Mapping of the binding frame for the chaperone SecB within a natural ligand, galactose-binding protein.

The chaperone SecB selectively binds polypeptides that are in a non-native state; however, the details of the interaction between SecB and its ligands are unknown. As a step in elucidation of the molecular mechanism of binding, we have mapped the region of a physiologic ligand (galactose-binding protein) that is in contact with SecB. The binding frame comprises approximately 160 aminoacyl residues and is located in the central portion of the primary sequence. Comparison to the binding frame within maltose-binding protein, which is similarly long and positioned around the center of that polypeptide, reveals no similarity in sequence or in folding motif. The results are consistent with the proposal that the selectivity in binding exhibited by SecB is based on the simultaneous occupancy of multiple binding sites, each of which demonstrates low specificity, by flexible stretches of polypeptide that are only accessible in non-native proteins.

Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli.

The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306. Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not. Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase. The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form.

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.

Calorimetric study of carbohydrate binding to concanavalin A.

Flow microcalorimetry has been used to examine the delta H of binding of two types of saccharides, a series of simple monosaccharides and a series of alpha-(1----4)-linked glucosides, to the lectin Concanavalin A. It has been found that the delta H decreases with any change in the stereochemistry of a hydroxyl group relative to methyl alpha-D-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The delta H's of binding for the alpha-(1----4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for alpha-(1----4)-linked glucopyranosides, in contrast to that proposed for alpha-(1----2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.

Kinetic analysis of tetracycline accumulation by Streptococcus faecalis.

Tetracycline transport by Streptococcus faecalis occurs by an energy-dependent, carrier-mediated process. The Michaelis constant for transport was unchanged, but the maximal velocity was increased when an energy source, glucose, was present. Accumulation levels, sometimes 25-fold greater than the external concentration, were controlled by the transport system.

Streptococcus faecalis proton gradients and tetracycline transport.

The transport of chlortetracycline by Streptococcus faecalis is energy dependent. Addition of glucose to energy-depleted cells enhances both the transport rates and accumulation levels. Transport rates can be altered independently of glucose by treating cells with ionophores that increase or decrease the proton gradient. The transport of the antibiotic is linked only to the transmembrane pH difference, delta pH, and not the transmembrane electrical potential, delta psi. This conclusion was verified by quantitative measurements of delta pH, delta psi, and tetracycline accumulation levels. A linear correlation between delta pH and the tetracycline electrochemical potential was observed. Tetracycline most likely accumulates by the symport of protons in which the protons are bound to an anionic form of the antibiotic to form an uncharged molecule.

A comparison of the cell-binding characteristics of the mitogenic and nonmitogenic lectins from lima beans.

Using the two lectins from lima bean, we have tested the model for mitogenic stimulation of lymphocytes proposed by Prujansky et al. (Prujansky, A., Ravid, A., and Sharon, N. (1978) Biochim. Biophys. Acta 508, 137-146). The lectins used, a tetramer with two saccharide-binding sites and an octamer with four binding sites, are specific for N-acetyl-D-galactosamine. Our results show that cooperative binding may not be a prerequisite for mitogenicity of all lectins. We found that neither the weakly mitogenic tetramer nor the potently mitogenic octamer bound cooperatively to bovine lymphocytes. The strong mitogen bound with a higher affinity than the weak mitogen and fewer mitogen molecules bound to the lymphocyte surface at saturation. Competitive binding experiments indicated that both lectins bound to the same receptors. Our results suggest that the mitogenic lectin is able to bind and cross-link more membrane receptors. We have also studied the binding of the lima bean lectins to human red blood cells of types A, AB, B, and O. Both lectins bound cooperatively to type A and type AB cells and our data indicate that the lima bean lectins bind predominantly to the type A determinant.

A comparison of the interactions of the mitogenic and nonmitogenic lima bean lectins with human lymphocytes.

Binding and mitogenic characteristics of the 2 Phaseolus lunatus (lima bean) lectins have been examined with human peripheral blood lymphocytes. Chemical cross-linking of the nonmitogenic lima bean lectin produced a species that stimulated human lymphocytes as well as or better than the mitogenic lima bean lectin, which is a T lymphocyte mitogen with requirement for monocyte participation. The maximal stimulation and the dose response to the cross-linked lima bean lectin did not significantly differ from that observed with the mitogenic lectin. We have used fluorescein-labeled lima bean lectins to show that both lectins share mutually exclusive binding sites on lymphocytes. Our results strongly support the concept that multiple valence of lectins is important in inducing mitogenesis. Both the mitogenic and nonmitogenic lectins demonstrated selective binding by labeling only 70% od human peripheral blood lymphocytes. The fraction not binding lectin is a population of T lymphocytes. Competitive binding studies with the lima bean lectins and other N-acetyl-D-galactosamine-specific lectins suggest that the cell surface receptors for these various lectins may be quite different. We have also studied the response of human lymphocytes to the lima bean lectins and soybean agglutinin after neuraminidase treatment. As previously demonstrated (22), neuraminidase treatment of the cells drastically altered the binding and mitogenic response to soybean agglutinin. Lima bean lectin binding to the treated cells was also markedly increased, but the mitogenic response was essentially unaffected.

Activation of concanavalin A by Cd2+.

Binding of Cd2+ to concanavalin A and the subsequent induction of saccharide-binding activity has been studied at pH 6.5. We found that Cd2+ bound to both metal sites, S1 and S2, and that Cd2+ alone would induce sugar binding in concanavalin A. Using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside we determined that full saccharide-binding activity was obtained only when the total bound Cd2+ stoichiometry reached 2 ions/concanavalin A subunit. We also report evidence suggesting that the binding of Cd2+ to S2 is the crucial step in activation and that Cd2+ binding to S1 induces a form of concanavalin A similar to that induced by Zn2+, Ni2+, or Co2+ and different from that induced by Mn2+.