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Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 685 records · Page 38Linked to original sources
PubMed1979

Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry.

The cellular location of the src gene product (p60src) of the Schmidt-Ruppin strain of avian sarcoma virus has been determined by electron microscopic immunocytochemistry in Schmidt-Ruppin ASV-transformed NRK cells, and the amount of the protein in different regions of the cell has been quantified. The protein is concentrated on the inner surface of the plasma membrane, particularly under ruffles, and it is highly concentrated on the inner surface of the membrane near junctions connecting adjacent cells. Small amounts of p60src were detected in the cytoplasm and in the perinuclear Golgi region of the cell. No significant localization was detected in control NRK cells or in NRK cells transformed by the Kirsten strain of murine sarcoma virus. The presence of p60src on the inner surface of the plasma membrane indicates that the changes in cell growth, cell shape and cell membrane structure noted in ASV-transformed cells are due to an initial action of p60src at the cell membrane.

Alpharetrovirus↗
PubMed1979

Epidermal growth factor stimulation of DNA synthesis is potentiated by compounds that inhibit its clustering in coated pits.

We have used inhibitors of receptor-mediated endocytosis to investigate the mechanism and function of epidermal growth factor uptake by cultured cells. When rhodamine-labeled epidermal growth factor is bound to cell surface receptors on confluent monolayers of BALB/c 3T3 cells, it rapidly collects in cell surface clusters and is internalized. The clustering of occupied receptors requires Ca(2+) and is inhibited by primary alkylamines; both of these properties are shared by the enzyme transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13). In Chinese hamster ovary cell extracts, methylamine inhibits 25-50% of the transglutaminase activity with a K(i) of 0.2 mM, and it inhibits the remaining transglutaminase activity with a K(i) of 20 mM. Clustering is almost completely inhibited by 10 mM methylamine. The polypeptide antibiotic bacitracin inhibits clustering of rhodamine-labeled epidermal growth factor or alpha(2)-macroglobulin at 0.7 mM, and it inhibits approximately 40% of the transglutaminase activity in Chinese hamster ovary cells with a K(i) of 0.03 mM. Fluorescent ligands bound to cell surface receptors in the presence of bacitracin form clusters within 30 min after bacitracin is removed from the culture medium. These results indicate that a transglutaminase-like enzyme may be required for the clustering and subsequent internalization of occupied receptors. The effects of 10 mM methylamine and 0.7 mM bacitracin on epidermal growth factor stimulation of DNA synthesis were examined. The stimulation of DNA synthesis by epidermal growth factor was increased 2- to 7-fold in the presence of methylamine or bacitracin. Alone, methylamine or bacitracin increased DNA synthesis 1.1- to 3-fold. The stimulation of DNA synthesis resulting from the simultaneous presence of the hormone and the clustering inhibitor was always greater than the sum of the stimulations produced by the hormone and the clustering inhibitors alone. The potentiation of epidermal growth factor activity by clustering inhibitors suggests that the hormone acts at the cell surface. We propose that rapid internalization of occupied receptors via coated pits may be a mechanism to limit the response to hormones.

Animals↗
PubMed1978

The stability of cell surface protein to surfactants and denaturants.

The effects of several denaturants and detergents on the structure and stability of cell surface protein have been evaluated by circular dichroism and fluorescence measurements. Cell surface protein undergoes a single broad transition in both urea and guanidinium chloride. Although guanidinium chloride is twice as effective as urea on a molar basis, both appear to eliminate all of the organized structure present in the native molecule. Nonionic surfactants and lysolecithin have little effect on cell surface protein. However, sodium dodecyl sulfate increases the alpha helical content and cetyltrimethylammonium bromide increases the beta structure of cell surface protein. The reorganization of the polypeptide backbone requires the loss of certain restraints imposed by tertiary interactions as evidenced by a decrease in ellipticity in the far ultraviolet and in the polarization of tryptophanyl fluorescence. These results along with the data of a previous paper (Alexander, S. S., Jr., Colonna, G., Yamada, K. M., Pastan, I., and Edelhoch, H. (1978) J. Biol. Chem. 253, 5820--5824) suggest the presence of structural domains distributed along the flexible polypeptide chain of cell surface protein.

Animals↗
PubMed1978

Molecular properties of a major cell surface protein from chick embryo fibroblasts.

The molecular structure of chick embryo fibroblast cell surface protein has been investigated by ultracentrifugation, circular dichroism, and fluorescence. Most measurements were restricted to alkaline solutions because of the limited solubility of this protein at more neutral pH values. A very high frictional ratio for the protein suggests an asymmetric structure. However, there are elements of organized structure since typical thermal transition curves were found by several methods. Consequently, a model in which ordered domains are connected by flexible polypeptide chains seems to account for all the hydrodynamic and optical data.

Animals↗
PubMed1978

Decreased levels of collagen mRNA in rous sarcoma virus-transformed chick embryo fibroblasts.

