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Biomedical subjects

K C Robbins

Publications and source records attributed to K C Robbins.

At least 109 records · Page 6Linked to original sources
PubMed1984

Gene product of v-fgr onc: hybrid protein containing a portion of actin and a tyrosine-specific protein kinase.

The nucleotide sequence of the region of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encoding its primary translation product, p70gag-fgr, has been determined. From the nucleotide sequence, the amino acid sequence of this transforming protein was deduced. Computer analysis indicates that a portion of P70gag-fgr has extensive amino acid sequence homology with actin, a eukaryotic cytoskeletal protein. A second region of P70gag-fgr is closely related to the tyrosine-specific kinase gene family. Thus, the v-fgr oncogene appears to have arisen as a result of recombinational events involving two distinct cellular genes, one coding for a structural protein and the other for a protein kinase.

Actins↗
PubMed1984

Expression of the PDGF-related transforming protein of simian sarcoma virus in E. coli.

The transforming gene of simian sarcoma virus (SSV), an acute transforming retrovirus, and human platelet-derived growth factor (PDGF), a potent mitogen for connective tissue cells, appear to have arisen from the same or very closely related cellular genes. In an effort to obtain sufficient quantities of the SSV transforming gene product for the detailed analysis of its structural and biologic properties, we placed the v-sis gene under the control of strong phage transcriptional and translational signals that provide for regulated expression of cloned genes in E. coli. When induced, the bacterial cells synthesized levels of the SSV transforming gene product that constituted at least 10% of their total protein. Differences in the structure and processing of the v-sis gene product in procaryotic and eucaryotic cells provided important insights concerning posttranslational modifications of this PDGF-related transforming protein in eucaryotic cells.

Animals↗
PubMed1984

Expression of the normal human sis/PDGF-2 coding sequence induces cellular transformation.

The human sis proto-oncogene contains the coding sequence for one of two polypeptide chains present in preparations of biologically active human platelet-derived growth factor (PDGF). A human clone, c-sis clone 8, which contains all of the v-sis-related sequences present in human DNA, was transcriptionally inactive when transfected into NIH/3T3 cells. When placed under the control of a retrovirus LTR, the clone was transcribed at levels comparable to that observed in cells transformed by SSV DNA. However, c-sis clone 8 DNA did not express detectable sis/PDGF-2 proteins and lacked biologic activity. A putative upstream exon was identified by its ability to detect the 4.2 kb sis-related transcript in certain human cells. When this sequence was inserted in the proper orientation between the LTR and c-sis clone 8, the chimeric molecule acquired high titered transforming activity, comparable to that of SSV DNA. Transformants containing this construct expressed human sis/PDGF-2 translational products. Thus the normal coding sequence for a human growth factor has transforming activity when expressed in an appropriate assay cell.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence analysis identifies the human c-sis proto-oncogene as a structural gene for platelet-derived growth factor.

The simian sarcoma virus transforming gene, v-sis, encodes a protein, p28sis , that is closely related to human platelet-derived growth factor (PDGF). The human locus related to v-sis was cloned and shown to contain at least five exons corresponding to the v-sis coding region. Nucleotide sequence analysis of these exons revealed that the predicted amino acid sequence of human c-sis differed by 6% from that of the woolly monkey-derived v-sis. These findings imply that the sis proto-oncogene has been well conserved during primate evolution. By comparison of the known amino acid sequences of PDGF peptides with the predicted human c-sis protein, it was possible to demonstrate that this human proto-oncogene is the structural gene encoding one of the two major polypeptides of this potent mitogen for connective tissue cells.

Base Sequence↗
PubMed1983

The human plasmin-derived light (B) chain X streptokinase complex: a second-generation thrombolytic agent.

