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S Gidwitz

Publications and source records attributed to S Gidwitz.

13 recordsLinked to original sources
PubMed2000

Expression and function of calcium binding domain chimeras of the integrins alpha(IIb) and alpha(5).

To further identify amino acid domains involved in the ligand binding specificity of alpha(IIb)beta(3), chimeras of the conserved calcium binding domains of alpha(IIb) and the alpha subunit of the fibronectin receptor alpha(5)beta(1) were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of alpha(IIb) with those of alpha(5), or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with beta(3) was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the K(d) or B(max) of any mutant. We conclude that chimeras of alpha(IIb) containing the second calcium binding domain sequences of alpha(5) are capable of complexing with beta(3), that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity.

Amino Acid Sequence↗
PubMed1997

Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants.

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase (pp125(FAK)) and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIbbeta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.

Amino Acid Sequence↗
PubMed1992

Conformational alteration of Sindbis virion glycoproteins induced by heat, reducing agents, or low pH.

Sindbis virions undergo a conformational rearrangement after attachment to cells but prior to entry, as detected by exposure of epitopes on virus-cell complexes which are not accessible to their cognate monoclonal antibodies on native virions (D. C. Flynn, W. J. Meyer, and R. E. Johnston, J. Virol. 64:3643-3653, 1990). The rearrangement did not appear to require transit of virions through a low-pH environment, and the altered virions participated in a productive infection. This naturally occurring structural alteration could be mimicked, although not precisely duplicated, by any of the three artificial treatments of purified virions in vitro: brief incubation at 51 degrees C, treatment with 1 to 5 mM dithiothreitol, or incubation of pH 5.8 to 6.0. Infectivity was maintained after all three treatments, suggesting that Sindbis virions are metastable and can exist in at least two infectious conformations. The integrity of external, neutralizing epitopes was maintained on cell-associated virions and in the altered conformations induced by heat and dithiothreitol, whereas these epitopes were unreactive under low-pH conditions that induced an analogous exposure of previously inaccessible epitopes. The pH at which the conformational change was induced and the pH at which virions could mediate cell-cell fusion from without were coordinately shifted when these two parameters were determined for another strain of Sindbis virus. This coordinate shift in pH optima suggests that the conformational change in virion structure observed at the cell surface may be causally related to fusion.

Antibodies, Monoclonal↗
PubMed1990

The genomic organization of platelet glycoprotein IIIa.

The platelet membrane glycoprotein (GP) IIb/IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix interactions, contains binding sites for fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Absence or defects of this receptor result in the platelet bleeding disorder Glanzmann's thrombasthenia. In this report, we describe the isolation of genomic DNA coding for the entire mature GPIIIa protein. Mature GPIIIa is encoded by 14 exons which range in length from 90 to 3618 base pairs, which are contained within an approximately 46-kilobase (kb) stretch of genomic DNA on chromosome 17. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. The coding region of the GPIIIa gene is identical with the previously described cDNA sequence except for three silent substitutions. One substitution creates a TaqI site which may be the site of a known GPIIIa polymorphism. A second substitution eliminates a SmaI site. Aside from the start of the first exon described, which begins at the second base of the first codon of the mature protein, there is no correlation between the organization of the exons in this gene and proposed functional domains of the protein based on analysis of the primary amino acid sequence. The less frequently used polyadenylation signal AAATTAAA was present at the 3'-end of the major RNA transcript. Recently, an alternatively processed GPIIIa transcript has been described. We demonstrate that this transcript results from nonsplicing of the final intron. The description of the GPIIIa gene organization should be of importance in understanding the evolution of the integrin family of receptors and should be useful in the molecular biology analysis of thrombasthenic patients who have a defect in the GPIIIa gene.

Amino Acid Sequence↗
PubMed1988

Differences in virion stability among Sindbis virus pathogenesis mutants.

