Microtubular protein catalytic interactions with nucleotides

Area of Science:

• Biochemistry
• Cell Biology
• Protein Chemistry

Background:

• Purified tubulin from pig brain naturally binds GDP and GTP.
• Tubulin plays a critical role in microtubule formation and cellular structure.

Purpose of the Study:

• To investigate the incorporation of inorganic phosphate into tubulin-bound guanine nucleotides.
• To elucidate the catalytic properties and nucleotide exchange mechanisms of purified tubulin.

Main Methods:

• Incubation of purified tubulin with inorganic [32P]phosphate.
• Analysis of labeled GDP and GTP formation and nucleotide hydrolysis.
• Investigation of the effects of added nucleotides (GDP, GMP, ADP) on labeling.

Main Results:

• 32P is incorporated into tubulin-associated GDP and GTP, indicating active nucleotide modification.
• A hydrolase activity associated with tubulin reduces the yield of labeled nucleotides.
• The formation of [32P]GDP occurs via an exchange mechanism, suggesting reversible protein guanidylation.
• Tubulin preparations also catalyze oxygen exchange between water and inorganic phosphate.

Conclusions:

• Purified tubulin exhibits catalytic activity, facilitating nucleotide exchange and phosphate incorporation.
• Tubulin possesses at least two distinct catalytic properties: nucleotide modification and oxygen exchange.
• These findings shed light on the dynamic nature of tubulin-bound nucleotides and their functional implications.