Purification and some properties of a membrane-bound dipeptidyl peptidase IV of guinea pig casein-induced intraperitoneal leukocytes

Dipeptidyl peptidase IV [EC 3.4.14.5] (DPP-IV) was demonstrated to exist in guinea pig casein-induced, peritoneal polymorphonuclear leukocytes (PMN) as well as contaminating macrophages and lymphocytes through the use of glycyl-L-prolyl-4-methylcoumaryl-7-amide (Gly-Pro-MCA) as a substrate. The DPP-IV of PMN was found to be located both on the plasma membrane and in the cytosol. For characterization, the plasma membrane-bound enzyme isolated from peritoneal cells mainly consisting of PMN was solubilized with 0.1% Triton N101 and purified by DEAE-cellulose chromatography, gel filtration, and affinity chromatographies on concanavalin A-Sepharose 4B and Gly-Pro-Sepharose 4B. The purification was more than 570-fold. The purified enzyme appears to be a noncovalent dimer of the 158K dalton DPP-IV. The pH optimum, isoelectric point and Km value for Gly-Pro-MCA were 7.2, 5.5, and 2.0 X 10(-4)M, respectively. The enzyme was inactivated by diisopropyl fluorophosphate and diprotin A. It was capable of cleaving dipeptidyl-MCA of an X-Pro-MCA type (X: free N-terminal amino acid). These properties are similar to those of DPP-IV purified from other tissues.     

Purification and characterization of rat urinary renin

Rat urinary renin was purified by a procedure involving ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B chromatography, ion exchange chromatography and gel filtration. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 39000 by SDS-gel electrophoresis and 40000 by gel filtration. The optimum pH determined with rat angiotensinogen was 7.0, and the Km was 3.6 microM. These properties agreed well with those of purified rat renal renin. The activity of urinary renin was specifically inhibited by anti-renin antibody. These results suggest that urinary renin may originate in the kidney.     

Dog renal kallikrein: purification and some properties.

The contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2,000-fold with an overall yield of 18% by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50. The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per A280, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8 X 10(4) from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3. The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein.     

Evaluating Khaya senegalensis for Dipeptidyl Peptidase-IV Inhibition Using in Vitro Analysis and Molecular Dynamic Simulation of Identified Bioactive Compounds

The dipeptidyl peptidase-IV (DPP-IV) inhibitory activity of Khaya senegalensis extracts was evaluated. The DPP-IV from a rat kidney was purified to a purification fold of 2.3. Among extracts from K. senegalensis, the hexane extract had the best DPP-IV inhibitory activity, with IC₅₀ value of 1.56±0.61 μg/mL and was fractionated to eleven fractions (A–K). Fraction I had the best DPP-IV inhibition via uncompetitive pattern. GC-MS analysis of fraction I showed that the major bioactive compounds were 3-amino-3-hydroxyimino-N-phenylpropanamide ( 1 ) and 11-(2-cyclopenten-1-yl)undecanoic acid ( 2 ), with good binding affinities toward DPP-IV, based on molecular docking,. They were then subjected to molecular dynamic simulation using WEBGRO and utilizing a GROMACS system for 100 ns. The 3-amino-3-hydroxyimino-N-phenylpropanamide-DPP-IV complex was more stable and compact than the other complex. K. senegalensis contains compounds like 1 that might be used for the design of new DPP-IV inhibitors.     

Purification and amino acid analysis of plasma acid stable trypsin inhibitor

Acid stable trypsin inhibitor (ASTI), with a molecular weight of about 85,000 by gel filtration, specific activity of 1,498 U/mg protein and pI of 1.6, from renal failure patient plasma was first purified. The amino acid composition of the purified ASTI was found to be that of a Gly- and Glu-rich protein which lacked His, closely resembling that of urinary trypsin inhibitor. The NH2-terminal amino acid sequence was Ala-Val-Leu-Pro-Gln-Glu- Glu-Glu-Gly-X-Gly-Gly-Gly-Gln-Leu-Val-Thr-Glu-Val-Thr-Lys-Lys-Glu- Asp-Ser-Ser-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro.     

