Phosphocellulose Column Research Articles

DNA-dependent ATPases from Escherichia coli K12

Four DNA-dependent ATPases have been isolated from E. coli extracts. ATPases I and III, both sensitive to NEM, require denatured DNA but differ in their heat sensitivity, elution from DEAE-cellulose, and sedimentation coefficient. ATPases II and IV are both resistant to NEM. ATPase II requires partially denatured DNA, whereas ATPase IV can be stimulated by SS DNA. ATPase I is a DNA-unwinding enzyme; ATPase II may be involved in recombination.

Biopterin. V. De novo synthesis of dihydrobiopterin: evidence for its quinonoid structure and lack of dependence of its reduction to tetrahydrobiopterin on dihydrofolate reductase.

AbstractSamples of quinonoid‐l‐erythrodihydrobiopterin (q‐BH2) and quinonoid‐6‐methyl‐dihydro‐pterin (q‐6‐MPH2) were prepared by oxidation of l‐erythro‐5,6,7,8‐tetrahydrobiopterin (BH4) and 5,6,7,8‐tetrahydro‐6‐methylpterin (6‐MPH4) and separated from D‐erythro‐7,8‐dihydrobiopterin (7,8‐BH2) and 6‐methyl‐7,8‐dihydropterin (7,8‐6‐MPH2) as well as from the tetrahydropterins on phosphocellulose column by high‐pressure liquid chromatography. The quinonoid dihydropterins were identified and quantitated by scan of their ultraviolet absorption and fluorescence emission spectra through their rearrangement to their 7,8‐tautomer and also by gas chromatography of their rapidly synthesized trimethylsilyl derivative. Identification was also achieved by the enzymatic reduction of [3H]q‐BH2to [3H]BH4 by dihydrofolate reductase (DHFR). Direct proof for the enzymatic synthesis of the q‐BH2 from GTP or from 2‐amino‐6‐(5′‐triphosphoribosyl)‐amino‐5‐ or ‐6‐formamido‐6‐hydroxypyrimi‐dine (FPyd‐P3) was obtained by isolation of the compound which was identical in all respects to the q‐BH2 obtained by chemical synthesis from BH4. The reduction of enzymatically synthesized q‐BH2 by dihydropteridine reductase (DHPR) to BH4 was not inhibited by methotrexate (MTX). When the enzymatically synthesized q‐BH2 was converted to 7,8‐BH2, it was reduced only by DHFR. This reduction, however, was inhibited by MTX. On the biosynthetic pathway from GTP to dihydrobiopterin, the enzyme responsible for the appearance of the quinonoid structure is the d‐erythro‐dihydroneopterin triphosphate synthetase, the product of which (quinonoid d‐erythro‐dihydroneopterin triphosphate) is converted to quinonoid dihydrobiopterin by l‐erythro‐dihydrobiopterin synthetase. Experiments in vivo established that DHFR does not participate in the reduction of dihydrobiopterin to tetra‐hydrobiopterin when the former is synthesized from GTP de novo. MTX at 5 × 10−6M exerted no inhibition on the reduction of the biosynthetic dihydrobiopterin to tetrahydrobiopterin in vivo, yet completely inhibited the reduction of intraventricularly injected tritiated dihydrofolate ([3H]FH2) to tritiated tetrahydrofolate ([3H]FH4).

Purification and characterization of the chick oviduct progesterone receptor a subunit

Abstract The DNA-binding A subunit of the chick oviduct progesterone receptor has been purified approximately 20,000-fold to apparent homogeneity. The procedure employs phosphocellulose, DNA-cellulose and DEAE-cellulose column chromatography. Purity has been assessed by polyacrylamide gel electrophoresis in two denaturing systems. The protein is obtained with about 20% of the available ligand sites complexed with labeled progesterone. Dissociation kinetics and displacement studies show the protein is identical to the crude starting material. Molecular weight (Mr) determined by gel electrophoresis in l% Na dodecyl SO 4 is 79,000. Values for Stokes radius (46 A) and sedimentation coefficient (3.6 S) are in agreement with the properties of the crude A protein and give an indicated molecular weight of 72,000 for the native protein. The purified protein retains its DNA-binding ability to both DNA cellulose columns and to free DNA using a nitrocellulose filter adsorption assay. The latter assay shows no saturability nor DNA sequence specificity under the conditions employed. The binding is enhanced 8-fold by heat denaturing the DNA.

The mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena are indistinguishable

The mitochondrial and cytoplasmic valyl tRNA synthetases from Tetrahymena pyriformis are indistinguishable. These synthetases cannot be differentiated through hydroxylapatite, DEAE-cellulose, or phosphocellulose column chromatography. Both enzymes show the same mean sedimentation coefficient of 5.9 S in sucrose gradient centrifugation analysis; when bound with tRNA, they are relatively stable and sediment at 7.8 S. The temperature optimum for aminoacylation reaction is 27.5 °C, the optimum Mg 2+ concentration is 4.4 m m, and substrate affinities ( K m values) for valine and ATP in aminoacylation are the same for both enzymes at 1.0 μ m and 2.5 m m, respectively. These enzymes show identical specificities for acylation of different tRNA species, i.e., Tetrahymena and rat liver tRNAs can be equally well recognized, but no significant acylation can be observed with Escherichia coli and Saccharomyces tRNAs. These observations suggest the probable molecular identity of mitochondrial and cytoplasmic valyl tRNA synthetases in Tetrahymena.

A low molecular weight DNA polymerase beta in the sea urchin Strongylocentrotus purpurantus. Partial purification, properties, and changes in development.

A low molecular weight DNA polymerase which sediments at 3.3 S on sucrose gradients has been purified from total cell homogenates of rapidly dividing embryos of the sea urchin Strongylocentrotus purpuratus. In the presence of 2 mM N-ethylmaleimide, it is the major polymerase activity in whole cell homogenates when assayed with an oligo(dT)10.poly(dA)200 template; a template which it uses about 200 times more efficiently than activated DNA. The requirement for N-ethylmaleimide exists only in crude cell fractions where it acts to inhibit a template digesting nuclease activity. The polymerase is highly stable if maintained in the presence of 20% glycerol, is completely dependent on added template, and shows no end addition activity. The physical and enzymatic properties of this enzyme clearly distinguish it from the DNA polymerase previously described by Loeb (Loeb, L. A. (1969) J. Biol. Chem. 244, 1672-1681) which sediments as a high moeluclar weight (5.6 to 6.6 S) enzyme and prefers the activated DNA template. In addition, these two DNA polymerase enzymes show distinctive chromatographic properties using DEAE-cellulose and phosphocellulose columns as well as their sensitivity to N-ethylmaleimide. The properties of the low molecular weight polymerase indicate close similarity to the beta-polymerase isolated from mammalian cells. These low molecular weight enzymes are both sensitive to phosphate salt and able to utilize the artificial ribohomopolymer template oligo(dT)10.poly(rA)200. A quantitative analysis of the low molecular weight DNA polymerase during early embryonic development indicates that the activity of this enzyme increases at least 2-fold immediately following fertilization and again during early blastula stage (hatching). Such quantitative changes in a beta enzyme activity are in contrast to findings with the alpha-polymerase which remains constant during early development.

Isolation and some properties of two deoxyribonucleases from a snail, Achatina fulica.

1. Two main DNases were found in the dried liver extract of a snail, Achatina fulica. They were purified by the phosphocellulose batch method and by phosphocellulose column chromatography. The enzyme eluted earlier from the phosphocellulose column was designated as Achatina DNase-1 and the other as Achatina DNase-2. DNase-1 was purified further by QAE-Sephadex A-25 column chromatography (twice) just before use because of the instability of the purified enzyme. By these procedures, DNase-1 and 2 were purified 200- and 130-fold, respectively. 2. Divalent or monovalent cations had no marked effect on either enzyme. The showed pH optima of 4.8 (DNase-1) and 5.2 (DNase-2). Ionic strength was found to be critical for the maximal activity. The isoelectric points of DNase-1 and 2 were both 6.9. On heating at 70--75 degrees C for 5 min, each enzymic activity fell to half of the initial value. 3. The enzyme preparations degraded native DNA 1.5--2.5 times faster than heat-denatured DNA. They both degraded heat-denatured DNA endonucleolytically, to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphoryl and 5'-hydroxy termini of the resulting oligonucleotides were analyzed. DNase-1 possessed marked specificity for dThd at 3'-termini and dAdo at 5'-termini in the early stages of degradation, but only for dAdo at 5'-termini in the later stages. DNase-2 showed some preference for purine nucleotides at both 3'- and 5'-termini in the later stages of degradation.

Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta.

DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.

In vitro HeLa cell DNA synthesis II. Partial characterization of soluble factors stimulating nuclear DNA synthesis

When HeLa cells are lysed in hypotonic buffer and the lysate is fractionated by centrifugation into particulate and soluble components, the particulate component (nuclei) is deficient compared to unfractionated lysate in the extent to which it can carry out in vitro DNA synthesis. In addition, rapidly labeled short DNA chains (Okazaki pieces) accumulate in purified nuclei, and are chased into higher molecular weight DNA to a lesser degree than in unfractinated lysate. When purified nuclei were reconstituted with soluble component, the capacity of the nuclei for in vitro DNA synthesis was fully restored, as was the capacity of the nuclei for conversion of Okazaki pieces to higher molecular weight DNA. This suggests that the soluble component contains a factor or factors necessary for normal DNA replication. The major incorporation-stimulating activity was partially characterized and partially purified from the soluble component. It is heat labile, non-dialyzable, partially recoverable in the supernatant after pH 5 precipitation, found mainly in a 55–85% saturated (NH 4) 2SO 4 fraction, and is included on Sephadex G-100. After passage through Sephadex G-100, the activity displays increased instability to storage at either 4°C or −70°C. Part of the activity does not bind to phosphocellulose at pH 7.2 and low salt; no additional activity can be recovered in a 0.5 M KCl wash of the phosphocellulose column. The Okazaki-piece-joining activity was found, along with the bulk of the incorporation-stimulating activity, in the 55–85% (NH 4) 2SO 4 fraction. These findings provide some of the groundwork for future attempts to completely purify and characterize those activities in the soluble component of cell lysates which are involved in DNA replication.

Poly(A) polymerases of rat liver nuclei Purification and specificity

Two poly(A) polymerases were isolated from rat liver nuclei and purified more than one thousand times by ion exchange chromatography on DEAE-Sephadex and phosphocellulose columns as well as affinity chromatography on a chromosomal RNA-Sepharose column. One of the two enzymes is bound to chromatin and uses as primer chromosomal RNA, while the second one is localized in the nucleoplasm and uses as primer poly(A) and hnRNA isolated from chromatin. The two enzymes seem to participate in the polyadenylation of chromosomal RNA in vitro, by a coupled mechanism. According to this mechanism, the chromatin bound enzyme adds 120–130 adenosine nucleotides to chromosomal RNA and consequently the nucleoplasmic enzyme completes the polyadenylation by adding 80–90 more AMP units to the polyadenylated end of chromosomal RNA.

Distribution of AMP-deaminase isozymes in rat tissues.

1. The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique employing specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kidney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B-C hybrids. 2. Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney and lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throughout development. In brain a shift of B to five isozymes is observed during development. Isozyme A is the predominant form in muscle throughout postnatal development. 3. AMP deaminase in the regenerating liver was analyzed, but the data indicated that there was no change of isozyme distribution during hepatic regeneration.

Purification of four pyruvate kinase isozymes of rats by affinity elution chromatography

1. 1. Purification of four isozymes of pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) L, M 1, M 2 and R was much improved to give good yields by affinity elution chromatography. The enzyme was eluted from a phosphocellulose column with 0.5 mM phosphoenolpyruvate. Type L, M 2 and R were stabilized with fructose 1,6-diphosphate throughout the purification procedures. 2. 2. The isozymes were crystallized under various conditions: types L and R were readily crystallized from medium of low ionic strength, types L, M 1, and M 2 were crystallized from ammonium sulfate solution in different forms in the presence and absence of phosphoenolpyruvate. Type M 1 was also crystallized in different forms in the presence and absence of fructose 1,6-diphosphate. 3. 3. Amino acid analyses showed that the compositions of types L and R, and of types M 1 and M 2, respectively, were very similar.

