Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

The Isolation and Physico-chemical Properties of Crystalline Quinolinate Phosphoribosyltransferase from Hog Liver

Quinolinate phosphoribosyltransferase (an intermediary enzyme in the de novo NAD biosynthetic pathway) was purified from hog liver and its crystallization from a mammal was successful for the first time. This crystalline enzyme preparation was certified to be homogeneous by ultracentrifugal analysis and polyacrylamide gel disc electrophoresis. Its molecular weight was 173,000 using the gel filtration method, and 172,000 using sedimentation velocity analysis. The subunit molecular weight was estimated at 33,500 with SDS polyacrylamide gel disc electrophoresis. Several physico-chemical parameters were also determined.     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.     

1, 4-alpha-Glucan phosphorylase from Klebsiella pneumoniae purification, subunit structure and amino acid composition.

1. A 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae has been purified about 80-fold with an over-all yield greater than 35%. The purified enzyme has been shown to be homogeneous by gel electrophoresis at different pH-values, by isoelectric focusing, by dodecylsulfate electrophoresis and by ultracentrifugation. 2. The molecular weight of the native enzyme has been determined to be 180 000 by ultra-centrifugation studies, in good agreement with the value of 189 000 estimated by gel permeation chromatography. 3. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The molecular weight of these polypeptide chains has been found to be 88 000 by dodecylsulfate polyacrylamide gel electrophoresis and 99 000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of two polypeptide chains. 4. The enzyme contains pyridoxalphosphate with a stoichiometry of two moles per 180 000 g protein, confirming that the 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is a dimeric enzyme. 5. The amino acid composition of the enzyme has been determined, and its correspondence to that of 1,4-alpha-glucan phosphorylases from other sources is discussed. 6. The pI of the enzyme has been shown to be 5.3 and its pH-optimum to be about pH 5.9. The enzyme is stable in the range from pH 5.9 to 10.5.     

Purification and properties of bromoperoxidase from Pseudomonas pyrrocinia

A bromoperoxidase was purified and partially characterized from Pseudomonas pyrrocinia ATCC 15958, a bacterium that produces the antifungal antibiotic pyrrolnitrin. The purified enzyme preparation was homogeneous as determined by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular mass of the enzyme was estimated to be 154 kDa +/- 3 kDa as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band with the mobility of a 76-kDa species. Therefore, in solution at neutral pH, bromoperoxidase exists as a dimeric species. The isoelectric point was 5.0. The prosthetic group of this procaryotic bromoperoxidase was ferriprotoporphyrin IX. The spectral properties of the native and reduced enzyme are reported. The purified enzyme showed brominating as well as peroxidase and catalase activity.     

Purification and properties of cyclic nucleotide phosphodiesterase from uterine tissue

Cyclic nucleotide phosphodiesterase from calf myometrium has been purified to a homogeneous state for the first time, as can be evidenced from polyacrylamide gel electrophoresis data. The purification procedure included ion-exchange chromatography on DEAE-cellulose, high pressure liquid chromatography on TSK 545 DEAE and gel filtration through Toyopearl HW-55. The molecular mass of the enzyme as determined by gel filtration and polyacrylamide gel electrophoresis is 110 kD. The purified enzyme hydrolyzes cAMP and cGMP with Km = 30 microM and 18 microM, respectively.     

Characterization of a soluble inorganic pyrophosphatase from Microcystis aeruginosa and preparation of its antibody.

A soluble inorganic pyrophosphatase was isolated from a crude extract of Microcystis aeruginosa by adsorption chromatography. The enzyme was purified to homogeneity as judged by sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide gel electrophoresis and N-terminal amino acid analysis. The molecular mass was estimated to be 80 kDa by gel filtration chromatography, 87 kDa by nondenaturing polyacrylamide gel electrophoresis, and 28 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an isoelectric point of 4.5, which is similar to the pI values reported for other soluble inorganic pyrophosphatases. The sequence of 29 N-terminal amino acids was determined; only 4 of these amino acids are identical to those in the sequence of Saccharomyces cerevisiae inorganic pyrophosphatase. M. aeruginosa inorganic pyrophosphatase is a Mg(2+)-dependent enzyme exhibiting a pH optimum of around 7.5. Its KM value for inorganic pyrophosphate was estimated to be 1.30 mM. A specific antibody was raised in chicken to M. aeruginosa inorganic pyrophosphatase. No immunological cross-reactivity was seen when Western blots of partially purified S. cerevisiae or Escherichia coli inorganic pyrophosphatase were probed with the antibody.     

Isolation and characterization of UDP-glucose dolichyl-phosphate glucosyltransferase from human liver

The enzyme UDP-glucose dolichyl-phosphate glucosyltransferase has been purified to near homogeneity from human liver microsomes. A 1100-fold enrichment over starting microsomal membranes was achieved by selective solubilization followed by anion- and cation-exchange chromatography, 5-HgUDP-thiopropyl-Sepharose affinity chromatography, butylagarose chromatography and hydroxyapatite chromatography. The glucosyltransferase was shown to be separated from other dolichyl-phosphate-dependent glycosyltransferases catalyzing the formation of dolichyl diphospho-N-acetylglucosamine and dolichyl phosphomannose. Sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis of the purified enzyme under reducing conditions revealed a protein band of Mr 36,000. Protection of the solubilized enzyme against rapid inactivation was achieved by its competitive inhibitor uridine. The purified glucosyltransferase activity exhibited a specific requirement for the presence of phospholipids. Phosphatidylethanolamine was the most effective activator of enzyme activity.     