Extraction of thromboxane B2 from urine using an immobilized antibody column for subsequent analysis by gas chromatography-mass spectrometry

This paper describes an antibody affinity (immunoaffinity) column which, in one step, extracts and sufficiently purifies urinary thromboxane B2 (TXB2) for quantitative analysis by high resolution gas chromatography-negative ion chemical ionization-selected ion monitoring-mass spectrometry (HRGC-NICI-SIM-MS). Polyclonal TXB2 antibody from rabbit was partially purified using immobilized Staphylococcus aureus Protein A. The purified IgG fraction was then immobilized using an N-hydroxysuccimidyl silica gel. The resulting matrix bound 570 ng TXB2 per ml of gel. TXB2 was quantitatively eluted with acetonitrile-water (19:1). Columns constructed from the gel could be used repeatedly since binding capacity was reconstituted using 0.01 M phosphate buffer (pH 7.4) with no apparent loss of activity. Using these columns, urinary TXB2 was sufficiently purified in one step such that in subsequent analysis by HRGC-NICI-SIM-MS interference free chromatograms were observed.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

Synthesis, biological evaluation and structural determination of beta-aminoacyl-containing cyclic hydrazine derivatives as dipeptidyl peptidase IV (DPP-IV) inhibitors

Inhibitors of dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes. A series of beta-aminoacyl-containing cyclic hydrazine derivatives were synthesized and evaluated as DPP-IV inhibitors. One member of this series, (R)-3-amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (10f), showed potent in vitro activity, good selectivity and in vivo efficacy in mouse models. Also, the binding mode of compound 10f was determined by X-ray crystallography.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Studies on rat renal cortical cell kallikrein. I. Separation and measurement.

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone.     

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.     

Studies on urinary kallikreins. I. Purification and characterization of human urinary kallikreins.

Human urinary kallikrein [EC 3.4.21.8] (HUK) was purified about 200-fold with an overall yield of 40 percent from crude powder by DEAE-cellulose chromatography, acetone fractionation, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 chromatography. Its activity was 200 kallikrein units (KU) per A280. HUK from active fractions obtained by DEAE-Sephadex A-50 chromatography was separated into three active components showing isoelectric points of 3.9 (HUK-1), 4.0 (HUK-2), and 4.2 (HUK-3) by isoelectric focusing: each HUK component was homogeneous on disc electrophoresis. The approximate molecular weights of HUK-1, -2 and -3 were estimated to be 2.7 X 10(4), 2.7 X 10(4), and 2.9 X 10(4), respectively, by gel filtration on a Sephadex G-100 column. The optimum pH's of HUK-1, -2, and -3 in esterolytic action were found to be 8.0, 8.3, and 7.5, respectively, and they were fairly heat stable in comparison with other glandular kallikreins. The three components of HUK were weakly inhibited by Trasylol, but were not affected by soybean and ovomucoid trypsin inhibitors. They were strongly resistant to treatment with urea and weakly resistant to treatment with guanidine. The activation energies of HUK-1, -2, and -3 were found to by 1.17 X 10(4), 5.1 X 10(3), and 1.45 X 10(4) cal per mole, respectively. The Km values were estimated toward N-alpha-tosyl-L-arginine methyl ester (TAME), N-alpha-benozyl-L-arginine ethyl ester (BAEE), and N-alpha-benozyl-L-arginine methyl ester (BAME).     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.     

