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A F Purchio

Publications and source records attributed to A F Purchio.

At least 73 records · Page 4Linked to original sources
PubMed1984

Cell-free translation of bovine viral diarrhea virus RNA.

Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to program cell-free synthesis, mature viral 80,000- and 115,000-molecular-weight proteins were detected; no precursor to the viral 55,000-molecular-weight glycoprotein was noted. The implications of these results with respect to virus-specific protein synthesis are discussed.

Animals↗
PubMed1984

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Animals↗
PubMed1983

Physical modification of purified Rous sarcoma virus pp60v-src protein after incubation with ATP/Mg2+.

Using partially purified enzyme preparations, we show that incubation of the Rous sarcoma virus transforming protein kinase, pp60v-src, with Mg2+ and ATP at concentrations near or above the enzyme's Km for ATP resulted in a physical modification of the pp60v-src polypeptide. Under such conditions, a portion of pp60v-src was converted to a form that migrated more slowly in SDS-polyacrylamide gels than enzyme incubated without ATP or with low concentrations of ATP. Comparative tryptic peptide mapping of pp60v-src incubated with low and high levels of ATP revealed that more extensive tyrosine phosphorylation of the pp60v-src polypeptide occurred at the higher concentrations of ATP. This more extensive phosphorylation was characterized by the appearance of several new phosphorylated tyrosine residues on both the amino-terminal and carboxy-terminal portions of the pp60v-src molecule. The possible consequences of these modifications on the protein kinase activity of pp60v-src, and the functional regulation of retrovirus transforming proteins in general, are discussed.

Adenosine Triphosphate↗
PubMed1983

Characterization of virus-specific RNA synthesized in bovine cells infected with bovine viral diarrhea virus.

Infection of bovine kidney cells with bovine viral diarrhea virus resulted in the synthesis of a single species of virus-specific RNA. Electrophoresis of this RNA on agarose-urea and agarose-formaldehyde gels indicated that it had a molecular weight of 2.9 X 10(6), corresponding to 8,200 bases (8.2 kilobases). This 8.2-kilobase RNA was resistant to RNase A treatment at 1 microgram/ml but was digested at higher concentrations of RNase (10 micrograms/ml). Sedimentation on neutral sucrose gradients indicated that the majority of this RNA (98%) sedimented at 21S, with a small amount sedimenting at 33S. Sedimentation on formaldehyde-containing sucrose gradients resulted in the conversion of all of the RNA to the faster-sedimenting form. At no time after infection were we able to detect virus-specific RNA species of lower molecular weight than the 8.2-kilobase RNA. The implications of these findings with respect to the means of replication of various togaviruses are discussed.

Animals↗
PubMed1983

Increase in the phosphotransferase specific activity of purified Rous sarcoma virus pp60v-src protein after incubation with ATP plus Mg2+.

pp60v-src, the product of the Rous sarcoma virus src gene, was partially purified by immunoaffinity chromatography from extracts of Rous sarcoma virus-transformed field vole cells. Incubation of this preparation with ATP plus Mg2+ and subsequent repurification by chromatography on hexylamine-agarose resulted in a net increase in the specific activity of the src protein kinase. This increase in phosphotransferase activity was detected by using a variety of substrates including casein, tubulin, and a 34,000-dalton protein presumed to be an in vivo target substrate of pp60v-src. In all cases, the phosphorylation was at tyrosine residues, and the kinase activity was inhibited by preincubation of the enzyme with immunoglobulin G prepared from tumor-bearing rabbit sera. The implications of these results for the regulation and control of pp60v-src-associated kinase activity are discussed.

Adenosine Triphosphate↗
PubMed1982

Evidence the pp60src, the product of the Rous sarcoma virus src gene, undergoes autophosphorylation.

pp60src, the product of the rous sarcoma virus src gene, was purified greater than 100,000-fold by a combination of ion-exchange and immunoaffinity chromatography. Incubation of pp60src purified in this fashion with [32P-gamma]ATP resulted in a single 32P-labeled protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of these gels with silver nitrate showed a predominant 60,000-dalton polypeptide which comigrated with the protein labeled with 32P in vitro. Partial digestion of this protein with V8 protease after in vitro iodination indicated that it was pp60src. These results suggest that pp60src is able to autophosphorylate.

Avian Sarcoma Viruses↗
PubMed1982

Synthesis of the viral glycoprotein of Rous-associated virus-2.

The synthesis of the viral glycoprotein of Rous-associated virus-2 was studied in vitro in a cell-free system programmed with viral RNA and supplemented with dog pancreas membranes. The protein synthesized was related structurally and immunologically to those found in Rous-associated virus-2-infected chicken embryo fibroblasts. This work confirms and extends earlier work on the nature and synthesis of viral glycoproteins.

Avian Leukosis Virus↗
PubMed1981

Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src).

