DNA polymerases of ascites hepatoma cells. I. Purification and properties of a DNA polymerase from soluble fraction

DNA polymerase from soluble fraction of ascites hepatoma cells has been purified about 490-fold. The polymerase requires template DNA, all four deoxyribonucleoside triphosphates, and magnesium ions for the reaction. Optimal activity was found at pH 7.0 – 7.5, with 3 – 8 mM magnesium chloride, and 20 – 40 mM potassium phosphate. The purified enzyme utilizes preferentially DNA treated with pancreatic DNase as template.     

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.     

DNA Polymerase of Murine Sarcoma-Leukemia Virus: Lack of Detectable RNase H and Low Activity With Viral RNA and Natural DNA Templates

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.     

Proteinase Enzyme System of Lactic Streptococci: I. Isolation and Partial Characterization1

Proteinase activity of Streptococcus lactis cells was maximal at pH 6.0, but after storage at 3 C the minimal activity was observed at this pH. Activity lost as a result of storage could be restored by adding glutathione. Whole cells were fractionated into soluble (intracellular) and particulate fractions by sonic disruption; both fractions contained enzymatic activity. Activity of the soluble (intracellular) fraction was found to be stable to storage at 3 C, but was inhibited progressively with increasing concentrations of p-hydroxymercuribenzoate (PHMB). The enzymatic activity of this fraction was not activated by ferrous or magnesium ions or by cysteine. In contrast, activity of the particulate fraction was labile to storage at 3 C, and the reduction was comparable to that of stored cells. Furthermore, proteinase activity in the cells and the particulate fraction was not affected by addition of PHMB. The particulate fraction was activated by ferrous and magnesium ions and by cysteine. After storage, only ferrous ion and cysteine promoted reactivation; magnesium ion was totally ineffective. The enzyme(s) contained in the particulate fraction may be involved in decreased proteinase activity observed in whole cells and in the effect on growth of cells after storage.     

CRYSTALLIZATION OF PEPSIN FROM ALCOHOL

1. Pepsin is soluble in 65 per cent alcohol and may be readily crystallized from 20 per cent alcohol. The crystals appear as needles or plates which may be transformed into the usual hexagonal bipyramids by recrystallization from water. The different crystals are probably two crystalline forms of the same chemical substance. 2. The enzyme is quite stable in 20 per cent alcohol at pH 2.0 but is inactivated by high concentrations of alcohol. 3. The enzyme is stable for several hours in 65 per cent alcohol at pH 4.0 to 5.0 but is rapidly inactivated in more acid solution. 4. No increase in activity could be noted after treatment with hydrogen peroxide. 5. No proteolytic activity either before or after treatment with hydrogen peroxide could be found in trichloracetic acid filtrates, butyl alcohol extracts of pepsin preparations, or oxidized phenylhydrazine solutions.     

DISTRIBUTION OF ENZYMES CLEAVING PYRIDINE NUCLEOTIDES IN ANIMAL TISSUES

1. The distribution of DPN and DPNH pyrophosphatases and DPNase in centrifugally prepared fractions of organs of several species of animals is reported. 2. A DPNH pyrophosphatase was found in the soluble fraction of pigeon and of rabbit liver. This enzyme did not split DPN but accounted for over 50 per cent of the DPNH pyrophosphatase activity of the whole homogenates. 3. All the organs tested, including the pigeon liver and rabbit liver, contained a microsomal pyrophosphatase that attacked both DPNH and DPN. This microsomal enzyme split DPNH faster than DPN in all cases. 4. DPN pyrophosphatase and DPNase activity were generally concentrated in the microsomal fraction of liver, of kidney, and of brain. 5. The DPNase of hamster liver was virtually inactive at pH 7.5 but was optimally active at pH 5.5. Considerable difference was found with respect to pH on the activity of DPNase from organs of different animals. 6. The inhibition of mitochondrial and microsomal DPNH oxidation by nicotinamide was noted during the course of these experiments. 7. The significance of some of the distribution patterns is discussed.     

Identification of gamma-like DNA polymerase from sea urchin embryos.

A gamma-like DNA polymerase devoid of DNA polymerase-alpha and -beta activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) . oligo(dT)12--18 followed by poly(dA) . oligo(dT)12--18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-gamma.     

