Influence of lectin concentration on the catalytic properties of S1 nuclease bound to Concanavalin A-sepharose.
Partially purified S1 nuclease was bound through its carbohydrate moiety to Con A-Sepharose containing increasing amounts of lectin. The retention of activity was high, varying essentially from 75% on the "low lectin" matrix (1 mg Con A/mL of Sepharose), to no detectable activity on the "high lectin" matrix (8 mg Con A/mL of Sepharose). However, approximately 50% activity could be restored in "high lectin" matrix when the coupling was carried out in the presence of glucose, suggesting that the loss of activity on the "high lectin" matrix is caused by conformational changes brought about by the multiple attachment of the enzyme to the matrix. Interaction of Con A with S1 nuclease was used to predict the nature of carbohydrate moiety and its location with respect to the active site of the enzyme. Immobilization resulted in an increase in the optimum temperature, pH, and temperature stabilities, but it did not affect the pH optimum. A marginal increase in the apparent Km was observed. The bound enzyme also showed enhanced stability toward 8 M urea. On repeated use, the bound enzyme retained more than 80% of its initial activity after 6 cycles. These results are discussed taking into consideration the factors affecting immobilized enzymes. In addition, the potential use of immobilized S1 nuclease as an analytical tool is discussed.
Reddy
L G
LG
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Shankar
V
V
D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004355:Drug Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005976:Glutaral / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D013696:Temperature
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; C028326:concanavalin A-sepharose; D012685:Sepharose; D015719:Single-Strand Specific DNA and RNA Endonucleases; D005976:Glutaral