Binding of Clostridium perfringens 125I-labeled delta-toxin to erythrocytes

Hemolytically active, 125I-labeled delta-toxin from Clostridium perfringens was used to study the binding of this cytolysin to sheep, goat, human, rabbit, horse, mouse, and guinea pig erythrocytes. The extent of toxin binding was correlated with the known hemolytic specificity of the toxin. Detailed studies of the binding were carried out on sheep erythrocytes which showed the highest sensitivity to lysis by delta-toxin. Simultaneous determination of toxin binding and release of intracellular 86Rb+ and hemoglobin suggested that toxin binding and membrane damage were separate sequential events. Toxin binding was rapid (2-5 min) and temperature-dependent. The extent of binding was temperature-independent. Binding was saturable, specific, relatively tight (Ka = 4.4 X 10(8) M-1) and largely irreversible. A single type of binding site (7,000/sheep erythrocyte) was found. Cell-bound toxin was extractable by chaotropic ions. Preincubation of the toxin with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM2 ganglioside) inhibited both binding and hemolysis. Toxin binding was affected by pretreatment of sheep erythrocytes with pronase but not with trypsin or chymotrypsin. Cell treatment with neuraminidase prevented toxin binding by 30%. Preincubation of the toxin with specific immune sera blocked its binding on target cells. It is suggested that GM2 ganglioside, a more complex membrane component containing this glycolipid or a structurally related molecule is the binding site for delta-toxin on the surface of sensitive erythrocytes.     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

Photolabeling of staphylococcal alpha-toxin from within rabbit erythrocyte membranes

Intrinsic membrane proteins of rabbit red blood cells were labeled with the photoreactive amphipatic reagent 12-(4-azido-2-nitrophenoxy) stearoyl (1-14C) glucosamine, which inserts into the hydrophobic membrane region and generates a reactive nitrene upon ultraviolet irradiation. Photolabeling of membrane-bound staphylococcal alpha-toxin after lysis of probe-treated rabbit red blood cells by this toxin implies its penetration into the hydrophobic region of the outer leaflet of the membrane. In contrast clostridial theta-toxin and staphylococcal delta-toxin were not labeled, but extraction of intrinsic membrane proteins by delta-toxin was evidenced.     

Immunoassay of leucovorin: use of 125I-labeled protein A to detect immunological binding.

An immunoassay method was developed for the quantitative determination of leucovorin. Leucovorin immobilized by covalent linkage to a solid support bound anti-leucovorin antibody produced in rabbits. The amount of ¹²⁵I-labeled Protein A bound specifically to IgG anti-leucovorin-coated beads served as a measure of antibody binding. The ability of increasing amounts of fluid-phase leucovorin to compete with solid-phase drug for specific antibody was reflected in a dose-dependent decrease in the amount of bound ¹²⁵I-labeled Protein A. This competition was used to obtain a standard curve to measure levels of leucovorin in the sera of rabbits treated with the drug. The relative abilities of methotrexate, 5-methyltetrahydrofolic acid, and other structurally related compounds to act as inhibitors demonstrated the serologic specificity of the leucovorin immune system.     

Physical states of staphylococcal alpha-toxin

At least three different forms of staphylococcal alpha-toxin have been shown to exist: soluble active alpha-toxin (alpha 3S), soluble inactive alpha-toxin (alpha(12s)), and insoluble inactive aggregate. Aggregation to the insoluble, biologically inactive form could be induced by brief heating to 60 C. The aggregate was dissociated by treatment with 8 m urea with reappearance of biological activity. Subsequent removal of urea by dialysis resulted in some spontaneous reaggregation to the insoluble state. The supernatant fluid obtained after dialysis contained soluble active alpha-toxin of high specific activity, possessing physical, toxic, and immunological properties closely resembling those of native toxin. The soluble biologically inert component (alpha(12s)) was identified as a third physical state. Negatively stained preparations of this material, when examined in the electron microscope, showed rings of approximately 100 A outside diameter containing 6 +/- 1 subunits.     

Approaches to sequence analysis of 125I-labeled RNA.

A method is described for the initial steps of sequence analysis of RNase T1-and pancreatic RN-ase-resistant oligonucleotides of RNA containing cytidylate residues labeled in vitro with 125I. In many cases an oligonucleotide sequence can be deduced from a consideration of (i) its relative position in the two-dimensional fingerprint (with DEAE thin layer homochromatographic second dimension), (ii) its electrophoretic mobility on DEAE paper at pH 1.9, and (iii) identification of its products of further enzymatic digestion by comparison with a set of marker oligonucleotides. Additional methods including analysis of oligonucleotides following chemical blocking of uridylate residues with CMCT and analysis of products of incomplete enzymatic digestion are also discussed.     

