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R R Meyer

Publications and source records attributed to R R Meyer.

At least 55 records · Page 3Linked to original sources
PubMed1978

Purification and properties of DNA polymerase-beta from guinea pig liver.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.

Animals↗
PubMed1978

The subliminal perception of movement and the course of autokinesis.

The course of autokinesis is shown to be sensitive to the real movement of a surrounding stimulus. With the supraliminal presentation of this stimulus, apparent movement in a direction opposite to that of the real movement is induced. With the subliminal presentation of the same stimulus the real movement serves to inhibit autokinesis by inducing brief periods of stationarity between the phases of upward and downward apparent movement. The results confirm previous findings that the movement of a stimulus may be discriminated without there being any perceptual (phenomenal) adjunct.

Adult↗
PubMed1977

Novikoff hepatoma deoxyribonucleic acid polymerase. Identification of a stimulatory protein bound to the beta-polymerase.

The Novikoff hepatoma DNA polymerase-beta sediments as a 7.3S form in crude extracts but during purification sediments as a 4.1S form (after diethylaminoethyl-Sephadex chromatography) or as a 3.3S form (after DNA-cellulose chromatography). If 0.25 M ammonium sulfate or 0.5 M NaCl is included in the sucrose gradients, the 7.3S form sediments at 3.3 S; after removal of the salt, it sediments again at 7.3 S, indicating the reversibility of the aggregation phenomenon. By careful adjustment of ionic strength in the gradient, four distinct and reproducible forms of the enzyme sedimenting at 7.3, 5.8, 4.1, and 3.3 S can be generated. The isoelectric point of the DNA polymerase also changes during purification; the 7.3S form has a pI of 7.5, while the 4.1S form isoelectrically focuses at a pH of 8.5. During DNA-cellulose chromatography, the Novikoff beta-polymerase is separated from a stimulatory factor designated as Novikoff factor IV. Factor IV is a protein as shown by its sensitivity to protease and resistance to nucleases. It is responsible for converting the 3.3S enzyme to the 4.1S form since the 3.3S homogeneous DNA polymerase-beta sediments at 4.1 S in the presence of factor IV. Factor IV confers stability to the polymerase in low ionic strength buffers as well as stability to heat denaturation. Factor IV has the ability to increase the activity of the 3.3S homogeneous polymerase by about fourfold.

Animals↗
PubMed1976

Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.

Animals↗
PubMed1976

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.

Animals↗
PubMed1975

Isolation and purification of mediators of cell proliferation.

An attempt was made to isolate and purify the important biological mediators that cause an increase in proliferative activity of fibroblasts following tissue injury. DNA synthesis and cellular growth, using cultured WI-38 fibroblasts, and DNA synthesis in an in vitro assay, using purified DNA polymerase, were stimulated by factors extracted from the lysosomal-mitochondrial fraction of normal guinea pig liver. These factors precipitated in 45 percent to 60 percent ethanol. They were insensitive to treatment with RNase, DNase and heating to 56 C for 30 minutes, but were inactivated at 100 C. isoelectric focusing of the active ethanol-precipitate resolved activity into five discrete fractions, one of which has been purified, using ion-exchange chromatography. The presence of these factors in normal tissue may explain the increase in proliferative activity of fibroblasts and other cells in the early stages of wound healing, via release caused by injury.

Animals↗
PubMed1975

Stimulation of DNA polymerase by factors isolated from Novikoff hepatoma.

Extracts of Novikoff hepatoma cells contain factors capable of stimulating in vitro DNA synthesis several fold. The activity can be resolved into three separate protein peaks on DEAE-Sephadex. Two of these, factors II and III, have been purified and partially characterized. Both factors increase the initial rate of DNA synthesis and allow synthesis to proceed much longer. If either factor is added after synthesis by the DNA polymerase has reached a plateau, resumption of synthesis occurs. The factors appear to have different modes of action or sites of action since they show an additive effect even when a single one is used at saturating conditions. These factors are present in normal rat liver but at a concentration less than 5% of that found in the tumor cells. When tested with several highly purified DNA polymerases (DNA nucleotidyltransferase, EC 2.7.7.7), the factors show a much greater stimulation of homologous, non-mitochondrial enzymes (rat liver nuclear-, rat liver cytoplasmic-, or Novikoff-DNA polymerases) when compared with rat liver or calf liver mitochondrial-, Escherichia coli I-, or sea urchin nuclear-DNA polymerases. The mechanism of action of these factors is not known at present. No enzymatic activity has been associated with factor III. Highly purified, but not homogeneous, preparations of factor II contain low levels of endonuclease; it has not been established whether endonuclease is a contaminant or is responsible for the stimulating activity.

Carcinoma, Hepatocellular↗