Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures ofDaucus carota L.

The major anthocyanins accumulated by an Afghan cultivar ofDaucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.Abbreviations CGT UDP-galactose: cyanidin galactosyltransferase - CGXT UDP-xylose: cyanidin 3-galactoside xylosyltrans-ferase - DEAE diethylaminoethyl This study was supported by a grant from the Deutsche Forschun-gsgemeinschaft and a fellowship (W.E.G.) from the Land Baden-Württemberg. The skilful technical assistance of Johannes Madlung is gratefully acknowledged.     

Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition

We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.     

Purification of a membrane-derived proteinase capable of activating a galactosyltransferase involved in volume regulation

UDPgalactose:sn-glycerol-3-phosphate α-D-galactosyltransferase (IFP-synthase, EC 2.4.1.96) shows low activity in extracts prepared from standard volume cells of Poterioochromonals malhamensis under certain conditions. This inactive enzyme has been partially purified by chromatography on DEAE-cellulose, Sephadex G-150 and α-lactalbumin-agarose. It can be activated by an auxiliary enzyme which can be eluted from membranes and which has been purified to homogeneity by chromatography on DEAE-Sephacel and immobilized hemoglobin and fetuin. The activating enzyme is inhibited by chymostatin, antipain and diisopropylfluorophosphate and does not require divalent ions. It consists of a single peptide chain of molecular weight 46 000, can split certain proteins and appears to be a serine proteinase operating around a pH of 6.0. The activating proteinase is irreversibly generated in the crude homogenates on addition of Ca²⁺ and also shows increased activity shortly after cell shrinkage. This might indicate that it represents one of the possibilities to render the galactosyltransferase active as a result of the physiological stimulus.     

Purification and partial characterization of 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase and 7,8-dihydropteroate synthase from Escherichia coli MC4100.

The enzymes 7,8-dihydroxymethylpterin-pyrophosphokinase (HPPK) and 7,8-dihydropteroate synthase (DHPS), which act sequentially in the folate pathway, were purified to homogeneity from crude extracts of Escherichia coli MC4100. The enzymes represent less than 0.01% of the total soluble protein. HPPK was purified greater than 10,000-fold; the native enzyme appears to be a monomer with a molecular mass of 25 kDa and a pI of 5.2. DHPS was purified greater than 7,000-fold; the native enzyme has an apparent molecular mass of 52 to 54 kDa and is composed of two identical 30-kDa subunits. The amino-terminal sequences for both enzymes have been determined.     

Biosynthesis of glycoproteins in pulmonary parenchyma. II. Galactosyltransferase activity in pneumocyte subcellular fractions]

A soluble galactosyltransferase exists in the cell sap of the alveolar cells of the lung. The optimal conditions of galactosyltransferase are: pH 6, 20 DEGREES C, 10 mM Mn2+, 1 mM Mg2+. Michaelis constant for substrate UDP-galactose is 390 nM. By electrofocusing on Ampholine column, galactosyltransferase appears in a unique peak, at pHi 4.8. The parameters of the purified enzyme are the same as that of the crude enzyme. UDP is a noncompetitive inhibitor for the purified enzyme.     

Isolation and characteristics of functionally active proteolytically modified forms of tyrosyl-tRNA synthetase from bovine liver

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).     

PEG-重组酵母尿酸酶结合物的基本特性研究

重组Candida utilis尿酸酶由含PET-Uricase表达质粒的重组E.coli JM109(DE3)经乳糖诱导表达,菌体破碎后依次经过硫酸铵沉淀、阴离子交换层析和凝胶过滤层析可以获得纯度95%的重组尿酸酶。还原性SDS-PAGE和HPLC测得其亚基表观分子量和天然分子量分别约为33 kDa和130 kDa。获得的纯酶与20 kDa (mPEG)2 -Lys-NHS在特定的条件下反应合成PEG-重组酵母尿酸酶结合物,考察了重组酵母尿酸酶PEG化前后的基本性质,结果显示PEG化尿酸酶的最适pH为7.5,较修饰前下降了1个pH单位,酸碱稳定范围与修饰前类似,都在pH 6-10范围内稳定;修饰前后最适温度均为40℃,重组酵母尿酸酶的热稳定性和抗蛋白酶水解能力较PEG修饰前有较大提高;PEG化尿酸酶可保留修饰前酶活力的87.5%;在最适条件下,PEG-尿酸酶结合物的Km为3.57×10-5 mol/L,而修饰前测得的Km为3.91×10-5 mol/L。研究结果为深入探讨PEG化尿酸酶的结构与功能奠定了基础。     

