Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization.

The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.     

Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.     

Translation of the human C3b/C4b receptor mRNA in a cell-free system and by Xenopus oocytes

The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190,000, 220,000, 250,000, and 280,000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220,000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CR1 and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CR1 monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.     

Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.     

A DNA binding protein from Xenopus laevis oocyte mitochondria

A DNA binding protein has been isolated, by affinity chromatography on DNA cellulose, from mitochondria and from purified mitDNA-protein complexes from oocytes of Xenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to DNA with a high efficiency. It exhibits an apparently preferential binding to the single-stranded fiber of the D loop structures.     

Catabolism of Ap3A and Ap4A in human plasma. Purification and characterization of a glycoprotein complex with 5'-nucleotide phosphodiesterase activity

A hydrolase splitting adenosine(5')triphospho(5')adenosine (Ap3A) to AMP and ADP has recently been detected in human plasma [Lüthje, J. and Ogilvie, A. (1984) Biochem. Biophys. Res. Commun. 118, 704-709]. The enzyme has been purified to apparent homogeneity, as stained in a native polyacrylamide gel. From gel filtration data a Stokes radius of 5.9 nm was calculated, suggesting a molecular mass of about 230 kDa. The presence of the non-ionic detergent Triton X-100 did not change the molecular mass. The hydrolase dissociated to three major protein components (66 kDa; 45 kDa; 16 kDa) during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. Binding of the native enzyme to concanavalin-A--Sepharose and specific inhibition of binding by methyl mannoside indicated that the hydrolase is a glycoprotein. Two of the subunits (66 kDa; 45 kDa) could be affinity-labeled with radioiodinated concanavalin A. Active hydrolase could be prepared in buffers without added metal ions. Treatment with EDTA, however, completely abolished the hydrolytic activity. The enzyme could be reactivated by incubation with Ca2+, Co2+ and, at best, with Zn2+, whereas Mg2+ was ineffective. The affinity of the enzyme for Ap3A was high (Km = 1 microM), with normal Michaelis-Menten kinetics. The homolog dinucleotide Ap4A was also substrate (Km = 0.6 microM) yielding AMP and ATP as products after the asymmetric split. Other dinucleotides, such as NAD, and also mononucleotides (ATP,UTP) were degraded to nucleoside monophosphates indicating a broad specificity of the enzyme. The synthetic compound thymidine 5'-monophosphate p-nitrophenyl ester was substrate with low affinity whereas its 3'-homolog was not hydrolyzed. Optimal activity of the hydrolase was found at pH 8.5.     

A multiprotein form of DNA polymerase alpha from HeLa cells. Resolution of its associated catalytic activities

The majority of the DNA polymerase alpha activity in HeLa cells has been isolated and purified as a multiprotein Mr 640,000 form. The multiprotein form of DNA polymerase alpha corresponds to DNA polymerase alpha 2 that was previously reported by us (Lamothe, P., Baril, B., Chi, A., Lee, L., and Baril, E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4723-4727). The highly purified DNA polymerase alpha 2 has in addition to DNA polymerase alpha-associated DNase, primase, and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A)binding activities and accessory primer recognition proteins C1 and C2. The DNA polymerase alpha and associated activities increase coordinately during the G1/S-phase transition of the cell cycle. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the electrophoretically homogeneous DNA polymerase alpha shows that it is composed of at least eight polypeptides in the molecular weight range of 180,000-15,000. Hydrophobic chromatography on butyl-agarose resolves the DNase and Ap4A-binding protein from a complex of DNA polymerase alpha, primase, and the primer recognition proteins C1 and C2. Hydrophobic chromatography of the latter complex on phenyl-Sepharose resolves the C1 protein from a DNA polymerase alpha-C2 protein-primase complex. Phosphocellulose chromatography of the DNA polymerase-primase-C2 protein complex resolves the C2 protein from a complex of DNA polymerase alpha-primase.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Extracellular Phytase (E. C. 3.1.3.8) from Aspergillus Ficuum NRRL 3135: Purification and Characterization

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85–100-KDa. On the basis of a molecular weight of 85–KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 × 10⁴ M-l cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0°C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68°C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58°C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85–100-KDa to about 76–KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.     

Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization

Diadenosine tetraphosphatase, an enzyme splitting diadenosine tetraphosphate to AMP and ATP, has been purified to apparent homogeneity from a permanent cell line derived from a leukemic child. The purification procedure consisted of fractionation by ammonium sulfate precipitation, followed by Sephacryl 200 and DEAE-cellulose chromatography, and finally a differential membrane filtration. The enzyme is a single polypeptide chain of Mr = 17,500 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weight of the native enzyme was calculated as 20,000 from gel filtration data. The apparent Km for Ap4A was 0.5 microM as determined by two independent kinetic assays. None of the following compounds were substrates of the enzyme: diadenosine triphosphate, NAD, nucleoside 5'-phosphates (AMP, ATP, GDP, GTP, and UTP). The enzyme had optimal activity in the presence of 1 mM Mg2+, showing no activity in the presence of EDTA.     

Purification of an antibacterial protein in the coelomic fluid of Nereis diversicolor (annelida,polychaeta). similitude with a cadmium-binding protein

1. Coelomic fluid of Nereis diversicolor shows antibacterial activity 24 hr after inoculation with Escherichia coll, while fluid from untreated control does not.2. This activity is detected by growth inhibition of E. coli or Micrococcus kristinae.3. The thermolabile protein has been purified by two distinct methods using gel filtration and exchange ion chromatography. By SDS-PAGE, the molecular weight of this protein has been estimated at about 10 kDa.4. The protein shows strong similitude with cadmium-binding protein (CBP) which is a dimer constituted by two 10 kDa subunits (Nejmeddine et al., 1988).5. In Western Blot, monoclonal antibody against CBP reacted with the antibacterial protein.6. Moreover, bacteriostatic activity of the coelomic fluid disappeared after incubation with polyclonal and monoclonal antibodies.     

Purification and characterization of maturation-promoting factor in fish.

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

A novel glycoprotein that binds to hyaluronic acid

Hyaluronic acid binding protein (HBP) has been purified to homogeneity from normal rat brain by using Hyaluronate-Sepharose affinity chromatography. It appears as a single band in non-dissociating gel electrophoresis. The molecular weight of native protein, as determined by gel filtration is found to be 68,000 daltons, and has a single subunit of molecular weight approximately 13,500 as determined under denaturing conditions in polyacrylamide gel electrophoresis, indicating that this protein is apparently composed of five identical subunits. Amino acid analysis shows the purified HBP to be rich in glycine and glutamic acid content, and is distinct from fibronectin, link proteins, and gelatin binding proteins which are known to bind to hyaluronic acid. This protein is further characterised as sialic acid containing glycoprotein.     

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).     

Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.     

Purification of bovine glia maturation factor and characterization with monoclonal antibody

Glia maturation factor (GMF) is purified 100 000-fold to apparent homogeneity from bovine brains by a procedure consisting of ammonium sulfate precipitation, column chromatography with diethylaminoethyl-Sephacel, Sephadex G-75, and hydroxylapatite, and a final step using C4 reverse-phase high-performance liquid chromatography. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 14 000 and an isoelectric point of pH 5.2. Purified GMF stimulates cultured astroblasts to proliferate and to grow out cell processes with half-maximal activity at 8 ng/mL. A monoclonal antibody raised against partially purified GMF adsorbs the activity of pure GMF and immunologically binds the putative GMF protein band.     

Purification of GVBD-Inducing Protein from the Ciliate Tetrahymenathermophila

Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.