Substrate specificity of alkaline phosphatase from human polymorphonuclear leukocytes

Area of Science:

• Biochemistry
• Enzymology

Background:

• Alkaline phosphatase (AP) is a crucial enzyme with diverse physiological roles.
• Understanding the substrate specificity and cofactor requirements of AP, particularly from human neutrophils, is essential for comprehending its function.

Purpose of the Study:

• To investigate the substrate specificity and magnesium (Mg2+) ion dependency of alkaline phosphatase from human neutrophils.
• To compare the properties of neutrophil alkaline phosphatase with other known forms of human alkaline phosphatase.

Main Methods:

• Purified alkaline phosphatase from human neutrophils and liver were used.
• Enzyme activity was assayed using various substrates including adenosine triphosphate (ATP), inorganic pyrophosphate, and p-nitrophenylphosphate at different Mg2+ concentrations.
• Inhibition studies using L-levamisole and analysis of substrate correlations were performed.

Main Results:

• Neutrophil alkaline phosphatase activity was highly dependent on Mg2+ concentration, with optimal activity for pyrophosphate hydrolysis occurring at 0.25-0.50 mmol/l Mg2+.
• p-Nitrophenylphosphatase activity showed activation up to 0.75 mmol/l Mg2+ and remained constant thereafter.
• Strong correlations were observed between enzyme activity using p-nitrophenylphosphate, ADP, and pyridoxal phosphate as substrates, suggesting hydrolysis by a single enzyme.
• L-levamisole, a specific inhibitor, abolished hydrolysis, confirming the enzyme's identity as alkaline phosphatase.

Conclusions:

• Human neutrophil alkaline phosphatase exhibits broad substrate specificity, similar to other human AP forms.
• The enzyme's activity is modulated by Mg2+ concentration, with limited activity towards Mg2+-complexed phosphoesters.
• No evidence for a distinct pyridoxal phosphatase activity was found in neutrophils.