Properties, intracellular localization, and stage-specific expression of membrane-bound beta-glucosidase, BglM1, from Physarum polycephalum.
Physarum polycephalum expresses a membrane-bound beta-glucosidase (BglM1) with a molecular mass of 130 kDa. The primary structure of BglM1 consists of a glycosyl hydrolase family 3 domain at an amino-terminal domain and a carboxyl-terminal region without homology to the sequence of known glycosidases. The latter region contains two calx-beta motifs known as Ca(2+)-binding sites; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. The molecular mass calculated from the amino acid sequence is 130 kDa, but that in the crude extract was estimated by SDS-PAGE to be 230 kDa, and decreased to 130 kDa during purification. However, when BglM1 was purified in the presence of calcium ion, the molecular mass remained 230 kDa. The biochemical characteristics of the 130- and 230-kDa BglM1 forms were analyzed: differences were found in the kinetic data for some substrates specific for both these enzymes; however, no difference was found in their intrinsic characteristics such as optimum pH and temperature. In addition, the molecular mass of native BglM1 with a calcium ion was estimated to be 1,000 kDa or larger by gel filtration. These results suggest that the calcium ion influences the conformation of BglM1. The evidence that BglM1 localizes on the plasma membrane of plasmodia was confirmed using immunofluorescence microscopy. Although Physarum BglM1 was expressed in microplasmodia and plasmodia, little expression was detected in other stages. BglM1 may have some function only in multinuclear cells.
Hayase
Masato
M
Department of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan.
Maekawa
Akinori
A
Yubisui
Toshitsugu
T
Minami
Yoshiko
Y
D000818:Animals; D000913:Antibodies, Protozoan; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology*; D002850:Chromatography, Gel; D005455:Fluorescent Antibody Technique; D042541:Intracellular Space / Q000201:enzymology*; D007700:Kinetics; D008018:Life Cycle Stages; D008970:Molecular Weight; D016839:Physarum polycephalum / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development* / Q000276:immunology; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000913:Antibodies, Protozoan; D015800:Protozoan Proteins; D001617:beta-Glucosidase