Changes in Enzyme Regulation during Growth of Maize

Abstract

The sensitivity of homoserine dehydrogenase (EC 1.1.1.3) to inhibition by the feed-back modifier, L-threonine, was examined in preparations derived from etiolated shoots, roots, and lightgrown tissues of Zea mays L. var. earliking. A progressive decrease in enzyme sensitivity was observed during seedling growth. Enzyme derived from internode tissue retained a greater sensitivity to the effector than enzyme derived from apical portions of etiolated shoots, whereas enzyme from root tips was characteristically more sensitive than that prepared from mature cells of the root. Enzyme desensitization occurred rapidly during culture of excised shoots and the activities of both homoserine dehydrogenase and aspartokinase (EC 2.7.2.4) declined during shoot culture under a variety of conditions. The initial enzyme levels and the characteristic sensitivity of homoserine dehydrogenase were preserved during culture at 5 to 7 C, but desensitization was not prevented by inclusion of cycloheximide in the culture medium.Results of control experiments provide evidence that desensitization occurs in vivo. No alteration of the enzyme properties was detected during extraction or concentration of sensitive or insensitive enzyme or during coextraction of enzyme from mixed populations of different age shoots; nor was a differential distribution of inhibitors or activators indicated during assay of mixed preparations. The change in enzyme sensitivity was apparent under a variety of assay conditions and was not accompanied by changes in the apparent affinity of the enzyme for the substrate, homoserine. It is suggested that systematic changes in the regulatory characteristics of certain enzymes could be an important level of metabolic regulation during cellular differentiation.Three forms of maize homoserine dehydrogenaase were detected after acrylamide gel electrophoresis of samples derived from 72-hr shoots. Similar analysis of samples from older shoots revealed a broad asymmetric band of enzyme activity, suggesting that changes in the relative distribution of specific forms of the enzyme could be related to the growth-dependent changes in the sensitivity of maize homoserine dehydrogenase. plants (4,19). In contrast to microbial systems, however, evidence of end-product control of the concentration of a number of key biosynthetic enzymes has not been obtained in higher plants (2,7,13,20,22,30). This may reflect significant differences in the range and extent of regulatory mechanisms which serve to balance metabolism in dissimilar organisms. A greater extent of metabolite compartmentation in structurally complex multicellular plants would be expected (24, 31), but unique mechanisms may also contribute to metabolic regulation in these organisms.The present experiments are concerned with alterations in the activities of homoserine dehydrogenase (EC 1 . 1 .1 . 3) and aspartokinase (EC 2.7.2.4) during development of Zea inays L. var. earliking. These enzymes contribute to the regulation of the biosynthetic pathway leading from aspartic...

Changes in Enzyme Regulation during Growth of Maize

“…Aspartic semialdehyde can also be metabolized to lysine in a separate branch of the pathway. During earlier investigations, evidence was obtained which suggested that the regulatory properties of homoserine dehydrogenase are progressively altered during maturation of a variety of maize tissues (7,19 In some experiments, chloroplast-enriched fractions were obtained by centrifugation of extracts for 1 min at 5OOg (maximum) to remove whole cells and debris and concentrating the plastids from the supernatant by centrifugation for 5 min at 1,500g (maximum). Enzymes were released by hypotonic shock from plastids isolated by differential centrifugation or obtained from sucrose gradients.…”

“…Disc gel electrophoresis was performed under the conditions described by Davis (6) except that 1 mM L-threonine was included in both large and small pore gels and the upper reservoir buffer. Bromphenol blue was used as the tracking dye and detection of the enzyme after electrophoresis was based on substrate-dependent dye precipitation (19). After visualization of the enzyme distribution, gels were scanned at 600 nm using a linear transport device attached to the Gilford spectrophotometer.…”

“…The mm extinction coefficients used were: catalase, 0.04; Cyt c oxidase, 18.5; fumarase, 2.44; triosephosphate isomerase and homoserine dehydrogenase, 6.22. Chl was measured as described by Arnon (2), and protein was determined spectrophotometrically after concentration with (NH4)2SO4 and dialysis (19).…”

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents

“…The HSDH activity was quantified by measuring the rate of NADP + reduction (Matthews et al, 1975;Paris et al, 2002). The reaction mixtures contained 100 mM Tris-HCl (pH 8.0), 150 mM KCl, 50 mM homo-Ser, 1 mM NADP + , and 112.5 mL of enzyme preparation in a final volume of 0.75 mL.…”

“…Protein samples for AK and HSDH activity assays were prepared as described by Di Marco and Grego (1975) and Matthews et al (1975). Because of the large amount of tissue required for enzyme extraction, the entire aboveground portion of 4-week-old plants was harvested (Supplemental Fig.…”