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Biomedical subjects

J M Buchanan

Publications and source records attributed to J M Buchanan.

At least 55 records · Page 3Linked to original sources
PubMed1977

Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 1. Labeling of the enzyme with iodoacetate.

A two-step method for labeling the glutamine active site of formyglycinamide ribonucleotide (FGAR) amidotransferase from chicken liver has been developed in which reaction of all other reactive groups with unlabeled iodoacetate is followed by specific labeling of the glutamine site with radioactive reagent. A study of the reaction as a function of duration, temperature, and pH of the incubation as well as concentration of iodoacetate has revealed that two nonessential groups of the enzyme react in the presence of glutamine and that this modified enzyme is relatively resistant to further carboxymethylation. When this modified enzyme was incubated with radioactive iodoacetate in the presence of FGAR, ATP, and Mg2+ after removal of glutamine by dialysis, about 1 mol of radioactive iodoacetate was incorporated per mol of enzyme with inactivation. This method permits labeling of the active site for glutamine without the use of glutamine analogues.

Animals↗
PubMed1977

Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 2. Amino acid sequence of labeled peptides.

The purification and characterization of two related peptides making up the glutamine binding site of formylglycinamide ribonucleotide amidotransferase from chicken liver have been presented. An amino acid residue(s) involved in binding glutamine to the enzyme was selectively labeled with [14C]iodoacetate. The labeled enzyme was reduced, carboxymethylated, and degraded by trypsin to a large radioactive peptide that yielded on acid hydrolysis only cysteine as a radioactive carboxymethylated derivative. The tryptic peptide was further digested with a protease from Streptomyces griseus. Two radioactive fractions (I and II) were obtained by gel filtration on Sephadex G-25. Furthermore, two 14C-containing peptides have been isolated from fraction I by the aid of ion exchange chromatography on AG 1-X2, AG 50W-X2 and DEAE-cellulose. Upon acid hydrolysis both peptides yielded only carboxymethylcysteine (CMCys), cystine, glycine, valine, aspartic acid, and glutamic acid. The partial sequences of the amino residues in these peptides, which are located at the glutamine binding site, have been established by the dansyl-Edman method. The sequences of amino acids of peptides a and b are Gly-Val-Cys([14C]CM)-Asp-Asx-Cys(CM)-Glx...and Gly-Val-Cys([14C]CM)-Asx-Asx..., respectively. The two peptides are undoubtedly derived from the same segment of the original protein.

Amino Acid Sequence↗
PubMed1977

Binding and internalization of thrombin by normal and transformed chick cells.

Thrombin stimulates cell proliferation in cultures of normal chick embryo fibroblasts but not in cells transformed with Rous sarcoma virus. Analysis of medium conditioned by Rous-sarcoma-virus-transformed cultures demonstrates that these cells do not secrete molecules that can inhibit or inactivate thrombin. The interaction of thrombin with these cells was investigated with enzymatically active 125I-thrombin. The amount of cell-associated 125I-thrombin was found to be three times greater with normal cells than with transformed cells. In both types of cell, greater than 50% of the total cell-associated 125I-thrombin was found as a component that was not dissociated from the cells by trypsin treatment, an observation suggesting that a significant portion was not on the cell surface. The amount of the trypsin-insensitive fraction increases with time up to 12 hr, whereas the trypsin-sensitive fraction is saturated after 1-4 hr. Autoradiography of thin sections of 125I-thrombin-treated cells observed by electron microscopy reveals that after 10 hr incubation greater than 70% of the label is localized in the cytoplasm of both normal and transformed cells. Autoradiograms of sodium dodecyl sulfate/polyacrylamide slab gels demonstrate that 40% of the intracellular label is the size of native thrombin with the remainder in two large fragments of 22,000 and 19,500 daltons.

Animals↗
PubMed1977

Cell-free synthesis of two proteins unique to RNA of transforming virions of Rous sarcoma virus.

