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Pyruvate carboxylase in Leptosphaeria michotü . Isolation, properties, intracellular localization and cyclic variations of its activity in relation to polypeptide level
Author(s) -
Tozo Koffi,
SéjalonDelmas Nathalie,
JerebzoffQuintin Simonne,
Jerebzoff Stephan
Publication year - 1992
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1992.tb01348.x
Subject(s) - pyruvate carboxylase , biochemistry , biotin , biology , cycloheximide , enzyme , protein subunit , microbiology and biotechnology , protein biosynthesis , gene
Pyruvate carboxylase (EC 6.4.1.1) was obtained from the fungus Leptosphaeria michotü (West) Sacc. and enriched 543‐fold by a 5‐step purification procedure as an a4‐β4 tetramer of M r 440000, composedof a M r 60000 α‐subunit, containing bound biotin, and a M r 50000 β‐subunit. The enzyme was active from pH 6.5 to 12.0, with a maximum between pH 8.0 and 8.5. Its specific activity was 125nkat (mg protein) −1 : it was not affected by acetyl CoA. A rabbit antiserum raised against the yeast pyruvate carboxylase was specifically reactive against the α‐subunits of the L. michotü enzyme. The enzyme was localized into the cytosol by gold‐labelled streptavidin and immunogold staining of thin sections of Lowicryl‐K4M‐embedded colonies. Pyruvate carboxylase and acetylCoA carboxylase in L. michotü had synchronous activity rhythms at constant temperature and in darkness; these rhythms were suppressed by cycloheximide or avidin supply. The pyruvate carboxylase level was quantified along the activity rhythm by gel electrophoresis using 35 S‐streptavidin. and by enzyme‐linked immunosorbent assay (ELISA) using serum against the yeast pyruvate carboxylase. The cyclic variations of pyruvate carboxylase activity were correlated with cyclic variations in the enzyme level. Suppression of pyruvate and acetyl CoA carboxylase activities by avidin had a no important effect on the transaminase rhythms of L. michotü .