Purification and characterization of human microsomal dipeptidase

Human microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase) [EC 3.4.13.11] was solubilized from the membrane fraction of kidney by treatment with octyl-beta-D-glucoside and purified by a procedure including ion exchange chromatography and affinity chromatography on cilastatin-immobilized Sepharose. The purified human MDP was found to be homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight (Mr) was estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions to be 130 kDa, comprising a homodimer of two subunits. After treatment with endoglycosidase F, human MDP showed a single band with an apparent Mr of 42 kDa on SDS-polyacrylamide gel electrophoresis. Human MDP was found to bind to Con A-Sepharose and the activity was eluted with methyl-alpha-D-mannopyranoside, suggesting that human MDP is a glycoprotein. We also examined the substrate specificity of human MDP and found that human MDP catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and S-N-ethylmaleimide-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to leukotriene E4. These results suggest that MDP might play an important role in the metabolism of glutathione and leukotriene.     

Structure based virtual screening of MDPI database: Discovery of structurally diverse and novel DPP-IV inhibitors

Inhibition of dipeptidyl peptidase IV (DPP-IV) has been emerged as a promising approach for the treatment of type 2 diabetes (T2D). Structure based virtual screening (SBVS) of Molecular Diversity Preservation International (MDPI) database was performed using Glide and Gold against DPP-IV enzyme. Six promising hits were identified and tested for DPP-IV inhibition. Three compounds were found to be active at low micromolar concentration. The 3-(1-hydrazinyl-1-(phenylamino)ethyl)-4-hydroxy-1-methylquinolin-2(1H)-one (compound A) was found to be the most potent hit with an IC₅₀ of 0.73 μM. These three compounds (A, B and D) were then assessed for their glucose lowering effects in glucose fed hyperglycemic female Wistar rats. The glucose lowering effects of compounds also confirms their potential as anti-diabetic agents. The present study demonstrates a successful utilization of in silico SBVS tools in identification of novel and potential DPP-IV inhibitor.     

Interaction between prostaglandin E2 and leukotriene D4 on the excretion of electrolytes by the dog kidney in vivo

Recent experiments indicate that prostaglandin E2 potentiates the vasodilatory properties of leukotrienes in the skin microcirculation. The present experiments were undertaken to study the effect of leukotriene D4 and prostaglandin E2 on renal hemodynamics and urinary electrolytes in the dog. Experiments were performed in three groups of anesthetized Mongrel dogs: the first group was studied under hydropenia, whereas the two remaining groups were studied during water diuresis with (Group 3) or without indomethacin (Group 2). LTD4 (100 ng/min) and PGE2 (3 ug/min) were infused in the left renal artery to minimize systemic effects of these compounds. LTD4 alone failed to influence urinary sodium excretion in all 3 groups. In Group 1, urinary sodium increased from 77 +/- 6 to 393 +/- 74 uEq/min during PGE2, and further increased to 511 +/- 52 uEq/min during LTD4 + PGE2. No change occurred in the contralateral right kidney. In this group, glomerular filtration as well as renal plasma flow were not statistically influenced. In Group 2, the same phenomenon was observed for urinary sodium. The combined infusion of LTD4 + PGE2 increased urinary sodium without significant changes in glomerular filtration and renal plasma flow. Finally, in Group 3, indomethacin was shown to reduce the natriuretic effects of LTD4 and PGE2: during PGE2 alone, urinary sodium increased from 90 +/- 14 to 260 +/- 66 uEq/min, and only rose from 80 +/- 10 to 175 +/- 19 uEq/min during the combined infusion of LTD4 and PGE2. In groups 2 and 3, free water clearance was utilized as an index of sodium chloride reabsorption in the thick ascending limb: this parameter increased from 2.35 +/- 0.25 to 4.70 +/- 0.30 ml/min, while urinary volume was increasing from 3.55 +/- 0.25 to 10.05 +/- 0.65 ml/min, during LTD4 + PGE2. Indomethacin, administered in Group 3, (3 mg/kg/hr) again abolished the effect of combined PGE2 + LTD4. These results indicate a potentiating effect of leukotriene D4 on the PGE2-induced natriuresis in the anesthetized dog. These phenomena occurred in the absence of significant changes in renal hemodynamics, therefore suggesting a direct tubular effect of these arachidonic acid metabolites. Finally, the water diuresis experiments suggest a proximal site of action of PGE2 and LTD4.     

Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.     

Inhibition of dipeptidyl-peptidase IV does not increase circulating IGF-1 concentrations in growing pigs

The enzyme dipeptidyl peptidase-IV (DPP-IV) inactivates a variety of bioactive peptides, including glucagon-like peptide-1 (GLP-1) and growth hormone releasing hormone (GHRH). Inhibiting DPP-IV in order to increase circulating GLP-1 is of interest as a treatment for Type II diabetes. Inactivation of DPP-IV may also increase circulating GHRH, potentially enhancing growth in domestic animals. To test the hypothesis that inhibition of DPP-IV activity will influence the growth hormone/ IGF-1 axis, growing pigs (Sus scrofa domesticus, 78 kg) were treated with a DPP-IV inhibitor (Compound 1, the 2,5-difluor-ophenyl analog of the triazolopiperazine MK0431, sitagliptin), and plasma concentrations of IGF-1 were monitored. Pigs were administered either sterile saline (0.11 ml/kg followed by a continuous infusion at 2 ml/hr for 72 hrs, controls, n = 2), Compound 1 (2.78 mg/kg followed by a continuous infusion at 0.327 mg/kg x hr for 72 hrs, n = 4) or GHRH (0.11 ml/kg sterile saline, followed by a continuous infusion of GHRH at 2.5 microg/ kg x hr for 48 hrs, n = 4). Plasma concentrations of Compound 1 were maintained at 1 microM, which resulted in a 90% inhibition of circulating DPP-IV activity. Relative to the predose 24-hr period, area under the IGF-1 concentration curve (AUC) tended to be lower (P = 0.062) with Compound 1 (.79 +/- 130 ng/ml x hr) than controls (543 +/- 330 ng/ml x hr). GHRH treatment increased the IGF-1 AUC (1210 +/- 160 ng/ml x hr, P = 0.049 vs. controls and P = 0.001 vs. Compound 1). We conclude that inhibition of DPP-IV does not alter the circulating levels of IGF-1 in the growing pig.     

Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells

Glucagon-like peptide-1 (GLP-1), a meal-stimulated gastrointestinal insulinotropic hormone inactivated by dipeptidyl peptidase-IV (DPP-IV), is reduced in type 2 diabetic patients. The present study shows that 2-week exposure of human glomerular endothelial cells to high glucose (22 mM) determines a highly significant increase in DPP-IV activity and mRNA expression, which cannot be entirely accounted for by hyperosmolarity. On the other hand, incubation of purified DPP-IV in a buffer solution added with high glucose does not affect enzyme activity. These results suggest that high glucose increases expression and activity of DPP-IV, possibly contributing to GLP-1 reduction in type 2 diabetic patients.     

Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium

An H2O2-requiring oxygenase found in the extracellular medium of ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium was purified by DEAE-Sepharose ion-exchange chromatography and gel filtration on Sephadex G-100. Sodium dodecyl sulfate (SDS)-disc gel electrophoresis indicated that the purified protein was homogeneous. The Mr of the enzyme as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 41,000. The absorption spectrum of the enzyme indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme (407 nm) shifted to 435 nm in the reduced enzyme and to 420 nm in the reduced-CO complex. The pyridine hemochrome absorption spectrum indicated that the enzyme contained one molecule of heme as iron protoporphyrin IX. Both CN- and N-3 bound readily to the native enzyme, indicating an available coordination site and that the heme iron was high spin. The purified enzyme generated ethylene from 2-keto-4-thiomethyl butyric acid, and oxidized a variety of lignin model compounds, including the diarylpropane, 1-(3'4'-diethoxyphenyl)1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I); a beta-ether dimer, 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (V); an olefin, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2 propene (III); and a diol, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2-propane diol (IV). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures.