DEAE Bio-Gel Research Articles

Stachyose with effect on anti-angiogenic activity from Salvia yunnanensis

Salvia yunnanensis is a traditional Chinese medicine for promoting blood circulation and eliminating blood stasis. Previous researches reported that Tanshinone IIA from Salvia yunnanensis had anti-angiogenic effect, however, whether its carbohydrates with similar effect is unknown. Here, we showed that a stachyose (Sta) from Salvia yunnanensis was purified by DEAE SepharoseTM Fast Flow anion-exchange chromatography and Bio-Gel P-2 gel permeation chromatography. Structure analyses suggested that Sta was tetrasaccharide as α-Gal-(1 → 6)-α-Gal-(1 → 6)-α-Glc-(1 → 2)-β-Fru. Further sulfated Sta was made and the degree of sulfate substitution was measured as 0.98. Bioactivity studies showed that Sta might block human microvascular endothelial cells tube formation in vitro and in vivo without significant cytotoxicity. Mechanism study suggested that Sta might bind to Galectin-3 (KD: 5.777E−6) and inhibit its expression to block angiogenesis linked to VEGFR/FAK and PDGFR/AKT/ERK signaling. Evidence also supported that Sta might be a potential compound for developing stachyose as an anti-angiogenesis functional food.

Specialized Hsp70 Chaperone (HscA) Binds Preferentially to the Disordered Form, whereas J-protein (HscB) Binds Preferentially to the Structured Form of the Iron-Sulfur Cluster Scaffold Protein (IscU)

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.

L-myo-Inositol-1-Phosphate Synthase Expressed in Developing Organ: Isolation and Characterisation of the Enzyme from Human Fetal Liver

L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0-60 % ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46 % of MIPS could be recovered. The human fetal liver MIPS specifically utilised D-glucose-6-phosphte and NAD(+) as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40 °C. The enzyme activity was remarkably stimulated by NH (4) (+) , slightly stimulated by K(+) and Ca(2+) and highly inhibited by Zn(2+), Cu(2+) and Hg(2+). The K (m) values of MIPS for D-glucose-6-phosphate and NAD(+) were found to be as 1.15 and 0.12 mM respectively while the V (max) values were 280 nM and 252 nM for D-glucose-6-phosphate and NAD(+) correspondingly. The apparent molecular weight of the native enzyme was determined to be 170 kDa.

Extracellular trypsin-like proteases produced by Cordyceps militaris

A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.

Effect of Proteolytic Cleavage on the Conformation of the Newcastle Disease Virus (NDV) Main Structural Glycoprotein

ABSTRACTThe structural NDV glycoproteins, the haemagglutinin-neuraminidase (HN) and the fusion protein (F) of La Sola strain of NDV were isolated from the pure virions by octyl glu-coside disruption, followed by ultracentifuge separation and chromatograhy on DEAE Bio gel column. Protein conformations measured by circular dichroism (CD) spectra (recorded using Jasko-J-500 A spectropolarimeter) were expressed as the percentage of a-helix and random coil on the base of mean ellipticity (θ). The HN NDV-glycoprotein has haemagglutinating and neuraminidase activities and it is responsible for the attachment of the virus to the cell surface, the penetration and the release of the virions from the cells. This gly-coprotein possesses one polypeptide chain and its secondary structure is represented by 15% a-helix and 85% random coil. The other glycoprotein, the F-protein, plays an essential role in the entry of virus into the cell by fusion of the viral envelope with the cell membrane. The active F-protein is presented in the infected cells as a disulfide-linked complex of two glycopeptides F1 and F2 derived from a precursor Fo by proteolytic cleavage. The active cleaved F-protein consists of 31% a-helix and 69% random coil. The treatment of pure HN with proteolytic enzymes enhances twice the activity of the protein and changes the conformation by increase of the a-helix percentage, i.e. 38% and diminishment of random coil to 62% In conclusion, the proteolytic cleavage of NDV-glyco-proteins, essential for their biological activities, is connected with changes of their conformation.

Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified alpha-mannosidase from Candida albicans.

A soluble alpha-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS-polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2) isomer B and mannose as a function of time of incubation up to 12 h at 37 degrees C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man(7)GlcNAc(2) and probably Man(6)GlcNAc(2). These two products were also observed when Man(8)GlcNAc(2) isomer B instead of Man(9)GlcNAc(2) was used as substrate. Other oligosaccharides, such as Man(6)GlcNAc(2)-Asn, Man(5)GlcNAc(2)-Asn, and the alpha1,3- and alpha1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an alpha1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.

Production, purification and properties of xylanase from a newly isolated Fusarium proliferatum

Fusarium proliferatum (NRRL 26517), was isolated by screening soil samples surrounding decaying corn and wood using corn fibre xylan as carbon source. The extracellular xylanase from this fungal strain was purified 975-fold to homogeneity from the culture supernatant by ammonium sulphate treatment, DEAE Bio-Gel A column chromatography, octyl-Sepharose column chromatography and Bio-gel A-0.5 m gel filtration. The purified xylanase (specific activity 591 U mg −1 protein) was a monomeric glycoprotein with a molecular weight (MW) of 22 400 as determined by SDS-PAGE. The optimum pH and temperature for the action of the enzyme were at 5.0–5.5 and 55 °C, respectively. The purified xylanase was fully stable at pH 5.0–7.5 and temperature up to 55 °C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability.

Purification and characterization of an extracellular beta-xylosidase from a newly isolated Fusarium verticillioides.

An extracellular beta-xylosidase from a newly isolated Fusarium verticillioides (NRRL 26518) was purified to homogeneity from the culture supernatant by concentration by ultrafiltration using a 10,000 cut-off membrane, ammonium sulfate precipitation, DEAE Bio-Gel A agarose column chromatography and SP-Sephadex C-50 column chromatography. The purified beta-xylosidase (specific activity, 57 U/mg protein) had a molecular weight (mol. wt.) of 94,500 and an isoelectric point at pH 7.8. The optimum temperature and pH for action of the enzyme were 65 degrees C and 4.5, respectively. It hydrolyzes xylobiose and higher xylooligosaccharides but is inactive against xylan. The purified beta-xylosidase had a K(m) value of 0.85 mM (p-nitrophenol-beta-D-xyloside, pH 4.5, 50 degrees C) and was competitively inhibited by xylose with a K(i) value of 6 mM. It did not require any metal ion for activity and stability.

Purification and properties of extracellular chitinases from the parasitic fungus Isaria japonica

Two chitinases (P-1 and P-2) induced with colloidal chitin were purified from the culture supernatant of Isaria japonica by chromatography on DEAE Bio-Gel, chromatofocusing and gel filtration with Superdex 75 pg. The enzymes were electrophoretically homogeneous and estimated to have a molecular mass of 43,273 (±5) for P-1 and 31,134 (±6) for P-2 by MALDI-MS. The optimum pH and temperature was 3.5–4.0 and 50°C for P-1 and 4.0–4.5 and 40°C for P-2. P-1 acted against chitosan 7B (degree of deacetylation, 65–74%) = glycol chitin > colloidal chitin = chitosan 10B (degree of deacetylation, above 99%) and P-2 against chitosan 7B > glycol chitin = chitosan 10B > colloidal chitin in order of activity. The products of hydrolysis of chitin and chitosan hexamer were analyzed by MALDI-MS. The products from the chitin hexamer obtained with P-1 were almost all dimers with only a small amount of trimer whereas those obtained with P-2 were mainly trimers with some dimer and tetramer. No hydrolysis of chitosan hexamer was observed. High homology in the amino-terminal sequence for chitinase P-1 was exhibited by chitinases from Trichoderma harzianum, Candida albicans and Saccharomyces cerevisiae in the range of 48–39%. The highest homology for Chitinase P-2 was shown by an endochitinase from Metarhizium anisopliae of 66%, while 44% homology was exhibited by chitinases of Leguminosae plants.