Specific assay methods for the human plasmin-derived light (B) chain X streptokinase (B X SK) complex, in terms of both streptokinase (SK) and urokinase (UK) International Units, are described. The kinetic properties of various SK activator complexes with plasminogen, Val442-plasmin, and the plasmin-derived light (B) chain were compared to SK in terms of their catalytic efficiencies and Lineweaver-Burk plots. Similar kinetic data, and Lineweaver-Burk plots, are described for both highly purified high-molecular weight UK and low-molecular weight UK, including different clinical UK preparations. The B X SK complex has the highest catalytic efficiency of all the activator species studied. The Lineweaver-Burk plots of each of the various activator species are "fingerprints" of the enzymatic character of the activator. The B X SK complex is more like UK than SK, as an activator, in activating non-human plasminogen species. The biological half-life of the B X SK complex, in a dog model, was determined to be about 4 hr which is longer than the biological half-life(s) of SK in the same animal model, namely 0.6 hr (47%) and 2.8 hr (53%). This new second-generation activator complex may prove to be a useful thrombolytic agent in the treatment of thromboembolic diseases.

Animals↗
PubMed1983

Identification of the plasminogen activator(s) produced by the transformed liver cell line, SK-HEP-1.

The availability of a cell line derived from an adenocarcinoma of the liver has made it possible to study the plasminogen activator(s) (PA) biosynthesized in culture by liver cells. Conditioned cultured media purified on fibrin-celite, benzamidine-Sepharose and immunoabsorbent anti-urokinase columns have shown the presence of multiple plasminogen activators when separated on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). These PAs differ in molecular weight but all are urokinase-like based on their reaction with goat anti-urokinase and rabbit anti-tissue activator. Subcellular fractionation of the cultured cells shows the presence of activator in both the cytoplasmic and membrane fractions, but the higher molecular weight forms appear primarily in the cytoplasm.

Adenocarcinoma↗
PubMed1983

Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor.

The transforming protein of a primate sarcoma virus and a platelet-derived growth factor are derived from the same or closely related cellular genes. This conclusion is based on the demonstration of extensive sequence similarity between the transforming protein derived from the simian sarcoma virus onc gene, v-sis, and a human platelet-derived growth factor. The mechanism by which v-sis transforms cells could involve the constitutive expression of a protein with functions similar or identical to those of a factor active transiently during normal cell growth.

Amino Acid Sequence↗
PubMed1983

A functionally active heavy chain derived from human high molecular weight urokinase.

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.

Amino Acids↗
PubMed1983

Comparative activation kinetics of mammalian plasminogens.

Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.

Amino Acids↗
PubMed1983

Analysis of the primary translational product and integrated DNA of a new feline sarcoma virus, GR-FeSV.

The Gardner-Rasheed strain of feline sarcoma virus (GR-FeSV), is a recent isolate of a naturally occurring cat sarcoma. The primary translational product of GR-FeSV (GR P70) was shown to be a phosphoprotein with associated tyrosine-specific protein kinase activity. The relationship between the GR-FeSV provirus and once genes of other transforming retroviruses known to code for tyrosine kinases was examined by molecular hybridization. Probes repesenting onc genes of Snyder-Theilen and McDonough strains of feline sarcoma virus, Rous sarcoma virus, and Abelson murine leukemia virus did not detectably hybridize integrated GR-FeSV. These findings suggest that GR-FeSV contains a distinct tyrosine kinase-coding onc gene.

Animals↗
PubMed1983

Nucleotide sequence of the simian sarcoma virus genome: demonstration that its acquired cellular sequences encode the transforming gene product p28sis.

The complete nucleotide sequence of the proviral genome of simian sarcoma virus (SSV), an acute transforming retrovirus of primate origin, has been determined. Like other transforming viruses, SSV contains sequences derived from its helper virus, simian sarcoma-associated virus (SSAV), and a cell-derived (v-sis) insertion sequence. By comparison with the sequence of Moloney murine leukemia virus, it was possible to precisely localize and define sequences contributed by SSAV during the generation of SSV. Comparative sequence analysis of SSV and SSAV showed that SSAV provides regulatory sequences for initiation and termination of transcription of the SSV transforming gene. Moreover, coding sequences for the putative protein product of this gene appear to initiate from the amino terminus of the SSAV env gene. Antibodies to synthetic peptides derived from the carboxy and amino termini of the putative protein predicted by the open reading frame identified within v-sis specifically detect a Mr 28,000 protein, p28sis, in SSV-transformed cells. These and other findings confirm the predicted amino acid sequence of this protein and localize it to the coding region of the SSV transforming gene.

