Calmodulin-dependent NAD Research Articles

Abstract: 1036 EFFECT OF CONVENTIONAL RISK FACTORS ON THE PRESENCE OF CORONARY ARTERY DISEASE AS ASSESSED BY 64-MDCT IN PATIENTS WITH LOW-STAGE CHRONIC KIDNEY DISEASE

Calmodulin-dependent NAD kinase has been purified more than 70fold from a crude plant (zucchini squash) homogenate by calmodulin-Sepharose affinity chromatography to a specific activity of 80 munits/mg protein. The enzyme could be activated about 8fold by calmodulin. Half-maximal activation was obtained with 6 ng of purified calmodulin from bovine brain. Together with NAD kinase other soluble plant proteins were retained specifically on the column. NaDodSo4 polyacrylamide gel electrophoresis of the proteins which were retained by the calmodulin-Sepharose column revealed at least 7 to 8 bands. Most of the intensively stained bands on the gels obtained from the crude homogenate had disappeared.

Tween-20 activates and solubilizes the mitochondrial membrane-bound, calmodulin dependent NAD+ finase of Avena sativa L.

Among different treatments assayed, a mix of a nonionic detergent (5% Tween-20) with 0.5 m NaCl was found to solubilize a large part of the calmodulin-dependent NAD+ kinase bound to the inner mitochondrial membrane. It also stimulated its activity by increasing 7 times the maximal velocity. Activity stimulation was also observed with phosphatidylcholine, phosphatidylethanolamine and with reductants (HSO3 and DTT). This solubilized NAD+ kinase and the calmodulin-dependent cytosoluble isoform displayed distinct molecular masses, as well as different kinetic parameters. We propose that solubilization of membrane-bound NAD+ kinase could occur in vivo in Avena sativa and could generate a soluble isoform.

Characterization and possible redox regulation of the purified calmodulin-dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)-dependent NAD+ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short-term NaCl stress (Delumeau, Morère-Le Paven, Montrichard, Laval-Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol-modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD+ kinase. A three-step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD+ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL−1 bovine CaM. The activity of NAD+ kinase was strongly inhibited by thiol-modifying reagents and oxidized glutathione. NAD+ kinase was also found to be air-inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five-fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD+ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP-thioredoxin reductase system.

NAD + kinase activity, calmodulin levels during the growth of isolated cells from Lycopersicon pimpinellifolium and kinetic constants of the calmodulin-dependent NAD + kinase

NAD + kinase activity and levels of active calmodulin (CaM), i.e. CaM able to activate NAD + kinase, were determined during the culture growth of isolated cells from Lycopersicon pimpinellifolium. Two peaks of CaM-dependent NAD + kinase activity occurred at times when the cells were not actively dividing: the first one, a few hours after medium inoculation and the second one, at the end of the exponential growth phase. These alterations in CaM-dependent NAD + kinase activity were not related to changes in active CaM levels, which remained nearly constant throughout the culture and were found to be sufficient enough to fully activate NAD + kinase in vivo in the presence of Ca 2+. Thus, the increases in NAD + kinase activity observed in the cell extracts during the culture growth, may result either from a de novo synthesis of the enzyme or from the presence of an additive effector, other than the Ca 2+–CaM complex. The CaM-dependent NAD + kinase from tomato cells was partially purified (870-fold). This enzyme displays a sequential addition of the substrates and a K m of 0.20 mM for NAD + and 0.08 mM for MgATP 2− were determined in the presence of tomato CaM, also purified during the course of this study.

Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species.

A strategy for elucidating specific molecular targets of calcium and calmodulin in plant defense responses has been developed. We have used a dominant-acting calmodulin mutant (VU-3, Lys to Arg115) to investigate the oxidative burst and nicotinamide co-enzyme fluxes after various stimuli (cellulase, harpin, incompatible bacteria, osmotic and mechanical) that elicit plant defense responses in transgenic tobacco cell cultures. VU-3 calmodulin differs from endogenous plant calmodulin in that it cannot be methylated post-translationally, and as a result it hyperactivates calmodulin-dependent NAD kinase. Cells expressing VU-3 calmodulin exhibited a stronger active oxygen burst that occurred more rapidly than in normal control cells challenged with the same stimuli. Increases in NADPH level were also greater in VU-3 cells and coincided both in timing and magnitude with development of the active oxygen species (AOS) burst. These data show that calmodulin is a target of calcium fluxes in response to elicitor or environmental stress, and provide the first evidence that plant NAD kinase may be a downstream target which potentiates AOS production by altering NAD(H)/NADP(H) homeostasis.

Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants.

Transgenic plants were constructed that express two foreign calmodulins (VU-1 and VU-3 calmodulins) derived from a cloned synthetic calmodulin gene. VU-1 calmodulin, similar to endogenous plant calmodulin, possesses a lysine residue at position 115 and undergoes posttranslational methylation. VU-3 calmodulin is a site-directed mutant of VU-1 calmodulin that is identical in sequence except for the substitution of an arginine at position 115 and thus is incapable of methylation. Both calmodulin genes, under the control of the cauliflower mosaic virus 35S promoter, were expressed in transgenic tobacco. Foreign calmodulin protein accumulated in plant tissues to levels equivalent to that of the endogenous calmodulin. All transformed lines of VU-1 plants were indistinguishable from untransformed controls with respect to growth and development. However, all transformed lines of VU-3 plants were characterized by decreased stem internode growth, reduced seed production, and reduced seed and pollen viability. The data suggest that these phenotypes are the result of the expression of the calmodulin mutant rather than the position of transferred DNA insertion or the overall alteration of calmodulin levels. Analyses of the activity of the purified transgenic calmodulins suggest that calmodulin-dependent NAD kinase is among the potential targets that may have altered regulation in VU-3 transgenic plants.

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.

Effect of Aluminium on Calmodulin-Dependent and Calmodulin-Independent NAD Kinase Activity in Wheat ( Triticum aestivum L.) Root Tips

Total NAD kinase activity is twice as high in the Al tolerant wheat strain (BHG) than in the Al sensitive variety (Grana). In the former the calmodulin-dependent enzyme constitutes about 50 % of the total activity, whereas in the latter it does not exceed 30 %. Aluminium induced NAD kinase activity 2.5 fold in the sensitive variety Grana and six fold in the Al tolerant BHG upon 10 h incubation in 0.74 mM Al. The induction, abolished by cycloheximide, involves in Grana both calmodulin-dependent and calmodulin-independent enzymes, whereas in the Al tolerant genotype the induction involves only the calmodulin-independent form, and the activity of the calmodulin-dependent enzyme becomes marginal. Changes in the activity of NAD kinase are paralleled by the shift in the NADP/NAD ratio. It is suggested that the mechanism of Al tolerance is manifested in proportion to calmodulin-dependent and calmodulin-independent NAD kinases and in the ability to replace the former by the latter enzyme in the tolerant genotype under Al stress.

Subchloroplastic localization of NAD kinase activity: evidence for a Ca 2+, calmodulin-dependent activity at the envelope and for a Ca 2+, calmodulin-independent activity in the stroma of pea chloroplasts

Chloroplasts were prepared from pea seedlings and tested for NAD kinase activity. More than half of a Ca 2+, calmodulin-dependent activity and most of a Ca 2+, calmodulin-independent activity of the homogenate were associated with chloroplasts. The Ca 2+, calmodulin-dependent activity could be detected by adding Ca 2+ and calmodulin to the incubation medium containing intact chloroplasts. This activity could not be separated from the chloroplasts by successive washes or by phase partition in aqueous two-polymer phase systems. After chloroplasts fractionation, the Ca 2+, calmodulin-dependent NAD kinase activity was localized at the envelope, and the Ca 2+, calmodulin-independent activity was recovered from the stroma. In view of these results and of a previous report [Simon (1982) Plant Cell Rep. 1, 119–122] the occurrence and presumed role of calmodulin in the chloroplast are discussed.

A Ca2+, Calmodulin-dependent NAD kinase from corn is located in the outer mitochondrial membrane.

NAD kinase activity from dark grown corn coleoptiles is shown to be almost totally dependent on Ca2+ and calmodulin. Nearly all of the enzyme activity is found in a particulate fraction. Upon differential and density gradient centrifugation the NAD kinase activity co-migrates with the mitochondrial cytochrome c oxidase whereas marker activities for nuclei, etioplasts, endoplasmic reticulum, and microbodies could well be separated, indicating that the NAD kinase is associated with mitochondria. This NAD kinase, associated with intact mitochondria, can be activated by exogenously added Ca2+ and calmodulin. In order to investigate the submitochondrial localization of the NAD kinase, the organelles were ruptured by osmotic treatment and sonication and the submitochondrial fractions were separated by density gradient centrifugation. The NAD kinase activity exhibits the same density pattern as the antimycin A-insensitive NADH-dependent cytochrome c reductase, a marker enzyme of the outer mitochondrial membrane. Marker enzymes for the mitochondrial matrix and the inner mitochondrial membrane reveal different density profiles. These results indicate that the Ca2+, calmodulin-dependent NAD kinase from coleoptiles of dark grown corn seedlings is located at the outer mitochondrial membrane. The physiological relevance of the location and the Ca2+, calmodulin-dependence of the NAD kinase will be discussed.