AXONAL TRANSPORT OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE IN TOAD SCIATIC NERVE

—The presence of phenylethanolamine-N-methyltransferase (EC 2.1.1.-) and dopamine-β-hydroxylase (EC 1.14.2.1) activities was demonstrated in the sciatic nerve of the toad, Bufo marinus. The rates of accumulation of phenylethanolamine-N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DBH) proximal to a ligation of the sciatic nerve were studied. DBH accumulated proximal to the ligation at a more than 10-fold faster rate than PNMT. By measuring the rate of loss of enzyme activity distal to a ligation, an estimate of per cent clearance of each enzyme was made. Based on the per cent of enzyme activity free to move, the absolute transport rates for each enzyme were estimated to be: PNMT, 3.6 mm/24 h; DBH, 102 mm/24 h. PNMT activity (89 per cent) was recovered in the soluble fraction of sciatic nerve homogenates with no change occurring in the subcellular distribution of the enzyme proximal to ligations. In contrast, 43 per cent of DBH activity was found in the soluble fraction of sciatic nerve homogenates; but a disproportionate increase in paniculate DBH activity was found proximal to sciatic nerve ligations. Reduction of toad body temperature to 4°C resulted in a complete but totally reversible block of the axonal transport of both PNMT and DBH.     

THE ENZYMIC HYDROLYSIS OF PHOSPHATIDYL INOSITOL BY GUINEA PIG BRAIN:

Abstract—

• 1 Phosphatidylinositol hydrolase activity of homogenates of guinea pig brain was studied by using [2-³H]inositol labelled substrate and measuring the release of radioactivity into the acid soluble fraction.
• 2 Inositol phosphate and diglyceride were found to be the main hydrolysis products. The principal enzyme involved, therefore, is a phosphatidylinositol inositolphosphohydrolase.
• 3 Most of the enzymic activity (61 per cent) was found in the soluble fraction. Osmotic shock of the high speed particulate fraction resulted in release of an additional 23 1 per cent into the soluble fraction. However, as contrasted to lactate dehydrogenase, significant activity remained particulate bound.

     

Inhibition of reverse transcriptase activity of RNA-tumor viruses by fagaronine+.

Fagaronine, a benzophenanthridine alkaloid from roots of $\text{Fagara zanthoxyloides}$ (Rutaceae), has been reported to possess anti-leukemic activity. It inhibited RNA-directed DNA polymerase activity from avian myeloblastosis virus, Rauscher leukemia virus and simian sarcoma virus. With poly rA·oligo dT, the alkaloid concentration for 50% inhibition of the enzyme activity from these viruses was in the range of 6–12 μg (15 – 31 nmoles) per ml of reaction mixture. The enzyme reaction was also inhibited with activated DNA and 70S RNA as templates; however, with poly rC·oligo dG no inhibition of enzyme activity was obtained. These results suggest that fagaronine inhibits enzyme activity by interaction with the A:T templateprimer.     

PROTEIN SYNTHESIS BY HIGHLY AGGREGATED AND PURIFIED POLYSOMES FROM YOUNG AND ADULT RAT BRAIN

The state of aggregation and the activity of polyribosomes as well as the activity of the pH 5 enzyme fraction were studied at two stages of postnatal brain development, 9 and 50 days after birth. When the polyribosomes were prepared at 0°C in the presence of 5 mm -Mg²⁺, more than 85 per cent of the polyribosome material exhibited a sedimentation coefficient higher than 110 S. High Mg²⁺ concentrations are, therefore, unnecessary to obtain highly aggregated brain polyribosomes. The basal amino acid incorporating activity of both 9- and 50-day-old rat brain preparations is at least equal to that of rat liver. When prepared by the same procedure as above, 9-day-old rat brain polyribosomes seem to be more active (20 per cent) than those of adult brain. However, this difference in activity depends on the presence of a non-ribosomal inactive contaminant which is always present in higher amounts in adult brain preparations. When purified from this contaminant, the preparations do not differ in activity. High Mg²⁺ concentrations are also not necessary for optimal protein synthetic activity and, in fact, are inhibitory. When assayed with both types of highly aggregated polyribosomes, the pH 5 enzyme fraction from adult brain is clearly less active than that of 9-day-old rats. These results suggest that the loss of brain protein synthesis during development does not depend on the stability of the messenger RNA-ribosome complex but only on the soluble pH 5 enzyme fraction.     

Adenosine triphosphate:creatine phosphotransferase of rat cerebral cortex: evidence for a bimodal subcellular localization

—(1) ATP: creatine phosphotransferase of rat cerebral cortex is soluble to the extent of 57 per cent when the tissue is homogenized in 0.25 M-sucrose and 80 per cent when distilled water is used for tissue dispersion. Among particulate fractions, the crude mitochondria] fraction contains the highest percentage of enzyme activity. (2) Discontinuous sucrose gradient fractionation of the crude mitochondrial fraction yields about 55 per cent of the particulate activity in the nerve ending fractions and 24 per cent in the mitochondrial pellet. (3) Rupturing of the nerve-ending particles by a moderate osmotic shock designed to spare the mitochondria results in about 60 per cent of the ATP:creatine phosphotransferase becoming soluble, the remainder preserving the association with heavy particles, presumably mitochondria. (4) Subfractionation of the microsomal fraction on a discontinuous sucrose gradient reveals that this particulate component of the enzyme is an adsorption artifact. (5) The overall evidence points to at least two distinct subcellular localizations of the enzyme in rat brain cortex, a major soluble component and a particulate component. It has not been unequivocally shown whether the latter, in turn, reflects the presence of a single, mitochondrial component or whether the soluble matrix of the nerve ending particles represents a third locale for the enzyme.     

