Influencia de alterações conformacionais na atividade da fosfatase acida de sementes quiescentes de soja

AUTOR(ES)
DATA DE PUBLICAÇÃO

2001

RESUMO

The central aim of the present work was to evaluate lhe conformational stability of a soybean seeds acid phosphatase. Through thermal denaturation studies, the transition temperature (T M) value determined, 65°C, has shown an ummsual resistance of the enzyme under high temperatures. The effects of strong binding compounds, such as concanavalin A (Iedin) and molybdate (inhibitor) on the denaturation profile and transition temperature values were also observed. Enzymatic denaturation by sodium dodecy! sulfate (SDS) was insJgnificant in presenca of phosphoenolpyruvate (PEP), which might be explained by the high affinity of the enzyme for lhe substrate in questiono Thermal denaturation studies in presence of PEP have shown both a significant change on denaturation profile and a reduction on the transition temperature value. Aminoacid analysis contributed to valuable informations that are in accordance to some properties of the enzyme, such as a relatively low isoelectric pcint (pl), equal to 5.0. Furthermore, the expressive number of acid residues may be related to the conformational stability of the polypeptidic chain. Systematic studies of reversible denaturation by urea and guanidinium chloride (GuaCI) were carried out using fluorescence as a monitoring technique. Conformational Stability calculations were made by taking the wavelengths corresponding to the maximum fluorescence emission as a para meter, and the obtained value was equal to 2.48 kcal.mol-1. Calculus of Centre of Mass was taken as a function of the denaturating agent concentration in both cases. Carbohydrate analysis were carried out by using High Performance Liquid Chromatography (HPLC) and lhe presence of mannose, glucosamine and galactosamine was detected as the main carbohydrates in the glycoprotein structure. Carbohydrate remova! was not succeeded, even afier treatment with 300 mU/ml of the Endo- beta -N-acetylglucosaminidasesH, F and F1

ASSUNTO(S)

carboidratos fosfatase acida soja enzimas

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