NAD Kinase Activity Research Articles

Effects of short‐term NaCl stress on calmodulin transcript levels and calmodulin‐dependent NAD kinase activity in two species of tomato

ABSTRACTNAD kinase is thought to play an important role in the plant cellular responses to biotic and abiotic stress as one of the isoforms of the enzyme is activated by the Ca2+–calmodulin (CaM) complex. NAD kinase activity was measured after short‐term NaCl stress applied to isolated cells from Lycopersicon esculentum, var. Volgogradskij (NaCl‐sensitive tomato) and L. pimpinellifolium, acc. PE2 (NaCl‐tolerant species). NAD kinase activity remained constant in the sensitive species, whereas a sharp decrease was observed in the tolerant one. After salt treatment, an induction of the calmodulin gene(s) was observed in the two species, together with a 30–50% decrease in ‘active’ CaM content, i.e. CaM able to activate purified NAD kinase, in L. pimpinellifolium. The decrease in NAD kinase activity could not, however, be fully explained by this decrease in active CaM content. A similar decrease in NAD kinase activity was also recorded with other ionic stresses and exposure to high temperatures, but not in the case of drought, exposure to low temperatures, hormonal (indole‐3‐acetic acid and abscisic acid) or H2O2 treatments. External Ca2+ certainly plays a role in the biochemical mechanism(s) leading to NAD kinase inhibition, while no role could be shown for intracellular Ca2+. In addition, after salt stress, a modification of the redox state of NAD kinase seems to be responsible for the inhibition of the enzyme.

Structural elements within the methylation loop (residues 112–117) and EF hands III and IV of calmodulin are required for Lys115 trimethylation

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114-->Ala; L116T, Leu116-->Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

Redox Changes during Fertilization and Maturation of Marine Invertebrate Eggs

Previous studies on sea urchin eggs indicate that activation of NAD kinase is one of the earliest Ca2+-mediated events of fertilization. The subsequently produced NADP is converted to NADPH by the pentose shunt pathway, and some of this NADPH is used by an NADPH oxidase for generation of H2O2. To examine whether these changes apply generally, we have analyzed changes in pyridine nucleotide content during meiotic maturation and fertilization in eggs from four phyla. Surprisingly, fertilization-associated increases in NAD kinase were found only in echinoid eggs. The ratio of NADPH/NADP (redox ratio) increased from 1–1.6 to 2.5–6 following fertilization of echinoid and also clam eggs. However, the ratio is already >2 for unfertilized asteroid, tunicate and echiuroid eggs, and this ratio is unaffected by fertilization. We conclude that activation of NAD kinase and shifts in pyridine nucleotide metabolism and thereby cellular redox status may have roles that vary between species. In echinoids, a major role is in providing NADPH for H2O2production, but there may be other yet unappreciated signaling functions for this change.

Ocular Lens NAD Kinase: Partial Purification and Metabolic Implications

The ocular lens displays a significant amount of NADP(H) dependent metabolic traffic, but the origin of this cofactor has not been established. Size exclusion chromatography of bovine lens crude extract on a Sephacryl S300-HR column fitted with an eluate concentrator revealed two bands with NAD kinase activity, based on enzymatic cycling with signal amplification of the column fractions using a Cobas-Fara II centrifugal fast analyzer.Ve/Voratios from the chromatographic runs suggest that the relative molecular weight values lie within the ranges 8.91–3.98 × 105and 2.04–1.26 × 105, respectively, for these two bands. An ∼10-fold enhancement of enzyme activity over the crude fraction is realized from the chromatography step. Results point to NAD kinase as the source generator of this anchoring and linking cofactor for the oxidative stress and pentose phosphate enzyme systems, respectively.

Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei

A monomeric acidic protein of 14 000 Da with an isoelectric point of 4.5 was isolated from Mycobacterium phlei, which stained poorly with Coomassie brilliant blue. This protein showed retardation in mobility in SDS-PAGE upon treatment with calcium, similar to eukaryotic calmodulin proteins. Activation of cAMP phosphodiesterase and NAD kinase by this protein was observed. The CD spectral analysis indicated that the CALP has 52% of β-conformation. The regular β-conformation of the calmodulin like protein was shifted to 46% α-helical structure when calcium ions reacted with the protein, however, 42% of the CALP still retained its original β-conformation. These observations indicated homology of this calcium binding protein with that of eukaryotic calmodulins in few structural and functional properties.

Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei.

A monomeric acidic protein of 14,000 Da with an isoelectric point of 4.5 was isolated from Mycobacterium phlei, which stained poorly with Coomassie brilliant blue. This protein showed retardation in mobility in SDS-PAGE upon treatment with calcium, similar to eukaryotic calmodulin proteins. Activation of cAMP phosphodiesterase and NAD kinase by this protein was observed. The CD spectral analysis indicated that the CALP has 52% of beta-conformation. The regular beta-conformation of the calmodulin like protein was shifted to 46% alpha-helical structure when calcium ions reacted with the protein, however, 42% of the CALP still retained its original beta-conformation. These observations indicated homology of this calcium binding protein with that of eukaryotic calmodulins in few structural and functional properties.

Proline metabolism and NAD kinase activity in soybean calli during short- and long-term exposures to light and NaCl

Calli of soybean (Glycine max Merr.) cv. Maple Arrow grew better and accumulated more proline when cultured for 5 d on 70 mM NaCl under darkness than at light. This rapid proline accumulation in salinized soybean calli appeared to play a protective role rather than to be a cause of growth failure. Throughout a 28 d-culture cycle (in control and NaCl-treated calli exposed to light or darkness), we followed the possible relationships between the proline contents and the activities of enzymes of proline biosynthesis [ornithine transaminase; NAD(P)H-pyrroline-5-carboxylate reductase], of proline catabolism [NAD(P) proline dehydrogenase], and of NAD kinase responsible of variations in NADP(H) contents. Enzyme activities of proline metabolism and NAD kinase were clearly light- and NaCl-regulated; nevertheless, relationships between enzyme activities and proline content existed only in calli grown for short-term under darkness and in presence of NaCl. The ornithine transaminase route, which was particularly enhanced in these calli during the first days of salt application, seemed to be involved in the initial proline accumulation in soybean.

Differential Activation of NAD Kinase by Plant Calmodulin Isoforms THE CRITICAL ROLE OF DOMAIN I

NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.

Developmental changes in NAD kinase of the Colorado potato beetle, Leptinotarsa decemlineata say (Coleoptera, Chrysomelidae)

The NAD kinase activity was measured throughout its development in whole body extracts of the Colorado potato beetle Leptinotarsa decemlineata. Systematic alterations in the level of enzyme activity that correlate with change of developmental phases were determined. Ten NAD kinase molecular forms with approximate M r values from 80,000 to 1,000,000 were revealed using polyacrylamide gel electrophoresis. Every developmental phase had its own profile of the enzyme forms, which changed after the transition to the next phase. The most striking differences in the spectrum of NAD kinase multiple forms occurred in postemryogenesis and during metamorphosis from larva to pupa. In L. decemlineata cells, NAD kinase is presented as an equilibrium system of dissociating and associating oligomers.

Pyridine nucleotide levels and activities of dehydrogenases in cambial derivatives ofRobinia pseudoacacia L.

