Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis.

Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.     

Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein

A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.     

A simple rapid purification scheme for hydroxymethylbilane synthase from human erythrocytes.

Hydroxymethylbilane synthase from human erythrocytes was purified 47,000-fold to greater than 95% homogeneity and 7.5% yield by a simple and rapid procedure using heat treatment (80 degrees C, in the presence of proteinase inhibitors, to convert one of two chromatographically separable forms into the other), DEAE-cellulose and Cibacron Blue F3G-A-Sepharose chromatographies and Sephadex G-75 gel filtration. The purified enzyme was similar to the enzyme purified from other species in showing hyperbolic dependence of velocity on substrate concentration, a non-linear progress curve for uroporphyrinogen appearance, and was monomeric, having an Mr of 44,000 by gel filtration on Sephadex G-100 and h.p.l.c. and an Mr of 45,000 on SDS/polyacrylamide-gel electrophoresis. The enzyme showed a sharp pH profile for Vmax, and various folates were shown to accelerate neither the enzymic formation of hydroxymethylbilane nor ring-closure of hydroxymethylbilane.     

Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes

Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.     

Rat liver uroporphyrinogen III synthase has similar properties to the enzyme from Euglena gracilis, including absence of a requirement for a reversibly bound cofactor for activity.

Uroporphyrinogen III synthase purified from rat liver is a monomer of Mr 36,000 by gel filtration and 28,000 by SDS/polyacrylamide-gel electrophoresis. The enzyme exists in two interconvertible forms separable on h.p.l.c. Both forms of the enzyme could be renatured with full activity after SDS/polyacrylamide-gel electrophoresis, demonstrating the absence of a reversibly bound cofactor. The enzyme activity could be inhibited by pyridoxal 5'-phosphate in the absence and in the presence of NaBH4, consistent with (an) essential lysine residue(s). The enzyme thus shows great similarity to that from Euglena gracilis.     

Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III.

Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.     

Purification and characterization of the beta-adrenergic receptor kinase

The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Isolation and characterization of buckwheat (Fagopyrum esculentum M.) chalcone synthase and its polyclonal antibodies

Chalcone synthase was isolated from illuminated buckwheat (Fagopyrum esculentum M.) hypocotyls and purified to electrophoretic homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using (NH)4SO4 fractionation, gel filtration on AcA 44, ion exchange chromatography on DEAE-Bio-Gel, and HPLC on hydroxylapatite. The properties of the enzyme were pH optimum, 8.0; Mr approximately 83,000 +/- 1000; Mr subunit, approximately 41,500 +/- 500; isoelectric point, pH 5.2; Km, 1 X 10(-6)M for malonyl-CoA, and 0.6 X 10(-6) M for p-coumaryl-CoA. Buckwheat chalcone synthase used p-coumaryl-CoA as substrate and also utilized caffeyl-CoA and ferulyl-CoA at 20 and 80%, respectively, of the rate of p-coumaryl-CoA in the chalcone synthase reaction. Antibodies against the buckwheat chalcone synthase were developed in a New Zealand white rabbit and characterized for specificity by enzyme-linked immunosorbent assay, Ouchterlony double immunodiffusion, and Western blotting.     

Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen.

Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Purification and properties of a soluble inorganic pyrophosphatase from Rhodopseudomonas palustris

A soluble inorganic pyrophosphatase from photolithoautotrophically grown Rhodopseudomonas palustris was purified to a state of apparent homogeneity applying high resolving liquid chromatography steps. Values of 65 500 and 64 500 were calculated for the relative molecular mass under non-dissociating conditions employing gel filtration and high-performance liquid chromatography, respectively. Dissociation sodium dodecyl sulfate gel electrophoresis resulted in a value of 32 000, indicating that the enzyme is composed of two subunits of equal molecular mass. Isoelectric focusing revealed a pI value of 4.7. The purified enzyme was specific for PPi and the activity was modified by divalent cations. Ca2+, Mn2+, Mg2+ and Co2+ were potent activators at a concentration ratio of [Me2+]/[PPi] less than 1. Ca2+ turned out to be the most potent activator. Free Me2+ was inhibitory on the PPiase activity. The (Me-PPi) complex is regarded as the functional substrate. Km and Ki values of the metal activation and inhibition were determined. An activation energy of Ea = 14.4 kJ/mol was derived from Arrhenius plots for the enzymatic reaction.     

Preparation of homogeneous pig liver thioltransferase by a thiol:disulfide mediated pI shift

An enzyme catalyzing thiol-disulfide exchange, thioltransferase, was purified to homogeneity from pig liver. By taking advantage of the relatively large pI shift of the enzyme between its reduced and disulfide forms, the purification procedure, which included a heat step, ammonium sulfate precipitation, Sephadex G-75 and G-50 gel chromatography, and two CM-Sepharose chromatography separations, resulted in a 32% overall yield. The purified enzyme was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography. The protein had a Mr of approximately 11,000 and, in the reduced form, a pI of 6.4. The amino acid composition of the enzyme was similar to that of rat liver thioltransferase and calf thymus glutaredoxin and the N-terminus of the protein was blocked. The optimal pH for the enzyme activity was 9.0. The plots of thioltransferase activity as a function of S-sulfocysteine, 2-hydroxyethyl disulfide, and reduced glutathione concentrations did not display Michaelis-Menten kinetics. The enzyme was very sensitive to a sulfhydryl alkylating reagent. Preincubation of the enzyme with its disulfide substrates prevented the inactivation of the enzyme by iodoacetic acid while the other substrate, GSH, did not provide such protection. The results suggest that the active center of thioltransferase is cysteine dependent.     

Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli

The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T-gel thiophilic interaction, Sephacryl S-300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg2+, and FAD.     

Purification and characterization of human placental aminopeptidase A

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.     

Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers

Uroporphyrinogen decarboxylase (EC 4.1.1.37) which converts uroporphyrinogen I or III into coproporphyrinogen I or III, respectively, was purified about 5,500-fold from chicken erythrocytes. Purification was accomplished by chromatography on DEAE-cellulose, ammonium sulfate fractionation, chromatography on Sephadex G-100, and chromatofocusing. The most purified preparation was homogeneous on polyacrylamide gel electrophoresis and had a specific activity of 1,420 units/mg of protein, the highest value so far reported. The molecular weight, as determined by Sephadex G-150 gel chromatography, is 79,000. The subunit molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 39,700, suggesting that uroporphyrinogen decarboxylase is dimeric in form. The purified enzyme had an isoelectric point of 6.2 and a pH optimum of 6.8. The SH reagents inhibited the enzyme activity, but neither metal ions nor cofactor requirements could be demonstrated. A new and simple method for the separation of free uroporphyrin, hepta-, hexa-, and pentacarboxylic porphyrins and coproporphyrin was developed using a high pressure liquid chromatograph equipped with a spectrofluorometric detector. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified enzyme were performed. 3,4,3',4'-Tetrachlorobiphenyl and 3,4,5,3',4'5'-hexachlorobiphenyl which specifically induce delta-aminolevulinic acid synthetase also strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III.     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Purification and characterization of the sesquiterpene cyclase patchoulol synthase from Pogostemon cablin

The sesquiterpene cyclase, patchoulol synthase, from Pogostemon cablin (patchouli) leaves was purified to apparent homogeneity by chromatofocusing, anion exchange, gel permeation, and hydroxylapatite chromatography. The enzyme showed a maximum specific activity of about 20 nmol/min/mg protein, and a native molecular weight of 80,000 as determined by gel permeation chromatography. The protein was very hydrophobic, as judged by chromatographic behavior on several matrices, and possessed a pI value of about 5.0, as determined by isoelectric and chromatofocusing. SDS-PAGE showed the enzyme to be composed of two apparently identical subunits of Mr approximately 40,000. Maximum activity was observed at pH 6.7 in the presence of Mg2+ (Km approximately 1.7 mM); other divalent metal ions were ineffective in promoting catalysis. The Km value for the substrate, farnesyl pyrophosphate, was 6.8 microM. Patchoulol synthase copurified with the ability to transform farnesyl pyrophosphate to cyclic olefins (alpha- and beta-patchoulene, alpha-bulnesene, and alpha-guiaene) and this observation, plus evidence based on differential inhibition and inactivation studies, suggested that these structurally related products are synthesized by the same cyclase enzyme. In general properties, the patchoulol synthase from patchouli leaves resembles fungal sesquiterpene olefin cyclases except for the ability to synthesize multiple products, a property more typical of monoterpene cyclases of higher plant origin.     

Purification of a soluble, sodium-nitroprusside-stimulated guanylate cyclase from bovine lung

A soluble, sodium-nitroprusside-stimulated guanylate cyclase as been purified from bovine lung by DEAE-cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL-6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000-fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non-denaturing conditions in acrylamide gels (5-12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20, w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15-fold and 140-fold to specific activities of 8.5 and 15.7 mumol of cGMP formed min-1 mg-1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half-maximal at about 2 microM, was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G-25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine and 3-morpholino-sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 mumol min-1 mg-1.