Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.     

Identification of an Epstein-Barr virus nuclear antigen by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis.

A 65,000-dalton (65K) antigen found in Raji cells by fluoroimmunoelectrophoresis and radioimmunoelectrophoresis has been identified as an Epstein-Barr virus nuclear antigen (EBNA). This identification is based on the following evidence. The 65K antigen is detected in Raji cells but not in three Epstein-Barr virus (-) human B cell lines. It is not detected with EBNA (-) sera. The 65K antigen is found predominantly in the nucleus and co-elutes with EBNA during partial purification by DNA-Sepharose and Blue Dextran-Sepharose chromatography. Finally, the partially purified 65K antigen is an effective absorbant of EBNA antibody as measured in an anticomplement immunofluorescence assay. Antigens with molecular weights of 72, 70, and 73K have been detected in B95-8, P3HR-1, and Namalwa cells, respectively. These antigens are the likely homologues of the 65K Raji EBNA. In addition, an Epstein-Barr virus-associated, 81K DNA-binding antigen has been detected in both B95-8 and Raji cells.     

Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen.

A 62,000-dalton (62K) cell protein reacts with antisera to the 72K polypeptide of the Epstein-Barr virus nuclear antigen (EBNA) in immunoblots. This protein was initially detected in EBNA-negative as well as EBNA-positive cell lines with anti-EBNA-positive human sera. A monoclonal antibody raised against the 72K EBNA and an antiserum from a rabbit immunized with the glycine-alanine domain of EBNA also reacted with the cellular protein. The cellular protein was partially purified from Epstein-Barr virus genome-positive and -negative cell lines. Absorption experiments identified a shared antigenic determinant between the 72K EBNA and 62K cellular protein. A comparison of the 62K protein and EBNA by protease digestion did not reveal similar peptides.     

Inhibition of calcium-dependent actin gelation by actin-binding protein from platelets

Various proteins related to cell contraction have been extracted from human platelets. Of these, a protein (48K) with the molecular weight of 48,000 and one with the molecular weight of 47,000 (P47) often migrate together with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We studied the biochemical characteristics of the 48K protein, purified by actin affinity and DEAE-Sepharose chromatography. The 48K protein did not react with anti-actin antibody or peroxidase-labelled actin. The protein inhibited the calcium-dependent gelation of actin. The 48K protein seemed to be a regulatory protein involving cell contraction not identified before.     

Identification of polypeptide components of the Epstein-Barr virus early antigen complex with monoclonal antibodies.

Three monoclonal antibodies were produced against the Epstein-Barr virus-induced early antigen complex. These antibodies were shown to be specific for the early antigen complex by the fact that they only reacted with cells supporting a permissive or abortive Epstein-Barr virus infection and their synthesis was not affected by inhibitors of viral DNA synthesis. One monoclonal antibody, designated R3, was directed against a diffuse component of the early antigen complex since it reacted by immunofluorescence with cells fixed in acetone or methanol. The other two monoclonal antibodies, designated K8 and K9, reacted with a methanol-sensitive restricted component of this complex. The appearance of the R3 antigen in P3HR-1 superinfected Raji cells occurred approximately 4 h earlier than the antigen detected by K8. By both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmunoelectrophoresis, it was determined that the R3 monoclonal antibody recognized two major polypeptides with molecular weights of approximately 50,000 to 52,000, whereas K8 and K9 precipitated a protein of approximately 85,000. The R3 monoclonal antibody also immunoprecipitated an in vitro primary translation product. It was, therefore, possible to map this product to the Epstein-Barr virus DNA BamH1 M fragment. These in vitro products were slightly smaller than the in vivo proteins, suggesting that these proteins probably undergo posttranslational modification during the virus replication cycle.     

Measles Virus-Specified Polypeptide Synthesis in Two Persistently Infected HeLa Cell Lines

Measles virus-directed protein synthesis was examined in two HeLa cell lines (K11 and K11A) that are persistently infected with wild-type measles virus. Four viral proteins (H, hemagglutination protein; P, nucleocapsid-associated protein; NP, the major nucleocapsid protein; and M, the matrix protein) were readily detected in both cell lines by immune precipitation of [(35)S]methionine-labeled cell extracts followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three (H, NP, and M) of the four viral proteins in both K11 and K11A cells differed from the corresponding viral proteins synthesized in HeLa cells acutely infected with the parental wild-type virus. In addition, the M protein from K11A cells migrated significantly more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the M protein from K11 cells, and there appeared to be slight differences in the H and NP proteins between these two persistently infected cell lines. The altered viral proteins detected in K11 and K11A cells appeared to be the result of viral mutations rather than changes in the host cell, since virus recovered from these cells directed the synthesis of similar aberrant viral proteins in HeLa cells. Virus recovered from K11 cells and virus recovered from K11A cells were both temperature sensitive and grew more slowly than wild-type virus. HeLa cells infected with virus recovered from K11 cells readily became persistently infected, resembling the original persistently infected K11 cells. Thus, viral mutations are associated with persistent measles virus infections in cell cultures.     

Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae.

We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.     

Polypeptides of the Epstein-Barr virus membrane antigen complex.

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.     

Partial purification of the Epstein-Barr virus nuclear antigen(s)

The Epstein-Barr virus nuclear antigen (EBNA) is speculated to be involved in cell transformation by the virus. Studies on the molecular properties of EBNA, however, have yielded conflicting results. In this study, three Epstein-Barr virus(EBV)-induced antigens were isolated and purified from extracts prepared from Raji cells. These antigens were able to block the anticomplement immunofluorescence reaction, indicating that all three were related to EBNA. The soluble antigen was found wholly in the cytosol fraction. An EBV-induced nuclear antigen I was found both in the cytosol and the nucleus. The EBV-induced nuclear antigen II was found associated with the chromatin. The soluble antigen and the nuclear antigen I were separated and partially purified using phosphocellulose chromatography. Each was further purified 1,400-fold with respect to the whole cell state by chromatography on CL-Sepharose 6B followed by blue dextran-Sepharose. subunit molecular weights of 70,000 were determined for each of these antigens, both in the crude and purified state, by radioimmunoelectrophoresis and gel filtration. The nuclear antigen II was purified 2,500-fold using hydroxylapatite, CL-Sepharose 6B, and blue dextran-Sepharose chromatographies. This antigen displayed two subunits by radioimmunoelectrophoresis with molecular weights of 65,000 and 70,000. Although all antigens shared similar molecular weights, the extent of their homology remains to be determined.     

Purification of a major sialoglycoprotein (SGP140) on P12/Ichikawa cells and its expression on differentiated HL-60 cells

SGP140 glycoprotein, a major cell surface sialoglycoprotein with an apparent m.w. of 140,000, was detected on the human T lymphoblastoid cell line P12/Ichikawa by labeling with periodate-tritiated sodium borohydride, followed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Then SGP140 was purified from P12/Ichikawa cells for study of its biochemical character and its distribution in various cell lines. The purification was performed by 0.2% Triton X-100 solubilization from crude membranes, DEAE-Sephacel column chromatography, WGA-agarose column chromatography, Blue-Sepharose 6MB column chromatography, and Sephadex G-150 gel filtration. Antiserum raised against SGP140 was then prepared, and immunoprecipitation and membrane immunofluorescence assay were performed on various cell lines. SGP140 was detected on P12/Ichikawa, Raji, P3HR-1, Daudi, Namalva, BALL-1, MOLT-4B, TALL-1, NALL-1, and K562 cells, but was not detected on HL-60 cells. When HL-60 cells were treated with dimethyl sulfoxide, retinoic acid, or 12-O-tetradecanoylphorbol-13-acetate, SGP140 was detected on cell surfaces. We discuss the possibility that SGP140 may be a differentiation antigen.     

Purification and Characterization of Glycerol Dehydratase from Lactobacillus reuteri

A coenzyme B₁₂-dependent glycerol dehydratase from Lactobacillus reuteri has been purified and characterized. The dehydratase has a molecular weight of approximately 200,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single major band with a molecular weight of 52,000. K_m values for substrates and coenzyme B₁₂ were in the millimolar and the submicromolar range, respectively.     

Component A2 of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum.

Component A2 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum has been purified 370-fold by liquid chromatography. Homogeneity was obtained by anaerobic preparative polyacrylamide gel electrophoresis. Component A2 is a colorless, air-stable protein consisting of a single polypeptide as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The relative molecular mass of the native protein was determined by high-performance, size exclusion chromatography to be Mr 52,000; on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a value of Mr 59,000 was obtained. When cell extract was subjected to N6-ATP-agarose affinity chromatography the methylcoenzyme M methylreductase system was resolved into two fractions; one of them was component A2. This work provides a new operational definition for component A2, i.e., its characteristic chromatographic behavior on N6-ATP-agarose. However, its functional definition is its ability to reconstitute the methylreductase activity with components A1, A3, and C. Several attempts to assign a role to component A2 are reported.     

Characterization of latex allergenic components by capillary zone electrophoresis and N-terminal sequence analysis

In a previous study, protein components purified from latex gloves that elicited allergenic reactions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and yielded apparent molecular weights of 14, 22, 30, 34, 46, and 58 kD. These allergenic components were isolated for further characterization by capillary zone electrophoresis and N-terminal amino acid sequence analysis. These components all migrated at approximately 25 and 35 min on capillary zone electrophoresis. Diode array spectral analysis detected indistinguishable characteristics between these two protein peaks. In addition, complex formation of these components with patients' immunoglobulin was demonstrated by capillary zone electrophoresis. Analysis of components separated by SDS-PAGE on a polyvinylidene difluoride membrane showed that the first 13 residues were identical to the sequence of hevein. Based on the criteria of charge-to-mass ratio and N-terminall amino acid sequence, our results suggest that these components of latex proteins are similar in the primary structure.     

