Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.     

Proteins in intracellular simian virus 40 nucleoportein complexes: comparison with simian virus 40 core proteins.

Intracellular nucleoprotein complexes containing SV40 supercoiled DNA were purified from cell lysates by chromatography on hydroxyapatite columns followed by velocity sedimentation through sucrose gradients. The major protein components from purified complexes were identified as histone-like proteins. When analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, complex proteins comigrated with viral core polypeptides VP4, VP5, VP6, and VP7. (3H) tryptophan was not detected in polypeptides from intracellular complexes or in the histone components from purified SV40 virus. However, a large amount of (3H) tryptophan was found in the viral polypeptide VP3 relative to that incorporated into the capsid polypeptides VP1 and VP2. Intracellular complexes contain 30 to 40% more protein than viral cores prepared by alkali dissociation of intact virus, but when complexes were exposed to the same alkaline conditions, protein also was removed from complexes and they subsequently co-sedimented with and had the same buoyant density as viral cores. The composition and physical similarities of nucleoprotein complex and viral cores indicate that complexes may have a role in the assembly of virions.     

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.     

Messenger RNA guanylyltransferase from Saccharomyces cerevisiae. I. Purification and subunit structure

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of the GMP moiety from GTP to the 5' end of the RNA to form a cap structure (G(5')pppN-), has been purified to an apparent homogeneity from Saccharomyces cerevisiae. The mRNA 5'-triphosphatase activity hydrolyzing the gamma-phosphoryl group from pppN-RNA was co-purified with mRNA guanylyltransferase activity through column chromatographies on CM-Sephadex and poly(U)-Sepharose, and centrifugation through glycerol gradients, suggesting that these two activities are physically associated. An 820,w value of 7.3, and Mr = 140,000 were estimated from the sedimentation behavior in glycerol gradients. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two major polypeptides, Mr = 45,000 (alpha) and 39,000 (beta), were detected with the purified enzyme preparation. Their molar ratios were close to unity when estimated by the relative density of silver staining. These results suggest that the yeast mRNA-capping enzyme is an oligomeric protein which may consist of two alpha and two beta chains (alpha 2 beta 2).     

Microtubule-associated protein tau. A component of Alzheimer paired helical filaments

Microtubule-associated protein tau was purified from bovine brain microtubules by either (1) phosphocellulose chromatography, (2) heat treatment at pH 6.4, (3) heat treatment at pH 2.7, (4) heat treatment at pH 2.7 followed by extraction with perchloric acid and precipitation with glycerol, or (5) by precipitation with ammonium sulfate followed by extraction with perchloric acid. All of these tau preparations reacted specifically with antibodies to Alzheimer paired helical filaments. Affinity purified antibodies to tau labeled both Alzheimer neurofibrillary tangles and plaque neurites but not amyloid in Alzheimer brain tissue sections and labeled paired helical filament polypeptides on Western blots. Human brain tau and paired helical filament polypeptides co-migrated on sodium dodecyl sulfate-polyacrylamide gels. These results suggest that tau is a major component of Alzheimer paired helical filaments.     

RNA-dependent DNA polymerase of an endogenous type C virus of mice: purification and partial characterization

An RNA-dependent DNA polymerase was isolated from purified virions of endogenous oncornaviruses released by the MOPC-315 murine myeloma cell line. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme was found to consist of two major polypeptides with molecular weights of about 28,000 and 26,500. The active enzyme had a molecular weight of approximately 56,000, as calculated from its sedimentation on glycerol density gradients, indicating that it is probably a dimer of the two subunit polypeptides. The isolated MOPC-315 virus polymerase exhibited all three activities known to be found in the DNA polymerase from oncornaviruses, namely, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and an RNase H. The RNA-dependent polymerase activity showed a prounced preference for Mn2+ over Mg2+, whereas the DNA-dependent and RNase H reactions were catalyzed by these two cations to an almost equal extent. The purified polymerase was found to be immunologically related to the polymerase of Rauscher murine leukemia virus.     

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).     

Isolation and characterization of an extremely basic protein from adenovirus type 5.