Kinetic Nature of Calmodulin-dependent NAD Kinase From Pea Seedlings

Summary The kinetic nature of calmodulin-dependent NAD kinase from pea seedlings was studied. Half-saturation concentration of Ca2+ and calmodulin for the enzyme were 70 μM and 19 ng ml-1, respectively. The optimum pH for the enzyme reaction was 8. MgATP2- was the true substrate for the enzyme, but free ATP and Mg2+ inhibited the enzyme. In the presence of saturating amount of Ca2+ and calmodulin, the Km values for MgATP2- and for NAD were 0.18 and 0.11 mM, respectively. The physiological role of the calmodulin-dependent NAD kinase in the light-induced conversion of NAD to NADP in higher plant leaves is discussed.

Calmodulin-dependent and independent NAD kinase activities from cytoplasmic and chloroplastic fractions of spinach (Spinacia oleracea L.)

NAD kinase activity has been found in a soluble, cytoplasmic fraction and in the chloroplasts prepared from green spinach leaves. A small amount of both the cytoplasmic and the chloroplastic NAD kinase activities was retained on a calmodulin-Sepharose affinity column. The cytoplasmic NAD kinase eluted from the affinity column was found to be enhanced by calmodulin in a Ca(2+)-dependent manner. The chloroplastic enzyme which is located exclusively in the stroma and not in the envelope and thylakoid fractions was not affected by Ca(2+) and calmodulin. The stromal fraction of purified chloroplasts contained only a negligible amount of calmodulin, most probably due to cytoplasmic contamination. Based on these data, two different mechanisms for the light-dependent modulation of spinach NAD kinase activity are suggested.

Activation of calmodulin-dependent NAD + kinase by trypsin

Sea urchin egg NAD + kinase (ATP;NAD + 2'-phosphotransferase, EC 2.7.1.23), a calmodulin-dependent enzyme, can be activated by a moderate treatment with trypsin in a similar fashion to calmodulin. Stimulation by trypsin is dependent on its concentration (hall-maximal dose: 1.5 μg/ml) but independent of the presence of calcium. This suggests that iimited proteolysis is able to activate NAD + kinase as described for several other calmodulin-activated enzymes and that these enzymes may interact with calmodulin in a similar way.

Plant and fungal calmodulin: Ca 2+-dependent regulation of plant NAD kinase

Although little is known about the role(s) of second messengers, including free Ca 2+, in plant cells there has been increasing evidence for a role for Ca 2+ in metabolic regulation in plants. The recent demonstration that the Ca 2+-binding protein, calmodulin exists in extracts of higher plants and basidiomycete fungi provides a basis for understanding Ca 2+-dependent metabolic regulation in plant cells. In this review we summarize the similarities and differences of plant, fungal and mammalian calmodulin. We also discuss the known in , vitro functions of calmodulin in higher plants. A Ca 2+-calmodulin-dependent NAD kinase has been purified to homogeneity from extracts of pea seedlings and shown to be absolutely dependent upon calmodulin and μM levels of free Ca 2+ for activity. The available evidence suggest that this Ca 2+-calmodulin-dependent NAD kinase is the major form of plant NAD kinase and that this regulatory enzyme is localized in the chloroplast. A model is presented which predicts that the rate of photosynthesis is regulated by a receptor-mediated change in the level of chloroplastic free Ca 2+ upon illumination. Free Ca 2+, acting as a second messenger, forms a Ca 2+-calmodulin complex thus converting calmodulin to its active conformation. This Ca 2+-calmodulin complex then activates chloroplastic NAD kinase resulting in an increased NADP NAD ratio.

Partial purification of plant NAD kinase by calmodulin-Sepharose affinity chromatography

Calmodulin-dependent NAD kinase has been purified more than 70fold from a crude plant (zucchini squash) homogenate by calmodulin-Sepharose affinity chromatography to a specific activity of 80 munits/mg protein. The enzyme could be activated about 8fold by calmodulin. Half-maximal activation was obtained with 6 ng of purified calmodulin from bovine brain. Together with NAD kinase other soluble plant proteins were retained specifically on the column. NaDodSo 4 polyacrylamide gel electrophoresis of the proteins which were retained by the calmodulin-Sepharose column revealed at least 7 to 8 bands. Most of the intensively stained bands on the gels obtained from the crude homogenate had disappeared.