Purification and metal requirements of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase of Phaseolus mungo seedlings was purified 4400-fold from the (NH₄)₂SO₄ fraction of a crude extract, the specific activity being 810 nkat per mg protein. When the purified enzyme was subjected to electrophoresis with or without sodium dodecyl sulfate, a single band was observed. The MW of the enzyme was estimated to be 67 000 by Sephadex G-100 gel chromatography and the minimum MW of the enzyme 43 000 by gel electrophoresis with sodium dodecyl sulfate. Atomic absorption analysis revealed that the purified enzyme contained small amounts of copper. Cobalt was not detected, although it has been implicated as a cofactor requirement.     

Alicyclic acid metabolism in plants 10. Partial purification and some properties of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase from Phaseolus mungo seedlings was purified120-fold by DE-23, hydroxylapatite and Sephadex G-100 columnchromatography. The final preparation was free of dehydroquinatehydro-lyase and NAD(P)H₂ oxidase. The dehydroquinate synthaserequired Co²⁺ and NAD as cofactors. Co²⁺ could be replaced byCu²⁺ at 0.1 mM, but Cu²⁺ at higher levels was inhibitory. Noneof the other metal ions tested activated the enzyme. Some activitywas observed in the absence of added Co²⁺ and this activitywas inhibited by EDTA but not by diethyldithiocarbamate, NaN₃or NaCN. Heavy metal ions, such as Ag⁺ and Hg²⁺, and p-chloromercuribenzoatestrongly inhibited the enzyme activity. Of the pyridine nucleotidestested only NAD was required for the maximum activity of theenzyme. In the absence of NAD, the enzyme retained 30 to 40%of the activity obtained with added NAD. The apparent Km valuefor DAHP at pH 7.4 was about 23 µM. The enzyme activityappeared to be maximum at about pH 8.5. However, the characteristicsof the enzyme were studied at pH 7.4, because of the labilityof the enzyme under alkaline conditions. An Arrhenius plot ofthe enzyme reaction showed a break at about 21?C, and belowthis critical temperature the activation energy increased. (Received March 4, 1977; )     

Organization of enzymes in the shikimate pathway of Phaseolus mungo seedlings

DEAE-cellulose and Sephadex G-100 column chromatography showedthe separability of 3-dehydroquinate synthase, 3-dehydroquinatehydro-lyase-shikimate: NADP oxidoreductase complex, shikimatekinase and 5-enolpyruvylshikimate-3-phosphate synthase of Phaseolusmungo seedlings. The approximate molecular weights of theseenzymes were estimated using a gel filtration column. (Received October 30, 1978; )     

Alicyclic acid metabolism in plants 7. Metabolism of 14G-labelled alicyclic acids in germinating Phaseolus mungo seeds

When Phaseolus mungo seeds were allowed to germinate in 5–20mM quinate solution in the dark, a marked increase in the endogenousshikimic acid level occurred in their tissues. The acid levelrose distincdy on the 2nd day of germination and reached a maximumon the 4th day. The quinate-¹⁴C fed to germinating seeds waspredominantly converted to shikimic acid, and little radioactivitywas found in 3-dehydroshikimic acid. When quinate-¹⁴C was suppliedsimultaneously with relatively high concentrations of 3-dehydroquinicacid or 3-dehydroshikimic acid, its conversionto shikimic acidwas restrained, but hardly any radioactivity was trapped ineither of the dehydro compounds. 3-Dehydroquinic acid-¹⁴C or3-dehydroshikimic acid-¹⁴C fed to the seeds was metabolizedpreferentially to shikimic acid. The experimental results arediscussed with respect to the metabolic relationship betweenquinic acid and odier alicyclic acids in the aromatic biosynthesisof P. mungo seedlings. (Received October 16, 1975; )     

3-Dehydroshikimate hydro-lyase ofPhaseolus mungo seedlings

3-Dehydroshikimate hydro-lyase was extracted and partially purified by ammonium sulfate fractionation from the hypocotyls of etiolatedPhaseolus mungo seedings. The enzyme was most active at pH 7.0 and the Km for DHS was 1.4 mM. Enzyme activity was inhibited byp-chloromercuribenzoate and arsenite, the inhibition being reversed by reduced glutathione. Enzyme activity was found in extracts from young hypocotyls and cotyledons, especially from 5–6 day-old hypocotyls and 3–4 day-old cotyledons. Changes in enzyme activity in both cotyledons and hypocotyls were measured during the early stage of germination. Participation of this enzyme in the catabolism of alicyclic acids is suggested.     

