reagent dithiothreitol (DTT) and was maximal at pH 6.0. ZnSO₄, an inhibitor of cathepsin B_I, inhibited the chemotactic factor formation likewise.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Isolation and biochemical characterization of grape malic enzyme

Malic enzyme (ME=L-malate: NADP oxidoreductase; E.C. 1.1.1.40) was extracted by Triton X-100-induced resolubilization of enzyme proteins which denaturize spontaneously upon homogenization of grape berry material. The purification procedure included fractionating with (NH₄)₂SO₄, preparative IEF, and Sephadex G-100 chromatography. ME was identified by TLC of the radioactive product after supplementing the assay mixture with [¹⁴C]malate. Cofactor dependence, pH-optimum and affinities for substrates and cosubstrates were determined. Enzymic pI was found to be 5.8, the Hill coefficients range from 1 to 3. In malate decarboxylating direction at pH 7.4, grape ME displayed positive cooperativity toward the substrate, the curve approaching normal Michaelis-Menten-kinetics at pH 7.0. Substituting Mn²⁺ for Mg²⁺ not only increased maximal turnover rates, but also enzymic affinity for malate. These features were considered indicative of the regulatory properties of the enzyme. Their relevance for grape malate metabolism and fruit ripening is discussed.Abbreviations EDTA ethylenediaminetetraacetic acid - IFF isoelectric focusing - MDH malate dehydrogenase - ME malic enzyme - OAA oxaloacetic acid - PAG polyacrylamide gel - TCA trichloroacetic acid - TLC thin layer chromatography     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Purification and metal requirements of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH₄)₂SO₄ fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement.