Purification and characterization of membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus

Membrane-bound 5'-nucleotidase from Vibrio parahaemolyticus was solubilized and purified using a nonionic detergent, heptyl-beta-D-thioglucoside, and was characterized. This enzyme required Mg2+ for activity, maximum activity being observed at 5 and 20 mM Mg2+ with AMP and ATP, respectively, as substrates. Of the divalent cations tested, Mn2+ and Co2+ were able to replace Mg2+ partially, whereas Ca2+ was ineffective. Zinc strongly inhibited the enzyme activity and Ni2+ caused partial inhibition. This enzyme required Cl- for activity, the optimal concentration being 20 mM or more. The order of effectiveness of anions was Cl- greater than Br- greater than I- approximately NO3-. Sulfate and acetate were ineffective. The optimal pH was 8.0. The activity of the purified enzyme was stimulated by the addition of lipid to the assay mixture. This enzyme hydrolyzed all 5'-nucleotides tested, but did not hydrolyze 3'-nucleotides or ribose 5-phosphate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a single polypeptide, with a molecular weight of 72 kDa.     

Purification and characterization of membrane-bound quinoprotein quinate dehydrogenase

Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3. This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Purification and characterization of a membrane-bound sialidase from pig liver

A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.     

Purification and characterization of membrane-bound endoglycoceramidase from Corynebacterium sp.

Endoglycoceramidase catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids. We found that a bacterial strain Corynebacterium sp., isolated from soil, produced endoglycoceramidase both intracellularly and extracellularly. The intracellular enzyme bound to the cell membrane was solubilized with 1% Triton X-100 and purified to homogeneity about 170-fold with 60% recovery. The molecular mass of the enzyme was approximately 65 kDa. The enzyme is most active at pH 5.5-6.5 and stable at pH 3.5-8.0. Various neutral and acidic glycosphingolipids were hydrolyzed by the enzyme in the presence of 0.1% Triton X-100. Ganglio- and lacto-type glycosphingolipids were readily hydrolyzed, but globo-type glycosphingolipids were hydrolyzed slowly.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Radiation sensitivity of membrane-bound glucose-6-phosphatase

A comparative study was made of the effect of X-radiation on the membrane-bound glucoso-6-phosphatase of the nuclear membrane and microsomal fraction of calf thymus cells. Dose- and concentration-dependencies of inactivation of glucoso-6-phosphatase are indicative of a higher radiosensitivity of glucoso-6-phosphatase of nuclear membranes than that of microsomes. This difference in radiosensitivity is associated with the peculiarities of the composition and structural organization of these two membrane systems of a cell.     

Purification of bovine brain inositol-1,4,5-trisphosphate 5-phosphatase.

In bovine brain, two soluble inositol-1,4,5-trisphosphate (InsP3) 5-phosphatases, which catalyse the dephosphorylation of InsP3 to inositol 1,4-bisphosphate, have been separated by DEAE-Sephacel. Type I, i.e. the first eluted enzyme, is the main soluble form and is reminiscent of the membrane-bound enzyme by multiple criteria. Type I was purified to apparent homogeneity by a method involving chromatography on DEAE-Sephacel, Blue-Sepharose, Sephacryl S-200, phosphocellulose, and C18 HPLC. A single protein band of 42-43 kDa was identified by SDS/PAGE, corresponding to the peak of maximal activity. InsP3 5-phosphatase was purified to apparent homogeneity to a final yield of 45-50 micrograms protein. The minimal estimate value of the Vmax for InsP3 5-phosphatase was in the range 20-35 mumol.min-1.mg protein-1.     

Purification and characterization of a membrane-bound protein kinase from spinach thylakoids

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.     

Purification and characterization of membrane-bound CO-reactive hemoprotein from Tetrahymena pyriformis mitochondria

Abstract A CO-reactive hemoprotein was purified from the mitochondrial membrane fraction of Tetrahymena pyriformis . It showed absorption peaks at 615 and 455 nm in the reduced form and an α peak at 565 nm in the pyridine ferrohemochrome spectrum. Although the spectral properties were apparently similar to those of 'cytochrome a ₆₂₀' which was previously proposed as a mitochondrial terminal oxidase in T. pyriformis , it did not contain any molecules of heme a or copper atoms. Further, it showed neither cytochrome c oxidase nor cytochrome c peroxidase activity. These results suggest that 'cytochrome a ₆₂₀' may not be the terminal oxidase in the mitochondrial respiratory chain of T. pyriformis .     

Purification and characterization of membrane-bound phospholipase A2 from rat platelets

Phospholipase A2 was solubilized from rat platelet membrane by 1 M KCl and purified to near homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC. The characteristics of the purified membrane-bound enzyme were compared with those of phospholipase A2 released from thrombin-stimulated rat platelets (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 625-631). The molecular weights, elution profiles on reversed-phase HPLC, and NH2-terminal sequences were identical for the two enzymes. Other characteristics of the two enzymes, such as specific activity, substrate specificity, pH optimum, Ca2+ requirement, heat lability, and sensitivity to p-bromophenacyl bromide were also indistinguishable. These findings suggest that both enzymes share a common structure.     

Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii.

The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme.     