The isolation and characterization of phosphofructokinase from the epithelial cells of rat small intestine.

1. Only a single phosphofructokinase isoenzyme is present in the mucosa of rat small intestine. 2. Mucosal phosphofructokinase was purified to yield a homogeneous preparation of specific activity 175 units/mg of protein. 3. The native enzyme is a tetramer, with monomer Mr 84 500 +/- 5000. 4. The native enzyme may be degraded by the action of endogenous proteinases to give two products with the same specific activity as the native enzyme: degradation occurs in the order native enzyme leads to proteolytic product 1 leads to proteolytic product 2. 5. Proteolytic product 1 has a greater mobility in cellulose acetate electrophoresis at pH8 and binds more strongly to DEAE-cellulose than does native enzyme; the converse is true for proteolytic product 2. 6. Proteolytic product 1 is a tetramer with a monomer Mr about 74 300; proteolytic product 2 is also a tetramer. 7. Native enzyme can only be prepared in the presence of proteinase inhibitors; partial purifications based on simple fractionation of crude mucosal extracts in the absence of proteinases inhibitors contain proteolytic product 2 as the main component and proteolytic product 1 together with little native enzyme. 8. Purified native mucosal phosphofructokinase displayed little co-operativity with respect to fructose 6-phosphate at pH 7.0 and was only weakly inhibited by ATP.     

Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.     

Purification and characterization of precarthamin decarboxylase from the yellow petals of Carthamus tinctorius L

Carthamin, a red quinochalcone pigment in safflower (Carthamus tinctorius L.), is enzymatically converted from a yellow precursor, precarthamin. The enzyme, which catalyzes the oxidative decarboxylation of precarthamin to carthamin, was purified to apparent homogeneity from yellow petals of safflower and named precarthamin decarboxylase. The molecular mass of the denatured enzyme was estimated as 33 kDa by SDS-PAGE. The molecular mass of the native enzyme was determined by gel filtration chromatography to be 24 kDa; thus, the native enzyme is a monomer. The optimum pH of the enzyme was 5.0. The enzyme activity was inhibited by Mn2+, Fe2+, and Cu2+ and sharply decreased at temperatures higher than 50 degrees C for 10 min. The activation energy and the Arrhenius frequency factor of the enzyme reaction were 19.7 kcal mol(-1) and 9.94 x 10(11) s(-1), respectively. The saturation curve of precarthamin showed that the enzyme follows Michaelis-Menten kinetics. The Km and Vmax of the enzyme were calculated as 164 microM and 29.2 nmol/ min, respectively. The turnover number (kcat) of the enzyme was calculated as 1.42 x 10(2) s(-1). The enzyme activity was severely inhibited by reducing agents such as glutathione and DTT at pH 5.0, suggesting that a disulfide bond may play an important role in enzyme function.     

Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor

In human neutrophils stimulated with phorbol myristate acetate (PMA) or with the chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLF) a number of proteins are phosphorylated, including proteins recovered in the membrane fraction corresponding to molecular masses of 130, 78, 46, 40, and 34 kDa and proteins recovered in the cytosol fraction corresponding to molecular masses of 65, 55, 48, 38, 36, 30, and 22 kDa. Phosphorylation of the membrane proteins was fourfold greater in cells stimulated with PMA, as compared to cells stimulated with fMLF, whereas both activators induced similar phosphorylation of proteins recovered in the cytosol fraction. Phosphorylation of membrane proteins appeared to be mediated by native protein kinase C (PKC) translocated from the cytosol to the plasma membrane. Thus phosphate incorporation was inhibited by retinal and a similar pattern of incorporation was reproduced in a reconstituted system composed of isolated cell membranes and purified PKC. Phosphorylation of cytosol proteins, on the other hand, appeared to be mediated by the proteolytically modified form of PKC. In this case, phosphate incorporation was inhibited by leupeptin, which prevents the conversion of native PKC to the proteolytically modified form, The phosphorylation pattern was reproduced when isolated cytosol fractions were incubated with the proteolytically modified form of the enzyme but not with the native PKC. These results demonstrate that responses to stimuli such as PMA or fMLF are mediated by different forms of PKC and that the proteolytically modified form is responsible for the major responses elicited by fMLF.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

The sensitivity of versican from rabbit lung to gelatinase A (MMP-2) and B (MMP-9) and its involvement in the development of hydraulic lung edema.

Large chondroitinsulphate-containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP-9 and proMMP-2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP-2 and MMP-9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

Human carbamylphosphate synthetase I. Stabilization, purification, and partial characterization of the enzyme from human liver

Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange chromatograhy and electrofocusing The native molecular weight of the enzyme was determined by gel filtration to be 190,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a monomeric molecular weight of 165,000. The isoelectric point of the purified enzyme was 6.05, and only one species of active enzyme was observed during electrofocusing of both purified enzyme preparations and crude liver homogenates. The enzyme exhibited a pH optimum of 7.8. The apparent Michaelis constants for NH4+, HCO3-, MgATP, and the activator, N-acetyl-L-glutamic acid, were 0.8, 6.7, 1.1, and 0.1 mM, respectively.     

A bacterial extracellular proteinase degrading silk fibroin

The bacterium Variovorax paradoxus, grown in a minimal medium in which silk fibroin represents the sole source of carbon and nitrogen, produces an extracellular protease that hydrolyzes fibroin as well as casein and, to a smaller extent, collagen and albumin. The optimal pH for activity was found to be in the acid range (optimum pH 5.8–6.4) and the enzyme activity was stimulated by the addition of divalent cations, either manganese or magnesium. Gel permeation chromatography and SDS-PAGE provided evidence that the native enzyme is a monomer with a M_r of ca. 21 kDa.     

In vitro phosphorylation of purified glycosylphosphatidylinositol-specific phospholipase D.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and by tyrosine kinase. Phosphorylation by PKA occurred in the 110 kDa native form of GPI-PLD as well as in multiple proteolytic degradation products and caused a significant decrease in enzyme activity. Dephosphorylation by treatment with alkaline phosphatase completely restored GPI-PLD activity. In addition, incubation of GPI-PLD with trypsin, which results in the generation of distinct peptide fragments, resulted in complete dephosphorylation of radiolabeled GPI-PLD. The site of phosphorylation by PKA was assigned to Thr-286. Tyrosine phosphorylation was only observed in a proteolytically processed fragment of GPI-PLD but not in the 110 kDa native form and had no effect on GPI-PLD activity.     

Purification and characterization of trimming glucosidase I from pig liver

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.     

Purification of a mitochondrial DNA polymerase from Crithidia fasciculata.

The mitochondrial DNA polymerase from Crithidia fasciculata has been purified to near homogeneity. SDS-PAGE analysis of the purified enzyme reveals a single polypeptide with a molecular weight of approximately 43,000. The protein is basic, with an isoelectric point between 7.6-8.0. Its Stokes radius of 22 A and its sedimentation coefficient of 4.1 S suggest a native molecular weight of 38,000, indicating that the protein is a monomer under our experimental conditions. Western blots and immunoprecipitations of crude extracts reveal a cross-reacting protein of 48 kDa, suggesting that the purified enzyme may be an enzymatically active proteolytic product. The mitochondrial origin of the polymerase was confirmed by cell fractionation. Our results indicate that the C. fasciculata enzyme may be among the smallest known mitochondrial polymerases.