Nucleoside phosphotransferase of chick embryo

Area of Science:

• Biochemistry
• Enzymology

Background:

• Nonspecific nucleoside phosphotransferase from chick embryo catalyzes phosphate ester transfer.
• This enzyme is crucial for deoxyribonucleotide and pyrimidine ribonucleotide metabolism.
• The enzyme exhibits instability to heat, dilution, dialysis, and common purification methods.

Purpose of the Study:

• To describe a purification procedure for chick embryo nucleoside phosphotransferase.
• To characterize the properties of the purified enzyme, including stability and molecular weight.
• To investigate the stabilizing effect of nucleotides on enzyme activity.

Main Methods:

• Purification of nucleoside phosphotransferase using deoxythymidine triphosphate (dTTP) as a nucleotide protector.
• Enzyme characterization including molecular weight determination, isoelectric point, and pH optimum.
• Stability assays at 37 degrees C and assessment of inactivation by gel filtration.

Main Results:

• A 920-fold purification of nucleoside phosphotransferase was achieved using dTTP (50 microM) as a stabilizer.
• The purified enzyme in the presence of dTTP has a molecular weight of approximately 270,000, an isoelectric point of 6.27, and a pH optimum of 8.8.
• The enzyme is stable at 37 degrees C for at least 10 minutes when protected by dTTP, but forms a smaller active form (MW ~30,000, pH optimum 7.6) without protection.

Conclusions:

• Nucleotide protection, particularly with dTTP, significantly enhances the stability and allows for the purification of chick embryo nucleoside phosphotransferase.
• The enzyme exists in different active forms depending on the presence of nucleotide protectors, influencing its molecular weight and optimal pH.
• Understanding these properties is vital for further research into DNA synthesis and nucleotide metabolism.