Binding of oligonucleotides to cell membranes at acidic pH.

Antisense oligodeoxynucleotides [oligo(dN)] have the ability to enter living cells and block the expression of specific genes. However, little is known about the mechanism of cellular uptake of oligo(dN). We have found that oligo(dN) can bind to the cell membranes of eukaryotic cells with much greater efficiency under acidic conditions (pH 4.0-4.5) than at neutral pH. The binding appears to be specific to poly nucleic acids since various sizes of oligo(dN), DNA and RNA, but not mononucleotides, compete for the binding. We have identified a 34 kDa membrane protein from T-cells, which binds to oligo(dT) cellulose at pH 4.5 and can be eluted at pH 7.5. This protein fraction blocked the binding of oligo(dN) to living T-cells in a competitive fashion. Our results suggest that eukaryotic cells have a receptor for oligo(dN) at acidic pH and that the 34 kDa dalton protein on the cell membrane may mediate such binding.     

Oxidative Enzymes, Ribonuclease, and Amylase in Lemon Buds Infested with Aceria sheldoni (Ewing) (Acarina: Eriophyidae)

In lemon buds the optimum for peroxidase activity was a reactionmixture of 0.03 M guiacol, 0.1 per cent enzyme (acetone powder),and 0.0086 per cent H²O², in 0.015 M acetate buffer pH 4.0.For polyphenoloxidase activity the optimum was a reaction mixtureof 0.3 per cent catechol and 0.025 per cent enzyme (acetonepowder), in 0.05 M phosphate buffer pH 7.0. Both enzymes werestrongly affected by mite infestation. The activity of polyphenoloxidaseand peroxidase in infested buds reaches respectively two andthree times that in uninfested buds. There are indications thatthe enhanced oxidative activity and corresponding increase inphenol level in the infested buds is part of a defence systemwhich may develop in the plant after infestation. The optimum for RNAase activity was a reaction mixture of 0.06per cent RNA and 0.02 per cent purified enzyme, in 0.035 M sodiumpotassium-buffer pH 5.6. An increase of about 30 per cent inRNAase activity occurred after mite infestation. Amylase activitywas affected slightly only after a heavy infestation.     

Identification of γ-like DNA polymerase from sea urchin embryos

A γ-like DNA polymerase devoid of DNA polymerase-α and -β activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) · oligo (dT)_12–18 followed by poly(dA) · oligo(dT)_12–18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-γ.     

DEGRADATION OF RIBONUCLEIC ACID IN MOUSE FIBROBLASTS TREATED WITH ACTINOMYCIN

The cellular RNA content of mouse fibroblasts incubated with actinomycin decreases at a rate of about 1 to 1.5 per cent per hour, while DNA and protein content remain unchanged. This degradation affects nuclear and cytoplasmic RNA, ribosomal and soluble RNA. The breakdown products appear quantitatively in the acid-soluble fraction of the cells and the medium. Polynucleotides synthesized a short period (120 minutes) prior to exposure to actinomycin are degraded before those synthesized 8 to 12 hours previously.     

Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger ribonucleoprotein particles

ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP.This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis.Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.     

Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase

A soluble RNA-dependent RNA polymerase was purified from the cytoplasm of poliovirus-infected HeLa cells. A single virus-specific protein designated as p63 (or NCVP4) copurified with this activity. The purified polymerase was free of ribonuclease activity and was shown to copy poliovirion RNA when oligo(U) was added to the in vitro reaction mixture. Characterization of the product RNA by electrophoresis in methylmercury (II) hydroxide-agarose gels showed that genome-sized copies of poliovirion RNA were synthesized in vitro by the purified polymerase. The product RNA was shown to be heteropolymeric, complementary to virion RNA, and covalently linked to oligo(U). The product RNA contained the expected distribution of UMP and GMP containing dinucleotide pairs which included a very low frequency of CpG pairs. The amount, size distribution, and rate of synthesis of product RNA was very dependent on the in vitro reaction conditions. Full sized product RNA was synthesized in about 6 min when reaction conditions were used that yielded maximum elongation rates (pH 8.0, 7 mM Mg2+, 37 degrees C). Under these conditions, most of the product RNA recovered from a 1-h reaction was full sized. Thus, the polymerase was found to specifically initiate synthesis at the 3'-end of the template using an oligo(U) primer and to carry out an elongation reaction at about 1250 nucleotides/min that resulted in the synthesis of full sized product RNA.