The kinetic and physical properties of the allosteric enzyme, aspartate transcarbamoylase from Escherichia coli, have been analyzed according to the two-state model (Monod, J., Wyman, J., and Changeux, J.-P. (1965), J . Mol. Biol. 12, 88). An internally consistent set of calculated parameters accounted quantitatively for the results from diverse experiments including: (a) enzyme kinetics as a function of the concentration of aspartate in the presence of saturating carbamoyl phosphate; (b) gross conformational changes of the enzyme, revealed by the decrease in the sedimentation coefficient and the increase in the reactivity of the sulfhydryl groups of the regulatory subunits, as a function of the extent of saturation of the active sites by the bisubstrate analogue, N-(phosphonacety1)-L-aspartate; (c) stimulation of enzymic activity at low concentrations of the substrate analogue, succinate; and (d) effects of the inhibitor, CTP, and the activator, ATP, on the kinetic and physical properties of the enzyme. The data were interpreted in terms of an equilibrium between a constrained or low-affinity (T) state and a relaxed or highaffinity (R) form of the enzyme and the perturbation of the equilibrium by the addition of various ligands. In the absence
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor ␣ chain (sGMR␣) and  chain (sc) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the c-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMR␣, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMR␣ and sc, and this complex could be disrupted by E21R. Significantly, sizeexclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one sc dimer with a single molecule each of sGMR␣ and of GM-CSF. In addition, a hitherto unrecognized direct interaction between c and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor. (Blood. 2003;101: 1308-1315)
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