Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2-5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50mug of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg(2+) and K(+) concentrations. 6. Maximal enzyme activity is approached with 40mug of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The (3)H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the (3)H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.     

DNA polymerases of rat liver. Partial characterization and effect of various inhibitors.

Three distinct DNA-dependent DNA polymerase activities have been partially purified from normal rat liver. Soluble activities are separable into two distinct fractions (P1 and P2) by phosphocellulose chromatography. A low-molecular-weight DNA polymerase was isolated from purified nuclei. The enzymes were characterized according to chromatographic and sedimentation behavior, enzymological properties, and response to various inhibitors. The results indicate that fraction P1 corresponds to the high-molecular-weight enzyme and suggest that polymerase P2 may be derived from partial dissociation of the high-molecular-weight enzyme. The molecular weight of polymerase P1 was estimated to be about 250 000 by Sephadex column chromatography. Both fraction P2 and nuclear DNA polymerase appeared to be low-molecular-weight enzymes. However, the molecular size of these activities was apparently different. The estimated molecular weights of nuclear and P2 enzyme are about 40 000 and 25 000, respectively. As with the nuclear enzyme, polymerase P2 (but not P1) appeared to be free of detectable exonuclease activity. All of these polymerases showed a marked preference for initiated polydeoxyribonucleotide templates. The rat liver polymerases differed in their ability to use poly[d(A-T)-A1 primer-template, as is shown by the ratios of their activity with this synthetic polymer to that with activated DNA: 0.5, 2.75, and 1.34 for P1, P2, and nuclear polymerase, respectively. Denatured DNA was a poor template for both enzymes P1 and P2, but it was inert as template for the nuclear enzyme. Although each of these polymerases required all four deoxynucleoside triphosphates for maximal activity, they catalyzed a high rate of synthesis in the absence of one or more deoxynucleoside triphosphates. Such a 'limited' synthesis was much more extensive for polymerase P2 and nuclear enzyme than for P1 was the most sensitive of the three to sulphydryl reagents, ehtidium bromide, heparin, and single-stranded DNA. The responses of P2 and nuclear enzymes to various inhibitors were very similar. However, these two enzymes respond differently to heat and high ionic strength.     

Nuclear Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerases from Saccharomyces cerevisiae

Two deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerases (I, II) have been solubilized from isolated Saccharomyces cerevisiae nuclei. The enzymes can be separated by chromatography on O-diethylaminoethyl Sephadex. Both enzymes are active with high-molecular-weight nuclear yeast DNA, although RNA polymerase I has a higher affinity for polydeoxy-adenylic-thymidylic acid and RNA polymerase II for denatured DNA. RNA polymerase I is active only with manganese. alpha-Amanitin inhibits only the activity of RNA polymerase II.     

Studies on deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Variations of the enzyme activity during growth

1. RNA polymerase activity of Escherichia coli extracts prepared from cells in exponential and stationary phases of growth, when measured in the presence and absence of external template, showed significant qualitative differences. 2. In both extracts, polymerase activity was higher when assayed with external template, suggesting the presence of a pool of enzyme not bound to cellular DNA. 3. In the crude extract, the fraction of enzyme bound to cellular DNA is higher during the exponential phase of growth. 4. A method is described for the purification of enzyme molecules not tightly bound to cellular DNA from exponential- and stationary-phase cultures. 5. Purified enzyme preparations showed differences in template requirement and subunit composition. 6. On phosphocellulose chromatography of stationary-phase enzyme, a major portion of polymerase activity eluted from the column with 0.25m-KCl. In the case of exponential-phase enzyme, polymerase activity eluted from a phosphocellulose column mainly with 0.35m-KCl. 7. Enzyme assays done with excess of bacteriophage T(4) DNA showed a strong inhibition of stationary-phase enzyme by this template. The exponential-phase enzyme was only slightly inhibited by excess of bacteriophage T(4) DNA.     

Acyl-CoA-ACP-transacylases of Mycobacterium smegmatis. An analytical study

Homogenates of Mycobacterium smegmatis have been prepared by sonication of bacteria in 0,1 M phosphate buffer, pH 7, containing 0,001 M EDTA and 0,01 M 2-mercaptoethanol. The 105 000 g supernatant of these homogenates has been fractionated by ammonium sulfate precipitation. The fraction precipitating between 25 and 65 per cent saturation in ammonium sulfate shows palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase activities. These activities are not associated, essentially, to a complexe of high molecular weight as they are retained on the column for 80 per cent or more upon gel filtration.By chromatographic procedures involving Sephadex G-150 gel filtration, and chromatography on DEAE-Sephadex A-50 the malonyl-CoA-ACP-transacylase activity is separated from the other two activities. The palmityl-CoA-ACP-transacylase activity and the acetyl-CoA-ACP-transacylase activity show two close but distinct major peaks either upon gel filtration on Sephadex G-150 or upon DEAE-Sephadex A-50 chromatography indicating thus that these two activities are attribuable to two distinct molecular entities. Besides the major peaks for these two activities, a supplementary minor peak for each activity is observed.An estimate of the approximate range of the apparent molecular weights for palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase has been made in using the curve of the K_av values of known proteins plotted against the logarithms of their molecular weights. The molecular weights thus estimated for these three enzymes are 100 000, 32 000 and 85 000 respectively.The void volume fractions of the chromatography of the homogenates on Sephadex G-150 present 20 per cent or less of the enzyme activity of the homogenates. Chromatography of the mixture of the void volume fractions on DEAE-Sephadex A-50 shows that the malonyl-CoA-ACP-transacylase activity associated to these fractions behaves differently from that of the fractions retained upon gel filtration; this may be taken as an indication for the presence of a complexe of the fatty acid synthetase present as a minor component of the homogenates.     

