Search PubMed⌕ Search

Biomedical subjects

I Pastan

Publications and source records attributed to I Pastan.

At least 613 records · Page 34Linked to original sources
PubMed1983

Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line.

Receptor-mediated endocytosis proceeds by transfer of receptor-ligand complexes from clathrin-coated pits at the cell surface to uncoated endocytic vesicles termed receptosomes (or endosomes). These vesicles have now been purified more than 37-fold based on their content of newly internalized epidermal growth factor. 125I-labeled EGF was bound to human KB carcinoma cells at 4 degrees C, and then the cells were warmed to 37 degrees C for 8 min and disrupted. The purification scheme involved density gradient centrifugation on colloidal silica and sucrose and gel filtration on Sephacryl S-1000. Relative to homogenate, receptosomes are enriched 4.3-fold in their cholesterol content and depleted in enzyme markers for plasma membranes (2- to 3-fold) and lysosomes (9-fold). Receptosomes have a polypeptide composition that is different from plasma membrane, lysosome, and other homogenate fractions. They are enriched in transferrin receptors (30-fold) and in unidentified Mr 70,000-75,000 glycoprotein(s); they contain phosphomannosyl receptors. They do not contain detectable amounts of clathrin.

Carcinoma↗
PubMed1983

Formation of receptosomes from plasma membrane coated pits during endocytosis: analysis by serial sections with improved membrane labeling and preservation techniques.

The early events in the entry of ligands into cultured fibroblasts through plasma membrane clathrin-coated pits were examined by using serial sections and electron microscopy. Swiss 3T3 cells were labeled at 4 degrees C with concanavalin A-horseradish peroxidase and warmed to 37 degrees C for 1 or 2 min to label organelles participating in endocytosis. Cells were fixed in glutaraldehyde and further preserved with a membrane contrast-enhancement technique that utilized sequential osmium/ferrocyanide, thiocarbohydrazide, and osmium/ferrocyanide treatment in situ. This technique provides exceptional preservation and enhanced contrast of membranous structures. Serial sections taken both parallel to and perpendicular to the substratum were examined by electron microscopy, and 263 clathrin-coated structures containing horseradish peroxidase were evaluated for their connection to the plasma membrane. All of these coated structures were found to be connected to the plasma membrane either directly or via necks approximately equal to 170 A in width and up to 7,000 A in length. These necks were often surrounded by a dense filamentous network. These results strongly suggest that isolated coated vesicles do not form during the endocytosis process in Swiss 3T3 cells.

Animals↗
PubMed1983

Inhibitors of endocytosis perturb phospholipid metabolism in rabbit neutrophils and other cells.

Dansylcadaverine, amantadine, and rimantadine, which have been shown to inhibit the endocytosis of alpha 2-macroglobulin, epidermal growth factor, and vesicular stomatitis virus [Schlegel, R., Dickson, R. B., Willingham, M. C. & Pastan, I. (1982) Proc. Natl. Acad. Sci. USA 79, 2291-2295], were found to decrease phosphatidylcholine synthesis, chemotaxis, and internalization of a formylated peptide but to stimulate the incorporation of inositol into phosphatidylinositol in rabbit neutrophils. Dansylcadaverine decreased phosphatidylcholine synthesis by both the CDP-choline and transmethylation pathways and also inhibited the synthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. Dansylcadaverine had no effect on the phosphocholine, CDP-choline, or S-adenosyl-L-homocysteine pools but increased 2-fold the S-adenosyl-L-methionine pool. These results suggest that dansylcadaverine in some manner inhibited the condensation of CDP-choline with diacylglycerol to form phosphatidylcholine. Dansylcadaverine also inhibited phosphatidylcholine synthesis in human neutrophils, human fibroblasts, chicken embryo fibroblasts, rat hepatocytes, osteosarcoma cells, and neuroblastoma cells. It did not stimulate phosphatidylinositol synthesis in chicken embryo fibroblasts.

