No abstract

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2' of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyame was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme.These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2' and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.Few reports have been devoted to the study of NAD kinase from bacteria. NAD kinase has been studied in a limited manner in crude, cell-free preparations from Escherichia coli (10). The enzyme has been purified up to 500-fold from Azotobacter vinelandii and has been found to have apparent Km values for NAD+ and ATP of 0.4 and 1 mM, respectively (7). NAD kinase has also been detected in immobilized Achromobacter aceris (25) and Brevibacterium ammoniagenes (9).Recently, we purified NAD kinase 180-fold from Bacillus licheniformis (31). Using this partially purified preparation, we have shown that this enzyme is subject to competitive inhibition by NADP+ and that it plays a key role in the regulation of NADP turnover in this organism (31). In addition, we have also purified NAD kinase from Bacillus subtilis and demonstrated that it is negatively modified by NADP+ and positively modified by NADPH (V. Lopez, E. Gomez, H. Kwong, and A. J. Andreoli, Fed. Proc. 44:2649, 1985).E. coli cells possess a functional NAD cycle that accounts for the turnover of NAD in this organism (2). In addition, E. coli cells also demonstrate NADP turnover (16; A. J. Andreoli, unpublished results). In the present investigation, we purified NAD kinase from E. coli to determine its role in the regulation of NADP turnover and pool size in this organism. Evidence is presented indicating that NADPH and NADH are potent negative modulators of this enzyme.

No abstract

Despite lacking a brain and having an apparent symmetrically pentaradial nervous system, echinoderms are capable of complex, coordinated directional behavioral responses to different sensory stimuli. However very little is known about the molecular and cellular mechanisms underlying these behaviors. In many animals, dopaminergic systems play key roles in motivating and coordinating behavior and although the dopamine receptor antagonist haloperidol has been shown to inhibit the righting response of the sea urchin Strongylocentrotus purpuratus, it is not known if this is specific to this behavior, in this species, or whether dopaminergic systems are needed in general for echinoderm behaviors. We found that the dopamine receptor antagonist haloperidol inhibited multiple different behavioral responses in three different echinoderm species. Haloperidol inhibited the righting response of the sea urchin Lytechinus variegatus and of the sea star Luidia clathrata. It additionally inhibited the lantern reflex of S. purpuratus, the shell covering response of L. variegatus, and the immersion response of L. variegatus, but not S. purpuratus or L. clathrata. Our results suggest that dopamine is needed for the neural processing and coordination of multiple different behavioral responses in a variety of different echinoderm species.