Properties of a Protein Activator of NAD Kinase from Plants

Abstract

Purification of pea (Pisum sativum) seedling NAD kinase by DEAEceilulose column chromatography resulted in loss of activity, due to dissociation of an activator from the enzyme. The purified enzyme preparation, which was almost completely inactive, regained the activity when the activator was added back.The activator was purified 320-fold by ion exchange chromatographies. The (23,24) reported that red light stimulated the conversion of NAD to NADP, and suggested that the NAD kinase activity was controlled by phytochrome.To elucidate the controlling mechanism of the NAD kinase activity in vivo, it is important to study the properties of the enzyme using a highly purified preparation. The most highly purified enzyme from plant has been obtained by Yamamoto (26). While trying to improve further the purity of plant NAD kinase, we found an activator of this enzyme. This communication describes some properties of the partially purified activator from pea seedlings, as well as the distribution of the activator in various green plants and Chlorella cells. ' This research was supported by a grant from the Japanese Ministry of Education. MATERIALS AND METHODSPlant Materials. Unless otherwise mentioned, the experiments were carried out with pea seedlings. Seeds of pea (Pisum sativum cultivar Gokuwase-akabana-tenashi-endo) were germinated in trays filled with vermiculite. The trays were placed in a greenhouse and irrigated once a day. The temperatures during daytime and the rest of the day were kept at 25 and 20 C, respectively. Seedlings grown under natural light conditions for 12 days were used for the experiments.To examine the distribution of NAD kinase and its activator among various plants, the respective seedlings of corn (Zea mays, cultivar, Nagano No. 1), and rice (Oriza sativa, cultivar Nihon-bare) grown in the greenhouse for 2 and 3 weeks were used. Chlorella vulgaris 11 hr cells were grown autotrophically with constant bubbling of air containing about 2% CO2 (15). Spinach (Spinacia oleracea L.) and Chinese cabbage (Brassica rapa var. Previdis) were purchased from the local market.Assay of NAD Kinase. The method adopted was essentially the same as described by Wang and Kaplan (25). The routine incubation medium contained 2 mm NAD, 3 mm ATP, 10 mM MgCI2, 100 mm tris-HCI (pH 8), an appropriate amount of enzyme, and 10 units (see below) of the activator where necessary, in a final volume of 0.5 ml. The reaction was initiated by adding NAD and terminated with 100 ,ul of N HCI. The reaction was run for 30 min at 37 C. The acidified suspension was neutralized with 100 Al of N NaOH and the coagulated protein was removed by centrifugation. The clear supernatant thus obtained was subjected to the determination of NADP according to the method described by Apps (2).The assay system of NADP cgntained 0.25 M tris-HCI (pH 8), 30 Ag of 2,6-dichlorophenol indophenol, 20 ,ug of phenazine methosulfate, 0.2 mm glucose-6-P, 0.06 unit of glucose-6-P dehydrogenase, and an appropriate amount of the supernatant (NADP), in a...

Structural Characterization of a Higher Plant Calmodulin

“…Vertical arrows point to-in "Materials and Methods." Peak 1 is R1T3 (residues 31-37), peak 2 is wards amino termini of trypsin peptides obtained from subdigests of R1T2 (residues [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30], and peak 3 is RITI (residues 1-13). Composiarginine peptides R I and R5.…”

“…several years ago (1,18) and subsequently calmodulins from spinach (28), barley (22), and peanut (1) have been chemically characterized. The amino acid compositions of plant calmodulins indicate some differences in tyrosine, threonine, leucine, arginine, and lysine content compared to bovine calmodulin.…”

Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform

“…Although a number of reports in the literature have described NADK activity in different plant species (Muto and Miyachi, 1977;Dieter and Marme, 1984;Delumeau et al, 2000;Pou De Crescenzo et al, 2001), evidence of an NADK in Arabidopsis has not previously been reported. Consequently, we examined Arabidopsis extracts for NADK activity.…”

“…Indeed, NADK was the first CaM-regulated enzyme identified in plants (Muto and Miyachi, 1977;Anderson et al, 1980) and remains among the most studied at the biochemical level (Zielinski, 1998). CaM is a ubiquitous and critical Ca 21 sensor found in all eukaryotes where it regulates a diverse array of targets involved in cytoskeletal rearrangements, signaling, metabolism, transcription, and ion homeostasis (Snedden and Fromm, 2001;Yang and Poovaiah, 2003).…”

Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae

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