Phosphatases and Kinases

Stanley Tabor1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.10
DOI:  10.1002/0471142727.mb0310s00
Online Posting Date:  May, 2001
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Abstract

The reaction conditions and applications of two phosphatases and one kinase are described in this unit. Bacterial alkaline phosphatase (BAP) from E. coli and calf intestine phosphatase (CIP) from veal are commonly used in nucleic acid research. Both enzymes catalyze the hydrolysis of 5′‐phosphate residues from DNA, RNA, and ribo‐ and deoxyribonucleoside triphosphates. The dephosphorylated products possess 5′‐hydroxyl termini which can subsequently be radioactively labeled using [γ‐32P]ATP and T4 polynucleotide kinase. T4 polynucleotide kinase has 3 activities: the forward reaction efficiently catalyzes the transfer of the terminal (γ) phosphate of ATP to the 5′‐hydroxyl termini of DNA and RNA. The exchange reaction catalyzes the exchange of 5'‐terminal phosphates. Lastly, T4 polynucleotide kinase is a 3′ phosphatase.

     
 
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Table of Contents

  • ENZYME: Alkaline Phosphatases: Bacterial Alkaline Phosphatase and Calf Intestine Phosphatase
  • ENZYME: T4 Polynucleotide Kinase
  • Basic Protocol 1: Labeling 5′ Ends by the Forward Reaction
  • Basic Protocol 2: Phosphorylating Synthetic Oligonucleotides by the Forward Reaction
  • Basic Protocol 3: Labeling 5′ Termini by the Exchange Reaction
  • Literature Cited
  • Figures
     
 
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Materials

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Literature Cited

Literature Cited
   Chaconas, G. and van de Sande, J.H. 1980. 5′ ‐ 32P labeling of RNA and DNA restriction fragments. Meth. Enzymol. 65:75‐88.
   Harrison, B. and Zimmerman, S.B. 1986. T4 polynucleotide kinase: Macromolecular crowding increases the efficiency of reaction at DNA termini. Anal. Biochem. 158:307‐315.
   Midgley, C.A. and Murray, N.E. 1985. T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product. EMBO J. 4:2695‐2703.
   Reid, T.W. and Wilson, I.B. 1971. Escherichia coli alkaline phosphatase. In The Enzymes, Vol. 4 (P.D. Boyer, ed.) pp. 373‐415. Academic Press, NY.
   Richardson, C.C. 1981. Bacteriophage T4 polynucleotide kinase. In The Enzymes, Vol. 14A (P.D. Boyer, ed.) pp. 299‐314 Academic Press, NY.
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