No abstract

Activation of the transcription factor NF-B is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IB. A large multiprotein complex, the IB kinase (IKK), catalyzes the phosphorylation of IB. The two kinase components of the IKK complex, IKK␣ and IKK␤, were overexpressed in insect cells and purified to homogeneity. Both purified IKK␣ and IKK␤ specifically catalyzed the phosphorylation of the regulatory serine residues of IB␣. Hence, IKK␣ and IKK␤ were functional catalytic subunits of the IKK complex. Purified IKK␣ and IKK␤ also preferentially phosphorylated serine as opposed to threonine residues of IB␣, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKK␣ and IKK␤ revealed that the kinase activity of IKK␤ on IB␣ is 50 -60-fold higher than that of IKK␣. The primary difference between the two activities is the K m for IB␣. The kinetics of both IKK␣ and IKK␤ followed a sequential Bi Bi mechanism. No synergistic effects on IB␣ phosphorylation were detected between IKK␣ and IKK␤. Thus, in vitro, IKK␣ and IKK␤ are two independent kinases of IB␣.The transcription factor NF-B plays a critical role in immune and inflammatory responses. It is regulated by the signaling of receptors for inflammatory cytokines such as TNF-␣, 1 IL-1, or other external stimuli (1). In resting cells, NF-B is sequestered in the cytoplasm through its association with inhibitory proteins termed IB. Two IB proteins, IB␣ and IB␤, are rapidly phosphorylated at Ser residues in the Nterminal region upon stimulation by TNF-␣ and IL-1. The regulated phosphorylation is at Ser-32 and Ser-36 of IB␣ and, correspondingly, Ser-19 and Ser-23 of IB␤ (2, 3). The more recently cloned IB isoform, IB⑀, also contains the two conserved Ser residues at the N terminus for signal-induced degradation (4). Phosphorylated IB␣ and IB␤ are subsequently ubiquitinated and undergo ubiquitin-dependent degradation by the 26 S proteasome (3, 5). Degradation of IB results in the release of NF-B, which then translocates to the nucleus, where it up-regulates the transcription of its target genes (1).A 500 -900-kDa protein complex that contains the TNF-␣-induced IB kinase (IKK) has been purified and characterized independently by two groups (6, 7). The IKK complex phosphorylates IB␣ at the specific Ser residues that target the protein for ubiquitination and degradation. Two kinase subunits of the IKK complex, denoted IKK␣ and IKK␤, have been cloned (6 -10). IKK␣ or IKK␤ overexpressed in mammalian cells specifically phosphorylates IB␣ and IB␤ after immunoprecipitation, and their kinase activities can be induced by TNF-␣ or IL-1 (6 -10). In HeLa cells, expression of antisense IKK␣ inhibited NF-B activation by TNF-␣ or IL-1 (6). Furthermore, overexpression of dominant-negative mutants of either IKK␣ or IKK␤ blocked TNF-␣/IL-1-induced NF-B activation (8, 10). Thus, both IKK␣ and IKK␤ contribute to the activity of the IKK complex and are involved in NF-B activation. An additional protein kinase, NF-B-inducing kina...

A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3-b:2',3'-e]- [1,4]diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1). An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation. BI-RG-587 and close structural analogues competitively protected RT-1 from inactivation by the photoaffinity label. A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds. Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation. A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1.

synopsisThe storage and loss shear moduli, G , and G", have been measured for solutions of three samples of poly-y-benzyl-L-glutamate with molecular weights from 16 to 57 X lo4, by use of the Birnboim-Schrag multiple-lumped resonator. The frequency range was 106 to 6060 Hz, the concentration range 0.0015-0.005 g/ml, and the temperature 25OC. Two helicogenic solvents with widely different viscosities, dimethylformamide and m-cresol, were used to provide a broader effective frequency range. The intrinsic moduli, extrapolated to infinite dilution, were compared with the predictions of the theory of Ullman for rigid rods; agreement was rather good at the lowest frequencies, but unsatisfactory at high frequencies. The data over the entire frequency range of three logarithmic decades could be described closely by a relaxation spectrum consisting of one terminal relaxation time separated by a gap from a sequence of relaxtion times spaced as in the Zmm theory. The terminal time agrees approximately with that calculated for end-overend rotation of a rigid rod. The additional relaxation mechanisms are tentatively attributed to modes of flexural deformation of the helix.

No abstract