Author

Santosh Misra

Date of Award

6-1983

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Professor B. Ann Oaks

Abstract

Metabolism of glutamine and asparagine was examined in developing endosperm of maize with emphasis on:

1. Characterization of the enzymes of amide metabolism.

2. A comparison of the levels of activity of enzymes of nitrogen assimilation in the endosperm of a normal variety of maize (W64A) and its isogenic high lysine mutants opaque-2 and_floury-2.

3. Investigations of the metabolism of (UL-¹⁴C) glutamine in the endosperm of cultured corn caryopsis.

Results showed that glutamate synthase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GDH), asparagine synthetase (AS), asparaginase (ASnase) and asparagine transaminase activities were present in the developing endosperm. The enzymes increased in activity just prior to the onset of zein biosynthesis, reached a maximum between 15 and 35 days after pollination and then declined. Two forms of GOGAT with molecular weights, estimated to be 171,000 and 270,000 for ferredoxin- and NADH-GOGAT, respectively, were identified. Almost equal levels of each enzyme were present at every stage of development. Either of these enzymes could mediate the transfer of the amide nitrogen of glutamine to α-amino nitrogen of glutamate. Glutaminase activity was not detected in the endosperm tissue.

The pattern of developmental changes in activity, and the actual levels of activity of GDH, GS, AS, and ASnase were similar in the control and the mutant varieties. In the mutants, the level of GOGAT activity was higher at all developmental stages examined. Twenty days after pollination the activity was 13 ± 0.6, 29 ± 0.5 and 18 ± 0.07 nmol NADH oxidized·minˉ¹·endospermˉ¹ in normal, opaque-2 and floury-2, respectively.

To estimate the capacity of the endosperm tissue to metabolize glutamine, (UL-¹⁴C) glutamine was injected into the endosperm of cultured caryopses. After 168 hr of incubation, 21% of the total label incorporated was recovered in starch, 38% in zein, 8% in glutelin and 13% in the free amino acid fraction. In the zein hydrolysate, glutamine accounted for 14% of the total label in this fraction. The results from tracer studies as well as the observations on the presence of enzymes lead to the conclusion that corn endosperm tissue is fully capable of metabolizing glutamine. The presence of label in the amino acid fraction as well as in carbohydrates suggests the presence of an enzyme system for the extensive metabolism of glutamine in the endosperm tissue.

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