By starch-gel electrophoresis and a staining method that is highly sensitive for argininyl residues, adenovirus type 5 was found to contain two minor basic polypeptides of extreme cathodic mobility in addition to the two known core proteins. The fastest-migrating polypeptide, named mu protein, and the second fastest polypeptide are found in adenovirions and virus-infected KB cells but not in top components or in uninfected cells. The top components and infected cells contain an additional basic polypeptide, presumably P-VII, that migrates slightly slower than polypeptide VII. None of the basic polypeptides of adenovirions was electrophoretically identical to the host histone. The basic proteins of adenovirions were purified by urea phosphocellulose column chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two minor basic core proteins, mu and another component, have similar mobilities in sodium dodecyl sulfate-polyacrylamide gels as a complex of polypeptides X-XII. After further purification on a Sephadex G-75 column, the mu protein was found to have a molecular weight of about 4,000. Amino acid analysis showed that the mu protein lacks tryptophan and 69% of the total amino acid residues are basic, that is, 54% arginine, 13% histidine, and 2% lysine. Only eight amino acids seem to contribute to make the mu polypeptide. There are 125 copies of the mu polypeptide per 1,000 copies of polypeptide VII in a virion.     

Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies

Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72- and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit.     

Purification and properties of eukaryotic initiation factor 2 and its ancillary protein factor (Co-eIF-2A) from yeast Saccharomyces cerevisiae

Two peptide chain initiation factor activities, eIF-2y and Co-eIF-2A20y, were purified from the high speed supernatant fraction of the yeast Saccharomyces cerevisiae and their properties were studied. 1) In sodium dodecyl sulfate-polyacrylamide gels, purified eIF-2y showed two major polypeptide bands corresponding to molecular weights of 54,000 and 36,000. The Mr 54,000 band was significantly more intense than the Mr 36,000 band, indicating the possible presence of two polypeptides of equal molecular weight in this band. The molecular weight of eIF-2y, determined using a density gradient centrifugation method, was approximately 140,000. 2) In sodium dodecyl sulfate-polyacrylamide gel, purified Co-eIF-2A20y showed a single polypeptide band corresponding to a molecular weight of 20,000. A similar molecular weight for Co-eIF-2A20y was also found using a density gradient centrifugation method. 3) In partial reactions, eIF-2y bound Met-tRNAf in the presence of Mg2+. The reaction required GTP. Co-eIF-2A20y stimulated Met-tRNAf binding to eIF-2y (2-3-fold) and also rendered the complex stable to 3 X 10(-5) M aurintricarboxylic acid. 4) This Co-eIF-2A20y activity was heat-labile and N-ethylmaleimide-insensitive. 5) Antibodies were prepared by injecting rabbits with homogeneous Co-eIF-2A20y. Such anti-Co-eIF-2A20y inhibited (60%) protein synthesis in a yeast cell-free protein synthesizing system and completely blocked Co-eIF-2A20y stimulation of Met-tRNAf. 40 S initiation complex formation. Protein synthesis inhibition by anti-Co-eIF-2A20y was almost completely reversed by preincubation of the antibodies specifically with homogeneous Co-eIF-2A20y.     

Characterization of matrix domains of the hamster acrosome

In this study we describe the purification and the structural and biochemical properties of a detergent-stable complex of the hamster sperm acrosome. This complex consists of two distinct acrosomal matrix domains and a layer of electron-dense material, termed the acrosomal lamina, derived from the luminal surface of the outer acrosomal membrane. This complex has been isolated by centrifugation of detergent-extracted sperm suspensions on Percoll density gradients. The complex contains two major polypeptides of Mr 29,000 and Mr 22,000 and minor polypeptides of Mr 64,000-62,000, 56,000 and 35,000. Gelatin-containing sodium dodecyl sulfate-polyacrylamide gels demonstrate that bands of proteinase activity are not the major polypeptide components of the complex. These data demonstrate that the matrix of the acrosome is compartmentalized into domains of differing structural properties that occupy specific locations in the intact acrosome and that matrix components are physically associated with the outer acrosomal membrane. These data indicate that a structural framework is present within the acrosome and we speculate that it may be involved in sequestering hydrolases into specific spatial domains and could affect the temporal release of activity of selected hydrolases during the acrosome reaction.     

