Purification and characterization of two protein kinases associated with Rous sarcoma virus

The two major phosvitin-utilizing kinases have been purified from virions of the Prague C strain of Rous sarcoma virus by the use of ion-exchange and affinity chromatography. The two kinases isolated may be differentiated by their molecular weights as well as by their ability to utilize GTP as a phosphate donor. Protein kinase G, which will use either GTP or ATP as a phosphate donor, has a molecular weight of 120 000 as determined under nondenaturing conditions by glycerol gradient centrifugation and 28 000 when assayed under denaturation in sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels. Protein kinase A, which will only efficiently use ATP as the phosphate donor, has an apparent molecular weight of 43 000 estimated by glycerol gradient sedimentation and 40 000 by NaDodSO4-polyacrylamide electrophoresis. Both kinases possess the ability to autophosphorylate. Phosvitin is the major, and casein the minor, phosphate-accepting substrate for both kinases in vitro; however, kinase G will also phosphorylate histones to an extent similar to that observed with casein.     

Characterization of the deoxyribonucleic acid polymerase associated with Kilham rat virus.

Purified preparations of the parvovirus, Kilham rat virus, have associated with them a protein with DNA polymerase activity. The enzyme has been separated from the other two or three viral proteins and purified 63-fold. The viral associated enzyme was found in a single peak of DNA polymerase activity after chromatography on DEAE-cellulose, DNA-cellulose, and phosphocellulose columns. It shares some properties in common with the host cellular DNA polymerases, described in the preceding paper (Salzman, L.A., and McKerlie, L. (1975) J. Biol. Chem. 250, 5589-5595), but also has some important distinguishing characteristics. The Kilham rat virus-associated DNA polymerase has increased enzyme activity in the presence of 0.02 M KCl and has a strong preference for a synthetic DNA polymer containing deoxyadenylate and deoxythymidylate. The enzyme has a molecular weight of approximately 75,000 plus or minus 3,000 and appears to contain endonuclease activity.     

Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus.

The vaccinia virus-induced DNA polymerase has been purified about 500-fold from a cytoplasmic extract of vaccinia-infected HeLa cells. Analysis of the purified fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide of 110,000 daltons, which is greater than 95% pure. This polypeptide co-sediments with polymerase activity through a glycerol gradient. The sedimentation coefficient of the enzyme is 6.3 S, and its Stokes radius is 4.6 nm. The molecular weight of the native enzyme derived from these values is 115,000. Vaccinia polymerase is therefore a single large polypeptide of 110,000 to 115,000 daltons. The purified fraction has no significant endonuclease activity, but a strong exonuclease activity co-purifies with polymerase activity through every step in the isolation. The polymerase and exonuclease activities are inactivated at 45 degrees C at the same rate. It is likely, therefore, that both activities are catalyzed by the same polypeptide. The exonuclease hydrolyzes DNA predominantly in the 3' leads to 5' direction, to produce 5' mononucleotides. The exonuclease degrades single-stranded DNA more rapidly than duplex DNA, and the rate of digestion of both single-stranded and double-stranded DNA increases as the size of the substrate decreases. Single-stranded circular DNA is a potent inhibitor of the exonuclease activity, but duplex circular DNA has no significant effect on its activity.     

Modulation of arachidonic acid metabolism by Rous sarcoma virus

Arachidonic acid (C20:4) metabolites were released constitutively from wild-type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF). 3H-labeled C20:4 and its metabolites were released from unstimulated and uninfected CEF only in response to stimuli such as serum, phorbol ester, or the calcium ionophore A23187. High-pressure liquid chromatography analysis showed that the radioactivity released from [3H]arachidonate-labeled transformed cells was contained in free arachidonate and in the cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha; no lipoxygenase products were identified. The release of C20:4 and its metabolites from CEF infected with pp60src deletion mutants was correlated with serum-independent DNA synthesis and with the expression of the mRNA for 9E3, a gene expressed in Rous sarcoma virus-transformed cells which has homology with several mitogenic and inflammatory peptides. 3H-labeled C20:4 release was not correlated with p36 phosphorylation, which argues against a role for this protein as a phospholipase A2 inhibitor. CEF infected with other oncogenic viruses encoding a tyrosine kinase also released C20:4, as did CEF infected with viruses that contained mos and ras; however, infection with a crk-containing virus did not result in stimulation of 3H-labeled C20:4 release, suggesting that utilization of this signaling pathway is specific for particular transformation stimuli.     

Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus.

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.     

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.     

Regulation of Rous sarcoma virus RNA-dependent DNA polymerase isoenzymes by in vitro phosphorylation-dephosphorylation

The activity of the αβ form of Rous sarcoma virus RNA-dependent DNA polymerase was stimulated upon treatment with the protein kinase purified from the same virus. This enhancement was observed for both DNA-dependent and RNA-dependent DNA polymerase activities, whereas the RNase H activity associated with the polymerase was not affected. On the other hand, the protein kinase did not induce detectable changes in the activities of the α-polymerase isoenzyme. Treatment with Escherichia coli alkaline phosphatase resulted in a reduction of the polymerase activities of the αβ isoenzyme with no effects on RNase H as well as on the α form of the DNA polymerase. Preincubations of the αβ- and α-oncornaviral polymerase isoenzymes with two other protein kinases—from avian myeloblastosis virus and from beef heart (catalytic subunit)—had no substantial effects on DNA polymerase and RNase H activities of both polymerase isoenzymes. Both α and β subunits of the polymerase isoenzymes were phosphorylated in vitro by all three protein kinases employed, although only the β subunit was shown previously to be phosphorylated in vivo.     

Studies of the Rous sarcoma virus RNA: characterization of the 5'-terminus

The 5′ terminus of the Rous Sarcoma Viral 30-40S RNA was characterized as follows: Unlabeled RNA was treated with polynucleotide kinase and (γ-³²P) ATP. Degradation of the 5′-(³²P) RNA with alkali yielded labeled pAp while degradation with venom phosphodiesterase yielded labeled 5′-AMP. Dephosphorylation with alkaline phosphatase was unnecessary for the RNA to accept³²P indicating the presence of 5′-OH ends. This establishes that the base at the 5′ end of Rous Sarcoma Viral 30-40S RNA is adenine.