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NAD kinase (NADK; ATP:NAD 2#-phosphotransferase, EC 2.7.1.23), an enzyme found in both prokaryotes and eukaryotes, generates the important pyridine nucleotide NADP from substrates ATP and NAD. The role of NADKs in plants is poorly understood, and cDNAs encoding plant NADKs have not previously been described to our knowledge. We have cloned two cDNAs from Arabidopsis predicted to encode NADK isoforms, designated NADK1 and NADK2, respectively. Expressed as recombinant proteins in bacteria, both NADK1 and NADK2 were catalytically active, thereby confirming their identity as NADKs. Transcripts for both isoforms were detected in all tissues examined and throughout development. Although the predicted catalytic regions for NADK1 and NADK2 show sequence similarity to NADKs from other organisms, NADK2 possesses a large N-terminal extension that appears to be unique to plants. Using recombinant glutathione-S-transferase fusion proteins and calmodulin (CaM)-affinity chromatography, we delineated a Ca 21 -dependent CaM-binding domain to a 45-residue region within the N-terminal extension of NADK2. Although recombinant NADK2 was not responsive to CaM in vitro, immunoblot analysis suggests that native NADK2 is a CaM-binding protein. In Arabidopsis crude extracts, CaMdependent NADK activity was much greater than CaM-independent activity throughout development, particularly in young seedlings. A native CaM-dependent NADK was partially purified from Arabidopsis seedlings (K m NAD 5 0.20 mM, K m Mg 21 2ATP 5 0.17 mM). The enzyme was fully activated by conserved CaM (S 0.5 5 2.2 nM) in the presence of calcium but displayed differential responsiveness to eight CaM-like Arabidopsis proteins. Possible roles for NADKs in plants are discussed in light of our observations.

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K m (PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K m (PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a .5-fold increase in the I 50 [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.Phosphoenolpyruvate (PEP) carboxylase (PEPC; E.C. 4.1.1.31) is a ubiquitous and tightly regulated cytosolic enzyme in vascular plants that is also widely distributed in green algae and bacteria. PEPC catalyzes the irreversible b-carboxylation of PEP to yield oxaloacetate and inorganic phosphate (Pi). The enzyme plays a pivotal photosynthetic role in primary CO 2 fixation by C 4 and Crassulacean acid metabolism leaves Izui et al., 2004;Nimmo, 2005). PEPC also has a variety of additional important functions in plants, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates that are withdrawn for biosynthesis and nitrogen assimilation. Most native plant PEPCs are homotetramers, composed of identical subunits of approximately 100 to 110 kD, with crystal structures having been established for both the maize (Zea mays) and Escherichia coli enzymes (Izui et al., 2004). All PEPCs show allosteric properties: Vascular plant PEPCs are typically inhibited by L-malate and activated by Glc-6-P. In addition, Asp and Glu are allosteric inhibitors of PEPCs in plant tissues active in nitrogen assimilation and/or transamination reactions, thus providing a link between carbon and nitrogen metabolism (Law and Plaxton, 1997;Golombek et al., 1999;Moraes and Plaxton, 2000;Blonde and Plaxton, 2003). It is also well esta...

NADH kinase (NADHK; ATP:NADH 2 -phosphotransferase; EC 2.7.1.86), an enzyme that preferentially utilizes NADH as the diphosphonicotinamide nucleotide donor, has been identified for the first time in plants. Low activity (0.4 nmol of NADPH produced/min per mg of protein) was observed in clarified protein extracts from Arabidopsis thaliana (thale cress) cell suspension cultures. However, unlike an NADHK from yeast (Saccharomyces cerevisiae) (POS5), the enzyme from Arabidopsis did not associate with the mitochondria. NADHK was cloned (gi:30699338) from Arabidopsis and studied as a recombinant protein following affinity purification from Escherichia coli. The enzyme had a pH optimum for activity of 7.9 and a subunit molecular mass of 35 kDa. Analytical gel filtration demonstrated that the recombinant enzyme exists as a dimer. Hyperbolic saturation kinetics were observed for the binding of NADH, ATP, free Mg 2+ and NAD + , with respective K m values of 0.042, 0.062, 1.16, and 2.39 mM. While NADHK could phosphorylate NADH or NAD + , the specificity constant (V max /K m ) for NADH was 100-fold greater than for NAD + . The enzyme could utilize UTP, GTP and CTP as alternative nucleotides, although ATP was the preferred substrate. PP i or poly-P i could not substitute as phospho donors. PP i acted as a mixed inhibitor with respect to both NADH and ATP. NADHK was inactivated by thiol-modifying reagents, with inactivation being decreased in the presence of NADH or ATP, but not NAD + . This study suggests that, in Arabidopsis, NADP + /NADPH biosynthetic capacity could, under some circumstances, become uncoupled from the redox status of the diphosphonicotinamide nucleotide pool.

A team of therapists from Minnesota and New York worked with labor union families of workers gone missing on September 11, 2001, after the attack on the World Trade Center, where they were employed. The clinical team shares what they did, what was learned, the questions raised, and preliminary evaluations about the multiple family meetings that were the major intervention. Because of the vast diversity, training of therapists and interventions for families aimed for cultural competence. The community-based approach, preferred by union families, plus family therapy using the lens of ambiguous loss are proposed as necessary additions to disaster work.