Cytotoxicity of commonly used nitroxide radical spin probes.

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.

Heparin modulates conformational states of plasma fibronectin: an electron spin resonance spin label approach.

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.

The lack of effects of alkylating agents on mammalian cell membranes.

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.

Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers.

Most (90 to 95%) human plasma fibronectin (PFn) molecules exist as 450-kDa disulfide-rich dimers comprised of two major types of subunits (A, 220 kDa; B, 215 kDa) that are joined near the COOH terminus by two disulfide bonds. Smaller PFn species (Zone II; 190-235 kDa) consist mainly of monomers and/or a monomeric subunit joined covalently to a smaller peptide remnant presumably derived by proteolysis of a parent 450-kDa molecule. A relatively simple and selective method for preparing functionally active, partially reduced monomeric fibronectin subunits (PR-PFn) by limited and selective reduction of dimeric plasma fibronectin (PFn) has been developed. PR-PFn was prepared by incubating PFn in phosphate-buffered saline, pH 7.4, for 2 h at room temperature in the presence of 17 mM dithiothreitol (DTT). Following S-carboxymethylation or S-carboxyamidomethylation, the material was passed through a gelatin-Sepharose column and nonbinding material was discarded; gelatin-bound material was eluted using a 0 to 2 M KSCN gradient. Residual dimeric species (10-20%) could be separated from monomers in high yield by gel-sieving chromatography on a Sepharose 6B-Cl in the presence of a chaotropic salt, 0.3 M KSCN. Most new SH groups (74-81%) in that fraction of PR-PFn binding to gelatin were localized in proteolytic fragments containing the COOH terminus, thus suggesting that selective cleavage of the interchain disulfide bridges had taken place. The binding affinity of PR-PFn to gelatin- and fibrin-Sepharose was lower than that of dimeric PFn, but the same as that of Zone II PFn and other monomeric gelatin-binding proteolytic derivatives. PR-PFn also bound to heparin-Sepharose and promoted cell attachment and spreading. We conclude that PR-PFn monomers possess the same functional activities as those of the parent chains.

Structure and flexibility of plasma fibronectin in solution: electron spin resonance spin-label, circular dichroism, and sedimentation studies.

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)

Spin label studies of sulfhydryl environment in plasma fibronectin.

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.

Effect of asparaginase on cell membranes of sensitive and resistant mouse lymphoma cells.

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.

Membrane fluidity of Chinese hamster ovary cells on plasma fibronectin-coated microcarriers. A spin label study.

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.

Comparative cytotoxic and biochemical effects of ligands and metal complexes of alpha-N-heterocyclic carboxaldehyde thiosemicarbazones.

Several alpha-N-heterocyclic carboxaldehyde thiosemicarbazones and their iron and copper complexes have been tested for their cytotoxicity and inhibiting activity against DNA synthesis under controlled metal conditions. No ligands show cytotoxicity against Ehrlich cells at the concentrations tested, while some iron and copper complexes are active. In contrast, the ligands inhibit DNA synthesis at much lower concentrations than used above. Similarly, the metal complexes are effective inhibitors at concentrations much below those necessary to demonstrate cytotoxicity. In addition, the iron complexes of 1-formylisoquinoline thiosemicarbazone, 2-formylpyridine thiosemicarbazone, and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone were shown to be three- to sixfold more active than their free ligands as inhibitors of partially purified ribonucleotide reductase to which no iron has been added. The copper complex of 2-formylpyridine thiosemicarbazone was slightly more active than the free ligand against the reductase.