We have utilized a reticulocyte lysate system to translate the 35S RNA of Rous sarcoma virus. Autoradiograms of the protein products separated on sodium dodecyl sulfate/polyacrylamide gels reveal a heterogeneous mixture of proteins of sizes ranging from 13,000 to 180,000 daltons. In comparing the translational products from 35S RNA of Prague B Rous sarcoma virus with those formed from the RNA of a transformation-defective deletion mutant derived from Prague B, we have found that two proteins, 25,000 and 18,000 daltons, are missing from the latter. Neither of these proteins is immunoprecipitated by monospecific antisera against the structural proteins of avian RNA tumor viruses. The combined atomic mass of 43,000 daltons corresponds to the amount of genetic coding capacity (40,000-50,000 daltons in terms of protein products) deleted from the RNA of the transformation-defective viruses. We propose that these proteins are coded for by the putative oncogene (onc) or sarc (src) gene and that one or both of them may be responsible for the oncogenic transformation caused by these viruses in infected cells.

Antigens, Viral↗
PubMed1976

Effects of protease treatment on growth, morphology, adhesion, and cell surface proteins of secondary chick embryo fibroblasts.

Several proteolytic enzymes have been studied with regard to their ability to induce DNA synthesis and cell proliferation in resting chick embryo fibroblasts. Of the enzymes examined, thrombin, bromelin, and trypsin exhibit potent mitogenic activity, elastase has significant but less marked activity, whereas thermolysin, papain, and alpha-protease are inactive. The enzymes were also tested for their ability to induce morphological change or to remove two iodinatable proteins of 250,000 and 205,000 daltons. Although the larger protein is removed by some but not all of the proteases examined, every protease tested removed the smaller cell surface proteins; however, loss of the smaller protein does correlate with the reduction of both cytoplasmic spreading and cell-cell interactions observed after protease treatment. A secondary, later event of migration of cells into clumps is observed in those instances when protease treatment did not result in a loss of the 250k protein. Arole for each of these proteins in the processes of cellular adhesion is discussed.

Bromelains↗
PubMed1975

Mitogenic activity of blood components. I. Thrombin and prothrombin.

When added to a culture medium of resting confluent chick embryo fibroblasts in the absence of serum, thrombin (EC 3.4.21.5) is able to stimulate DNA synthesis 12 hr later and to cause a substantial increase in cell number over a period of 4 days. As compared to thrombin, prothrombin exhibits low mitogenic activity. However, in the presence of purified Factor Xa (EC 3.4.21.6) and Factor V, prothrombin is converted to thrombin by "thromboplastin activity" supplied by the fibroblasts. Prothrombin, either purified or as a constituent of plasma or serum, may thus be considered to be a reservoir of mitogenic activity in tissue culture unless antithrombin is present in the culture medium in amounts sufficient to neutralize the thrombin formed. By use of a specific inhibitor of proteases, and by separation of prothrombin by absorption on BaSO4, we estimate that the potential mitogenic activity of prothrombin is approximately 30-50% of the total activity that can be obtained by treatment of fibrinogen-free plasma with thromboplastin. In addition to its mitogenic activity, thrombin can also stimulate the migration of cells. These experiments with thrombin illustrate that well-characterized proteases of blood can act as potent mitogens and suggest that they may play a role in the process of wound healing.

Animals↗
PubMed1975

Plasminogen-independent fibrinolysis by proteases produced by transformed chick embryo fibroblasts.

The fibrinolytic activity of proteases secreted by chick embryo fibroblasts infected with Rous sarcoma virus was studied by use of a procedure in which a fibrin clot was formed with highly purified fibrinogen and thrombin above the cell layer. This procedure results in the formation of fibrin that is apparently a more suitable substrate for studies on fibrinolysis than is fibrin prepared by other methods. Since neither plasminogen nor serum were included in the assay system in the present studies, the fibrinolytic activity observed cannot be ascribed to the conversion of the plasminogen in serum to plasmin by a plasminogen activator produced by transformed cells. Our procedure, therefore, measures proteolytic activities other than those reported by previous investigators. Maintenance of some of the transformed phenotypes of Rous sarcoma virus transformed chick embryo fibroblasts such as morpholigical change and increased rate of glucose uptake apparently does not depend on the presence of plasminogen in the culture medium.

