Functional Differences Between Normal and Mutant Alkaline Phosphatase

Functional Differences Between Normal and Mutant Alkaline Phosphatase

Abstract

Alkaline phosphatase (AP), an enzyme that transfers phosphate groups into bone, is inhibited by acidic conditions, which is why blood acidosis in Fanconi Syndrome causes a loss of bone density. This lab studied the effect of a mutation in a certain amino acid in AP, and why these patients with hypophosphatemia, characterized by low blood phosphate levels, had almost normal bone densities. Here, the optimal wavelength, extinction coefficient, and incubation time for measuring AP enzyme activity were used to determine the optimum pH for mutant AP enzyme activity. First, an absorbance spectrum was created for p-nitrophenol, the product of the reaction, to determine optimum wavelength. Absorbances were plotted, and the extinction coefficient was calculated using the slope of the resulting line. Next, the optimum incubation time for AP and PNP substrate was determined by testing a range of times and finding the greatest increase in absorbance on a resulting graph. Finally, by incubating enzyme-substrate mixtures in different pH buffers and measuring absorbance, optimum pH was determined. It was found that the mutant AP enzyme showed a higher rate of activity in acidic versus basic conditions. When run in a polyacrimide gel electrophoresis, the functional pH differences were visually detected in the resulting bands. These observations demonstrate that it is important to find out how certain altered genes can affect symptoms of a disease, because it helps further the ability for doctors and other scientists to treat their patients.

Introduction

In the human body, alkaline phosphatase is an enzyme used for regulating the amount of hydrozyapatite deposited into bone. From there, phosphate and calcium can be integrated or removed from bone when needed (Sogabe 2008). AP functions best in basic conditions, resulting in normal phosphate levels in the blood and in bone. When someone has Fanconi...