The in vivo feeding of 2-'4C-ABA to embryonic bean axes (Phaseolus vulgaris L.) has established PA2 and DPA as products in one ABA metabolic pathway (21,23). The accumulation of DPA in bean seeds (24), as well as the continuous formation of PA and DPA in water-stressed leaves (5), indicates that this pathway has an active role in regulating total ABA concentration within the plant. The decreased activity of PA in bioassay systems for growth inhibition (9, 22), abscission promotion (1, 4), and stomatal closure (7) suggests that the DPA pathway has a role in regulating the hormonal activity of ABA. The recent observation that PA inhibits photosynthesis, while ABA does not, indicates that the DPA pathway may also play a role in the activation of physiological activities (7). The actual role which the DPA pathway plays in regulating ABA levels requires the investigation of the enzymes involved. The goal of the present investigation was to establish a cell-free enzyme system capable of metabolizing ABA by the DPA pathway.

Glucuronokinase from Llum lougMorum polen was purified 30-to 40-fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and al were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a Ki value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mM ATP, while the rates for 10 mM GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl a-glucuronoside, methyl 8-glucuronoside, 8-glucuronic acid-i-phosphate, and 4-0-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The ,8-glucuronic acid-i-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depend on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.Glucuronokinase (EC 2.7.1.43) catalyzes the reaction glcUA2 + ATP --a-glcUA-1-P + ADP (13) and is an enzyme of the myoinositol oxidation pathway (4, 8). The enzyme is inhibited by the pathway intermediates, a-glcUA-I-P and UDP-glcUA, suggesting it may have a regulatory role in the formation of cell wall uronic acids and pentoses (7). A number of sugars and sugar acids were evaluated as enzyme inhibitors, but none were effective (5, 7). Derivatives of glcUA have not been tested and there is no information concerning the possible inhibitory effects of nucleotides.The present work deals with the selectivity of glucuronokinase toward glucuronic acid analogs and nucleotides other than ATP. The enzyme was purified by a new one-step procedure which utilized affinity chromatography on blue dextran-Sepharose resin. MATERIALS AND METHODSReagents. The nucleotides, the CNBr-activated Sepharose, Escherichia coli /3-glucuronidase (type VII), calf intestine alkaline phosphatase (type I), and fl-glucose-1-P dicyclohexylammonium salt were obtained from Sigma Chemical Co for 10 min. The supernatant was decanted through two layers of cheesecloth and used as the crude enzyme preparation. The crude enzyme was purified on a blue dextran-Sepharose affinity column. The column (55 x 6 mm) was loaded with 45 to 55 ml of crude enzyme preparation (-8 mg of protein/ml) at a peristaltic pump flow rate of 0.4 ml/min. The column was washed with 50 ml of 10 mm HEPES (pH 7.2) containing 1 mm DTT. The enzyme was eluted from the column with the same buffer containing 100 mm KCI. The KCI eluate was made up to 50 ml with wash buffer and concentrated to less than I ml using an Amicon XM-50 ultrafilter. The salts were further removed by reconstituting the concentrate to 50 ml with the wash buffer and again concentrating it to I ml. The second concentrate was divided into 100-,ul portions and stored in liquid N2. Under these storage conditions no loss of enzyme activity was observed over...

Soluble proteins from excised Phaseolus vulgaris axes incubated for 1 hour in 3H or 14C-amino acid mixtures at different times during the period leading up to initiation of cell elongation were compared by acrylamide gel electrophoresis. Differences in electrophoretic patterns were found when proteins from axes incubated during the 1st hour of imbibition were compared with proteins from axes incubated during the hour when cell elongation was initiated. These differences greatly diminished by the 2nd hour of imbibition which suggests that they were due primarily to incomplete axis imbibition. A 5-hour actinomycin D treatment which reduced amino acid incorporation by 40% in the 5th hour had no apparent effect on the electrophoretic pattern during that hour.An apparent requirement for protein synthesis prior to initiation of cell elongation has been demonstrated for several embryonic axes (2,3,9,11). Amino acid incorporation studies suggest that protein synthesis is initiated upon embryo hydration and increases in rate during the period leading to cell elongation (3, 10). Studies with protein synthesis inhibitors give some evidence for qualitative changes in the proteins synthesized prior to cell elongation (2, 10). The dependence of early protein synthesis on de novo nucleic acid synthesis appears to vary with seed type (2, 3, 7, 9, 1 1).The purpose of the present investigation was to determine if qualitative changes occur in protein synthesis prior to cell elongation in excised Phaseolus vulgaris axes. Soluble proteins labeled by in vivo incorporation of a "4C-L-amino acid mixture during the hour in which cell elongation was initiated were compared by acrylamide gel electrophoresis to soluble proteins labeled at earlier times by the in vivo incorporation of a 3H-Lamino acid mixture. 50 ,ug of chloramphenicol in 1 ml of sterile water. The flasks were shaken in a Dubnoff metabolic incubator at 26 C. After various incubation periods, the axes were transferred to flasks containing the above solution plus either a 50 tuc 'H-or a 10 ,.tc "4C-amino acid mixture of the same composition (New England Nuclear 'H amino acid mixture NET-250 or 14C amino acid mixture NEC 445). After a 1-hr incubation with the radioactive amino acid mixture, the axes were rinsed in 250-mg portions and ground with a mortar and pestle in 2 ml containing 0.01 M NaPO, buffer, pH 7, 0.4 M sucrose, 1 mM MgCl2, and 10 mm KCl. The homogenates were filtered through miracloth and centrifuged at 20,000g for 10 min. The supernatants were then centrifuged for 60 min at 104,000g, and the resulting supernatants, minus the lipid layer, were used for electrophoresis.The soluble protein from the two treatments were mixed so that 'H and 14C cpm were approximately equal. The mixtures were reduced for 1 hr at 60 C with dithiothreitol at a concentration of 700 jg/mg soluble protein. After reduction, the mixtures were dialyzed for 12 hr at room temperature against 0.1 M NaPO4, pH 7, containing 0.1% sodium dodecyl sulfate.The dialyzed mixtures were brought to ...