Regulatory factors produced by lymphocytes. II. Inhibition of cellular DNA synthesis associated with a factor inhibiting DNA polymerase alpha activity.

Supernatants of phytohemagglutinin-stimulated human tonsil cells contain two growth inhibitory factors. These factors, called inhibitors of DNA synthesis (IDS), reduce (3)H-thymidine incorporation into mitogen-stimulated lymphocytes and into growing HeLa cells. By Sephadex chromatography, these factors have volumes of distribution corresponding to about 80,000 and 40,000 daltons. Both factors inhibit the activity of calf thymus DNA polymerase alpha in cell-free assays (termed inhibitor of DNA polymerase, IDP). The larger factor, which is chromatographically separable from alpha-lymphotoxin (alpha-LT), is completely inactivated by heating at 70 degrees C for 15 min. This treatment does not destroy alpha-LT. Using supernatants from PHA-stimulated tonsil cells cultured for 5 days in serum-free medium, we attained a 150-fold purification with a succession of molecular sieving, ion exchange, and adsorption chromatographic procedures. Although not purified to homogeneity, the extensive copurification of IDS and IDP activities and their identical heat inactivation profiles suggest that they are the same entity. IDP separated free of alpha-LT inhibits thymidine incorporation into HeLa cells without causing cell death. alpha-LT purified free of IDS does not inhibit thymidine incorporation into HeLa cells, not even at concentrations 7000 times that necessary to kill 50% of growth-inhibited L cell cultures.     

Alterations of DNA-dependent DNA polymerase activities in the immature quail oviduct in response to estrogen stimulation.

Administration of diethylstilbestrol, an estrogen analogue, to immature female quails causes an increase of extractable DNA-dependent DNA polymerase activities from the oviduct. At least two forms of polymerases have been determined, a high molecular weight polymerase (210,000 daltons) and a low molecular weight polymerase (34,000 daltons) calculated from column chromatography Sephadex G-200. During the primary hormone stimulation the amount of extractable enzyme reaches a maximum on the fifth day after daily injections of the hormone. In the period of withdrawal the activities decrease and reach values similar to those determined in the unstimulated oviducts. During secondary stimulation the polymerase activities increase again the first day; subsequently the values decrease drastically. The alterations in enzyme activity correlate with the DNA synthesis in the oviduct, as measured by analytical determination of the DNA content.     

Multiple forms of rat liver mitochondrial DNA polymerase.

Mitochondrial DNA polymerase (DNA polymerase mt) exists in two active forms. DNA polymerase present in crude extract (M-I) and ammonium sulfate precipitate (M-II) stages of purification sediments at 12.1S. The enzyme at the M-II stage of purification has a molecular weight of approximately 250,000 as determined by Sephadex G-200 chromatography in buffers of low ionic strength. In buffers containing 0.15 m NaCl, the enzyme sediments at 9.4S and has a molecular weight of approximately 190,000. When the enzyme is further purified on diethylaminoethyl cellulose (M-III stage of purification), the 9.4S activity predominates. Addition of a polymerase-free fraction from the M-III stage of purification changes the sedimentation coefficient of the enzyme from 9.4 to 12.1S.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Multiple forms of nuclear deoxyribonucleic acid polymerases and their relationship with the soluble enzyme

1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE-cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution of activity between these depends on: (a) the source of the enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed.     

Nonidentity of Some Simian Virus 40-induced Enzymes with Tumor Antigen

The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.     

Biosynthesis of chondroitin sulfate. Solubilization of chondroitin sulfate glycosyltransferases and partial purification of uridine diphosphate-D-galactose:D-xylose galactosyltrans.

UDP-D-Galactose:D-xylose galactosyltransferase, a membrane-bound enzyme which catalyzes the second glycosyl transfer reaction in the biosynthesis of chondroitin sulfate chains, has been solubilized and partially purified from embryonic chick cartilage. Solubilization was effected by treatment of a particulate fraction of a homogenate (sedimenting between 10,000 and 100,000 times g) with the nonionic detergent Nonidet P-40 (0.5%) and KCl (0.5 M) or by the alkali-detergent method described previously (Helting, T. (1971) J. Biol. Chem. 246, 815-822). The applicability of the salt-detergent procedure as a general method for solubilization of membrane-bound glycosyltransferases was tested by assay of four other glycosyltransferases involved in chondroitin sulfate synthesis (UDP-D-xylose:core protein xylosyltransferase, UDP-D-galactose:4-O-beta-D-galactosyl-D-xylose galactosyltransferase, UDP-D-glucuronic acid: 3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine: (GlcUA-GalNAc-4-sulfate)4 N-acetylgalactosaminyltransferase). In each case, greater than 70% of the activity was solubilized and, on gel chromatography on Sephadex G-200, the enzymes appeared largely in included positions and partially separated from each other. After partial purification by gel chromatography on Sephadex G-200, UDP-D-galactose:D-xylose galactosyltransferase was purified further by chromatography on one of several affinity matrices, i.e. xylosylated core protein of cartilage proteoglycan coupled to CNBr-activated Sepharose, a core protein matrix saturated with UDP-D-xylose:core protein xylosyltransferase or UDP-D-xylose:core protein xylosyltransferase covalently bound to Sepharose. The specific activities of the enzyme preparations obtained by these procedures were approximately 1000-fold greater than that of the crude homogenate.     

