Separation of a New Deoxyribonucleic Acid Polymerase from Vaccinia-infected HeLa Cells

HeLa cell deoxyribonucleic acid (DNA) polymerase was purified about 100-fold by sequential column chromatography on phosphocellulose, hydroxylapatite, and Bio Rex 70. A new form of DNA polymerase found in vaccinia virus-infected cells was separated from HeLa DNA polymerase by chromatography on diethylamino-ethyl cellulose. The new form was also purified approximately 100-fold in the same manner as the HeLa DNA polymerase. In addition to chromatographic differences, the two enzymes differed with regard to primer response, relative activity at high pH, inactivation by heat and p-chloromercuribenzoate, and inhibition by vaccinia antiserum.     

Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography.

The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.     

Separation of the Two Constituent Polypeptide Chains of Ricin D

Two constituent polypeptide chains were isolated from performic acid-oxidized ricin D by DEAE-cellulose column chromatography in phosphate buffer, pH 7.0, containing 8m urea or from reduced-carboxymethylated ricin D by gel filtration on Sephadex G-75 followed by DEAE- cellulose column chromatography in Tris-HCl buffer, pH 8.5. Amino acid analyses of the isolated two chains revealed that they were distinct molecules possessing similar molecular weights of near 30,000 and linked by one disulfide bond in ricin D.     

Reversible Inactivation of the Deoxyribonucleic Acid Polymerase of Rauscher Leukemia Virus

Rauscher leukemia virus deoxyribonucleic acid polymerase is reversibly inactivated by 6 m guanidine-hydrochloride. Gel filtration in 6 m guanidine-hydrochloride reveals that the viral deoxyribonucleic acid polymerase consists of a single polypeptide chain of approximately 70,000 molecular weight.     

Phosphorylation of specific polypeptides in isolated murine splenocyte nuclei which is controlled by cyclic nucleotides

Murine splenocyte nuclei were phosphorylated with a less than 10(-5) M concentration of [gamma-32P]ATP at 0 degrees C and the phosphorylated nuclear proteins were analyzed by SDS-polyacrylamide gel slab electrophoresis and Sephadex gel filtration column chromatography. Two polypeptides of 10K and 11K daltons were predominantly phosphorylated. These polypeptides were likely linked by a disulfide bond to form a nonhistone protein of 21K daltons. Both phosphoserine and phosphothreonine were detected in the hydrolysate of the 10.5K dalton polypeptide, while phosphoserine was predominant in the 10K dalton polypeptide. Maximal activation of phosphorylation by cAMP of both polypeptides was shown at a concentration of 10(-6) M. On the contrary, cGMP activated phosphorylation of the 10K dalton polypeptide at 10(-8) M and at 10(-4) M. The phosphorylation of the 10.5K polypeptide was not activated by 10(-4) M cGMP and suppression of the phosphorylation was seen in both polypeptide chains by cAMP at higher concentrations.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Insulin-stimulating peptide from a tryptic digest of bovine serum albumin: purification and characterization

A procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulin-stimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8,496.     

Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high- speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.     

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma.

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpirdium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractioned by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Glycosaminoglycans and proteoglycans of human chondrosarcoma

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpyridium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractionated by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and a proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture

A growth-factor-like substance capable of inducing nontransformed mouse AKR-2B, rat NRK, and EGF-receptorless mouse NR6 cells to form progressively growing colonies in soft agar was identified in acid/ethanol extracts of 17-day mouse embryos. This "mouse embryo factor" (MEF) is similar to previously described transforming growth factors in that it is capable of stimulating DNA synthesis and conferring a reversible transformed morphology on nontransformed cells in vitro. Passage of crude embryo extracts over a Bio-Gel P-60 column gave a major peak of soft agar growth-stimulating activity in the 15,000 molecular weight range with a minor peak at about 22,000. This biological activity was sensitive to treatment with either trypsin or dithiothreitol, but was unaffected by heat (56 degrees C for 30 minutes or 100 degrees C for 3 minutes), indicating that the activity is due to a heat-stable polypeptide(s) with disulfide bonds. Separation of these polypeptide(s) by chromatography on carboxymethyl cellulose revealed two peaks of soft agar growth-stimulating activity which did not cochromatograph with a peak of epidermal growth factor receptor-competing activity. The similarities of this mouse embryo-derived growth factor to previously identified transforming growth factors suggest that both fetal development and neoplastic transformation may be affected by similar mechanisms.     

Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex.

Donor deoxyribonucleic acid strands in the eclipse phase of genetic transformation of pnuemococcus (Streptococcus pneumoniae) are purified as a complex with a cf the deoxyribonucleic acid strand in this complex to digestion by nucleases was shown to be 50- to 1,000-fold less than that of uncomplexed single strands of deoxyribonucleic acid. Deoxyribonuclease I, micrococcal nuclease, Neurospora endonuclease, nuclease P1, and the major endogenous nuclease of cell-free extracts were studied. Sensitivity to nuclease attack was not uniform along the deoxyribonucleic acid strand; sequences of strongly protected bases were separated by more sensitive regions. The minimum size of protected fragments was about 70 bases. A complex of protein with the protected deoxyribonucleic acid segments was obtained after partial digestion. The sizes of these complexes, of the protected deoxyribonucleic acid segments, and of the protein subunit released by complete nuclease digestion, are all approximately identical, as determined by gel exclusion chromatography. Deoxyribonucleic acid strands of eclipse complex were also shown to be particularly well protected from attack by the major pneumococcal endonuclease in cell extracts.     

Expression and purification of hexahistidine-tagged human glutathione S-transferase P1-1 in Escherichia coli

The bacterial expression and purification of human pi class glutathione S-transferase (hGST P1-1) as a hexahistidine-tagged polypeptide was performed. The expression plasmid for hGST P1-1 was constructed by ligation of the cDNA which codes for the protein into the expression vector pET-15b. The expressed protein was purified by either glutathione or metal (Co(2+)) affinity column chromatography, which produced the pure and fully active enzyme in one step with a yield of more than 30 mg/liter culture. The activity of the purified protein was 130 units mg(-1) from the GSH affinity column and 112 units mg(-1) from the Co(2+) affinity column chromatography. The purity of the protein was assessed by electrospray ionization mass spectrometry and size-exclusion chromatography. It showed that the real molecular weight of the hexahistidine-tagged hGST P1-1 polypeptide chain agreed with the calculated value and that the purified protein eluted as an apparent homodimer on the gel filtration column. Our expression system allows the expression and purification of active hexahistidine-tagged hGST P1-1 in high yield with no need of removal of the hexahistidine tag and gives pure protein in one purification step allowing further study of this enzyme.     

Purification and characterization of a murine cellular retinoic acid-binding protein

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE9-4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14,600 in SDS--polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins. However, it is immunologically distinct from the cellular retinol-binding protein.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. II. Products formed by lysis with sperm enzymes and dithiothreitol.

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.