The purification of a human mismatch-binding protein and identification of its associated ATPase and helicase activities.

A mismatch-binding protein has been purified an estimated 4500-fold from HeLa nuclear extracts using four different chromatographic steps. Two polypeptides of apparent molecular weight of 160,000 and 100,000 were present in the final affinity-purified fraction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolytic clipping of the protein-DNA complexes visualized after UV treatment indicated that the 100-kDa polypeptide is most likely a degradation product of the 160-kDa polypeptide. UV cross-linking experiments have shown that both these polypeptides bind specifically to oligonucleotide duplexes containing G/T mismatches. Direct DNA binding studies and band-shift competition assays showed that although the mismatch-binding protein binds with highest affinity to oligonucleotides containing G/T mismatches, it is also capable of binding to oligonucleotides containing other mispairs. The purified protein has an associated Mg(2+)-dependent ATPase activity, which is markedly enhanced in the presence of single-stranded DNA. A helicase capable of unwinding a 34-mer oligonucleotide, annealed to a complementary sequence in single-stranded M13, also copurified with the mismatch-binding protein. This reaction occurs in an ATP- and magnesium-dependent manner.     

Multiple forms of endothelial cell growth factor. Rapid isolation and biological and chemical characterization

Endothelial cell growth factor (ECGF) can be rapidly purified from bovine brain to high specific activity using heparin-Sepharose affinity chromatography. Purification of the mitogen by this method results in relatively high yields of the polypeptide (10 to 100 micrograms/kg of tissue) with biological activity on murine and human endothelial cells in the picogram range. The product obtained is a mixture of two single-chain polypeptides with apparent molecular weights of 17,000 (alpha-ECGF) and 20,000 (beta-ECGF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two forms of ECGF can be separated by either NaCl gradient elution from heparin-Sepharose or reversed-phase high pressure liquid chromatography. The two polypeptides are related on the basis of similar: amino acid compositions, affinity for heparin-Sepharose, cyanogen bromide and trypsin-derived cleavage products, and biological activity. Furthermore, the cyanogen bromide fragments derived from the two forms of ECGF also possess similar amino acid compositions and mobilities on sodium dodecyl sulfate gels. These data suggest that there are at least two discrete molecular forms of ECGF in bovine brain and that these two molecules are structurally related.     

A novel Ca2+-dependent protein kinase from Paramecium tetraurelia

The ciliated protozoan Paramecium tetraurelia contained two protein kinase activities that were dependent on Ca2+. We purified one of the enzymes to homogeneity by Ca2+-dependent affinity chromatography on phenyl-Sepharose and ion exchange chromatography. The purified enzyme contained polypeptides of 50 and 55 kDa, with the 50-kDa species predominant. From its Stokes radius (32 A) and sedimentation coefficient (3.9 S), we calculated a native molecular weight of 51,000, suggesting that the active form is a monomer. Its specific activity was 65-130 nmol X min-1 X mg-1 and the Km for ATP was 17-35 microM, depending on the exogenous substrate used. Kinase activity was completely dependent upon Ca2+; half-maximal activation occurred at approximately 1 microM free Ca2+ at pH 7.2. Phosphatidylserine and diacylglycerol did not stimulate activity, nor did the addition of purified Paramecium calmodulin. The enzyme phosphorylated casein and histones, forming primarily phosphoserine and phosphothreonine, respectively. It also catalyzed its own phosphorylation in a Ca2+-dependent reaction; the half-maximal rate of autophosphorylation occurred at approximately 1-1.5 microM free Ca2+, and both the 50- and 55-kDa species were autophosphorylated. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation in situ, the 50-kDa protein retained its Ca2+-dependent ability to phosphorylate casein, suggesting that Ca2+ interacts directly with this polypeptide. This was confirmed by direct binding studies; when the enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred to nitrocellulose, and renatured, there was 45Ca2+-binding in situ to both the 50- and 55-kDa polypeptides. The Paramecium enzyme appears to be a new and unique type of Ca2+-dependent protein kinase.