Purification and Properties of Extracellular Chitinases from the Parasitic Fungus Isaria japonica

Two chitinases (P-1 and P-2) induced with colloidal chitin were purified from the culture supernatant of Isaria japonica by chromatography on DEAE Bio-Gel, chromatofocusing and gel filtration with Superdex 75 pg. The enzymes were electrophoretically homogeneous and estimated to have a molecular mass of 43,273 (+/-5) for P-1 and 31,134 (+/-6) for P-2 by MALDI-MS. The optimum pH and temperature was 3.5-4.0 and 50 degrees C for P-1 and 4.0-4.5 and 40 degrees C for P-2. P-1 acted against chitosan 7B (degree of deacetylation, 65-74%) = glycol chitin > colloidal chitin = chitosan 10B (degree of deacetylation, above 99%) and P-2 against chitosan 7B > glycol chitin = chitosan 10B > colloidal chitin in order of activity. The products of hydrolysis of chitin and chitosan hexamer were analyzed by MALDI-MS. The products from the chitin hexamer obtained with P-1 were almost all dimers with only a small amount of trimer whereas those obtained with P-2 were mainly trimers with some dimer and tetramer. No hydrolysis of chitosan hexamer was observed. High homology in the amino-terminal sequence for chitinase P-1 was exhibited by chitinases from Trichoderma harzianum, Candida albicans and Saccharomyces cerevisiae in the range of 48-39%. The highest homology for Chitinase P-2 was shown by an endochitinase from Metarhizium anisopliae of 66%, while 44% homology was exhibited by chitinases of Leguminosae plants.

Production of Aspergillus terreus xylanase in solid-state cultures: application of the Plackett–Burman experimental design to evaluate nutritional requirements

Xylanase was produced by Aspergillus terreus cultivated on finely ground wheat straw in solid-state fermentation. The optimal medium composition was developed by applying the Plackett–Burman experimental design. Best enzymic activity was obtained in a medium containing 10 g wheat straw/flask moistened with a concentrated nutrient salt solution to 75% initial water content and incubated for 4 days at 30°C. A. terreus xylanase was fractionated by ammonium sulfate precipitation and purified by chromatography on DEAE Bio-Gel A followed by gel-filtration on Sephadex G-75. The enzyme was characterized by apparent V max and K m values of 333.3 U/mg protein and 16.7 mg xylan/ml, respectively, obtained for xylanase with oat spelt xylan as substrate. The optimal pH and temperature for maximum activity were 7 and 50°C, respectively. The enzyme showed high specificity towards oat spelt xylan and minute activities were observed with carboxymethyl cellulose and cellobiose. About 48.02% of the activity remained after the enzyme had been incubated at 60°C for 30 min. Metal ions such as Hg 2+, Cu 2+, Co 2+, Fe 3+, Pb 2+ strongly inhibited xylanase, whereas, Ca 2+ activated the enzyme.

Purification and Characterization of a Novel α-L-Arabinofuranosidase fromPichia capsulataX91

An intracellular alpha-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized. The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography. The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE. The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50 degrees C. The alpha-L-arabinofuranosidase was active at ethanol concentrations of wine. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition.

Purification and characterization of a novel thermostable alpha-L-arabinofuranosidase from a color-variant strain of Aureobasidium pullulans.

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced alpha-L-arabinofuranosidase (alpha-L-AFase) when grown in liquid culture on oat spelt xylan. An extracellular alpha-L-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75 degrees C, displayed optimal activity at 75 degrees C and pH 4.0 to 4.5, and had a specific activity of 21.48 mumol min-1. mg-1 of protein against p-nitrophenyl-alpha-L-arabinofuranoside (pNP alpha AF). The purified alpha-L-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The K(m) values of the enzyme for the hydrolysis of pNP alpha AF, arabinan, and debranched arabinan at 75 degrees C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The alpha-L-AFase activity was not inhibited at all by L-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM).