Animals↗
PubMed1983

Receptor-mediated endocytosis of alpha 2-macroglobulin: solubilization and partial purification of the fibroblast alpha 2-macroglobulin receptor.

Recent studies in our laboratory have been aimed at biochemically characterizing the alpha 2M receptor present on fibroblast membranes. The approach we have taken is to develop a means of assessing binding to solubilized alpha 2M binding sites. The binding activity was not removed by treatment with high salt concentration or treatment with chaotropic agents. Removal of the binding activity from membranes did occur using a variety of detergents which suggests that the receptor molecules may be "intrinsic" membrane proteins. The most useful detergent for solubilizing the alpha 2M receptor was octyl-beta-D-glucoside. The alpha 2M binding activity could be removed from NRK fibroblasts and Vero cells using this detergent and was found to remain in solution at 100,000 X g. Removal of the octyl-beta-D-glucoside by extensive dialysis resulted in formation of protein-lipid aggregates that bind to 125I-alpha 2M specifically and with high affinity. Such binding sites were not generated when KB cells (which lack receptors) were extracted with the detergent. Significantly, the observed affinities detected for both high- and low-affinity binding sites were similar to those reported with intact cells or membranes. In addition, binding to the solubilized sites could be inhibited using compounds known to block the receptor-mediated endocytosis of alpha 2M (bacitracin, IBHNA). Other compounds (monensin, dansylcadaverine), which did not directly inhibit the high-affinity binding sites, may exert their effects at different stages in receptor-mediated endocytosis (i.e., receptor recycling). alpha 2M binding sites from NIH-3T3 (spontaneous) tumors have been purified approximately 95-fold by a combination of ion exchange and gel permeation chromatography. The receptor appears to be an acidic protein that approximately coelutes with aldolase (45 A, 158,000 daltons) on gel filtration. Ion exchange chromatography appears to remove an endogenous inhibitor of alpha 2M binding and may also remove binding sites having lower affinity for 125I-alpha 2M. Recent studies using immobilized alpha 2M as an affinity resin suggest that the high-affinity alpha 2M receptor may have a subunit molecular weight of approximately 85,000. Studies are now in progress aimed at further characterizing these high-affinity binding sites. Once bound to the alpha 2M receptor, alpha 2M enters cells via coated pits and is rapidly transferred to receptosomes. These organelles carry the ligand into the Golgi region, from which it is transferred to lysosomes where it is slowly degraded.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system.

Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts. The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template. At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts. The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos. The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease. A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 degrees C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 degrees C. These data suggest that it is a small, hydrolphilic compound probably organic in nature. The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick alpha 2 (Type I) collagen-promoter as DNA templates. The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II. In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.

Animals↗
PubMed1982

Properties of the src kinase purified from Rous sarcoma virus-induced rat tumors.

We recently described a method for the purification of a protein kinase related to pp60src from Rous sarcoma virus-induced rat tumors (Blithe, D. L., Richert, N. D., and Pastan, I. H. (1982) J. Biol. Chem. 257, 7135-7142). In this report, we describe some of the properties of the 7200-fold purified enzyme. The purified kinase phosphorylates casein, vinculin, actin, and histone H2B, but not bovine serum albumin or ovalbumin. Protein substrates are phosphorylated exclusively on tyrosine residues. Casein was used as a substrate for more detailed analysis. The phosphorylation reaction proceeds at a linear rate for at least 40 min at 22 degrees C. Maximum enzyme activity occurs at pH 6.5 to pH 6.8 and requires the presence of either Mg2+ (5 to 10 mM) or Mn2+ (1 to 5 mM). The Km for ATP is 30 microM and the Vmax 0.03 mumol/min/mg using 0.4 mg/ml of casein as a substrate. The enzyme utilizes ATP or dATP, but not GTP as a phosphate donor in the reaction. The enzyme is inhibited by adenosine and deoxyadenosine and by their corresponding mono- and diphosphates. No inhibition of enzyme activity is observed with adenine, GTP, UTP, TTP, or CTP. The enzyme is very sensitive to increased ionic strength. Addition of 0.1 M KCl, 0.1 M NaCl, 50 mM KPO4, or 50 mM NaPO4 inhibited casein phosphorylation by 90 to 95%. Analysis of the products of the phosphorylation reaction by thin layer chromatography revealed that the src kinase phosphorylates glycerol in addition to casein or the enzyme itself.