Despite the importance of the vascular cambial differentiation, little is known about its regulation. In order to address this problem we attempted to biochemically characterize differentiating xylem and phloem elements during the early stages of development. By applying techniques of quantitative histochemistry we show that the total pool size of pyridine nucleotides is similar in the phloem (PD) and xylem (XD) oriented derivatives of the cambial zone of trees ofRobinia pseudoacacia L. Within the PD zone, the amount of NAD + NADH exceeded that of NADP + NADPH [around 600 versus 200 pmol (mg dry weight)-1], possibly indicative of a preponderance of catabolic pathways (ratio of NADH∶NAD about 1). In contrast, the NADP(H) system dominated in the XD zone. This coincided with a high activity of NAD kinase. In addition, the extractable activities of the key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were greatly increased. At a ratio of NADPH∶NADP of approximately 1, this could be indicative of increased rates of reductive biosyntheses, and could thus well be involved in early steps of the formation of phenols and lignin monomers. Taken together, this first approach clearly shows that phloem-oriented and xylem-oriented cambial descendents exhibit distinct differences in their biochemical patterns even in early stages of differentiation.

Extracellular calmodulin-binding proteins in plants: purification of a 21-kDa calmodulin-binding protein.

A 21-kDa calmodulin (CaM)-binding protein and a 19-kDa calmodulin-binding protein were detected in 0.1 M CaCl2 extracts of Angelica dahurica L. suspension-cultured cells and carrot (Daucus carota L.) suspension-cultured cells, respectively, using a biotinylated cauliflower CaM gel-overlay technique in the presence of 1 mM Ca2+. No bands, or very weak bands, were shown on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels overlayed with biotinylated cauliflower CaM when 1 mM Ca2+ was replaced by 5 mM EGTA, indicating that the binding of these two CaM-binding proteins to CaM was dependent on Ca2+. Less 21-kDa CaM-binding protein was found in culture medium of Angelica dahurica suspension cells; however, a 21-kDa protein was abundant in the cell wall. We believe that the 21-kDa CaM-binding protein is mainly in the cell wall of Angelica dahurica. Based on its reaction with periodic acid-Schiff (PAS) reagent, this 21-kDa protein would appear to be a glycoprotein. The 21-kDa CaM-binding protein was purified by a procedure including Sephadex G-100 gel filtration and CM-Sepharose cation-exchange column chromatography. The purity reached 91% according to gel scanning. The purified 21-kDa CaM-binding protein inhibited the activity of CaM-dependent NAD kinase and the degree of inhibition increased with augmentation of the 21-kDa protein, which appeared to be the typical characteristic of CaM-binding protein.

Evidence for two NAD kinases in Salmonella typhimurium

The electron-carrying cofactor NADP is formed by phosphorylation of NAD. A strategy for the isolation of NAD kinase mutants revealed two classes of temperature-sensitive mutations, nadF and nadG, mapping at min 13 and 72 of the Salmonella chromosome. Both mutant types grew on nutrient broth at both 30 and 42 degrees C but on minimal medium showed a temperature-sensitive growth defect which was not corrected by any of the single nutritional supplements tested. A nadF deletion mutant grew on nutrient broth but not on minimal medium. A double mutant with the nadF deletion and a nadG(Ts) mutation showed temperature-sensitive growth on all media. We propose that Salmonella typhimurium has two NAD kinases, one encoded by the nadF and one by the nadG gene. This is supported by the fact that temperature-sensitive mutants of both genes produce kinase activity with altered heat stability. Results suggest that either one of two NAD kinases is sufficient for growth on rich medium, but that both are needed for growth on minimal media. Enzyme assays show that the nadF gene is responsible for about 70% of total NAD kinase activity, and that the nadG gene dictates the remaining 30%. While testing nutritional phenotypes of nadF and nadG mutants, we found that the biosynthetic intermediate, quinolinic acid (QA) inhibited growth of nadF mutants on nutrient broth. This suggested that the NadG enzyme might be inhibited by QA. Enzyme assays demonstrated that QA inhibits the NadG but not the NadF enzyme. This suggests the existence of a regulatory mechanism which controls NADP levels.

