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I Pastan

Publications and source records attributed to I Pastan.

At least 757 records · Page 42Linked to original sources
PubMed1974

Myosin is a component of the cell surface of cultured cells.

Antisera to myosin from mouse L929 cells, mouse uterus, and rabbit skeletal muscle have been prepared in goats. Each antiserum shows specific reaction by immunodiffusion only to the myosin against which it was prepared. Antiserum against L cell myosin agglutinates L929 cells and shows surface localization in unfixed cells by indirect immunofluorescence. This reaction is prevented by immunoabsorption with L cell myosin, but not by absorption with mouse uterine or rabbit skeletal muscle myosins. Elution of the antibody from L cell myosin antiserum that absorbs to fixed L cells yields antibody reactive to L cell myosin in immunodiffusion. At the ultrastructural level, surface localization is also evident with a peroxidase bridge immunocytologic method as a uniform distribution of surface label. Antiserum against L cell myosin also shows localization on the surface of other cells of rodent origin (3T3-4, NRK, MNRK, KNRK, and MEF) and HeLa cells, but not with some other cells of human origin (ALL and WI-38). The reactivity with antibody of the plasma membrane myosin is unaffected by trypsin treatment or fixation with 0.5-2.0% formalin. These results strongly suggest that myosin in cultured nonmuscle cells is a component of the plasma membrane, a finding of possible significance in studying the mechanisms regulating the motility, morphology, and growth of these cells.

Agglutination↗
PubMed1974

The 5'-terminal nucleotide sequence of galactose messenger ribonucleic acid of Escherichia coli.

The 5'-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA.DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T(1), pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5' terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].

Amino Acid Sequence↗
PubMed1974

Cyclic AMP mediates the concanavalin A agglutinability of mouse fibroblasts.

We have devised a quantitative way to measure the agglutination of cells which utilizes the size discrimination feature of an automatic particle counter. With this method we have studied the agglutinability by concanavalin A of 3T3 cells, a mutant of 3T3 cells (3T3cAMP(tcs)) in which cyclic AMP levels fall when the cells are subjected to temperature change or fresh serum, and L929 cells. We find with 3T3cAMP(tcs) cells that low levels of cyclic AMP correlate with increased agglutinability and that high levels of cyclic AMP correlate with decreased agglutinability. Prior treatment of these cells with a cyclic AMP phosphodiesterase inhibitor or Bt(2)cAMP blocks the increase in agglutinability induced by temperature change. When 3T3 cells are treated with fresh serum, their agglutinability also increases although to a much smaller extent than with 3T3cAMP(tcs) cells. Cells change their agglutinability very rapidly. Treatment of L929 cells for 15 min with 1-methyl-3-isobutyl xanthine at 1 mM decreases their agglutinability to the level of normal 3T3 cells. We conclude that in normal and transformed cells the level of cyclic AMP regulates agglutinability.

Agglutination↗
PubMed1973

Interrelationship between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels, and growth of cells in culture.

To investigate how cell population density influences the intracellular concentration of cyclic AMP we have measured adenylate cyclase and cyclic AMP phosphodiesterase activities and cyclic AMP levels at various stages of cell density in normal rat-kidney (NRK) cells, which exhibit contact-inhibition of growth, and in normal chick-embryo fibroblasts (CEF), which do not show contact inhibition of growth under our conditions. Until NRK cells reach confluency, both activities increase with increasing cell population and cyclic AMP levels are low. As NRK cells reach confluency, cyclic AMP phosphodiesterase activity decreases somewhat whereas adenylate cyclase activity continues to rise. This increase in synthetic ability is accompanied by the increase in cyclic AMP levels which occurs in these cells at confluency. In CEF grown in 5% serum where density-dependent inhibition of growth is not observed, both adenylate cyclase and cyclic AMP phosphodiesterase activities increase proportionately with increasing cell population density. No significant alteration occurs in the ratio between these two enzyme activities and no change is observed in cyclic AMP levels. The NaF-stimulated activity in NRK cells increases with increasing cell density until the cells reach confluency; thereafter the NaF-stimulated activity remains constant. In contrast, the NaF-stimulated activity observed in CEF does not vary appreciably between light and heavy density. The observed changes in the enzymes of cyclic AMP metabolism accurately reflect the changes in cyclic AMP concentration as a function of cell population density. The data indicate that these two enzyme activities respond to increasing cell density to elicit a rise in intracellular cyclic AMP levels. The elevated cyclic AMP levels are thought to be involved in the regulation of cellular growth rate and the mediation of contact inhibition of growth.

Adenylyl Cyclases↗
PubMed1973

In vitro repression of the transcription of gas operon by purified gal repressor.

We have studied the in vitro repression of gal mRNA synthesis by the gal repressor from Escherichia coli. By use of a four-step purification procedure involving chromatography on phosphocellulose, DEAE-cellulose, and an affinity resin, the gal repressor has been purified about 1600-fold from a crude cell extract. The purification was aided by use of a cell extract made after prophage induction of cells lysogenic for bacteriophage lambda that carries the gal repressor gene (galR). The highly purified gal repressor is an effective and specific repressor of in vitro synthesis of gal mRNA with lambda gal DNA as template. Both D-fucose and D-galactose overcome the action of gal repressor; the half-maximal concentrations of D-fucose and D-galactose for overcoming the action of repressor are 1 mM and 0.5 mM, respectively. The repressor fails to repress gal-specific transcription when the gal DNA contains a cis-dominant operator constitutive (O(c)) mutation. We conclude that the gal repressor recognizes the gal operator site and acts by preventing gal transcription.

Ammonium Sulfate↗
PubMed1973

Transformation of chick-embryo fibroblasts by wild-type and temperature-sensitive Rous sarcoma virus alters adenylate cyclase activity.

The activity of the enzyme adenylate cyclase, a component of the plasma membrane, has been determined in chick-embryo fibroblasts and in cells transformed by either Bryan high-titer strain of Rous sarcoma virus (RSV-BH) or a temperature-sensitive mutant of this virus (RSV-BH-Ta). Adenylate cyclase activity is reduced in cells transformed by the wild-type virus and also by the temperature-sensitive mutant when the cells are grown at the permissive temperature (37 degrees ). Transformation results in an altered affinity (K(m)) for the substrate (Mg ATP). The apparent K(m) ATP is 0.23 mM in normal cells and 1.1 mM in cells transformed with wild-type virus. The K(m) ATP of the cells infected with RSV-BH-Ta is 0.67-1.0 mM at 37 degrees and 0.28 mM at 42 degrees . The enzyme from normal cells appears to have two binding sites for Mg(++), one at the catalytic site and a second at a regulatory site. Transformation by RSV-BH or RSV-BH-Ta (37 degrees ) apparently alters this second Mg(++) site. A decrease in adenylate cyclase activity occurs within 10 min after cells infected with RSV-BH-Ta are shifted from 42 degrees to 37 degrees ; the activity falls to one-half that of normal cells 30 min after the temperature shift. Our observations indicate that a viral function lowers cyclic AMP content by lowering the activity of adenylate cyclase, probably through some modification of the plasma membrane.

Adenosine Triphosphate↗