Effects of Adenosine Triphosphate and Magnesium Chloride on Affinity Elution of Aminoacyl-Transfer Ribonucleic Acid Synthetases from Phosphocellulose with Transfer Ribonucleic Acids1

Aminoacyl-tRNA synthetases of bakers' yeast (Saccharomyces cerevisiae) were adsorbed to a phosphocellulose (P-cellulose) column, and those specific for tyrosine [EC 6.1.1.1], threonine [EC 6.1.1.3], valine [EC 6.1.1.9], and isoleucine [EC 6.1.1.5] were eluted with several specific tRNAs. Elutions of these synthetases were affected by ATP and/or MgCl2. The effects of ATP and MgCl2 differ with synthetases. Elutions of tyrosyl- and valyl-tRNA synthetases with their cognate tRNAs were more specific in the presence of MgCl2. Isoleucyl-tRNA synthetase was eluted with its cognate tRNA in the presence of both ATP and MgCl2. On the other hand, threonyl-tRNA synthetase was eluted in the absence of ATP and MgCl2 with unfractionated tRNA but not with some non-cognate tRNAs. This suggests that elution of threonyl-tRNA synthetase is highly specific. The present data on the effects of ATP or MgCl2 or both on this affinity elution will be useful for simple and rapid purification of the synthetases.

Purification of cholera toxin and its subunits: new methods of preparation and the use of hypertoxinogenic mutants.

Cholera toxin was obtained in pure form by fractionation on two phosphocellulose columns successively. Cholera toxin and choleragenoid were quantitatively and selectively adsorbed to the first column in 10 mM phosphate buffer, pH 7.0, and were subsequently eluted with buffer of high ionic strength. The toxin was then separated from choleragenoid on the second column by chromatography at pH 8.3. The toxin obtained was highly active and pure as judged by electrophoresis, isoelectric focusing, and various immunological and chemical tests. Pure choleragenoid was by-product of the procedure. The A1 chain of the toxin was obtained in pure form by treating phosphocellulose-bound toxin with urea and a reducing agent. The anionic A1 peptide was thereby released, leaving a complex of the B and A2 chains (A25B) bound to the resin. The latter was then eluted and further purified to obtain nontoxic antigen. The overall yields of cholera toxin and choleragenoid were increased two- to threefold by the use of hypertoxinogenic mutants of Vibrio cholerae.

Purification and characterization of varicella-zoster virus-induced DNA polymerase

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.

Hydrocortisone receptors of hormone-responsive and irresponsive embryonic chick retina

The activated cytoplasmic hydrocortisone-receptor complexes from both hormoneinsensitive (irresponsive to induction of glutamine synthetase by hydrocortisone) 7-day retina and hormone-sensitive (responsive to induction of glutamine synthetase by hydrocortisone) 12-day retina have been fractionated and identified by chromatography on phosphocellulose columns. The sedimentation patterns of these complexes are similar and indistinguishable from their nuclear counterparts. The retention pattern, salt extractability and the sedimentation properties of the nuclear complexes from both 7-day and 12-day retina also appear to be very similar. These results strongly suggest that the majority of the hydrocortisone receptors involved in the interaction with the nuclear acceptor sites are common to both the 7-day and the 12-day retina and that the factors controlling the age-related response of the retina to hydrocortisone lie at a step beyond the receptor-acceptor interaction.

Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity.

