Calmodulin-dependent NAD Kinase 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.

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.

Calmodulin dependent NAD-kinase is associated with both the outer and inner mitochondrial membranes in maize roots

Maize root mitochondrial have been subfractionated after osmotic rupture. A calcium-calmodulin-dependent NAD-kinase activity has been shown to be present in both inner and outer membrane fractions. Cytochrome c-reductase activities are also associated with outer and inner membrane fractions but whereas the former is entirely insensitive to 50 μmol·1(-1) antimycin A the latter is reduced by 60% in its presence. This residual antimycin A-insensitive cytochrome c-reductase activity cosediments with the major portion of NAD-kinase activity and equilibrates in sucrose gradients at densities around 1.146 g·cm(-3). Rate zonal centrifugation with renografin allows an excellent separation of both cytochrome c-reductase and NAD-kinase activities. We have no evidence for allocating NAD-kinase activity to endo- or plasma membranes.

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.

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.