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Purification and characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase from human placenta
Abstract:
5'-Methylthioadenosine phosphorylase has been purified to homogeneity (30,000-fold) from human full-term placenta by a procedure involving covalent chromatography on organomercurial-agarose as the major step. The specific activity of the homogeneous enzyme is 10.2 mumol of 5'-methylthioadenosine cleaved per min per mg of protein, and the overall yield is about 20%. The enzyme has a molecular weight of 98,000, as determined by gel filtration on Sephacryl S-200 and Superose 6B, and is composed by three apparently identical subunits with a molecular weight of 32,500. The isoelectric point is 5.5, and the optimal pH ranges from 7.2 to 7.6. The resistance of the enzyme to thermal inactivation is increased remarkably by the addition of 5'-methylthioadenosine or phosphate. The homogeneous enzyme shows an absolute requirement for -SH-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds. The reaction catalyzed by the enzyme is fully reversible with a Keq of 1.39 X 10(-2) (in the direction of phosphorolysis) at 37 degrees C and pH 7.4. The Km values for 5'-methylthioadenosine, phosphate, adenine, and 5-methylthioribose 1-phosphate are 5, 320, 23, and 8 microM, respectively.
Insights
Human placenta 5'-methylthioadenosine phosphorylase was purified to homogeneity. This enzyme plays a role in purine metabolism and requires reducing agents for activity.
Area of Science:
- Biochemistry
- Enzymology
Background:
- 5 '-Methylthioadenosine phosphorylase (MTAP) is an enzyme involved in purine metabolism.
- Understanding MTAP's properties is crucial for its potential therapeutic applications.
Purpose of the Study:
- To purify and characterize 5 '-methylthioadenosine phosphorylase from human placenta.
- To determine the enzyme's kinetic and physical properties.
Main Methods:
- Purification using covalent chromatography on organomercurial-agarose.
- Gel filtration (Sephacryl S-200, Superose 6B) for molecular weight determination.
- Enzyme activity assays and kinetic analysis.
Main Results:
- Homogeneous enzyme purified 30,000-fold with 20% yield.
- Molecular weight of 98,000 Da, composed of three 32,500 Da subunits.
- Optimal pH 7.2-7.6; stable with substrate/phosphate; requires reducing agents; inactivated by thiol-blocking agents.
Conclusions:
- The study successfully purified and characterized human placental MTAP.
- Kinetic parameters (Km values) and stability factors were elucidated, providing insights into enzyme function.