Energetic contribution of solvent-exposed ion pairs to alpha-helix structure.

Understanding the role of amino acid side-chain interactions in forming secondary structure in proteins is useful for deciphering how proteins fold and for predicting folded structures of proteins from their sequence. Analysis of the secondary structure as a function of pH in two designed synthetic peptides with identical composition but different sequences, affords a quantitative estimate of the free energy contribution of a single ion pair to the stability of an isolated alpha-helix. One peptide contains repeated blocks of Glu4Lys4. The second has repeated blocks of Glu2Lys2. The former contains significant helical structure at neutral pH while the latter has none, based on ultraviolet light circular dichroism measurements and 1H nuclear magnetic resonance spectroscopy. The difference is attributed to formation of helix-stabilizing salt-bridges between Glu- and Lys+ spaced at i, i + 4 intervals in the former peptide. The free energy of formation of a single Glu(-)-Lys+ salt-bridge can be evaluated by using a statistical model of the helix-coil transition that explicitly includes salt-bridges: the result is -0.50(+/- 0.05) kcal/mol at 4 degrees C and neutral pH in 10 mM salt, in agreement with a value derived for a single salt-bridge in a helix on the surface of a globular protein.

The helix-coil transition in heterogeneous peptides with specific side-chain interactions: theory and comparison with CD spectral data.

Natural and synthetic peptides that contain detectable intramolecular alpha-helical structure in aqueous solution have been used to evaluate the helical propensities for the common amino acids. Experimental spectroscopic data must be fit to a model of the helix-coil transition in order to determine quantitative stability constants for each amino acid. We present here a statistical mechanical description of helix formation in peptides or protein fragments that takes into account multiple internal conformations, heterogeneity in the stabilizing effects of different side chains, and specific side-chain-side-chain interactions. The model enables one to calculate values of [theta]222 for a given peptide using the length dependence of the helix signal computed by a quantum mechanical treatment of the n pi * transition that dominates the 222-nm band. In addition, the helical probability at any residue in the chain is readily computed, and should prove useful as nmr spectral data become available. The free energy of specific side-chain interactions, including ion pair formation, can be evaluated. Application of the analysis to experimental data on a pair of isomeric peptides, only one of which contains ion pairs, indicates that forming a single glutamate-lysine ion pair stabilizes the alpha-helix by 0.50 kcal/mole in 10 mM sodium ion and pH 7. A survey of the CD data measured for a variety of model peptides is presented, indicating that a single set of s values and sigma constant can account for some but not all of the available results.

The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro.

In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro. We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA. Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA. However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA. To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short-term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium. Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others. Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.

The synthesis and secretion of granulocyte-monocyte colony-stimulating activity (CSA) by isolated human monocytes: kinetics of the response to bacterial endotoxin.

We studied the kinetics of the synthesis and secretion of granulocyte-monocyte colony-stimulating activity (CSA) by human monocytes stimulated by S. typhi endotoxin. We found that these cells initially secrete copious quantities of CSA when exposed to endotoxin but rapidly become refractory to its stimulatory effect. When monocytes were incubated in liquid suspension cultures, large amounts of CSA were generated during the first 24 hr of culture after the addition of as little as 10 ng/ml of endotoxin to previously unstimulated monocytes. After the addition of endotoxin, CSA secretion abruptly took place after an initial 1 to 2 hr lag phase, and occurred primarily within the first 6 to 12 hr of culture. The addition of puromycin or cycloheximide to the cultures significantly inhibited CSA secretion in response to endotoxin, suggesting that CSA production by stimulated monocytes requires de novo protein synthesis and does not solely result from the release of preformed active CSA. After initial exposure to endotoxin, CSA production by monocytes steadily decreased after 24 hr and ceased after 72 hr. Subsequent reexposure to the same concentration of endotoxin resulted in little CSA production, whereas the refractory state could be overcome by increasing the quantity of endotoxin added. Additional studies demonstrated that refractoriness of monocytes to further CSA production after initial response to endotoxin was not due to a loss of cell viability, degradation of endotoxin, or inhibition of CSA synthesis by soluble metabolites generated by stimulated monocytes. Our data suggest that the capacity of human monocytes to synthesize CSA in response to endotoxin rapidly becomes blunted after initial exposure of the cells to the lipopolysaccharide, but that the refractory state of these cells can be overcome by increasing the concentration of endotoxin. Acquired hyporesponsiveness of cells that produce CSA may in part account for the phenomenon of immediate endotoxin tolerance observed in vivo.

Fractionation of human bone marrow cell suspensions in nylon fiber columns: an efficient method for the removal of cells that produce colony stimulating factor (CSF).

In this report, we describe an efficient technique for the extraction of CSF-producing cells from human marrow suspensions. Prior to plating in agar cultures, we incubated buoyant human marrow cells for 45 min in columns packed with nylon fiber or subjected the cells to two one-hour incubations in glass petri dishes. Recoveries of total cells, differential marrow elements, and committed granulocyte-monocyte progenitor cells (CFUc) were similar after each separative procedure. However, spontaneous CFUc proliferation was more effectively eliminated when cells were fractionated in nylon fiber columns. After the removal of cells which were adherent to glass, spontaneous CFUc proliferation in cultures containing no exogenous CSF accounted for 2.1% of total CFUc at a plating concentration of 10(5) cells/ml and 7.8% at a concentration of 3 X 10(5) cells/ml. After the fractionation of marrow cell suspensions in nylon fiber columns, spontaneous CFUc growth was completely obliterated at a plating concentration of 10(5) cells/ml, and at a concentration of 3 X 10(5) cells/ml accounted for only 0.09% of total CFUc. Further experiments were undertaken which demonstrated that buoyant marrow cells after incubation in nylon fiber columns may be employed to assay CSF in extremely dilute concentrations. Because of the simplicity and efficiency of this procedure, nylon fiber chromatography appears to be a highly useful technique for the rapid semi-purification of marrow suspensions for use in the assay of human CSF.