Prevotella intermedia が産生する酸性ホスファターゼに関する研究

Prevotella intermedia (P. intermedia), an anaerobic gram-negative bacterium, has been implicated as a pathogen in adult periodontitis, in acute necrotizing ulcerative gingivitis and in pregnancy gingivitis. P. intermedia has been known to have a high acid phosphatase (ACP) activity, one of the potential virulence factors in periodontopathogenic bacteria. However, little is known about the structure and function of ACPs from oral bacteria. In this study, in order to examine the potential role of ACP, the ACP from P. intermedia ATCC 25611 was purified to homogeneity and characterized. The ACP from P. intermedia was solubilized by extraction with 1% Triton X-114, and the crude extract was purified by ion-exchange chromatography on DEAE Bio-Gel, CM Bio-Gel and cellulose phosphate, followed by hydrophobic chromatography on Phenyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme revealed a single protein band with a molecular weight of Mr &ap;30 kDa. The molecular weight of the native enzyme from P. intermedia was estimated to be &ap;30 kDa using a gel filtration column with TSK gel G3000SW in the HPLC system. These findings suggest that the purified ACP is a monomer. The optimum pH for the enzyme was about 4.9 and the optimum temperature was 46℃. The enzyme activity was inhibited by sodium orthovanadate, sodium molybdate, sodium fluoride and Zn^<2+>, which are known as the inhibitors of protein tyrosine phosphatase (PTP) activity. On the other hand, it was not inhibited by okadaic acid, which is known to be a specific inhibitor of protein serine/threonine phosphatase (PP) activity. The enzyme had high activities against o-phospho-L-tyrosine and phosphotyrosine-containing peptides. The purified ACP was found to dephosphorylate the tyrosine-phosphorylated protein of a molecular weight &ap;170 kDa in an A431 cell lysate. The N-terminal amino acid sequence was determined for the purified ACP, and significant homology (64.3% identical amino acid residues) was found using the Swiss Prot Protein database between the purified ACP and the Pho C, the acid phosphatase of Morganella morganii. Thus, these findings suggest the possibility that the ACP from P. intermedia constitutes a novel class of ACPs, which has a PTP-like activity.

Actinobacillus actinomycetemcomitans が産生する酸性ホスファターゼのプロテインチロシン/セリンホスファターゼ様の性質に関する研究

Actinobacillus actinomycetemcomitans has been implicated as one of the causative organisms of localized juvenile periodontitis and adult periodontitis. A. actinomycetemcomitans has been known to have a high acid phosphatase (ACP) activity, which is previously known to be one of the virulence factors in periodontopathic bacteria. Little is known about ACP from oral bacteria. In order to examine the potential role of ACP, the ACP of A. actinomycetemcomitans was purified to homogeneity and characterized. The ACP in the cell-associated form of A. actinomycetemcomitans was released from the membranes with Triton X-114, then purified by ion-exchange chromatography on DEAE Bio-Gel and Gel filtration, followed by affinity chromatography on Hi-Trap Blue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single protein band with a molecular weight of Mr &ap; 40, 000. The molecular weight of the native enzyme was estimated to be 80, 000 by gel filtration with a Superdex-200 column. The pH optimum for the purified enzyme was about 5.4 and the temperature optimum was 37℃. The purified enzyme was inhibited by sodium orthovanadate, which is one of the inhibitors of protein tyrosine phosphatase (PTP) activity. However, it was not inhibited by okadaic acid, which is a specific inhibitor of protein serine/threonine phosphatase (PP) activity. The purified enzyme dephosphorylated o-phospho-L-tyrosine, phosphotyrosine-containing peptides and Raytide, which are used as substrates for PTP. Also, the purified enzyme dephosphorylated O-phospho-L-serine and phosphoserine-containing peptides and casein, which is good substrate for PP. On the other hand, phosphorylase kinase, which is used as a substrate for PP, was not dephosphorylated by the purified enzyme. It turned out that ACP of A. actinomycetemcomitans is classified in the PTP family, while it is not an SH-enzyme because it does not completely need the thiol reducing agent. The purified enzyme exhibited substantial activity against both Tyr and Ser-containing substrates. Thus, these findings suggest for the first time the existence of a novel tyrosine/serine protein phosphatase in oral bacteria and that ACP of A. actinomycetemcomitans has unique biochemical properties compared with any known PTPs.

