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Nuclear factor kappa B (NF-kappaB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB, initiated by an IkappaB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. To better understand the role of IKKs in NF-kappaB 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 IkappaBalpha peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 micrometer, 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) micrometer(-1) using an IkappaBalpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) micrometer(-1)), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) micrometer(-1)), or rhIKK1 (0.02 h(-1) micrometer(-1)). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IkappaBalpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K(i) of 0.77 micrometer may play a physiological role in regulation of the enzyme activity.

The effects of rose bengal, erythrosin B and fluorescein on ruminant digestion were evaluated by an in vitro rumen technique. Rose bengal reduced in vitro dry matter disappearance (IVDMD) values starting at .5 mM in the nutrient media and erythrosin B reduced IVDMD values starting at .05 mM in the nutrient media. Maximum reduction in IVDMD values was observed at 3.0 mM of rose bengal and erythrosin B. Fluorescein depressed digestion but not as severely as rose bengal or erythrosin B. Dry matter digestibility (DMD) values for steers given erythrosin B at a daily dosage of 6.5 mg/kg body weight were higher (P<.05) than DMD values for control steers. DMD values were higher for steers given 16.3 or 26.1 mg erythrosin B/kg body weight daily than for the control steers, but the differences were not significant. There were no significant differneces among animals given the different dosages in digestible energy. Recovery of erythrosin B from feces of treated steers varied with time, indicating that steers fed erythrosin B at a dosage of 6.5 mg/kg body weight might excrete feces during some periods of the day which would not control facefly development; however, administration of erythrosin B at a dosage of 16.3 mg/kg body weight would provide enough erythrosin B in feces to control face fly development throughout a 24-h period.