The structure of closely related Sindbis virus strains, which differ in their virulence for neonatal mice, has been probed by measuring the sensitivity of virions to heat, varying concentrations of dithiothreitol (DTT), phenylglyoxal or low pH. Attenuated mutants (SB-RL and SB-FP) were much more sensitive to loss of infectivity after heat or DTT treatment than either the prototype virulent strain (SB) or same-site virulent revertants of SB-RL. Incubation of SB-RL virions in the presence of DTT increased their density and exposed sulfhydryl groups on both E1 and E2 glycoproteins as determined by [14C]iodoacetamide labeling. Incubation of SB-RL at pH 6.1 for 1 h at 37 degrees C resulted in a 50% decrease in titer, whereas an equivalent decrease in SB titer was obtained at pH 6.0. Attenuated (SB-RL) and virulent (SB) strains could not be distinguished on the basis of phenylglyoxal sensitivity. Comparison of the nucleotide sequences of these strains and analysis of strains derived from recombinant full-length cDNA clones demonstrated that reduced virion stability under these conditions was a consequence of an arginine for serine substitution at position 114 of the surface glycoprotein E2. This same amino acid substitution is responsible for attenuation in neonatal mice. However, experiments with a second-site virulent revertant of SB-RL, which retained E2 arginine 114 and the reduced stability phenotype, indicated that virion instability in itself was not directly responsible for reduced virulence in vivo.

Animals↗
PubMed1988

Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction.

Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders.

Blood Platelets↗
PubMed1985

Binding and endocytosis of alpha 2-macroglobulin-plasmin complexes.

The clearance of 125I-labeled alpha 2-macroglobulin-plasmin complexes (125I-alpha 2M-PM) from mouse circulation is slower than that of 125I-labeled alpha 2M-methylamine complexes (125I-alpha 2M-CH3NH2). In addition, clearance of 125I-alpha 2M-PM is biphasic, but that of 125I-alpha 2M-CH3NH2 follows simple first-order kinetics. Treatment of alpha 2M-PM with trypsin yields a complex that clears like alpha 2M-CH3NH2. Complexes of alpha 2M with Val442-plasmin (alpha 2M-Val442-PM) were prepared; alpha 2M-Val442-PM has a stoichiometry of 2 mol of Val442-PM to 1 mol of alpha 2M and also clears like alpha 2M-CH3NH2. In vitro 4 degrees C binding inhibition studies with mouse peritoneal macrophages show that alpha 2M-CH3NH2, alpha 2M-PM, trypsin-treated alpha 2M-PM, and alpha 2M-Val442-PM bind with the same affinity, apparent Kd = 0.4 nM. The binding isotherms at 4 degrees C are the same for 125I-alpha 2M-CH3NH2, 125I-alpha 2M-PM, and 125I-trypsin-treated alpha 2M-PM in both mouse peritoneal macrophages and 3T3-L1 fibroblasts. The Scatchard plots for the binding isotherms in macrophages were curved; those in 3T3-L1 fibroblasts were linear with an apparent Kd of 0.48 nM and a receptor activity of 140 fmol/mg of cell protein for alpha 2M-CH3NH2, an apparent Kd of 0.29 nM and a receptor activity of 110 fmol/mg of cell protein for alpha 2M-PM, and an apparent Kd of 0.35 nM and a receptor activity of 210 fmol/mg of cell protein for trypsin-treated alpha 2M-PM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Changes in the binding of "fast"-form alpha 2-macroglobulin to 3T3-L1 cells after differentiation to adipocytes.

Human alpha 2-macroglobulin (alpha 2M)-CH3NH2 specifically binds to 3T3-L1 fibroblasts and adipocytes with an apparent Kd of 0.3 nM at 4 degrees C. Binding to fibroblasts follows first-order kinetics only for the first 20-30 min of reaction, k1 = 160 microM-1 h-1, and then proceeds in a non-first-order reaction that takes 28 h to reach steady state. Receptor activity is 120 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is nondissociable. In contrast, binding to adipocytes follows first-order kinetics, k1 = 720 microM-1 h-1, and reaches steady state in 6-8 h. Receptor activity is 35 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is reversible with a k2 of 0.4 h-1. Control studies with 3T3-C2 cells, which do not differentiate after hormone treatment, indicate that these differences are not due to hormone treatment alone. Binding to both fibroblasts and adipocytes is specific for "fast"-form alpha 2M but not for native alpha 2M. Inhibition studies with neoglycoproteins demonstrate that binding does not occur via any of the known carbohydrate receptors. Some cross-reactivity with antithrombin III-trypsin complexes is demonstrated. Both fibroblasts and adipocytes take up and degrade alpha 2M-CH3NH2 at 37 degrees C. For both cell types, the concentration of alpha 2M-CH3NH2 needed for half-maximal uptake is 65 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1982

Facile enzymatic synthesis of fatty acylcoenzyme A thioesters.