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of glucagon resistance in large rat adipocytes: 125I-labeled glucagon binding and lipolytic capacity

This study is concerned with potential modifications of large fat cells from adult rats (400-450 g) that make them resistant to stimulation by glucagon. The lipolytic capacity and (125)I-labeled glucagon-binding capability of these cells were compared with these properties of small glucagon-sensitive cells from young rats (130-160 g). As determined by maximal stimulation with theophylline, dibutyryl cAMP, or epinephrine, the lipolytic capacity of large cells was not markedly different from small cells, which suggests that an alteration contributing to glucagon insensitivity is not present in the enzymes involved with hormone-mediated lipolysis. Glucagon-binding studies did indicate a difference between the two cell types. Both large cells and particulate fractions from large cells bound less (125)I-labeled glucagon than small cells or small-cell particles. That diminished binding is not a consequence of glucagon degradation is indicated by the similar amounts of (125)I-labeled glucagon degraded by both cell types. The decrease in (125)I-labeled glucagon binding was not as marked as the decrease in lipolytic response to glucagon stimulation. This lack of correlation and the relationship between elevated phosphodiesterase levels and glucagon insensitivity described in the accompanying report suggest that diminished binding explains only in part the marked resistance to glucagon found in large cells.     

Interaction of staphylococcal alpha-toxin with biomembranes

The recent data on staphylococcal alpha-toxin, its functions, methods for its purification, forms are presented in the review. The problem of a target for toxin molecule is discussed.     

Autoradiographic analysis of proestrous changes in the binding of 125I-labeled prolactin to the hamster ovary

Autoradiographic histochemistry was employed to examine changes in the binding of 125I-labeled prolactin (Prl) to ovaries from proestrous hamsters before (at 1200 h), during (at 1600 h), and after (at 2000 h) the preovulatory gonadotropin surge. In untreated control hamsters, there was a marked and progressive loss of Prl binding, first in the interstitial cells and follicular thecae by 1600 h, and then in the granulosa cells of the preovulatory follicles by 2000 h. When proestrous hamsters were treated with ergocryptine to significantly lower serum Prl, or injected with exogenous Prl, Prl binding to their ovaries did not differ from controls, suggesting that decreased Prl binding was due to neither increased occupancy of binding sites by endogenous Prl nor down regulation of Prl receptors by Prl itself. Conversely, when proestrous hamsters were treated with phenobarbital to block the luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, the loss of Prl binding sites in the ovaries was prevented, suggesting that the LH/FSH surge might initiate a down regulation of Prl receptors in the ovary. Such a down regulation of Prl receptors may serve as a mechanism by which the ability of Prl to affect periovulatory events in the ovary might be regulated.     

Iodination of a tyrosyl residue in staphylococcal alpha-toxin.

Iodination of staphylococcal alpha-toxin by the lactoperoxidase method resulted in the maximal incorporation of about 2.5 atoms of iodine per molecule of alpha-toxin. The iodination primarily involved a single tyrosine residue as shown by analysis of both cyanogen bromide and tryptic peptides. Iodination at a level of 1.2 iodine atoms per alpha-toxin molecule led to a dramatic decrease in the hemolytic and lethal activities, although no decrease in the binding of iodinated toxin to rabbit erythrocytes was observed (Cassidy and Harshman (1976), Biochemistry, the following paper in this issue). Monoiodinated alpha-toxin was found to have 15% of the specific hemolytic activity of native alpha-toxin. Incubation of rabbit erythrocytes with iodinated alpha-toxin led to a significant protection from the hemolytic activity of native alpha-toxin added later. The results show the modification of a single unique tyrosyl residue in alpha-toxin permits the resolution of alpha-toxin's biological activities from its cell binding activity.     

Isolation and characteristics of staphylococcal alpha-toxin

A method for isolation of staphylococcal alpha-toxin preparations has been elaborated. Characteristics of the toxin isolated by the method are as follows: mol. mass = 35 Kd; HU = 0.1 microgram; DnD= 0.1 microgram; LD50 = 2 micrograms. It is for the first time that alpha-toxin was fragmented by papain and digested by alpha, gamma-chemotrypsin. The papain fragments (18.5 and 15 Kd) retained lethal activity but lost hemolytic and dermonecrotic activities. Alpha, gamma-chemotryptic digested fragments (18 and 15 Kd) retained hemolytic and lethal effects, but lost their dermonecrotic activity.     

Transport and processing of staphylococcal alpha-toxin

Two larger precursors to staphylococcal alpha-toxin were identified and partially characterized. Both precursor proteins were present on the cell membrane at very low levels and appeared to be rapidly processed to the mature form. Dinitrophenol inhibited processing such that the two precursors accumulated in the membranes, whereas little extracellular (mature) alpha-toxin is formed. The peptide maps of the 35S-labeled peptides from extracellular alpha-toxin and the two precursors were almost identical. The larger precursor protein contained four additional peptides and the smaller precursor protein contained three additional peptides not found in the extracellular toxin.