Subcellular localization of the NAD+-dependent alcohol dehydrogenase in Entamoeba histolytica trophozoites.
The protozoan parasite Entamoeba histolytica is an ancient eukaryotic cell that shows morphologically atypical organelles and differs metabolically from higher eukaryotic cells. The aim of this study was to determine the subcellular localization of ameba NAD+-dependent alcohol dehydrogenase (ADH2). The enzyme activity was present in soluble and mainly in particulate material whose density was 1.105 in a sucrose gradient. By differential centrifugation, most of the ADH activity sedimented at 160,000 g (160,000-g pellet), similar to the Escherichia coli polymeric ADHE. In the Coomassie staining of the 160,000-g pellet analyzed by electrophoresis, a 96-kDa protein was more prominent than in other fractions; this band was recognized by antibodies against Lactococcus lactis ADHE. By gold labeling, the antibodies recognized the granular material that mainly constitutes the 160,000-g pellet and a material that sedimented along with the internal membrane vesicles. By negative staining, the 160,000-g fraction showed helical rodlike structures with an average length of 103 nm; almost no membrane vesicles were observed in this pellet. In internal membrane fractions, no rodlike structures were found, but protomerlike round structures were observed. These results indicate that the main amebic NAD+-dependent ADH2 activity is naturally organized as rodlike helical particles, similar to bacterial ADHE. Detection of ADH2 in membrane fractions might be explained by cosedimentation of the multimeric ADH during membrane purification.
Avila
Eva E
EE
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, México. edilia@quijote.ugto.mx
Martínez-Alcaraz
Edith R
ER
Barbosa-Sabanero
Gloria
G
Rivera-Baron
Elda I
EI
Arias-Negrete
Sergio
S
Zazueta-Sandoval
Roberto
R
D000135:Acid Phosphatase / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001845:Bone Cysts / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002500:Centrifugation, Isopycnic; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron
D000426:Alcohol Dehydrogenase; D000135:Acid Phosphatase