Enhanced Activity of a Ca-Calmodulin-Dependent Isozyme of NAD Kinase and Changes in Nicotinamide Coenzyme Levels in Lettuce Half-Seeds Caused by Red Light

Summary The hypothesis was tested that Pfr enhances formation of Ca-calmodulin during red-light-induced axis elongation accompanying dormancy breaking in seeds of Grand Rapids lettuce ( Lactuca sativa L.). The Ca-calmodulin complex was measured indirectly by measuring its known activation of NAD kinase and by assaying tissue amounts of NAD + and other nicotinamide coenzymes. Half-seeds containing hypocotyl/radicle sections were imbibed, treated with red or far-red light, then kept in darkness for various times up to 17 h. Two isozymes of NAD kinase were resolved by chromatography on a DEAE-Sephacel column, one Ca-calmodulin-dependent and one-independent. Activity of the dependent isozyme was enhanced by red compared with far-red at most times following irradiation, whereas the independent isozyme was not affected. Red light decreased the level of NAD + (compared with far-red) as early as 30 min after irradiation and at all subsequent times. Furthermore, the ratio of NADP + plus NADPH to NAD + plus NADH was enhanced by red relative to far-red at all sampling periods, consistent with in vivo activation of NAD kinase. Data are consistent with the hypothesis that Pfr causes formation of Ca-calmodulin that activates the kinase, but the simplest explanation is that Pfr induces formation of the dependent but not the independent kinase isozyme.

Enhanced Activity of a Ca-Calmodulin-Dependent Isozyme of NAD Kinase Caused by Gibberellic Acid in Photodormant Lettuce Seeds

Summary We tested the hypothesis that GA3, like red light (Zhang et al., 1994), enhances activity of a Ca-calmodulin- dependent isozyme of NAD kinase and causes lower levels of NAD+ and higher levels of NADP+ in half-seeds of Grand Rapids lettuce (Lactuca sativa L.). Half-seeds containing hypocotyl/radicle sections were imbibed for 1 h in H2O or 560 μM GA3, treated with far-red light, then kept in darkness for various times up to 17 h. NAD kinase activity from crude extracts was measured, and activities of a partially purified Ca-calmodulin-independent and a Ca-calmodulin-dependent isozyme were determined. In all experiments kinase activity increased with time after treatment. GA3 enhanced activity in crude extracts and of the Ca-calmodulin-dependent isozyme 30 min after irradiation, but it had no effect on the Ca-calmodulin-independent isozyme at any time period. GA3 caused increased amounts of NADP+ and NADPH and decreased amounts of NAD+, all effects significant 30 min after irradiation. Although these results might be explained partially by GA3-enhanced formation of Ca-calmodulin in vivo, it appears that GA3 additionally or alternatively induced formation of the Ca-calmodulin-dependent isozyme of NAD kinase. These effects of GA3 are similar to those of red light, and they support the hypothesis that Pfr somehow promotes synthesis of a gibberellin as it overcomes photodormancy of the seeds.

Activation of enzymes by calmodulins containing intramolecular cross-links

We have reacted calmodulins containing cysteines substituted at positions 3 and 146 or 5 and 146 with bismaleimidohexane (BMH) to generate intramolecularly cross-linked proteins termed BMHCM or BMHCM1, respectively. Reactions were also performed with N-ethylmaleimide (NEM) in place of BMH to generate corresponding S-ethylsuccinimidylated proteins termed NEMCM or NEMCM1. The abilities of these proteins to activate plant NAD kinase, erythrocyte Ca2±ATPase and bovine brain calcineurin activities were assessed. The BMH- or NEM-reacted proteins activate calcineurin activity as does control calmodulin. K act values for Ca2±ATPase activation by BMHCM and BMHCM1 are increased 10-fold relative to the control value, with no corresponding change in V max values. Activation of this enzyme by NEMCM or NEMCM1 is not different from the control. In NAD kinase activation experiments BMHCM and BMHCM1 are associated with a 10 to 20-foid increase in K act values and a 60–75% reduction in V max values relative to the control. NEMCM1 is not associated with any apparent changes in NAD kinase activation, however, NEMCM is associated with a 10-fold increase in the K act value. NEM-reacted calmodulin containing a cysteine only at position 3 is not associated with an increased K act value, implying that this change is due to interactions between S-(ethylsuccinimido)cysteines at positions 3 and 146. In conclusion, cross-linking and associated distortions in the structure of calmodulin appear to have little or no effect on activation of calcineurin enzyme activity. However, bending in the central helix and/or steric restrictions associated with cross-linking increase significantly the K act value for Ca2±ATPase and NAD kinase activation, and dramatically reduce maximal activation of NAD kinase activity.

