Structural Biochemistry/Calf Intestinal Alkaline Phosphatase

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Alkaline Phosphatase

Alkaline phosphatase is the enzyme that catalyzes a dephosphorylation of DNA, RNA, ribo-, and deoxyribonucleoside triphosphates. It can also remove phosphates from nucleotides and proteins. They are most active at a basic pH. This enzyme has become very useful in molecular biology because this allows us to cleave the 5' end of DNA and prevent any further ligation thus keeping the DNA molecules linear until they are prepared for analysis. We can then use radiolabeling to easily track a certain section of a DNA through the various processes of DNA replicaton.

This has its uses in vector recombination and cloning genes. After restrictive enzymes cleave a plasmid we have to make sure that this circular DNA doesn't reconnect and form the original circular DNA. So before we can actually insert our gene of interest to allow for its cloning, alkaline phosphatase can remove any phosphate group that might be susceptible to attack by the other end of the DNA or even by an external nucleophile. It's also very valuable for its high rate of activity even in harsh conditions.

There are several main sources of alkaline phosphatase, these are: bacterial alkaline phosphatase(BAP), calf intestinal alkaline phosphatase(CIP), and shrimp alkaline phosphatase. Not to mention it is also present in human tissues and most concentrated at organs such as the kidneys and liver. BAP is the most active of all these enzymes but also is hard to get rid of after the dephosphorylation reaction. CIP is of great interest and is extracted from bovine intestine. It can easily be denatured and destroyed by protease digestion. It has been shown that CIP can be used as antiflammatory agents in human bodies and have been tested on mice.

Alkaline phosphtase must be monitored in the human body because it if the concentration goes too low or too high, health problems may arise. Elevated levels caused by factors that are still not completely clear can cause liver damage and bone disease. However, it is normal for levels to spike during puberty when bone growth is occurring rapidly. Lowered levels of alkaline phosphatase is characterized by different kinds of anemia and leukemia. Postmenopausal women are also more likely to have higher levels.

ALP is associated with cell membranes and present in many tissues in animals and humans. ALP is connected to the membranes by glycoproteins. When these are cleaved, phosphate groups are recycled throughout the cells and the amount of ALP levels increases in the plasma. Glucocorticoids can significantly raise the amoutns of ALP present in the body while anticonvulsants secrete less but still a significant amount. Glucocorticoids generate ALp in the C isomer while anticonvulsants generate ALP in an L isomer. Alkaline phosphtase is essential for the mineral deposition in teeth and bones, Bone can become weak and deficient if alkaline phosphatase is not present, a disease called hypophosphatasia. ALP is nonspecific and mainly cleaves phosphate esters.