Antibody inhibition of ferripyochelin binding to Pseudomonas aeruginosa cell envelopes

A 14K molecular weight protein which has been shown to bind ferripyochelin has been purified from cell envelopes of Pseudomonas aeruginosa low iron grown cells. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to be free of contamination by lipopolysaccharide or carbohydrate. Antiserum to this protein was made in rabbits and was shown to react with the purified protein by immunoblot assay. The immunoglobulin G fraction of this antiserum blocked binding of [59Fe]pyochelin to isolated cell envelopes of P. aeruginosa in a dose-dependent fashion.     

Purification of phospholipase D from citrus callus tissue

Phospholipase D in extracts of soluble proteins from callus cultures derived from cotyledons of Citrus sinensis (L.) Osbeck is activated by Ca2+ and anionic detergents and has a pH optimum of 6.5. The enzyme was purified 703-fold over the crude protein extract with a yield of 15% by ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic interaction chromatography, and preparative acrylamide gel electrophoresis. Preparative electrophoresis was carried out using conventional slab gel equipment and electroelution of the sliced gel. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified phospholipase revealed two bands of the same staining intensity running at 94.2K and 90.5K.     

Phosphodiesterase-phosphomonoesterases from Fusarium moniliforme. VI. A modified method of purification and identification of isozymes.

Fusarium phosphodiesterase-phosphomonesterase was purified 1,630-fold with 19% yield from dried powder of the culture medium by a modified method consisting of seven steps. The purified preparation was shown to be devoid of inactive protein by disc electrophoresis. The preparation was homogeneous with respect to size as demonstrated by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weights determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 106,000 and 100,000, respectively. The sedimentation coefficient at infinite dilution was 5.71 S. Isoelectric focusing of the purified preparation showed the presence of at least four isozymes with isoelectric points of 6.6, 6.3, 6.2, and 5.9.     

Immunochemical characterization of Epstein-Barr virus-associated early and late antigens in n-butyrate-treated P3HR-1 cells.

Sodium butyrate induces the Epstein-Barr virus cycle in latently infected P3HR-1 cells with a high efficiency. This fact was utilized for the metabolic labeling of the Epstein-Barr virus antigens. Nonproducer Raji cells, lacking both early antigen and viral capsid antigen, were used as controls. Immunoprecipitation patterns were compared with 13 anti-Epstein-Barr virus (viral capsid antigen) - positive and 3 negative sera. Sixteen polypeptides were identified as being associated with the lytic Epstein-Barr virus cycle. Their molecular weights ranged from 31,000 (31K) to 275K on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides, 158K and 165K, could be classified as late viral products on the basis of their sensitivity to cytosine arabinoside. Six of the polypeptides, i.e., 90K, 95K, 134K, 165K, 236K, and 275K, were detected by [(3)H]glucosamine labeling. Among the early, cytosine arabinoside-insensitive polypeptides detected by [(35)S]methionine labeling, a 152K component appears to be a major constituent of early antigen. This polypeptide was precipitated by all anti-Epstein-Barr virus-positive sera tested. As a rule, together with the 103K and 134K polypeptides, the 152K component is precipitated by anti-early antigen, R (restricted) antibodies. In addition, anti-early antigen D (diffuse) antibodies precipitate 31K, 51K, 65K, and 90K components.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Tyrosine hydroxylase purification from rat PC12 cells.

Tyrosine hydroxylase was purified in high yield from rat PC12 cells. This three-day procedure consisted of differential ammonium sulfate precipitation, anion-exchange chromatography, and heparin-Sepharose affinity chromatography. It yielded an average of 15 mg of purified protein from 100 flasks of PC12 cells, with greater than 40% recovery of tyrosine hydroxylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single protein band with a molecular weight of approximately 60,000. The protein had a specific activity of 670 nmol/min/mg and had a Km for its reducing cofactor tetrahydrobiopterin of 1.8 mM. The purified protein can be phosphorylated and activated by cyclic AMP-dependent protein kinase.     

Purification and properties of an endothelial cell growth factor from human platelets

An endothelial cell growth factor has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the factor consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about 20 ng/ml (440 pM). Heparin did not potentiate the activity, nor did the factor bind to heparin immobilized on Sepharose. The purified factor was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin fibroblasts. Chromatofocusing revealed that the pI of the factor was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell growth factor are distinct from previously characterized endothelial cell mitogens with affinities for heparin.