Aspartate aminotransferase isoenzymes in Leptosphaeria michotii. Properties and intracellular location.
Two forms of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five-step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 9110 nkat oxalacetate/mg protein-1 and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers of Mr 92,000. They differed mainly in their Km for 2-oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer of Mr 25,000; form B with an unidentified protein of Mr 40,000-44,000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form B [corrected] and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl-K4M-embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasm. Moreover, mitochondria were isolated and purified by Percoll-density-gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.
Romestant
M
M
Centre de Physiologie Végétale et Unité associée 241 du Centre National de la Recherche Scientifique, Université P. Sabatier de Toulouse, France.
Jerebzoff
S
S
Noaillac-Depeyre
J
J
Gas
N
N
Dargent
R
R
D001203:Ascomycota / Q000201:enzymology*; D001219:Aspartate Aminotransferases / Q000302:isolation & purification*; D002473:Cell Wall / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000201:enzymology; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight
D007527:Isoenzymes; D001219:Aspartate Aminotransferases