Identification of novel calcium binding proteins of heart and brain 100,000 x g supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.     

Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.     

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.     

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.     

Heme deficiency of soluble guanylate cyclase in rat platelets]

Analysis of soluble guanylate cyclase of rat platelets (105,000 g supernatant) revealed no activating effect of sodium nitroprusside on the enzyme activity. Dithiothreitol (2 x 10(-4) H) added to the sample stimulated the basal activity of guanylate cyclase in the presence of Mg2+ but did not induce the enzyme activation by sodium nitroprusside. Hemoglobin added to the enzyme did not influence its basal activity or the activating effect of sodium nitroprusside. DEAE-Cellulose chromatography of the 105,000 g supernatant revealed two protein peaks, I and II, of which only peak II possessed a guanylate cyclase activity. Fraction I added to a partly purified enzyme did not change the enzyme activity, nor did it enhance the sodium nitroprusside-induced activation of guanylate cyclase. Spectral analysis of the 105,000 g supernatant revealed that the presence of a maximum at 415-425 nm (Soret band) depended on the degree of plasma hemolysis. In the absence of hemolysis the Soret band was unobserved either in the 105,000 g supernatant or in fractions I and II. It is suggested that rat platelet guanylate cyclase is present in these cells in a heme-deficient state.     

Staphylococcal Hyaluronate Lyase: Purification and Characterization Studies

Staphylococcal hyaluronate lyase (hyaluronidase) derived from a pathogenic strain of staphylococcus was purified by means of salt fractionation with ammonium sulfate and gel filtration through Sephadex G-100. Most of the enzyme activity from concentrated culture supernatant fluids of staphylococci was obtained in a fraction precipitated by 90 to 100% saturation with ammonium sulfate. A small amount of enzyme was also precipitated by 80 to 90% saturation with the salt. The hyaluronidase-rich fractions did not contain other staphylococcal enzymes, such as coagulase, protease, lipase, and staphylokinase. These enzymes were present in the original concentrates. Molecular sieving chromatography of the partially purified enzyme by filtration through Sephadex G-100 resulted in a further increase in specific enzyme activity. However, more than one active peak was obtained after gel filtration, thus suggesting that there may be more than one molecular form of the enzyme. Immunodiffusion in agar gel of the chromatographically purified enzyme fraction, with immune serum from rabbits injected with concentrated staphylococcal culture supernatant fluids, indicated that there was one major antigen. A similar antigen, giving reactions of identity with the purified material, was present in the original culture supernatant fluid.     

Tetrahymena alpha-type DNA polymerase A: purification and subunit analysis

DNA polymerase A (an alpha-type polymerase) from the ciliate, Tetrahymena pyriformis, has been purified 260,000-fold (40,000 units/mg protein). The polymerase A did not show any heterogeneity in terms of size and charge during purification. Enzymatic properties of the DNA polymerase A remained unchanged during the purification. Two-dimensional gel electrophoresis revealed that in the first dimension (isoelectric focusing agarose gel), the activity of the purified enzyme was focused at around pH 5.5 and that in the second dimension (sodium dodecyl sulfate-polyacrylamide gel), 135,000- and 66,000-dalton polypeptides emerged from the activity peak at a stoichiometric ratio of about 1:3. The native molecular weight of the DNA polymerase A estimated from the stoichiometric subunit ratio approximately coincided with that estimated from gel filtration on Sephadex G-200 under low ionic strength conditions. The present results strongly suggest the existence of a common high-molecular-weight catalytic core subunit in alpha-type polymerases of eukaryotes.     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Conversion of DNA polymerase extracted from rat ascites hepatoma cells

DNA polymerase extracted fresh from rat ascites hepatoma cells possesses high molecular weight, maximal activity at neutral pH, and high sensitivity to N-ethylmaleimide (NEM). After physical and chemical treatment of the enzyme fraction, the appearance of low molecular weight DNA polymerase was detected by means of Sephadex gel filtration or sucrose density gradient centrifugation. This low molecular weight DNA polymerase possesses alkaline pH optimum, preference of native DNA as template/primer, and relative resistance to NEM.     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

Fractionation of RNA polymerase subspecies from soybean hypocotyls by isoelectric focusing in Sephadex

RNA polymerase enzymes isolated from chromatin of 6-day-old soybean hypocotyls are resolved into two major and one minor species of activity by DEAE-Sephadex chromatography. A soluble form of the enzyme, isolated from the postchromatin supernatant fraction, shows a broad peak of activity when fractionated by this method. The elution characteristics and α-amanitin sensitivity data indicate the two major chromatin-bound activities to be Class I and III enzymes, while the minor chromatin-bound activity and the soluble enzyme are representative of the Class II enzymes. In contrast to these profiles, fractionation of these enzyme preparations by the new method of isoelectric focusing in Sephadex G-15 yields five distinct chromatin-bound and four soluble subspecies. The relationships of these observed activities to their parent DEAE classes are investigated, showing two subspecies within the Class I and III RNA polymerase enzymes, respectively, and four subspecies within the Class II enzymes.