Amino Acid Sequence↗
PubMed1983

Molecular cloning of integrated Gardner-Rasheed feline sarcoma virus: genetic structure of its cell-derived sequence differs from that of other tyrosine kinase-coding onc genes.

Gardner-Rasheed feline sarcoma virus (GR-FeSV) is an acute transforming retrovirus which encodes a gag-onc polyprotein possessing an associated tyrosine kinase activity. The integrated form of this virus, isolated in the Charon 21A strain of bacteriophage lambda, demonstrated an ability to transform NIH/3T3 cells at high efficiency upon transfection. Foci induced by GR-FeSV DNA contained rescuable sarcoma virus and expressed GR-P70, the major GR-FeSV translational product. The localization of long-terminal repeats within the DNA clone made it possible to establish the length of the GR-FeSV provirus as 4.6 kilobase pairs. The analysis of heteroduplexes formed between lambda feline leukemia virus (FeLV) and lambda GR-FeSV DNAs revealed the presence of a 1,700-base-pair FeLV unrelated segment, designated v-fgr, within the GR-FeSV genome. The size of this region was sufficient to encode a protein of approximately 68,000 daltons and was localized immediately downstream of the FeLV gag gene coding sequences present in GR-FeSV. Thus, it is likely that this 1.7-kilobase-pair stretch encodes the onc moiety of GR-P70. Utilizing probes representing v-fgr, we detected homologous sequences in the DNAs of diverse vertebrate species, implying that v-fgr originated from a well-conserved cellular gene. The number of cellular DNA fragments hybridized by v-fgr-derived probes indicated either that proto-fgr is distributed over a very large region of cellular DNA or represents a family of related genes. By molecular hybridization, v-fgr was not directly related to the onc genes of other known retroviruses having associated tyrosine kinase activity.

Animals↗
PubMed1982

In vivo identification of the transforming gene product of simian sarcoma virus.

Simian sarcoma virus (SSV) deletion mutants were constructed from a molecular clone containing the entire infectious provirus. Transfection analysis of these mutants localized the SSV transforming gene to a small region of the viral genome encompassing its cell-derived sequence (v-sis). Antiserum to a peptide synthesized on the basis of the predicted amino acid sequence of the SSV transforming gene detected a 28,000-dalton protein that was specifically expressed in SSV transformed cells and that corresponded in size to that predicted from the v-sis coding sequence. The v-sis gene product designated p28sis was not a phosphoprotein, nor did it possess detectable protein kinase activity. These findings distinguish p28sis from a number of other retroviral onc proteins.

Animals↗
PubMed1982

Methods for studying fibrinolytic pathway components in human plasma.

Methods have been developed to quantitatively measure the major plasma components of the human fibrinolytic system. Plasminogen is measured functionally with a 9M excess of streptokinase and immunochemically by rocket immunoelectrophoresis; the normal range was found to be 16.7-23.8 mg/dl and 17.4-21.6 mg/dl, respectively. Alpha 2-plasmin inhibitor is measured functionally and immunochemically; the normal range for the major plasma plasmin inhibitor was found to be 5.30-6.60 mg/dl by both methods. Plasminogen activator concentrations, as well as, free, and complexed, protease activities are measured along with plasmin generation rates by spectrophotometric assays with chromogenic substrates. Both activator and free protease activities are zero in plasma samples from normal human subjects. Plasmin generation rates are 0.25-0.47% with urokinase and 5.30-9.70% with streptokinase; these values are the percentages of the respective initial velocities of activation in purified systems.

Fibrinolysin↗