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1.
FEBS Lett ; 173(2): 283-6, 1984 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-6086404

RESUMEN

The local environment of the free sulfhydryl groups in plasma fibronectin has been investigated by ESR techniques using a series of maleimide spin labels, varying in chain length between the maleimide and nitroxide free radical groups. Chemical modification with these analogs does not affect either the CD spectra or the cell adhesion activity of the protein molecule. The ESR results show that the free sulfhydryl group of plasma fibronectin is in a cleft about 10.5 A in length. The significance of this finding is discussed.


Asunto(s)
Fibronectinas/sangre , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Maleimidas , Conformación Proteica , Marcadores de Spin , Compuestos de Sulfhidrilo/análisis
2.
Life Sci ; 40(5): 495-8, 1987 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-3027487

RESUMEN

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 microM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30-50 microM) none of the lipid soluble spin probes tested was cytotoxic. At 100 microM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.


Asunto(s)
Supervivencia Celular/efectos de los fármacos , Marcadores de Spin/toxicidad , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Fluidez de la Membrana
3.
Int J Tissue React ; 8(5): 347-54, 1986.
Artículo en Inglés | MEDLINE | ID: mdl-3023251

RESUMEN

The importance of cell membrane components as target sites for the action of 2,3,5-tris(ethyleneimino)-benzoquinone (Trenimon), mechlorethamine hydrochloride (HN2) and tris(2-chloroethyl) amine hydrochloride (HN3) was investigated. Uptake of 2-aminoisobutyric acid (AIBA) was studied under nonsaturating conditions where the transport system was rate-limiting for the uptake. Uptake of AIBA into L5178Y leukaemic cells was either inhibited or stimulated, depending on the type of the drug, the drug concentration and the length of incubation. Treatment of human erythrocytes with 10(-3) M HN3 produced new high-molecular-weight protein bands on SDS-polyacrylamide gel electrophoresis. Conversely, HN3 had no effect on L5178Y cell membrane proteins. Neither HN2 nor Trenimon produced any detectable changes in membrane proteins of L5178Y cells or human erythrocytes. None of the three drugs at concentrations and incubation conditions which inhibited cell replication changed the stoichiometry or dissociation constant of concanavalin A (Con A) binding sites on L5178Y cells. Trenimon at highly toxic concentrations had no effect on the fluidity of phospholipid membranes or of membranes of Ehrlich ascites tumour cells as analysed by ESR spin-label methods. The results presented here do not support the hypothesis that cell membranes are the primary target sites for alkylating drugs.


Asunto(s)
Alquilantes/farmacología , Membrana Celular/efectos de los fármacos , Mecloretamina/farmacología , Compuestos de Mostaza Nitrogenada/farmacología , Ácidos Aminoisobutíricos/metabolismo , Animales , Carcinoma de Ehrlich/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Espectroscopía de Resonancia por Spin del Electrón/métodos , Membrana Eritrocítica/metabolismo , Humanos , Leucemia L5178/metabolismo , Ratones , Tretinoina/farmacología
4.
Biochemistry ; 23(26): 6393-7, 1984 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-6099139

RESUMEN

Human plasma fibronectin has been investigated by electron spin resonance (ESR) spin-label methods in conjunction with circular dichroism (CD) and sedimentation techniques to investigate its structure and flexibility in solution. The buried sulfhydryl groups of fibronectin were modified with a maleimide spin-label [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. Both conventional and saturation transfer ESR spectra give a rotational correlation time of about (2-3) X 10(-8) s for plasma fibronectin, a value that is at least 40 times faster than the rotational correlation time calculated from the minimal molecular dimensions. This argues that plasma fibronectin is not a compact, globular protein and suggests that the regions of ordered structural domains have a relatively high degree of independent mobility. ESR, CD, and sedimentation measurements showed that many structural features of plasma fibronectin remain unchanged when the pH is decreased from 7.4 to 3.0. On the other hand, ESR results indicate an unfolding of the protein molecule either at pH 11 or in 4 M urea solution. Similarly, the sedimentation coefficient decreases from about 13 to 8.4 S when the pH is raised to 10.8. At pH values above 11, the CD spectrum resembles a random coil; however, some ordered structure is retained either at pH 11 or in 4 M urea. It is likely that the sulfhydryl-containing regions of the molecule are more sensitive to urea or alkali than are portions of the molecule stabilized by intrachain disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)


Asunto(s)
Fibronectinas , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Concentración de Iones de Hidrógeno , Conformación Proteica , Soluciones , Ultracentrifugación , Urea
5.
Exp Cell Res ; 150(1): 77-83, 1984 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-6319163

RESUMEN

Purified plasma fibronectin promotes the spreading of Chinese hamster ovary (CHO) cells on microcarriers in a serum-free medium. The promotion of 50% of cell spreading on microcarriers requires about 3.4 X 10(8) fibronectin molecules per bead. CHO cells spreading on plasma fibronectin-coated microcarriers had a more rigid cell membrane compared to CHO cells in suspension as determined by using 5-doxylstearate spin label. No detectable differences in the electron spin resonance (ESR) spectra of 12-doxylstearate spin-labeled CHO cells on plasma fibronectin-coated microcarriers and in suspension were observed, suggesting that the effects of plasma fibronectin on membrane fluidity are restricted to the polar head group region of the cell surface membrane. In addition, no significant differences in membrane fluidity and cell spreading were found between CHO cells spreading on plasma fibronectin-coated cytodex 1 (without denatured collagen) and cytodex 3 (with denatured collagen) microcarriers, indicating that a surface layer of denatured collagen is not required for plasma fibronectin to promote cell spreading and to rigidify the cell surface membrane.


Asunto(s)
Movimiento Celular , Fibronectinas/fisiología , Fluidez de la Membrana , Absorción , Animales , Línea Celular , Colágeno , Cricetinae , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Calor , Microesferas , Ovario
6.
In Vitro ; 20(5): 376-84, 1984 May.
Artículo en Inglés | MEDLINE | ID: mdl-6724617

RESUMEN

High concentrations of Escherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containing Acinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells. Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolated and maintained in asparagine depleted or asparaginase containing medium. The E. coli asparaginase preparation inhibited protein and glycoprotein biosynthesis to comparable degrees. It did not have proteolytic or glycolytic activity. Escherichia coli asparaginase did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentrations of E. coli asparaginase have a specific effect on the Con A receptor in the sensitive line.


Asunto(s)
Asparaginasa/farmacología , Membrana Celular/efectos de los fármacos , Leucemia L5178/fisiopatología , Leucemia Experimental/fisiopatología , Aminoácidos/metabolismo , Animales , Membrana Celular/fisiología , Medios de Cultivo , Resistencia a Medicamentos , Glicoproteínas/biosíntesis , Ratones , Proteínas de Neoplasias/biosíntesis , Biosíntesis de Proteínas , Receptores de Concanavalina A/análisis
7.
Arch Biochem Biophys ; 244(1): 50-6, 1986 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-3004341

RESUMEN

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.


Asunto(s)
Fibronectinas/sangre , Heparina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Heparina/sangre , Humanos , Fragmentos de Péptidos , Unión Proteica , Conformación Proteica/efectos de los fármacos , Marcadores de Spin , Ultracentrifugación
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