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.

A continuous spectrophotometric assay for the activation of plant NAD kinase by calmodulin, calcium(II), and europium(III) ions

A continuous spectrophotometric assay has been developed to quantify the calmodulin, calcium(II) ion, and europium(III) ion dependence of the activation of NAD kinase from pea seedlings. Experimental enzyme activation data are compared with the theoretical curves for the binding of calcium(II) ions to the individual calcium binding sites of calmodulin. These results indicate that the binding of three calcium(II) ions is necessary for activation of plant NAD kinase. Further studies demonstrate that europium(III) ions can replace calcium(II) ions in calmodulin with retention of its ability to activate NAD kinase.

Response of Calmodulin-Dependent and Calmodulin-Independent NAD Kinase to Aluminium in Root Tips from Various Cultivated Plants

The activity of NAD kinase (E.C. 2.7.1.23.) was studied in root apical meristems of various crops as well as aneuploid lines of Chinese Spring wheat cultivar growing for 4d in nutrient solution with or without alluminium. The level of aluminium tolerance in cereals (rye, wheat, barley and oat) corresponds to the level of total NAD kinase activity. In the most tolerant oat this activity is five times higher than in Al sensitive barley. Al stress causes a three-fold increase of the total NAD kinase activity in oat and two-fold in other cereals. Under these conditions calmodulin-dependent activity increases in sensitive genotypes, while it decreases in Al tolerant ones. In the two legume cultivars studied (lupine and pea) the response of total activity is exactly the reverse of that in cereals; however, the calmodulin-dependent activity follows the same course of reaction as in cereals. In Chinese Spring wheat cultivar the factors responsible for extinguishing the increase of calmodulindependent NAD kinase activity are located on the long arms of chromosomes 4D and 2D, whereas the genes located on the short arm of chromosome 5A control other mechanisms of Al tolerance.

Rhythmic changes in the activities of NAD kinase and NADP phosphatase in the achlorophyllous ZC mutant of Euglena gracilis klebs (strain Z)

NAD kinase and NADP phosphatase activities were detected in the supernatant and the pellet fractions prepared by sonication and centrifugation of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis. A detailed study of substrate concentration-velocity curves enabled us to define the saturating substrate concentrations that were used in the enzyme assays. An analysis of the reproducibility of the entire assay procedure indicated that the pooled standard error was about 14%. We report circadian variations in the activities of NAD kinase and NADP phosphatase in the soluble and membrane-bound fractions of both synchronously dividing and nondividing cultures maintained in constant darkness. Bimodal circadian rhythms in total NADP phosphatase activity were found in dividing cells (peaks at circadian times [CT] 00 and 12). The peak observed at CT 00–03 disappeared when the cells had ceased dividing, a result that suggests that it might be regulated by the cell division cycle. NAD kinase activity displayed unimodal circadian rhythms (peak at CT 12) in dividing cells, which persisted with the same phase after the culture entered the stationary phase of growth. Results are discussed with reference to a model ( K. Goto, D. L. Laval-Martin, and L. N. Edmunds, Jr., 1985, Science 228, 1284–1288 ) in which we have proposed that the Ca 2+-transport system, Ca 2+, calmodulin, NAD kinase, and NADP phosphatase could represent clock “gears” that might constitute a self-sustained circadian oscillating loop.

Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+and Nadp+in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant ofEuglena GracilisKlebs (Strain Z)

We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP+ and NAD+, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CT), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2(+)-calmodulin) or for its substrate, NAD+. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.