Guanidoacetate methyltransferase. Purification and molecular properties

Area of Science:

• Biochemistry
• Enzymology

Background:

• Guanidoacetate methyltransferase (GAMT) is a key enzyme in creatine biosynthesis.
• Understanding GAMT's properties is crucial for metabolic studies.

Purpose of the Study:

• To purify and characterize guanidoacetate methyltransferase from pig liver.
• To determine the enzyme's molecular weight and subunit composition.
• To investigate potential inhibitors of GAMT activity.

Main Methods:

• Enzyme purification using standard biochemical techniques.
• Polyacrylamide gel electrophoresis (PAGE) under native and denaturing conditions (pH 3, 8 M urea).
• Sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) for molecular weight estimation.
• Sephadex G-75 chromatography for molecular weight determination.
• Enzyme inhibition assays using specific compounds.

Main Results:

• Guanidoacetate methyltransferase was purified approximately 140-fold from pig liver.
• Native PAGE revealed four protein bands with GAMT activity, but under denaturing conditions (pH 3, 8 M urea), it migrated as a single component.
• SDS-PAGE indicated a single protein component with GAMT activity and estimated the molecular weight at 31,000.
• Sephadex G-75 chromatography yielded a similar molecular weight estimate.
• The enzyme was inhibited by adenosylhomocysteine (Ki = 16 µM), 3-deazaadenosylhomocysteine (Ki = 39 µM), and sinefungin (Ki = 18 µM).

Conclusions:

• Pig liver guanidoacetate methyltransferase appears to be a single polypeptide chain of approximately 31,000 molecular weight.
• The enzyme's activity is sensitive to specific S-adenosylmethionine analogs, suggesting potential regulatory mechanisms or drug targets.