Quinate:NAD oxidoreductase of germinating Phaseolus mungo seeds: Partial purification and some properties

Quinate:NAD oxidoreductase, which catalyzes the interconversionof quinic acid and 3-dehydroquinic acid, has been extractedfrom liquid N₂-frozen powders of 2-day-old etiolated seedlingsof Phaseolus mungo. The enzyme was partially purified by ammoniumsulfate fractionation and by DEAE-cellulose and gel filtrationcolumn chromatographies, and was separable from shikimate: NADPoxidoreductase and 3-dehydroquinate hydrolyase. The activityappeared to be maximal at pH 8.6–9.0. The apparent Kmvalues at pH 8.6 were 0.48 mM for quinic acid and 0.043 mM forNAD. The involvement of sulfhydryl group in the reaction wasdemonstrated by the potent inhibitory action of both heavy metalions and sulfhydryl inhibitors. The purified preparation ofthe enzyme was reasonably stable for storage in the presenceof dithiothreitol. The metal ions tested, except Hg²⁺ and Ag⁺,showed practically no inhibitory action on the enzyme activity.Aromatic amino acids and other aromatic and alicyclic compoundstested had little or no effect on the activity. ¹ Part 9 of "Alicyclic acid metabolism in plants". (Received January 20, 1977; )     

Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate:NADP+ oxidoreductase in Phaseolus mungo seedlings

Two associated enzymes, 3-dehydroquinate hydro-lyase (EC 4.2.1.10) and shikimate:NADP+ oxidoreductase (EC 1.1.1.25), have been purified from Phaseolus mungo seedlings. These enzymes were purified 6900- and 9700-fold, respectively, but they were not separable. Moreover, two activity bands of the shikimate:NADP+ oxidoreductase were detected after polyacrylamide gel electrophoresis and the two peaks also have 3-dehydroquinate hydro-lyase activity. The two forms of the associated enzymes showed only small differences in molecular weight, Km value, pH optimum and the responses to some inhibitors.     

Cytokinins in Root Nodules of Phaseolus mungo

Four cytokinins have been separated from extracts of root nodulesof Phaseolus mungo by thin-layer chromatography. Their activitywas determined on the basis of their ability to induce betacyaninsynthesis in cotyledons of Amaranthus caudatus. Zeatin and itsriboside showed greater activity than N⁶ (²-(isopentenyl)) aminopurine and its riboside in the bioassay. Phaseolus mungo, mung bean, cytokinins, isopentenyl, amino-purine, zeatin, betacyanin synthesis, Amaranthus caudatus     

Alicyclic acid metabolism in plants 12. Partial purification and some properties of shikimate kinase from Phaseolus mungo seedlings

Shikimate kinase from Phaseolus mungo seedlings was partiallypurified by DEAEcellulose, hydroxyapatite and Sephacryl S-200column chromatographies. The activity was completely inhibitedby EDTA and the requirement for Mg²⁺ could be partially replacedby Mn²⁺, Ca²⁺; Co²⁺ and Cd²⁺. Sulfhydryl inhibitor did not inhibitthe enzyme activity. The apparent Km values for shikimic acidand ATP at pH 8.6 were 0.25 mM and 0.38 mM, respectively. Theactivity appeared to be maximal at pH 8.6–9.0. Shikimate-3-phosphateand ADP inhibited the activity slightly. Aromatic amino acids,quinic acid and dehydroquinic acid had no significant effecton the activity. (Received January 11, 1979; )     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