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp60v-src and pp60-c-src are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v-src and pp60c-src. We have therefore characterized the sites of tyrosine phosphorylation in the two proteins. Tyrosine phosphorylation of pp60v-src in infected cells occurs mainly (if not entirely) at residue 419 in the deduced amino acid sequence of the protein. Surrounding this residue is the sequence Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg. This peptide is distinguished by the fact that three out of the four amino acids that precede the phosphorylated tyrosine are acidic in nature. These results define what may prove to be a widely used site for tyrosine phosphorylation in the regulation of cellular function. The same site was phosphorylated when partially purified pp60v-src was used in a phosphotransfer reaction in vitro. The results with pp60c-src were more complex. The site of tyrosine phosphorylation in vitro appeared to be the same as that found in pp60v-src. By contrast, phosphorylation of pp60c-src in vivo apparently occurred at a different, and currently unidentified, tyrosine residue. It is therefore possible that pp60v-src and pp60c-src respond differently to regulatory influences in the intact cell.

Amino Acid Sequence↗
PubMed1980

Towards a molecular description of cell transformation by avian sarcoma virus.

The avian sarcoma virus transforming gene product has been identified and partially purified from extracts of transformed cells. It is a phosphoprotein with a relative molecular mass of 60 000 (pp60src) with two major sites of phosphorylation. pp60src appears to be a cyclic-AMP-independent protein kinase as judged by protein phosphorylation with partly purified fractions. The specificity of the phosphorylation observed was judged by inhibition with anti-pp60src IgG but not by normal IgG and by the fact that the protein kinase activity isolated from ts transformation-mutant infected cells was more thermolabile than that from wild-type transformed cells, thus showing more directly the origin of the enzymic activity. A cellular protein substrate of pp60src has been identified as a 34 000 molecular mass protein. These data together suggest that protein phosphorylation by pp60src may be a function of the molecule that plays a major role in transformation.

Alpharetrovirus↗
PubMed1980

Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine.

The protein responsible for malignant transformation by avian sarcoma viruses (ASVs) has been identified as a phosphoprotein of molecular weight 60,000 designated pp60src (refs 1--4). It has been suggested that this protein has a functional role in cellular transformation involving the phosphorylation of cellular proteins, for it was discovered that specific immunoprecipitates from ASV-transformed cells that contain pp60src catalysed the transfer of phosphate from [gamma-32P]ATP to the heavy chain of rabbit immunoglobulin. Additional studies involving the cell-free synthesis of the ASV src protein further demonstrated that the presence of the src polypeptide correlated with that presence of a phosphotransferase activity. Our studies, involving the biochemical purification of this protein, have demonstrated that the ASV-transforming gene product, pp60src, is itself a protein kinase. We have purified the pp60src protein approximately 5,000-fold using either conventional ion-exchange chromatography or immunoaffinity chromatography. The resultant partially purified preparations contain a cyclic AMP-independent protein kinase activity. We report here that the soluble phosphotransferase activity of partially purified pp60src results in the phosphorylation of exclusively tyrosine residues in a variety of proteins that serve as substrates.

Actins↗
PubMed1980

Molecular events in cells transformed by Rous Sarcoma virus.

The Rous sarcoma virus (RSV) transforming gene product has been identified and characterized as a phosphoprotein with a molecular weight of 60,000, denoted pp60src. Partially purified pp60src displays a closely associated phosphotransferase activity with the unusual specificity of phosphorylating tyrosine residues in a variety of proteins. That the enzymatic activity observed is actually encoded by the RSV-transforming gene is indicated by the comparison of the pp60src-protein kinase isolated from cells tranformed by a wild-type RSV or by a RSV temperature-sensitive transformation mutant; these experiments revealed that the latter enzyme had a half-life of 3 min at 41 degrees C, whereas that of the wild-type enzyme was 20 min. Evidence is now beginning to accumulate showing that viral pp60src expresses its protein kinase activity in transformed cells as well as in vitro because at least one cellular protein has been identified as a substrate for this activity of pp60src. Although the protein kinase activity associated with pp60src is itself cyclic AMP (cAMP) independent, the molecule contains at least one serine residue that is directly phosphorylated by the cellular cAMP-dependent protein kinase, thus suggesting that the viral transforming gene product may be regulated indirectly by the level of cAMP. The significance of this latter observation must be regarded from the point of view that the RSV src gene is apparently derived from a normal cellular gene that seemingly expresses in normal uninfected cells a phosphoprotein structurally and functionally closely related to pp60src. This celluar protein, found in all vertebrate species tested, also is a substrate for a cAMP-dependent protein kinase of normal cells, and, therefore, may be evolved to function in a regulatory circuit involving cAMP.

Animals↗
PubMed1980

Sites of synthesis of viral proteins in avian sarcoma virus-infected chicken cells.

