Isolation and characterization of IIAChb, a soluble protein of the enzyme II complex required for the transport/phosphorylation of N, N'-diacetylchitobiose in Escherichia coli.

N,N'-Diacetylchitobiose is transported/phosphorylated in Escherichia coli by the (GlcNAc)(2)-specific Enzyme II permease of the phosphoenolpyruvate:glycose phosphotransferase system. IIA(Chb), one protein of the Enzyme II complex, was cloned and purified to homogeneity. IIA(Chb) and phospho-IIA(Chb) form stable homodimers (). Phospho-IIA(Chb) behaves as a typical epsilon2-N (i.e. N-3) phospho-His protein. However, the rate constants for hydrolysis of phospho-IIA(Chb) at pH 8.0 unexpectedly increased 7-fold between 25 and 37 degrees C and increased approximately 4-fold with decreasing protein concentration at 37 degrees C (but not 25 degrees C). The data were explained by thermal denaturation studies using CD spectroscopy. IIA(Chb) and phospho-IIA(Chb) exhibit virtually identical spectra at 25 degrees C (approximately 80% alpha-helix), but phospho-IIA(Chb) loses about 30% of its helicity at 37 degrees C, whereas IIA(Chb) shows only a slight change. Furthermore, the T(m) for thermal denaturation of IIA(Chb) was 54 degrees C, only slightly affected by concentration, whereas the T(m) for phospho-IIA(Chb) was much lower, ranging from 40 to 46 degrees C, depending on concentration. In addition, divalent cations (Mg(2+), Cu(2+), and Ni(2+)) have a dramatic and differential effect on the structure, depending on the state of phosphorylation of the protein. Thus, phosphorylation destabilizes IIA(Chb) at 37 degrees C, potentially affecting the monomer/dimer transition, which correlates with its chemical instability at this temperature. The physiological consequences of this phenomenon are briefly considered.

The structural stability of the co-chaperonin GroES.

The structural stability of the co-chaperonin GroES has been studied by high sensitivity differential scanning calorimetry and circular dichroism under different solvent conditions. The thermal folding/unfolding of GroES is a spontaneous reversible process involving a highly cooperative transition between folded heptamers and unfolded monomers. During the denaturation process folded monomers are energetically unfavourable and consequently never become populated to an appreciable degree. Analysis of the high resolution structure indicates that isolated folded monomers of GroES bury a significantly smaller fraction of their total surface than typical globular proteins of similar molecular mass. For this reason the intramolecular interactions within each GroES monomer appear not to be sufficient for thermodynamic stabilization. The stabilization of the heptameric structure is due primarily to intersubunit interactions rather than intrasubunit interactions. These interactions favor oligomerization both enthalpically and entropically. Despite the high density of charged residues, the stability of GroES shows no measurable dependence on salt concentration at pH 7. On the other hand, millimolar concentrations of magnesium stabilize GroES, presumably by specific binding. The stabilization elicited by Mg2+ is consistent with a dissociation constant of the order of 0.5 mM and approximately three binding sites per heptamer. These results emphasize the role of quaternary structure in the stabilization of small oligomeric proteins.

Detection and characterization of ceramide-1-phosphate phosphatase activity in rat liver plasma membrane.

A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R.N., and Hemer, M.R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) J. Biol. Chem. 264, 14354-14360). We now demonstrate that the addition of both Cer-1-phosphate (Cer-1-P) and a short-acyl chain analog of Cer-1-P,N-hexanoylsphingosine-1-phosphate (C6-Cer-1-P) to cultured cells and a variety of subcellular fractions results in rapid degradation to Cer and C6-Cer, respectively. The Cer-1-P phosphatase activity is enriched in a rat liver plasma membrane fraction and appears to be distinct from the phosphatase that hydrolyzes phosphatidic acid (PA), PA phosphohydrolase, as shown by the difference in sensitivity of Cer-1-P and PA hydrolysis to propranolol, detergent, and heat treatment. Moreover, the Km of Cer-1-P hydrolysis is 10-fold lower than the Km of PA hydrolysis in plasma membrane. PA is a noncompetitive inhibitor of Cer-1-P hydrolysis, with an inhibition constant 1-1.5-fold higher than the Km of Cer-1-P hydrolysis. In contrast, Cer-1-P does not inhibit PA hydrolysis. Finally, we describe the synthesis of a novel analog of Cer-1-P which is not hydrolyzed in vitro and in vivo and is internalized in cultured cells by endocytosis. These results are discussed in relation to the possible roles of Cer-1-P in regulating intracellular levels of Cer.