Purification of the ADP-ribosylating enzyme from S. solfataricus by SDS-polyacrylamide gel electrophoresis and electroelution

The ADPribosylating enzyme from the thermophilic archaeon S. solfataricus was purified by a simple procedure which included preparative electrophoresis on a 0.1% SDS- polyacrylamide gel. The gel slice containing the enzymatic protein was cut out and the enzyme was solubilized by electroelution. The pure enzyme was obtained by chromatography of the electroeluted sample on a DNA-Sepharose column. The purified enzyme retained both its full activity and the structuring ability as a function of temperature increase.     

Purification, some properties and quaternary structure of the D-ribulose 1,5-diphosphate carboxylase of Alcaligenes eutrophus.

D-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacterium Alcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000. Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2. Michaelis constant (Km) values for ribulose 1,5-diphosphate, Mg2+, and CO2 were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.     

Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle

A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.     

露花叶片PEP羧化酶的纯化及某些分子特性

经硫酸铵分部沉淀,DEAE-纤维素(DE52),DEAE-Sephadex A-50,SephacrylS-200和二次羟基磷灰石等柱层析,从露花叶片中分离得到纯化63.9倍、电泳均一的磷酸烯醇式丙酮酸羧化酶。此酶的天然分子量经聚丙烯酰胺梯度凝胶电泳测定为260kD,经SephadexG-200凝胶过滤法测定为240kD。用SDS-聚丙烯酰胺梯度凝胶电泳测得酶的亚基分子量为115kD,表明此酶是个二同聚体。此酶的等电点为PI=5.6。免疫双扩散的结果表明此酶与高梁PEPG的抗原决定簇呈部分同一性。     

Aspartic proteinase from wheat seeds: isolation,properties and action on gliadin

Wheat endosperm was shown to contain an aspartic proteinase capable of hydrolyzing the wheat storage protein, gliadin, in vitro. The enzyme was purified to homogeneity by affinity chromatography on bacilliquin-silochrome, diethylaminoethyl-Toyopearl ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide gel electrophoresis. The sedimentation constant of the enzyme was 3.4 S and the relative molecular mass (M_r), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 58000 dalton (Da). The purified enzyme was completely inhibited by pepstain whereas other enzyme inhibitors did not affect its activity. The enzyme was found to hydrolyze mainly - and -gliadins with M_r's of 67000–95000 Da, with maximal activity at pH 4.5. The data make it possible to suggest that the enzyme has an endogenous function by initiating proteolysis of storage proteins in germinating wheat seeds.Abbreviations BSA bovine serum albumin - Da dalton - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Characterization of a Mg2+-dependent phosphatase from turkey gizzard smooth muscle

A Mg2+-dependent phosphatase has been purified to apparent homogeneity from turkey gizzard smooth muscle. The enzyme has a Mr = 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 44,500 as determined by sedimentation equilibrium centrifugation under nondenaturing conditions. Using polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate all of the phosphatase activity was found to migrate as a single band, subsequently shown to have an Mr = 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is inactive in the absence of Mg2+ and maximum activity is reached at a free concentration of 12 mM Mg2+. Mn2+ can replace Mg2+, but the activity is only about one-fifth of that found with 12 mM Mg2+. NaF and the nucleotides ATP, ADP, and AMP inhibit phosphatase activity. This inhibition appears to be independent of their ability to bind Mg2+. The phosphatase purified from turkey smooth muscle appears to be identical with that purified from canine heart (Binstock, J. F., and Li, H. C. (1979) Biochem. Biophys. Res. Commun. 87, 1226-1234) and rat liver (Hiraga, A., Kikuchi, K., Tamura, S., and Tsuiki, S. (1981) Eur. J. Biochem. 119, 503-510).     

Purification and properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyacrylamide gel electrofocusing, about 710,000-fold with a 35% yield from the liver cytosol of thioacetamide-treated rats. The final specific activity was approximately 24,400 nmol/min/mg of protein. The apparent molecular weight of the enzyme determined by gel filtration analyses on Sephacryl S-200 was 55,000 in the presence of 0.25 M NaCl and 145,000 in its absence. The minimum molecular weight of the enzyme was determined to be 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was estimated as 5.7 in the presence of 8 M urea. Some catalytic properties of the enzyme were also studied.     

Purification and properties of maize polyamine oxidase: a flavoprotein

Polyamine oxidase was purified from maize shoots to homogeneityby the criteria of polyacrylamide gel electrophoresis and ultracentrifugation.The purified yellow enzyme showed absorption maxima at 278,380 and 460 nm. The molecular weight estimated by gel filtrationwas about 65,000 and the sedimentation coefficient was 5.95S. Sodium dodecylsulfate gel electrophoresis yielded a singleband at a molecular weight of 65,000. The enzyme contained 1mole of FAD per mole of enzyme. Amino acid composition and kineticproperties of the enzyme are presented. (Received April 30, 1980; )     

Malignancy-associated DNA-binding protein C3DP from human serum. Further characterization and purification of C3DP retaining its DNA-binding affinity.

C3DP, a malignancy-associated DNA-binding protein from human serum[1], was purified to homogeneity without loss of its DNA-binding affinity. For this purpose normal human serum was submitted to affinity chromatography on Con A-Sepharose and DNA-cellulose and to preparative polyacrylamide gel electrophoresis. The purified C3DP was identified by immunodiffusion and sodium dodecylsulfate polyacrylamide gel electrophoresis and it was shown to bind to DNA by DNA-cellulose chromatography. The isoelectric point of C3DP was determined to 4.9 by isoelectric focusing.