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Adenosine deaminase from bovine brain: purification and partial characterization
G Lupidi1, M Falasca, F Marmocchi
1Dept. of M.C.A. Biology, University of Camerino, Italy.
Biochemistry International
|May 1, 1992
Summary
Bovine brain adenosine deaminase was purified and characterized, revealing a stable enzyme with unique properties. This research provides insights into enzyme kinetics and inhibition for adenosine deaminase.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Adenosine deaminase (ADA) is a crucial enzyme in purine metabolism.
- Understanding mammalian ADA forms is vital for biochemical and medical research.
Purpose of the Study:
- To purify and characterize the cytoplasmic form of bovine brain adenosine deaminase.
- To investigate the enzyme's kinetic properties, stability, and inhibition patterns.
Main Methods:
- Purification using salt fractionation, DEAE-cellulose, octyl-sepharose 4B, and affinity chromatography.
- Characterization via disc gel electrophoresis, molecular mass determination, and isoelectric focusing.
- Enzyme kinetics determined using adenosine and 2'-deoxyadenosine.
- Inhibition studies with adenosine analogs and coformycin derivatives.
Main Results:
- The enzyme was purified ~450-fold and found to be homogeneous with a molecular mass of 65 kDa and pI of 4.87.
- Km values for adenosine and 2'-deoxyadenosine were determined.
- The enzyme exhibited remarkable thermal stability (t1/2 = 15 hours at 53°C).
- Aza and deaza analogs of adenosine were effective inhibitors.
Conclusions:
- The bovine brain adenosine deaminase displays unique thermal stability compared to other mammalian forms.
- Inhibitor profiles are similar to other ADA enzymes, suggesting conserved active site features.
- Kinetic data for coformycin and deoxycoformycin binding are reported.