Identification and characterization of a sac domain-containing phosphoinositide 5-phosphatase

We have characterized a novel Sac domain-containing inositol phosphatase, hSac2. It was ubiquitously expressed but especially abundant in the brain, heart, skeletal muscle, and kidney. Unlike other Sac domain-containing proteins, hSac2 protein exhibited 5-phosphatase activity specific for phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. This is the first time that the Sac domain has been reported to possess 5-phosphatase activity. Its 5-phosphatase activity for phosphatidylinositol 4,5-bisphosphate (K(m) = 14.3 microm) was comparable with those of Type II 5-phosphatases. These results imply that hSac2 functions as an inositol polyphosphate 5-phosphatase.     

Identification and characterization of a novel inositol polyphosphate 5-phosphatase

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.     

Purification and characterization of the lipid A 1-phosphatase LpxE of Rhizobium leguminosarum

LpxE, a membrane-bound phosphatase found in Rhizobium leguminosarum and some other Gram-negative bacteria, selectively dephosphorylates the 1-position of lipid A on the outer surface of the inner membrane. LpxE belongs to the family of lipid phosphate phosphatases that contain a tripartite active site motif and six predicted transmembrane helices. Here we report the purification and characterization of R. leguminosarum LpxE. A modified lpxE gene, encoding a protein with an N-terminal His6 tag, was expressed in Escherichia coli. The protein was solubilized with Triton X-100 and purified to near-homogeneity. Gel electrophoresis reveals a molecular weight consistent with the predicted 31 kDa. LpxE activity is dependent upon Triton X-100, optimal near pH 6.5, and Mg2+-independent. The H197A and R133A substitutions inactivate LpxE, as does treatment with diethyl pyrocarbonate. In a mixed micelle assay system, the apparent Km for the precursor lipid IV(A) is 11 microm. Substrates containing the 3-deoxy-d-manno-oct-2-ulosonic acid disaccharide are dephosphorylated at similar rates to lipid IV(A), whereas glycerophospholipids like phosphatidic acid or phosphatidylglycerol phosphate are very poor substrates. However, an LpxE homologue present in Agrobacterium tumefaciens is selective for phosphatidylglycerol phosphate, demonstrating the importance of determining substrate specificity before assigning the functions of LpxE-related proteins. The availability of purified LpxE will facilitate the preparation of novel 1-dephosphorylated lipid A molecules that are not readily accessible by chemical methods.     

The membrane-bound hydrogenase from Paracoccus denitrificans. Purification and molecular characterization

The membrane-bound hydrogenase from Paracoccus denitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 mumol H2 min-1 (mg protein)-1 (methylene blue reduction). Purification involved solubilization by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denaturing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100 kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 20 degrees C was 8 h. The Paracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloroindophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V = 161.9 U/mg; Km = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised against the membrane-bound hydrogenase from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.     

Purification and characterization of a detergent-requiring membrane-bound metalloendopeptidase from porcine brain.

A detergent-requiring metalloendopeptidase cleaving a progastrin-C-terminal peptide (progastrin-(88-101)) mainly at the Arg95-Gly96 bond was solubilized from porcine cerebral vesicular membranes and purified to homogeneity as examined by PAGE. The purified enzyme had a molecular mass of approximately 76 kDa as estimated by both SDS/PAGE and Sephacryl S-300 gel filtration. It hydrolyzed progastrin-(88-101) peptide, BAM-12P, and bradykinin fairly specifically, and more efficiently than various other neuropeptides and related oligopeptides examined as substrates. It was inactive in the absence of detergents, and required certain detergents such as Triton X-100 or Lubrol PX for activity. Its optimum pH was about 6.5 and was strongly inhibited by metal-chelating agents such as EDTA, EGTA, and o-phenanthroline. It was extremely sensitive to EDTA and was completely inhibited even by 0.3 microM EDTA; the activity was fully restored by addition of a 10-fold higher concentration of Zn2+, CO2+, or Mn2+ ions over EDTA. On the other hand, dynorphin A-(1-13) peptide, a strong inhibitor of neurolysin, failed to inhibit the enzyme. The various characteristics indicated that the present enzyme is a unique membrane-bound metalloendopeptidase.     

Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain.

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C(16)-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K(m) of 1.29 mol % and a V(max) of 4.4 micromol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 micromol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC(50) of 5-10 microM. These results indicate that the purified enzyme is a novel ceramidase.     

Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart.

Prostaglandin (PG) E synthase was solubilized with 6 mM sodium deoxycholate from the microsomal fraction of bovine hearts. The enzyme was purified by about 800-fold to apparent homogeneity. The specific activity of the purified enzyme was about 830 mU/mg of protein, and the K(m) value for PGH(2) was 24 microM. The molecular weight of the enzyme was about 31000 on SDS-polyacrylamide gel electrophoresis and was about 60000 by gel filtration. The enzyme was separated from glutathione (GSH) S-transferase by DEAE-Toyopearl column chromatography, and did not exhibit any GSH S-transferase activity towards four different substrates. The purified enzyme was active in the absence of GSH, but it was activated by various SH-reducing reagents including dithiothreitol, GSH, or beta-mercaptoethanol. This is the first reported purification of membrane-bound PGE synthase to apparent homogeneity.     

Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets

Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.