The fatty acid:CoA ligase (acyl-CoA synthetase, EC 6.2.1.3) of rat liver microsomes was solubilized with Triton X-100 and bound to Matrex Gel Red A. Fatty acid:CoA ligase immobilized on Matrex Gel Red A was active and proved useful for the synthesis of fatty acyl-CoA thioesters. The immobilized activity was characterized by a 3-fold higher apparent Km for ATP than the soluble activity, similar apparent Km values for CoA and palmitate, and a shift in the pH dependence. Quantitative incorporation of fatty acid or CoA was possible. Long chain-fatty acyl CoA thioesters were purified in a single step by hydrophobic chromatography on Octyl-Sepharose. In addition to producing the thioesters of typical fatty acids (e.g., myristic, palmitic, stearic, oleic, cis-vaccenic, linoleic, and arachidonic), analogs such as the fluorescent molecules beta-parinaroyl-CoA and palmitoyl-(1-N6-etheno-)CoA were easily synthesized. These procedures should be generally applicable for both the small-scale, i.e., 1 to 10 mumoles, and large-scale, i.e., 50 to 250 mumoles, scale preparation of numerous fatty acyl-CoA's and related compounds.

Acyl Coenzyme A↗
PubMed1980

Effect of membrane phospholipid composition changes on adenylate cyclase activity in normal and rous-sarcoma-transformed chicken embryo fibroblasts.

Adenylate cyclase specific activities in membranes isolated from chicken embryo fibroblasts transformed by Rous sarcoma virus are significantly lower than the specific activity of the enzyme in normal membranes. Since normal and transformed membranes have different phospholipid and fatty acid compositions, adenylate cyclase activities were examined in normal and transformed membranes which had been supplemented with polar head groups or fatty acids. Basal, fluoride, and prostaglandin E1-stimulated activities changed systematically with phospholipid composition. Increases in the primary amino group of the phospholipid polar head groups or the average degree of fatty acid unsaturation both inhibited adenylate cyclase activity. In general, adenylate cyclase activities in normal membranes were more sensitive to phospholipid compositional changes compared to adenylate cyclase in transformed membranes. The data indicate that the lower adenylate cyclase activities in transformed membranes are not solely attributable to phospholipid changes but do suggest that increases in the percentage of phosphatidylethanolamine may contribute to the lower adenylate cyclase activities in transformed membranes.

Adenylyl Cyclases↗
PubMed1980

A comparison between adenylate cyclase solubilized from normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

The specific activity of adenylate cyclase in membrane preparations obtained from Rous Sarcoma virus-transformed chicken embryo fibroblasts is two to four times lower than that found in untransformed membranes. Adenylate cyclase was solubilized from normal and transformed membranes in order to evaluate the influence of the membrane phase on the properties of the enzyme. Adenylate cyclase in normal and transformed membranes differed in specific activity, V for ATP, activation entropies, sensitivity to Ca2+, and stability at 37 degrees C. Solubilization with Brij 96 abolished or greatly reduced these differences. These data suggest that the differences between adenylate cyclase activities in normal and transformed chicken embryo fibroblasts are due either to differential modulation of enzyme activity by an effector which requires intact membranes for its effects, or indirect effects due to altered membrane properties.

Adenylyl Cyclases↗
PubMed1976

Solubilization of adenylate cyclase from normal and Rous sarcoma-transformed chicken embryo fibroblasts.

Adenylate cyclase activities in membranes prepared from Rous sarcoma-transformed chicken embryo fibroblasts are 2 to 4 times lower than in membranes prepared from normal chicken embryo fibroblasts. Adenylate cyclase activities were solubilized from normal and transformed membranes with five different nonionic detergents. In all cases, the specific activities of the enzyme solubilized from normal and transformed preparations were essentially identical. These data suggest that the microenvironment of adenylate cyclase in transformed membranes may be wholly or partially responsible for the decreased activities of this enzyme.

Adenylyl Cyclases↗