We determined the sites of synthesis of avian sarcoma virus-specific proteins in infected chicken cells by immunoprecipitation of the products synthesized in vitro by free and membrane-bound polyribosomes; 85% of Pr76, the precursor of the viral internal structural proteins (group-specific antigens), was synthesized on free polyribosomes, and 15% was synthesized on membrane-bound polyribosomes. Pr92, the lycosylated precursor of the viral glycoproteins (gp85 and gp35), was synthesized exclusively on membrane-bound polyribomes, which is consistent with its role as a membrane protein. When we investigated the site of synthesis of pp60src, the product of the avian sarcoma virus src gene, we found that 90% was synthesized on free polyribosomes, whereas 10% was detected on membrane-bound polyribosomes. The implications of these results with respect to the subcellular location of pp60src are discussed.

Alpharetrovirus↗
PubMed1980

Transforming gene product of avian sarcoma viruses and its homolog in uninfected cells.

The product of the avian sarcoma virus (ASV) transforming gene is a 60,000-dalton phosphoprotein, pp60src. Sera from mice and from an occasional rabbit bearing ASV-induced tumors are capable of immunoprecipitating a phosphoprotein of similar, but not identical, structure from normal avian and mammalian cells. This protein is presumed to be the product of the cellular sarc gene, and has been tentatively designated pp60sarc. Analysis of the tryptic phosphopeptides reveals that the viral and avian proteins have an apparently identical phosphoserine-containing peptide. They evidently also both have an analogous phosphothreonine residue surrounded by a different amino acid sequence. Immunoprecipitates containing either the viral or normal cellular pp60 protein catalyze the transfer of radiolabeled phosphate from [gamma-32P]-ATP to the heavy chain of immune rabbit IgG, therefore suggesting that both viral and cellular phosphoproteins may be protein kinases.

Alpharetrovirus↗
PubMed1979

Cloning the argF gene from Escherichia coli K-12 with simian virus 40.

We have inserted a gene coding for ornithine transcarbamylase (OTCase) from Escherichia coli K-12 into the late gene region of simian virus 40 (SV40) DNA and propagated the hybrid molecules as free episomes or by co-infection with an SV40 tsA helper virus. In the first case, the E. coli argF gene was inserted via the EcoRI and BamHI termini in the late gene region of SV40 and the recombinant molecules were used to transfect monkey kidney cells. The hybrid DNA, which was too large to be encapsidated, was replicated for a short time (14 days) but was eventually lost from the surviving cells. In order to allow the argF gene to be packaged into virions, we purified two SV40 vectors containing large deletions of late gene region sequences. One was a 3325 base pair segment from a HaeII + BamHI digest. The argF gene was joined to both vectors at the BamHI site and these linear molecules were used to transfect monkey cells in the presence of SV40 tsA58 DNA as helper. These hybrid DNAs were replicated and packaged into virions. Late in the lytic infection of monkey cells, polyadenylated, cytoplasmic argF transcripts were detected, but significant translation of these trancripts was not observed.

Cloning, Molecular↗
PubMed1979

Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase.

The avian sarcoma virus transforming gene product pp60src has been partially purified by using ion exchange or immunoaffinity chromatography. These preparations contain a cyclic AMP-independent protein kinase activity capable of transferring radiolabel from [gamma-32P]ATP to immune rabbit IgG, casein, and the heavy chain purified from normal rabbit IgG. The casein kinase activity is specifically inhibited by anti-pp60src IgG. Comparison of the pp60src-protein kinase isolated from cells transformed by a wild-type ASV or by an ASV temperature-sensitive transformation mutant revealed that the latter product had a half-life of 3 min at 41 degrees C whereas that of the wild-type product was 20 min.

Animals↗
PubMed1979

A normal cell protein similar in structure and function to the avian sarcoma virus transforming gene product.

This report extends our previous studies concerning the identification and characterization of a protein from normal cells that is closely related to the avian sarcoma virus (ASV) transforming gene product pp60src. This normal cellular protein, which we have found in both avian and mammalian cells and have tentatively designated pp60sarc, was detected by immunoprecipitation of radiolabeled cell extracts with serum derived from both mice and rabbits bearing ASV-induced tumors. The normal cell pp60sarc is a 60,000-dalton phosphoprotein that is structurally similar, but not identical, to viral pp60src. The phosphorylation patterns of the normal cell and viral proteins are also similar: both contain two major phosphorylated residues, a phosphoserine located on the NH2-terminal 60% of the polypeptide and a phosphothreonine present on the COOH-terminal 40% of the molecule. In addition, the normal cell pp60sarc from both chicken and mammalian cells appears to have an associated protein kinase activity analogous to that previously described for the viral pp60src. The possible roles played by the normal cell protein pp60sarc and the ASV transforming protein pp60src in normal cellular growth and neoplastic disease, respectively, are discussed.

Alpharetrovirus↗