Extensive charging of transfer ribonucleic acid by bean leaf extracts in vitro

1. Phenol was effectively removed from aqueous extracts of RNA by chromatography on Sephadex G-50. 2. Elution of tRNA from Sephadex G-50 columns at pH7.6 was shown to remove 91% of the endogenously bound amino acids. 3. tRNA prepared without recourse to ethanolic precipitation was capable of accepting much greater amounts of amino acids than could redissolved samples of precipitated tRNA. 4. Aminoacyl-tRNA synthetase enzymes were partially purified with calcium phosphate gel. Elution of enzymes from the gel at pH6.5 yielded a fraction having phenylalanine- and alanine-charging activity, but no aspartate-, lysine- or proline-charging activity, whereas elution at pH7.6 gave a fraction having aspartate-, lysine- and proline-charging activity but no phenylalanine- or alanine-charging activity. 5. By using partially synthetase enzymes and tRNA eluted from DEAE-Sephadex A-50 columns, 52% of the theoretical maximum of aminoacyl-tRNA synthesis was obtained in vitro.     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells.

Epidermal growth factor and insulin need a low concentration of serum to effectively stimulate quiescent 3T3 cells into DNA synthesis. We partially purify a polypeptide component of serum which has no activity by itself but which acts synergistically with epidermal growth factor and insulin to stimulate cultures of 3T3 cells into DNA synthesis as effectively as whole serum. The active fraction is separated from serum by gel chromatography on Sephadex G-100, under acid dissociating conditions, and chromatographs with a molecular weight of 18,000 daltons.We suggest as a general technique, the use of pure growth factors to assay for growth promoting fractions from serum or other sources. Fractions which are not mitogenic by themselves can be detected when assayed together with their complementary pure factors.     

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Small peptides controlling transcription in vitro are bound to chromatin DNA

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 μg/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.     

Characterization of multiple forms of histone phosphatase in rat liver.

By using chromatography on DEAE-cellulose, aminohexyl-Sepharose 4B and Sephadex G-200, rat liver extract was shown to contain at least three fractions, IA, IB and II, of histone phosphatase. Fractions IA and II are probably the same enzymes as the previously described glycogen synthase phosphatase and phosphorylase phosphatase, respectively, but IB exhibits noticeable activities only with phosphohistone as substrate. Approximate molecular weights of 69 000, 300 000 and 160 000 were determined by gel filtration on Sephadex G-200 for IA, IB and II, respectively.     

Low molecular weight mitogenic factor produced by BRL-3A cultured rat liver cells

A new mitogenic factor has been isolated from medium conditioned by BRL-3A rat liver cells. The factor has been partially purified by a two step procedure involving ion exchange chromatography on Dowex 50 followed by gel filtration chromatography on Sephadex G-75 in 1 M acetic acid. The factor is eluted from the Sephadex G-75 column in the low molecular weight region, behin three peaks of multiplication stimulating activity. The factor is inactivated by treatment with trypsin and dithiothreitol, suggesting that it is a peptide that contains a disulfide linkage. Unlike multiplication stimulating activity, the new factor only weakly stimulates DNA synthesis in quiescent chick fibroblasts, whereas it strongly stimulates DNA synthesis in quiescent NIL8 hamster cells, $\text{BALB}\text{c}$ 3T3 cells, and IMR-90 human fibroblasts.     

DNA polymerase of a basidiomycete fungus, Coprinus cinereus.

DNA polymerase activity was studied in Coprinus cinereus, a basidiomycete fungus. Only one from of the enzyme could be demonstrated, whether by affinity or ion-exchange chromatography; this enzyme had a molecular weight of 185000 on Sephadex G-200, and was inhibited by mercaptoethanol. Coprinus, a representative of the most advanced type of the filamentous fungi, resembles other eukaryotic micro-organisms in its lack of a mammalian beta-type DNA polymerase. The properties of the polymerase are compared with those of two other fungi, and found to resemble most closely the yeast polymerase A in Mg2+ requirements and template preference.