Animals↗
PubMed1982

Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts.

We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.

Animals↗
PubMed1982

Inhibition of the nuclear entry of 3,3',5'-triiodo-L-thyronine by monodansylcadaverine in GH3 cells.

We have recently reported that the entry of 3,3',5-triiodo-L-thyronine (T3) into mouse 3T3 fibroblasts occurs by receptor-mediated endocytosis (Cheng, S. Y., Maxfield, F. R., Robbins, J., Willingham, M. C., and Pastan, I. (1980) Proc. Natl. Acad. Sci. U. S. A. 77: 3425-3429). In this communication, we evaluated the functional significance of this mode of entry using GH3 cells, a growth hormone-producing cell line which has a high number of T3 nuclear receptors. T3-specific, saturable membrane uptake systems were demonstrated in GH3 cells. Monodansylcadaverine, an inhibitor of alpha 2-macroglobulin, epidermal growth factor, vesicular stomatitis virus, and T3 uptake in fibroblasts, blocked virtually all of the cellular uptake of T3, with a half-maximal concentration of 29 microM. Concomitant with the inhibition of the cellular uptake of T3, the accumulation of T3 into nuclei was reduced. The inhibitory effect of monodansylcadaverine on the reduction of T3 incorporation into nuclei was not due to the inhibition of binding to nuclear receptors, but probably was due to a decrease in the cytoplasmic availability of T3 as a result of inhibition of cellular entry. These results indicate that the uptake of T3 by receptor-mediated endocytosis is a physiologically significant process.

Animals↗
PubMed1982

Rous sarcoma virus transformed cells are resistant to cyclic AMP.

A nontransformed line of Chinese hamster ovary (CHO) cells (Pollard and Stanners, 1979) has been transformed by the Schmidt-Ruppin subgroup D strain of Rous sarcoma virus (SR-RSV). SR-RSV transformed CHO cells are shown to differ from spontaneously transformed cells in that the virally transformed cells are more resistant to growth inhibition or changes in cell shape by 8-Br-cyclic AMP or cholera toxin. SR-RSV transformed rat (NRK) cells also have a reduced sensitivity to growth inhibition by 8-Br-cyclic AMP. Cyclic AMP-dependent protein kinase was examined in SR-RSV transformed CHO cells, but no differences in enzyme level, activation by cyclic AMP, chromatographic behavior, or its ability to phosphorylate endogenous proteins in whole cells could be detected. It is concluded that transformation of CHO and NRK cells by SR-RSV alters the cells in a manner different from spontaneous transformation, and that this alteration does not affect cAMP-dependent protein kinase activity.

Animals↗
PubMed1982

Binding and internalization of alpha 2-microglobulin by cultured fibroblasts. Effects of monovalent ionophores.

Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.

Animals↗
PubMed1982

The morphologic pathway of exocytosis of the vesicular stomatitis virus G protein in cultured fibroblasts.

Cultured fibroblasts were infected with vesicular stomatitis virus (VSV) and the pathway of exocytosis of G protein, the transmembrane glycoprotein of VSV, was followed by immunofluorescence and electron microscopy. G protein was detected within the endoplasmic reticulum, within smooth vesicles and stacks in the Golgi region and on the cell surface. No G protein was detected in the coated regions of the Golgi. Our data are consistent with the hypothesis that coated regions of the Golgi are involved in transfer of lysosomal enzymes and other substances to lysosomes and not in exocytosis.

