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Purification and Characterization of Adenosine Triphosphate: Ribonucleic Acid Adenyltransferase from Escherichia coli

  1. Albrecht E. Sippel†

Article first published online: 3 MAR 2005

DOI: 10.1111/j.1432-1033.1973.tb02953.x

Issue

European Journal of Biochemistry

European Journal of Biochemistry

Volume 37, Issue 1, pages 31–40, August 1973

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How to Cite

Sippel, A. E. (1973), Purification and Characterization of Adenosine Triphosphate: Ribonucleic Acid Adenyltransferase from Escherichia coli. European Journal of Biochemistry, 37: 31–40. doi: 10.1111/j.1432-1033.1973.tb02953.x

Author Information

  1. †

    Institute of Cancer Research and the Department of Biochemistry, Columbia University College of Physicians and Surgeons, 99 Fort Washington Avenue, New York City, New York, U.S.A. 10032

  1. †

    Institute of Cancer Research and the Department of Biochemistry, Columbia University College of Physicians and Surgeons, 99 Fort Washington Avenue, New York City, New York, U.S.A. 10032

Publication History

  1. Issue published online: 3 MAR 2005
  2. Article first published online: 3 MAR 2005
  3. (Received March 5, 1973)

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Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

A primer-dependent poly (A)-polymerizing activity using ATP as substrate (ATP: RNA adenyltransferase) was isolated in high yield from Escherichia coli and purified to apparent homogeneity. For this purpose an assay system had to be used which restricted a variety of infering enzyme activities. Since the enzyme aggregates to macromolecular cell components or precipitates when kept in low salt conditions (<0.4 M NaCl), it was necessary to perform the entire purification procedure in high salt conditions. This was accomplished by using a high salt ribosomal supernatant, a polyethylenglycol-dextran-NaCl phase partition step and a very efficient final high salt phosphocellulose chromatography. At 0.5 M NaCl the enzyme has a molecular weight of approximately 58000. Dodecylsulfate gel electrophoresis shows a single polypeptide chain of molecular weight × 50000. The enzyme has a high preference for ATP as substrate. Manganese ions show higher activity as cofactors than magnesium ions. All classes of natural RNAs are used as primers. The free 3′terminal hydroxyl group of the RNA is required. The enzyme is unable to catalyze a pyrophosphorolysis or a phosphorolysis reaction. It is shown that ATP: RNA adenyl-transferase preferentially synthesizes rather long chains of poly(A) attached to the RNA primers. No relation between the enzyme protein and subunits of the DNA-dependent RNA polymerase could be detected.

Abbreviations
A-A

adenyl-(5′-3′)-adenosine

poly(A)

polyadenylate

poly(dA-dT)

copolymer of alternating de-oxyadenylate and deoxythymidilate

3′:5′-AMP

cyclic adenosine 3′:5′-monophosphate

Enzymes
 

ATP: RNA adenyltransferase or polyadenylate nucleotidyltransferase (EC 2.7.7.19)

 

DNA-dependent RNA polymerase or RNA nucleoside triphosphate: RNA nucleo-tidyl transferase (EC 2.7.7.6)

 

ribonuclease A (EC 2.7.7.16)

 

ribonuclease T1 (EC 2.7.7.26)

 

snake venom phosphodiester-ase (EC 3.1.4.1)

 

deoxyribonuclease I (EC 3.1.4.5)

 

pyruvate kinase (EC 2.7.1.40)

REFERENCES

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  2. Abstract
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