Alicyclic acid metabolism in plants 8. Association, of two enzymes of the shikimate pathway in shoots of Phaseolus mungo seedlings

The association of two enzymes involved in the shikimate pathway,3-dehydroquinate hydro-lyase (EC 4.2.1.10 [EC] ) and shikimate: NADPoxidoreductase (EC 1.1.1.25 [EC] ), was studied with shoots of etiolated4-day-old Phaseolus mungo seedlings. The enzymes were not separableby ammonium sulfate fractionation, sucrose density gradientcentrifugation, polyacrylamide gel electrophoresis and chromatographyon Sephadex G-100 and DEAE-Sephadex A-50. The results are discussedin relation to the channelling function of metabolites in thealicyclic acid metabolism in higher plants. (Received October 28, 1975; )     

Alicyclic acid metabolism in plants 4. Effect of external supplies of shikimate and quinate to excised hypocotyls of Phaseolus mungo seedlings

When excised hypocotyls of etiolated Phaseolus mungo seedlingswere allowed to take up shikimate solutions of various concentrationsfor 4 to 32 hr at 25°C in the dark, a marked rise in endogenousquinic acid level appeared in the organ. A similar effect wasobserved, to a lesser extent, in other organs, i.e. roots andcotyledons. When quinate solution was supplied to hypocotyls,the effect on the increase of endogenous shikimic acid concentrationwas discernible, although it was not as remarkable as the profoundeffect of shikimate on quinic acid level. A supply of eitherof the acids to excised hypocotyls in a culture solution resultedin a significant increase in dehydroquinate dehydratase activityin the organ, whereas shikimate dehydrogenase activity was slightly,if at all, affected by this treatment. On the basis of theseexperimental results, the metabolic role of quinic acid in aromaticbiosynthesis in this plant is discussed. (Received February 26, 1972; )     

Lipase activity in germinating sunflower seedlings

Studying lipase in germinating sunflower seedlings, we looked for an activator of the lipolytic activity. In the presence of 1.25 mM ATP, the enzyme activity increased 2-fold. Lipid-body lipase solubilization was realized using two detergents: Tween 80 and CHAPS. Lipolytic activity was increased 10-fold in the presence of 2% (w/v) CHAPS, showing the probable 'complexity' of the enzyme. Looking for the possible lipolytic activity of the 10000 g pellet we detected the presence of the enzyme. The pellet extract was mixed, in a range of concentrations, with the oil-body fraction. The resulting lipolytic activity was 4-fold higher. These results give clues as to the subcellular distribution of lipase and its intracellular transport.     

Cytological studies of the hybrid,Phaseolus aureus×P. mungo

The two closely related species,Phaseolus aureus andP. mungo were crossed successfully using the former as the female parent. A detailed cytological study of the hybrid, which showed morphological characters of both the parents, was carried out.At the pachytene stage, the pairing behaviour of 9 out of the 11 chromosomes belonging to the haploid complement of the two parents was investigated. The remaining two chromosomes are represented by morphologically distinct bivalents. The two species differ by a translocation, a deletion and a duplication. A consideration of the data from comparative morphology, geographical distribution, protein characters and genetical studies in the F₂ and back cross generations, indicates a very close relationship between the two diploid species. Chromosome differentiation seems to constitute the major difference. The karyological evidences seem to indicate thatmungo is derived fromaureus.     

Expression of a-Amylase in Phaseolus vulgaris and Vigna mungo Plants

Levels of starch and soluble sugar in pods of Phaseolus vulgarisand Vigna mungo plants were analyzed during the course of maturationof fruits. The results suggest that the immature pods of P.vulgaris function to some extent as temporary reservoirs ofcarbohydrates for growth of seeds. A less clear pattern of accumulationof starch was observed in pods of maturing fruits of Vigna mungo.Measurements of a-amylase activites in pods of maturing fruitsand immunoblotting with an antiserum against     

Selenium as a novel regulator of porphyrin biosynthesis in germinating seedlings of mung bean (Phaseolus vulgaris)

5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.