A boron-rhamnogalacturonan-II complex from bamboo shoot cell walls

A boron-polysaccharide complex was isolated from a Driselase digest of bamboo ( Phyllostachys edulis) shoot cell walls by successive DEAE Sepharose FF, Bio-Gel P-10 and Mono Q HR 5/5 chromatography. The complex contained 0.15% boron (w/w). The glycosyl residue and linkage composition analyses of the polysaccharide moiety of the complex identified the polysaccharide as a rhamnogalacturonan-II (RG-II), a structurally complex pectic polysaccharide present in the primary cell walls of all higher plants. 11B NMR spectroscopy showed that boron was present as a tetrahedral borate-diol diester. Removal of boron from the complex decreased the M r by half without any loss of glycosyl residues, suggesting that boron cross-links two RG-II molecules. The boron-RG-II complex from bamboo (a monocot) shoot cell walls has almost the same structure as that of sugar beet (a dicot) cell walls. The results demonstrate that the structure of boron-RG-II complex is very similar in dicots and monocots.

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).

Enzymic sulphation of dopa and tyrosine isomers by HepG2 human hepatoma cells: stereoselectivity and stimulation by Mn2+.

HepG2 human hepatoma cells, labelled with [35S]sulphate in media containing L-3,4-dihydroxyphenylalanine (L-dopa), (D-dopa), DL-m-tyrosine or D-p-tyrosine, were found to produce the [35S]sulphated forms of these compounds. Addition to the labelling media of m-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor, greatly enhanced the production of L-dopa O-[35S]sulphate and DL-m-tyrosine O-[35S]sulphate, with a concomitant decrease in the formation of dopamine O-[35S]sulphate and m-tyramine O-[35S]sulphate. With 3'-phosphoadenosine 5'-phospho[35S]sulphate as the sulphate donor., HepG2-cell cytosol was shown to contain enzymic activity catalysing the sulphation of L-dopa, D-dopa, L-m-tyrosine, D-m-tyrosine, L-p-tyrosine and D-p-tyrosine. The pH optimum of the enzyme, designated dopa/tyrosine sulphotransferase, was determined to be 8.75 with D-m-tyrosine as the substrate. The enzyme exhibited stereoselectivity for the D-form of dopa or tyrosine isomers. Addition of 10mM MnCl2 to the reaction mixture resulted in a remarkable stimulation of dopa/tyrosine sulphotransferase activity, being as high as 267.8 times with D-p-tyrosine as the substrate. Quantitative assays revealed L-dopa, D-dopa and D-m-tyrosine to be better substrates than L-p-tyrosine. When the HepG2-cell cytosol was subjected to DEAE Bio-Gel and hydroxyapatite column chromatography, dopa/tyrosine sulphotransferase was co-eluted with the thermolabile 'M-form' phenol sulphotransferase. Furthermore dopa/tyrosine sulphotransferase displayed properties similar to that of the M-form phenol sulphotransferase with respect to thermostability and sensitivity to 2,6-dichloro-4-nitrophenol. Whether the M-form phenol sulphotransferase is truly (solely) responsible for the dopa/tyrosine sulphotransferase activity present in HepG2 cells remains to be clarified.

Purification, Characterization, and Molecular Cloning of A Novel Rat Liver Dopa/Tyrosine Sulfotransferase

A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II). The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000. Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form. The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine. Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates. The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM. The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing. Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases. The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed.

Purification of Chlorpromazine-Sensitive GTPase from Rat Cerebral Cortex

The chlorpromazine-sensitive GTPase from the cell membrane of rat cerebral cortex was purified to homogenity by using DEAE Bio-Gel A agarose, hydroxyapatite and heparin agarose chromatography. The purified chlorpromazine-sensitive GTPase was purified 370-fold to obtain a final specific activity of 40 mumol GTP hydrolyzed2min/mg protein. The purified enzyme was inhibited by chlorpromazine but not by compound 48/80. Magnesium was required for its activity instead of calcium. The purified enzyme had an apparent pH optimum of 8.0, and molecular weight was estimated to be 58,000.