NF-B is sequestered in the cytoplasm by the inhibitory IB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IBs leading to their degradation that results in NF-B activation. IKK-1 and IKK-2 are two direct IB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IB␣ peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.

Nuclear factor kappa B (NF-B) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-B activation depends on phosphorylation and degradation of its inhibitor protein, IB, initiated by an IB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IB kinase 1 (IKK1) and IB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-B essential modulator. To better understand the role of IKKs in NF-B activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K m values for ATP and IB␣ peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 M, respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency (k cat / K m ) of 47.50 h ؊1 M ؊1 using an IB␣ peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h ؊1 M ؊1 ), its mutant rhIKK2 (S177E, S181E, 1.18 h ؊1 M ؊1 ), or rhIKK1 (0.02 h ؊1 M ؊1 ). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IB␣ peptide, exhibited competitive inhibitory kinetics, only ADP with the low K i of 0.77 M may play a physiological role in regulation of the enzyme activity.

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A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.