Purified avian myeloblastosis virus reverse transcriptase contains two subunits that are structurally related. The large subunit, beta (molecular weight, 95,000), was converted in vitro by chymotrypsin into a polypeptide of molecular weight 63,000. This polypeptide was indistinguishable from the small subunit, alpha (molecular weight, 65,000), in its chromatographic behavior on the phosphocellulose column and its tryptic peptide composition. During this proteolytic conversion, a polypeptide of molecular weight 32,000 (fragment B) was obtained. It was composed of tryptic peptides unique to beta and appeared to be derived from the portion of the beta subunit that was cleaved off during the conversion of beta into alpha. Upon continued proteolysis, a smaller polypeptide of molecular weight 24,000 (fragment A) was generated. This polypeptide manifested only RNase H activity and shared common amino acid sequences with beta and alpha subunits. Fragment A did not share any amino acid sequence homology with fragment B.

Isolation and some properties of a deoxyribonuclease from Turbo cornutus.

1. Four DNases were found in the dried liver extract of a top shell, Turbo cornutus. The major one was purified 120-fold by phosphocellulose column chromatography, sulfoethylcellulose column chromatography and gel-filtration on Sephadex G-150. The yield was 2.7%. 2. The enzyme activity was not affected by Mg2+ (10(-3)--10(-2)M), EDTA (10(-3)--10(-2)M), or NaCl (10(-1)M). It showed a pH optimum of 4.7--4.8. Ionic strength was found to be critical for the maximal activity. The isoelectric point was 8.5--9.0. On heating at 50 degrees C C for 5 min the enzymic activity fell to half the initial value. 3. The enzyme preparation degraded native as well as heat-denatured DNA, but not RNA. It degraded heat-denatured DNA endonucleolytically to give oligonucleotides with 3'-phosphates. 4. The 3'-phosphate and 5'-hydroxy termini of oligonucleotides were investigated. At both the 3'- and 5'-terminal positions, purine nucleotides were predominant.

Nuclear and nucleolar protein phosphokinases in novikoff hepatoma

1. 1. Two chromosomal protein phosphokinases and their substrates were partially characterized in nuclei and nucleoli of Novikoff hepatoma. 2. 2. Two nucleolar protein knase peaks eluted from a phosphocellulose columns with 0.5 and 0.7 M NaCl. These two peaks of enzymatic activity differed in their divalent cation optima and substrate specificities from the corresponding nuclear enzymes. 3. 3. It was concluded that Novikoff hepatoma nucleoli contain at least two specific protein phosphokinase-substrate systems.

T7 gene 6 exonuclease has an RNase H activity

T7 gene 6 exonuclease has been shown to have an RNase H activity as well as a double-strand specific DNase activity by the following experiments: The RNase H activity coelutes with the DNase activity from DEAE-cellulose, phosphocellulose, hydroxyapatite, and Sephadex G-200 columns. Gene 6 exonuclease specified by a T7 strain with a temperature sensitive mutation in gene 6 has an extremely heat-labile RNase H activity as well as a heat-labile DNase activity. T7 gene 6 exonuclease degrades the RNA region of a poly(A) . poly(dT) hybrid polymer exonucleolytically from the 5' terminus, releasing a ribonucleoside 5'-monophosphate product. When the RNA strand of a 0X174 RNA . DNA hybrid molecule synthesized with E. coli RNA polymerase is degraded, a ribonucleoside triphosphate is produced from the 5'-triphosphate terminus. Participation of T7 gene 6 exonuclease in the removal of primer RNA in discontinuous replication of T7 DNA is discussed.

Chapter 10. Purification and Assay of Nuclear Protein Kinases

This chapter discusses methods for fractionation and assay of the chromatin-associated protein kinases involved in catalyzing the phosphorylation of these proteins. The general approach involves purification of phosphorylated nonhistone chromatin proteins through salt extraction, chromatography on phosphocellulose columns involving a salt/pH step gradient, and assay for protein kinase activity in the absence of exogenous substrate. In addition, a step in the column elution increases the complexity of the protein kinase profile by a substantial amount. These enzyme activities are presumably left on the column under conditions employed by others. Thus, the methodology described in the chapter has several distinct advantages over the techniques previously used. The results indicate the existence of a large number of distinctly different enzymes involved in the phosphorylation of chromatin-associated proteins.