The enzymes nitrate reductase (NR), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS) and asparagine synthetase (AS) have been assayed in various regions along the seedling root ofZea mays L. In the intact attached root and calculated on a protein basis NR, GOGAT, and GS are found to have slightly higher specific activities in the apical 5 mm than in more mature regions of the root. GDH and AS, on the other hand, are much more active in extracts prepared from mature regions of the root than in the apical region. In excised root tips incubated in the presence of NH4 (+) and NO3 (-) there was a marked increase in GDH and AS, and a slight decrease in GOGAT and GS. Additions of NO3 (-) are required for NR activity but neither NO3 (-) nor NH4 (+) additions altered the activity levels of the other four enzymes. Additions of glucose to the medium inhibited the development of AS and GDH activities and resulted in higher activity levels of NR, GS and GOGAT. Glucose additions also enhanced the incorporation of acetate-(14)C and leucine-(14)C into protein. Additions of cycloheximide inhibit the development of NR, AS and GDH activities and also the incorporation of acetate-(14)C and leucine into protein.

Additions of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase (GS), result in an increase in NH13 in seedling leaves of Cs ( (9) suggested that the major reaction leading to the release of CO2 in photorespiration was the conversion of glycine to serine. If this were true, the release of NH3 by photorespiration would be as significant as the release of CO2. In C3 plants, the rates of photorespiration can be considerable, up to 25% the rate of photosynthesis (9). Thus, in a system where NH3 accumulation can be measured, NH3 derived from photorespiration could be far in excess of the NH3 produced by the reduction of N03 . Under normal conditions, however, little NH3 is recovered from leaf tissue (1, 21). Miflin and Lea (1 1 NH3, and subsequently additions of MSX, an inhibitor of GS, were shown to result in accumulations of NH3 (4,5,10,12,18) and in the inhibition of photosynthesis (13,14). In 1978, Keys et al. (7) proposed a photorespiratory nitrogen cycle whereby NH3 released from glycine would be efficiently reassimilated by GS in the cytosol. The resultant glutamine (20) would be transferred to the chloroplast where it would serve as a substrate for GOGAT, and the resultant glutamate would serve as the N-donor in the transaminase reaction leading to glycine formation in the peroxisome. The overall reactions and potential sites of inhibition are illustrated in Figure 1. Sommerville and Ogren (16) showed that Arabidopsis mutants lacking GOGAT accumulated NH3 under conditions which permitted photorespiration. In mutants lacking serinetranshydroxymethylase activity, NH3 was necessary for the continued synthesis of glycine, again under conditions which permitted photorespiration (17). Thus, their results support the hypothesis of Keys et al (7).Recently, Platt and co-workers (13, 14, and personal communication) observed an accumulation of NH3 in spinach leaf discs and an inhibition of photosynthetic activity in the presence of MSX. Under conditions where photosynthesis was inhibited, they still saw an accumulation of NH3 in experiments where the 02 content was reduced to 2%. These conditions should have inhibited photorespiration (15,19) and hence NH3 accumulation. These observations agree with neither the Arabidopsis-mutant studies (16,17) nor with the original hypothesis of Keys et al (7,20) as it suggests alternate and significant sources of NH3 in leaf tissue. Explanations for this apparent contradiction could be the excessively high levels of MSX (8 mM) used in Platt's experiments and to the use of leaf discs rather than whole leaves.In view of the fact that corn leaf pieces showed an accumulation of NH3 in the presence of MSX (12) and that high levels of MSX (2.5 mM) caused a general inhibition of root metabolism (A. Oaks, unpublished), it seemed to us that the role of MSX in leaves needed to be reexamined. In this paper, we show that a maximum level of NH3 is released by relatively low concentrations of MSX

The enzymes nitrate reductase (NR), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS) and asparagine synthetase (AS) have been assayed in various regions along the seedling root ofZea mays L. In the intact attached root and calculated on a protein basis NR, GOGAT, and GS are found to have slightly higher specific activities in the apical 5 mm than in more mature regions of the root. GDH and AS, on the other hand, are much more active in extracts prepared from mature regions of the root than in the apical region. In excised root tips incubated in the presence of NH4 (+) and NO3 (-) there was a marked increase in GDH and AS, and a slight decrease in GOGAT and GS. Additions of NO3 (-) are required for NR activity but neither NO3 (-) nor NH4 (+) additions altered the activity levels of the other four enzymes. Additions of glucose to the medium inhibited the development of AS and GDH activities and resulted in higher activity levels of NR, GS and GOGAT. Glucose additions also enhanced the incorporation of acetate-(14)C and leucine-(14)C into protein. Additions of cycloheximide inhibit the development of NR, AS and GDH activities and also the incorporation of acetate-(14)C and leucine into protein.

ABSTRACrCarboxypeptidase activity (hydrolysis of N-carbobenzoxy-L-phenylalanyl-L-alaine) is higb in a number of temperate zone cereals, orignting in Asia Minor (wheat, barley, oats, wild oats, rye, triticale) compared to other cereals origiting in central America or Asia (maize, sorghum, rice). However, endopeptidase activity (hydrolysis of azocasein or hemoglobin) is relatively much higher in the latter group. Comparison of trichloroacetic acid (TCA)-soluble products derived from the hydrolysis of hemoglobin showed that carboxyterminal amino acids (itidine, arginine, and tyrosine), are released when extracts from wheatt and barley endosperms are used. With extracts from corn endosperms, much more TCA-soluble ultraviolet-absorbing material is released, but very little is released as free amino acids within the first 2 hours and the expected Cterminal amino acids of hemoglobin are not detected in si nt amounts. These results suggest that the method of hydrolysis of the storage proteins may be signiicantly different in these two classes of cereals.Endo-and exopeptidases (1-7, 9, 10, 13-22) are involved in the hydrolysis of the storage proteins in cereals. One major problem in examining peptide hydrolases in cereal endosperms is the unusual solubilities of the major storage proteins. The prolamins, for example, are soluble in 70% ethanol. Because of this problem investigators have often used proteins such as hemoglobin or azocasein to assay the proteases which develop in the cereal endosperm after imbibition. In maize endosperm, the major protease active against hemoglobin, denatured zein, glutelin or the a-chain of insulin has been identified as a endopeptidase (2, 6, 9, 15). It is maximally active at acid pH. In wheat, on the other hand, one of the major proteases active against hemoglobin or gluten (18, 19) is a carboxypeptidase which is also active at an acid pH. In other cereals the relative importance of these general activities has not yet been clearly defined.In Enzyme Extrction. The endosperm and scutellum were removed from seedlings harvested at appropriate times and rinsed three times with disfilled H20. After blotting dry, the endosperm was separated from the scutellum, frozen in liquid N2 and subsequently stored at -20°C. Extraction and assay systems were optimized for maize, barley, and wheat.Frozen endosperms were homogenized with a mortar and pestle on ice. The buffer, 0.2 M sodium acetate (pH 3.8), contained 5 mm (i-mercaptoethanol. Two ml ofbuffer were used per g of maize tissue. Four ml/g were used for other cereals. The homogenate was filtered through Miracloth and centrifuged at 20,000g for 15 min at 4C. The supernatant was used directly for endopeptidase determinations, or dialyzed overnight against 0.1 M sodium acetate buffer (pH 5.2) for the carboxypeptidase analysis.Enzyme Assays. Endopeptidase was measured as described previously (9). The assay mixture contained 1.0 ml of 5% hemoglobin in water, 1.0 ml of 0.05 M sodium acetate buffer (pH 3.8), containing 2.5 mm EDTA, and up to 0.5 ml of e...

No abstract