Science Fair Project Encyclopedia
Alkaline phosphatase (ALP) (EC 22.214.171.124) is a hydrolase enzyme responsible for removing phosphate groups in the 5- and 3- positions from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment.
In bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. Since this space is much more subject to environmental variation than the actual interior of the cell, bacterial alkaline phosphatase is comparatively resistant to inactivation, denaturation, and degradation, and also has a higher rate of activity. Although the actual purpose of the enzyme is still not fully understood, the simple hypothesis, that it is a means for the bacteria to generate free phosphate groups for uptake and use, is supported by the fact that alkaline phosphatase is usually only produced by the bacteria during phosphate starvation and not when phosphate is plentiful. However, other possibilities exist; for instance, the presence of phosphate groups usually prevents organic molecules from passing through the membrane, therefore dephosphorylating them may be important for bacterial uptake of organic compounds in the wild. Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production.
The most common alkaline phosphatases used in research are:
- Bacterial alkaline phosphatase (BAP), from Escherichia coli C4 cells
- Shrimp alkaline phosphatase (SAP), from a species of arctic shrimp (Pandalus borealis)
- Calf intestine alkaline phosphatase (CIAP), from calf intestine
Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end), thereby stabilizing the DNA until the next step of the process for which it is being prepared; also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the calf intestine alkaline phosphatase is the most useful, as it is the easiest to inactivate once it has done its job.
The contents of this article is licensed from www.wikipedia.org under the GNU Free Documentation License. Click here to see the transparent copy and copyright details