We have found that double-stranded cDNA synthesized in extended reactions by avian myeloblastosis virus reverse transcriptase is suitable substrate for a variety of restriction endonucleases. Experiments in which rabbit reticulocyte mRNA was reverse-transcribed and restricted to generate beta-globin-specifihe nucleotide sequence of beta-globin mRNA. This method has been applied to study collagen mRNA synthesis in normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts. Characteristic sets of collagen cDNA restriction fragments were produced from RNA fractions rich in collagen message activity. These sets of cDNA fragments, generated by the restriction endonucleases Hae III and Hap II, provided a convenient marker for the presence of collagen mRNA sequences. Equal quantities of high molecular weight mRNA from chick embryo fibroblasts (CEF) and RSV-CEF were reverse-transcribed and the resulting cDNA was restricted. The relative yields of collagen cDNA fragments from such reactions strongly suggest that the decrease in functional collagen RNA following RSV-induced transformation of CEF represents a decrease in the copy number of collagen messenger sequences. The potential of this approach for the study of regulation in other systems is discussed.

Animals↗
PubMed1978

Cellular transformation and the 'morphologic phenotype' of transformed cells.

Expression of the product of the transforming gene (src) of RNA tumour viruses promotes growth and usually alters the adhesion, appearance and surface properties of cultured fibroblasts. The latter group of properties termed the 'morphologic phenotype' of transformed cells is largely due to diminished cell-to-substratum adhesion. The role of cyclic AMP, cell surface protein (CSP), and other factors in producing the 'morphologic phenotype' are discussed. The effects of src expression bear a striking resemblance to the action of peptide hormones such as insulin on appropriate target cells.

Animals↗
PubMed1978

Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle.

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.

Actins↗
PubMed1978

Transformation-sensitive cell surface protein: isolation, characterization, and role in cellular morphology and adhesion.

Cell surface protein is the major cell surface glycoprotein of chick embryo fibroblasts. We have isolated and purified this glycoprotein and find that it is an adhesive protein that increases cell-cell and cell-substratum adhesiveness in a variety cellular adhesion assays. Transformation of chick fibroblasts results in decreased quantities of CSP due primarily to a fivefold reduction in CSP biosynthesis, although increased proteolytic degradation and shedding from the cell surface also contribute. The decreased biosynthesis is apparently due to a fivefold reduction in translatable mRNA for CSP. Reconstitution of isolated purified CSP on 14 transformed cell lines from several species results in reversion to a more normal fibroblastic morphology, adhesiveness, cell surface architecture, microfilament bundle organization, motility, and alignment at confluence. Cell surface protein does not restore growth control. The effects of CSP appear to be due to at least two actions, increased cell-substratum adhesion plus altered cell-cell interactions. Untransformed chick cells treated with affinity-purified antibodies to CSP develop the rounded morphology characteristic of many transformed cells that are deficient in CSP (LETS protein). Cell surface protein is found primarily in fibrillar aggregates on the cell surface. These CSP fibrils are relatively immobile and do not affect the mobility of other cell surface components. However, CSP can be eventually redistributed to caplike structures with anti-CSP. Isolated CSP consists of highly asymmetric disulfide-linked dimers and multimers. The interchain disulfide bridges are confined to a short terminal fragment that is readily removed by trypsin. Cell surface protein and cold-insoluble globulin have similar compositions but differ in solubility and amino termini. Cell surface protein contains primarily asparagine-linked oligosaccharides that appear to be responsible for CSP's concanavalin A receptor activity. Inhibition of CSP's glycosylation by treatment with tunicamycin results in decreased CSP due to marked increases in its degradation rate, without inhibition of synthesis or secretion. Studies of this major cell surface glycoprotein have provided insight into the biochemical mechanisms of cellular adhesion, morphology, and social interaction and provide an approach to analyze the dynamics and regulation of protein synthesis, glycosylation, secretion, and turnover.

Animals↗
PubMed1978

Filamin-actin interaction. Dissociation of binding from gelation by Ca2+-activated proteolysis.

Chicken gizzard filamin has been digested with purified Ca2+-activated protease. The subunits of (Mr = 250,000) of the protein are cleaved asymmetrically into two fragments, heavy merofilamin, Mr = 240,000, and light merofilamin, Mr = 9,500. Digestion is complete at substrate to enzyme ratios of 100:1 and requires Ca2+ concentrations in excess of 0.3 mM. Heavy merofilamin binds to F-actin as evidenced by cosedimentation with F-actin, by direct observation under the electron microscope, and by its ability to inhibit actin activation of heavy meromyosin ATPase. Heavy merofilamin does not form a gel when mixed with actin, except at very low concentrations of KCl. Thus, actin binding and gelation are separable activities of filamin. We speculate that Ca2+-stimulated proteolysis may play a role in the regulation of actin-filamin interactions.

Actins↗
PubMed1978

Purification of mammalian filamin. Similarity to high molecular weight actin-binding protein in macrophages, platelets, fibroblasts, and other tissues.

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.

Actins↗
PubMed1978

Activation of adenylate cyclase in cultured fibroblasts by trypsin.

Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.

Adenylyl Cyclases↗
PubMed1978

Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts.

Liposomes prepared with cholesterol and dipalmitoyl phosphatidylcholine were incubated with a clone of normal rat kidney fibroblast of cells in culture. The cells took up [14C]cholesterol in proportion to the concentration of liposomes in the incubation medium, and the uptake increased with time over the four hours of study. Two cell membrane enzymes, adenylate cyclase and (Na+ + K+)-ATPase, exhibited decreased activity after treatment with cholesterol-containing liposomes. The decrease in adenylate cyclase activity was directly proportional to the uptake of [14C]cholesterol. When a variety of subclones of NRK 5W were examined some were found to respond to cholesterol treatment and some did not. These data are consistent with the view that membrane cholesterol content plays a role in controlling the activity of some plasma membrane enzymes.

Adenosine Triphosphatases↗