Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity

• Autorzy: Sakowicz M. , Grden M. , Pawelczyk T.
• Języki publikacji: EN

Abstrakty

EN

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot anal­ysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with β-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measure­ment of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.

Słowa kluczowe

EN

expression level; cell; 5'-nucleotidase; enzyme activity; tissue distribution; rat; phosphate ion; phosphate effect; Escherichia coli; Ni-nitrilotriacetate; tissue; adenosine kinase; recombinant protein

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2001
• Tom: 48
• Numer: 3
• Opis fizyczny: p.745-754,fig.

Twórcy

autor

Sakowicz M.

• Medical University of Gdansk, Debinki 7, paw.29, 80-211 Gdansk, Poland

autor

Grden M.

autor

Pawelczyk T.

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