Tunicamycin inhibition of N-glycosylation of α-glucosidase from Aspergillus niveus: partial influence on biochemical properties.
Treatment of Aspergillus niveus with 30 μg tunicamycin/ml did not interfere with α-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, ~56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65°C). The K(m) and V(max) values of under- and fully-glycosylated forms of α-glucosidase were similar when assessed for hydrolysis of starch (~0.6 mg/ml, ~350 μmol glucose per min per ml), maltose (~0.54 μmol, ~330 μmol glucose per min per ml) and p-nitrophenyl-α-D: -glucopyranoside (~0.54 μmol, ~8.28 μmol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.
Silva
Tony Márcio
TM
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, 14040-901, Ribeirão Preto, SP, Brazil.
Almeida
Fausto Bruno Dos Reis
FB
Damásio
André Ricardo de Lima
AR
Maller
Alexandre
A
Michelin
Michele
M
Jorge
João Atílio
JA
Hanna
Ebert Seixas
ES
Roque-Barreira
Maria Cristina
MC
Terenzi
Héctor F
HF
Polizeli
Maria de Lourdes Teixeira de Moraes
Mde L
D001230:Aspergillus / Q000187:drug effects* / Q000201:enzymology*; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D065089:Glycoside Hydrolase Inhibitors; D006031:Glycosylation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008970:Molecular Weight; D009598:Nitrophenylgalactosides / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D013696:Temperature; D014415:Tunicamycin / Q000494:pharmacology*; D000520:alpha-Glucosidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D065089:Glycoside Hydrolase Inhibitors; D014415:Tunicamycin; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D008320:Maltose; D013213:Starch; D000520:alpha-Glucosidases