Animals↗
PubMed1975

Surface ruffles as markers for studies of cell transformation by Rous sarcoma virus.

Confluent chick embryo fibroblasts infected with the Ts68 mutant of Rous sarcoma virus were examined by scanning electron microscopy at the permissive (36 degrees) and nonpermissive (41 degrees) temperatures for transformation. Infected cells shifted from 41 degrees to 36 degrees undergo a change in shape from elongated to rounded. This process is preceded by the appearance of surface ruffles on the cell. These surface ruffles are not observed on cells maintained at 41 degrees, appear as early as 0.5 hr after a shift to 36 degrees, and are common on cells maintained at 36 degrees. By 3.5 hr after the shift from 41 degrees to 36 degrees, cultures appear fully transformed by the criteria of cell roundedness and the presence of surface ruffles. This surface alteration of cells is the earliest event of those so far reported during the transformation process and is not dependent upon protein synthesis and extracellular plasminogen during the period of temperature shift.

Animals↗
PubMed1974

Initiation characteristics for the synthesis of five T4 phage-specific messenger RNAs in vitro.

The involvement of the nucleoside triphosphates in the initiation of the synthesis of the messenger ribonucleic acid of five T4 specific enzymes has been studied. Only one of these, the messenger RNA for deoxynucleosidemonophosphate kinase, can be initiated in the presence of one nucleoside triphosphate, namely ATP. All of the remaining four require the presence of at least two nucleoside triphosphates during the initiation period. The combination of ATP and UTP was best for the initiation of messenger RNA for dihydrofolate reductase, ATP and CTP for deoxycytidylate hydroxymethyltransferase and beta-glucosyltransferase, and ATP and GTP for alpha-glucosyltransferase. We have concluded that there is a great variation in the nucleotide composition and sequence of the initiation sites in T4 DNA. No correlation in the requirements of nucleoside triphosphates during the initiation period could be observed among the five systems studied according to their classification as one type or another of "early" T4 messenger RNA.

Adenosine Triphosphate↗
PubMed1974

Synergistic interactions of T4 early proteins concerned with their binding to DNA.

Under appropriate conditions, 19 distinct early proteins produced by bacteriophage T4 bind DNA with various affinities. They include products of genes that have been implicated in T4 DNA metabolism, such as DNA negative, DNA arrest, and DNA delay genes, and others whose roles have not yet been defined. The identifiable DNA-binding proteins are the products of genes 43, rIIA, 46, 30, 39, 52, betagt, 32, and internal protein III. In some cases, protein-protein interactions are involved in the formation of the DNA-protein complexes.

Centrifugation, Density Gradient↗
PubMed1974

Transcriptional regulation of T4 bacteriophage-specific enzymes synthesized in vitro.

In contrast to dihydrofolate reductase and four other phage-specific enzymes, the initiation of deoxynucleotide kinase is essentially prevented if rifampin is added to a culture of Escherichia coli B cells within 1.5 min after infection with T4. Deoxynucleotide kinase thus belongs to a group of so-called delayed-early enzymes that is not initiated from an immediate-early promoter site. We prepared crude extracts from infected cells in a manner designed to maintain the integrity of the complexes of native, endogenous T4 DNA with bacterial structural and enzymatic units concerned with RNA synthesis. The initiation of the synthesis of the mRNA for dihydrofolate reductase, an example of an immediate-early enzyme, and deoxynucleotide kinase, a special type of delayed-early enzyme, was studied with these extracts prepared from cells infected in the absence or presence of chloramphenicol. Initiation of transcription of the dihydrofolate reductase gene is immediate when programmed by extracts made either from cells treated with chloramphenicol prior to infection (CM extracts) or from cells 3 min into the normal infection cycle (3-min extracts). However, initiation of transcription of the deoxynucleotide kinase gene programmed by CM extracts is delayed 2 min relative to the immediate initiation of transcription of the deoxynucleotide kinase gene programmed by 3-min extracts. These experiments duplicated in vitro effects of the antibiotics on the synthesis of phage-specific mRNA previously noted only in vivo.

Carbon Radioisotopes↗