Lupin enzyme activity

Figure 2. Effect of 1,4-cineole (circles), cis-2-hydroxy-1,4-cineole (triangles) and the commercial herbicide cinmethylin (squares) on the activity of asparagine synthetase from lupin. The dotted line represents 50% inhibition of enzyme activity.

The catalytic activity of glutamate synthases from a variety of sources has been found to be unstable (Miller and Stadtman, 1972 Boland and Benny, 1977 Wallsgrove et al., 1977). The E. coli enzyme was found to be more stable in the presence of glutamine or reducing agents (Miller and Stadtman, 1972). Very high concentrations of mercaptoethanol or dithiothreitol were found to be necessary during purification of the lupin nodule enzyme and routine assays of this enzyme are carried out in the presence of 1% mercaptoethanol (Boland and Benny, 1977). The lupin enzyme is reported to be more stable (20% loss per week) in phosphate compared with Tris buffers. 

Chloride ions selectively activate the glutamine-dependent AS reaction of the yellow lupin enzyme and decrease the of glutamine 50-fold (Rognes, 1980). 

In soil, the chances that any enzyme will retain its activity are very slim indeed, because inactivation can occur by denaturation, microbial degradation, and sorption (61,62), although it is possible that sorption may protect an enzyme from microbial degradation or chemical hydrolysis and retain its activity. The nature of most enzymes, particularly size and charge characteristics, is such that they would have very low mobility in soils, so that if a secreted enzyme is to have any effect, it must operate close to the point of secretion and its substrate must be able to diffuse to the enzyme. Secretory acid phosphatase was found to be produced in response to P-deficiency stress by epidermal cells of the main tap roots of white lupin and in the cell walls and intercellular spaces of lateral roots (63). Such apoplastic phosphatase is safe from soil but can be effective only when presented with soluble organophosphates, which are often present in the soil. solution (64). However, because the phosphatase activity in the rhizo-sphere originates from a number of sources (65), mostly microbial, and is much higher in the rhizosphere than in bulk soil (66), it seems curious that plants would have a need to secrete phosphatase at all. 

Seedlings are a rich source for nonspecific acid phosphatase. Newmark and Wenger (114) have reported on a 1000-fold purification from lupine seedlings. The purified enzyme hydrolyzes phosphate monoesters and pyrophosphate with p-nitrophenyl phosphate as substrate. The optimal activity was at pH 5.2-5.5, and Km was 3 X 10 4 M. Fluoride inhibition was noncompetitive. 

Alkaloid metabolism in lupine was proved by Wink and Hartmann to be associated with chloroplasts (34). A series of enzymes involved in the biosynthesis of lupine alkaloids were localized in chloroplasts isolated from leaves of Lupinus polyphylls and seedlings of L. albus by differential centrifugation. They proposed a pathway for the biosynthesis of lupanine via conversion of exogenous 17-oxosparteine to lupanine with intact chloroplasts. The biosynthetic pathway of lupinine was also studied by Wink and Hartmann (35). Two enzymes involved in the biosynthesis of alkaloids, namely, lysine decarboxylase and 17-oxosparteine synthetase, were found in the chloroplast stoma. The activities of the two enzymes were as low as one-thousandth that of diaminopimelate decarboxylase, an enzyme involved in the biosynthetic pathway from lysine to diaminopimelate. It was suggested that these differences are not caused by substrate availability (e,g., lysine concentration) as a critical factor in the synthesis of alkaloids. Feedback inhibition would play a major role in the regulation of amino acid biosynthesis but not in the control of alkaloid formation. 

Glutamate synthase activity has been detected in the plant fraction of lupin nodules (Robertson et al., 1975b Radyukinaer al., 1977) and the level of this enzyme also increases during nodule development. Glutamate synthase has been purified from the plant fraction of lupin nodules (Boland and Benny,... 