Animals↗
PubMed1982

Unusual methylation pattern of the alpha 2 (l) collagen gene.

We studied the methylation pattern of the alpha 2 (type 1) collagen gene in DNA from five cell types with varying rates of type I collagen synthesis: chick embryo fibroblast (CEF), CEF transformed by Rous sarcoma virus, erythrocyte, brain and sperm. The methylation-sensitive restriction enzymes, Msp I, Hpa II, Ava I and Sma I were used to detect methylation in three regions of the alpha 2 (type I) collagen gene: a 5.7 kb region, which includes the start site of transcription and the first two exons of the collagen gene; a 5.2 kb region containing exons in the middle of the gene; and a 3.5 kb region containing exons in the 3' portion of the gene. The DNA around the start site of transcription is not methylated whether or not the cells synthesize collagen. In contrast, the DNA from the central and 3' region of the gene is methylated to about the same extent whether or not the cells make collagen. Our data indicate that a gene that is methylated can be actively transcribed and that the level of expression of the alpha 2 (type I) collagen gene seems to be independent of methylation.

Animals↗
PubMed1982

Phospholipids stimulate phosphorylation of vinculin by the tyrosine-specific protein kinase of Rous sarcoma virus.

The phosphorylation of vinculin by a highly purified tyrosine-specific protein kinase was enhanced more than 10-fold by anionic phospholipids: the phosphorylation of casein, actin, and alpha-actinin was inhibited. The effect of phospholipid was dependent on the divalent cation used. Stimulation was observed by phosphatidylinositol or phosphatidylglycerol in the presence of either 0.5 mM Mn2+ of 5 mM Mg2+; with either phospholipid, more enzyme activity was observed with Mn2+. Maximal stimulation by phosphatidylinositol was observed at about 400 micrograms/ml. In contrast, marked stimulation by phosphatidylserine was observed only with Mn2+ and marked stimulation by phosphatidic acid was observed only with Mg2+. These results raise the possibility that phospholipids modulate vinculin phosphorylation in Rous sarcoma virus-transformed cells.

Avian Sarcoma Viruses↗
PubMed1982

Affinity labeling of the plasma membrane 3,3',5-triiodo-L-thyronine receptor in GH3 cells.

The binding of 3,3',5-triiodo-L-thyronine (T3) to GH3 rat pituitary tumor cells was studied at 15 degrees C and was shown to be saturable, reversible, and stereospecific. Least-squares analysis of the binding data showed two classes of binding sites with dissociation constants of 1.8 +/- 0.2 nM and 260 +/- 30 nM and binding capacities of (5.2 +/- 0.2) X 10(4) and (1.6 +/- 0.2) X 10(6) sites per cell, respectively. Affinity labeling of intact cells was carried out by incubation of cells with 0.3 nM N-bromoacetyl-[125I]T3 at 15 degrees C for 1 hr. Analysis of the cellular extracts by sodium dodecyl sulfate gel electrophoresis showed three labeled protein bands with apparent molecular masses of 55, 47, and 33 kilodaltons (kDal) in a ratio of 86:7:7. The labeling of only the 55-kDal protein band was selectively reduced to 50% by 20 microM unlabeled T3. Highly purified plasma membranes of GH3 cells were prepared and shown to be free of nuclei. Affinity labeling of the purified plasma membranes gave the same labeling pattern as with intact cells. Peptide mapping by Staphylococcus aureus V8 digestion of the 55-kDal protein from cells or plasma membranes gave the identical peptide fragments. Thus the 55-kDal protein labeled from intact cells is the same protein as that from purified plasma membranes. These results together with our earlier findings [Horiuchi, R., Cheng, S.-y., Willingham, M. & Pastan, I. (1982) J. Biol. Chem. 257, 3139-3144] suggest that the 55-kDal protein may be involved in mediating the uptake of T3 in GH3 cells.

Affinity Labels↗
PubMed1982

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

Acetyltransferases↗