2-3.1.2 Alkaline phosphatase:

• Homodimeric enzyme which catalyzes reactions like hydrolysis and transphosphophorylation of phosphate monoester.
  
  
• They show their optimal activity at pH of about 10.
  
  
• Alkaline phosphatase was the first zinc enzyme discovered having three closed spaced metal ion. Two Zn⁺² ions and one Mg⁺² ion, in which Zn⁺² ions are bridges by Asp 51. The mechanism of action is based on reaction where a covalent serine – phosphate intermediate is formed to produce inorganic phosphate and an alcohol.
  
  
• In human body it is present in four isoforms, in which three are tissue specific isoform i.e. placental, germ cell, intestinal and one is non tissue specific isoform. The genes that encode for tissue specific isoforms are present on chromosome -2 p37-q37, while the genes for one non tissue specific are present on chromosome -1 p34- p36.1.
  
• During post-translational modification, alkaline phosphatase is modified by N-glycosylation. It undergoes a modification through which uptake of two Zn⁺² ion and one Mg⁺² ion occurs which is important in forming active site of that enzyme. Alkaline phosphatases are isolated from various sources like microorganisms, tissue of different organs, connective tissue of invertebrate and vertebrate, and human body.
  

There are several AP that are used in gene manipulation-

•  Bacterial alkaline phosphatase (BAP) - Bacterial alkaline phosphate is a phosphomonoester that hydrolyzes 3' and 5' phosphate from nucleic acid (DNA/ RNA). It more suitably removes phosphate group before end labeling and remove phosphate from vector prior to insert ligation. BAP generally shows optimum activity at temperature 65°C. BAP is sensitive to inorganic phosphate so in presence of inorganic phosphates activity may reduce.

•  Calf intestinal alkaline phosphatase (CIP) – It is isolated from calf intestine, which catalyzes the removal of phosphate group from 5' end of DNA as well as RNA. This enzyme is highly used in gene cloning experiments, as to make a construct that could not undergo self-ligation. Hence after the treatment with CIP, without having a phosphate group at 5' ends a vector cannot self ligate and recircularise. This step improves the efficiency of vector containing desired insert.