The specific activity of these enzymes in the plant fraction of lupin nodules increases over the same time period as the increase in Ne-fixing activity in the bacteroids (Scott et al., 1976 Bolandet ai, 1979 Reynolds and Farnden, 1979). Asparagine synthetase has only been reported to date in nodules of lupin (Scott et ai, 1976 Radyukina et ai, 1977 Boland et al., 1979), however, numerous reports of aspartate aminotransferase activity in the plant fraction of several legume species have appeared (Table I). 

Neither lupin nodule cytosol or V. faba glutamate synthases are reported to utilize ammonia (see Boland and Benny, 1977 Wallsgrovee/ al., 1977) and neither of these enzymes appear to have been examined for glutaminase activity. 

In bacteria (Jackson and Handschumacher, 1970), j8-cyanoalanine hydrolysis and asparagine hydrolysis are carried out by the same enzyme. However, Castric et al. (1972) found no evidence for asparaginase activity in the presence of j3-cyanoalanine hydrolase. It is interesting to note that although j8-cyanoalanine is not a substrate for purified asparaginase from maturing lupin cotyledons, the amino acid is a strong inhibitor (Lea et ai, 1978). 

It is known that RNA polymerase I is associated in animal cells with protein kinase, which phosphorylates RNA-polymerase and induces an increase of its activity [20]. RNA polymerase I isolated from barley leaf chromatin is also associated with protein kinase activity [19], which is dramatically increased by the addition of BA into the reaction medium (Fig. 1). The dose response curve of this effect was typical of other phytohormone-induced reactions. The effect was specific for biologically active cytokinins (Z, kinetin, BA) and could not be demonstrated with their non-active analogues, adenine or 6-methyladenine. These results were confirmed in experiments with RNA-polymerase I and II isolated from nuclei of lupin cotyledons. Both enzymes possessed low protein kinase activity, but this was increased in vitro 10-fold by BA. 

Other scientists proposed the removal of QAs fromL. albus flours using bacteria able to cataboUze lupanine and to degrade other lupin alkaloids in culture media [58]. The obtained results suggest that enzymatically active bacterial lysates or purified enzyme complex may possess a high potential for the detoxification of QAs, being an alternative debittering process. 

Route c involves the conversion of aspartate to asparagine by glutamine-dependent asparagine synthetase (EC 6.3.5.4). The enzyme has had a checkered career and it has proved extremely difficult to prepare extracts with high activity. Joy and Ireland (1990) have described in detail suitable assay methods and discussed possible factors that may prevent the determination of maximum rates of activity. A heat-stable, dialyzable inhibitor was found in pea leaf homogenates that inhibited the lupin cotyledon enzyme (Joy et al. 1983). The presence of this inhibitor may well account for the current lack of detection of asparagine synthetase in green leaf tissue. 

The cotyledons of germinating seeds have proved to be a major source of asparagine synthetase activity and the enzyme has been studied in lupins (Rognes, 1975,1980 Lea and Fowden, 1975X soybean (Streeter, 1973), cotton... 

Dilworth and Dure, 1978), and Vigm (Kern and Chrispeels, 1978). The enzyme from lupins requires Cl for activity and is inhibited by the presence of Ca ". In maize roots the enzyme is also able to use ammonia as a substrate, which may be of physiological importance in this tissue (Stulen et al, 1979 Oaks and Ross, 1984). The enzyme in root nodules is discussed by C. P. Vance in the second article in this volume. 

Enzymes ofpurine oxidation High levels of xanthine oxidase, uricase, and allantoinase were reported in soybean nodules by Tajima and Yamamoto (1975). The levels of several of these enzymes were elevated in soybean nodules when compared to lupin (Reynolds et al, 1982a) or pea (Christensen and Jochimsen, 1983). Subsequent work has demonstrated that xanthine oxidase (which is in fact an NAD -dependent dehydrogenase), uricase, and allantoinase activities in the nodules increase dramatically during the onset of N2 fixation and NH4" assimilation in soybean (Schubert, 1981 Reynolds et al, 1982b) and cowpea (Atkins ct fl/., 1980 1984b). 

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