Quantitative Estimates of the Distribution of Homoserine Dehydrogenase Isozymes in Maize Tissues

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Homoserine dehydrogenase (EC 1.1.1.3) was extracted from shoots of etiolated seedings of Zea mays L. which had been grown for periods ranging from three to thirteen days. Both the amount of enzyme extracted and its regulatory properties, as measured by the sensitivity of the enzyme to inhibition by the feedback modulator, L-threonine, were found to be a function of seedlng age and extraction conditions. Equivalent amounts of enzyme with similar properties could be isolated from young seedlings under a variety of conditions. Extraction media containing comparatively low concentrations of the buffer component and a high concentration of dithioerythritol were found to be required for optimum extraction of the enzyme from shoots of seedlngs grown longer than four days and from leaves of light-grown plants. In the absence of dithioerythritol, diminished regulatory control was observed to be a direct function of seedling age.

Section: Methodsmentioning

confidence: 99%

Effects of Plant Age and Extraction Conditions on the Properties of Homoserine Dehydrogenase Isolated from Maize Seedlings

Bryan

Lochner²

1981

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“…The resulting preparations were purified over 100-fold. HSDH-II was completely separated from HSDH-I during gel filtration chromatography, as judged by native PAGE and by treatment with 4 M urea which selectively inactivates HSDH-II (9). Experimental results ob-tained with these enzyme preparations were qualitatively similar to those obtained with homogeneous enzyme that had been purified by immunoaffinity chromatography (22).…”

Section: Enzyme Preparation and Assaymentioning

confidence: 52%

“…In contrast, synthesis could be sharply curtailed in unilluminated chloroplasts by diminished substrate availability and by stringent inhibition by threonine. The extent to which this form of regulatory control occurs in vivo will depend upon a variety of factors, and additional modulation of amino acid biosynthesis could be achieved by changes in enzyme levels (9).…”

Section: Other Physiological Factorsmentioning

confidence: 99%

Differential Regulation of Maize Homoserine Dehydrogenase under Physiological Conditions

Bryan

1990

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Homoserine dehydrogenase is associated with the multibranched pathway of amino acid biosynthesis originating with aspartic acid. Like most of the related pathway enzymes, this enzyme is localized in chloroplasts. The activity and regulatory properties of the threonine-sensitive isozyme of homoserine dehydrogenase isolated from Zea mays var earliking were examined under variable conditions that could exist within chloroplasts. Catalytic activity is not significantly altered within the range of pHs that occur within these organelles, but inhibition of the enzyme by the pathway product, L-threonine, is markedly diminished at the alkaline pHs characteristic of illuminated chloroplasts. Inhibition by threonine is also subject to modulation by physiological levels of NADPH. Under conditions considered to represent the environment within unilluminated chloroplasts, the enzyme is severely inhibited by micromolar concentrations of threonine, but significant enzyme activity is retained under conditions that are likely to occur during illumination, even in the presence of millimolar levels of threonine. These results indicate that homosenne dehydrogenase may be subject to environmentally mediated regulation in vivo. Other observations support this concept and suggest that the intrinsic catalytic and regulatory properties of key enzymes could facilitate a direct link between light-dependent carbon and nitrogen assimilation and amino acid biosynthesis in chloroplasts of higher plants.Aspartic acid serves as a common precursor of several nutritionally essential amino acids, including lysine, methionine, threonine, and isoleucine, with most of the requisite enzymes being localized in chloroplasts of higher plants (8). Homoserine dehydrogenase (EC 1.1.1.3) is necessary for the synthesis of methionine, threonine and isoleucine, and catalyzes the NAD(P)H-dependent reduction of aspartic semialdehyde to homoserine (7). Of the two isozymes of HSDH2 that have been identified in several plant species, HSDH-II is localized within chloroplasts and is clearly involved in amino acid biosynthesis, while the physiological function of the cytoplasmic, isozyme, HSDH-I, remains unclear (8). HSDH-II isolated from Zea mays L. has been purified to homogeneity and studied in some detail (22,29). It is characterized by the ability to undergo ligand-induced hysteretic transitions among four physically distinct states, each of which exhibits different catalytic and regulatory properties (20,21 (19,26,28). Using this and other information, the present study was conducted to evaluate the extent to which variable physiological conditions might influence the properties of maize HSDH-II. The results suggest that changes in pH and substrate concentrations that could occur in vivo can have a profound influence on the regulatory properties of this enzyme. These results differ from those obtained under conventional assay conditions and suggest a novel regulatory role of the enzyme within the context of amino acid biosynthesis in higher plants. MATERIA...

“…In many higher plants (17-19, 22, 26), this enzyme activity is feedback inhibited by the pathway end product, L-threonine, and is stimulated in the presence of potassium ions. HSDH has been extensively studied in crude or partially purified extracts of maize (4,5,9,17), soybean (20,22), carrot (19,21), barley (1, 26), and pea (2, 26). Only the enzyme in maize, however, has been subjected to rigorous purification and to some characterization (13,14,17,27).…”

mentioning

confidence: 99%

Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures

Matthews¹,

Farrar²,

Gray³

1989

Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 ± 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K@. When the enzyme is dialyzed in the presence of K+ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K+. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K+ concentrations increase. The change in threonine and K+ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonineinsensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.