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Y-Z Le, M Agbaga, RE Anderson; Molecular Cloning and Expression of a Phosphatidylinositol Phosphate Kinase From Rat Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1438.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Phosphatidylinositol phosphate kinase (PIPK) is an enzyme responsible for the synthesis of PI-4,5-P<sub≷2</sub≷, an important intermediate in the phosphoinositide second messenger pathway. To study the mechanism of replenishment of PI-4,5-P<sub≷2</sub≷ and the activation of PIPK in rod outer segments, we cloned the PIPK gene that is expressed in rat retina. Methods: The rat PIPK cDNA was cloned using a RT-PCR based approach. The entire open reading frame was sequenced, fused to the glutathione S-transferase (GST) gene, and expressed in <i≷E. coli</i≷. Results: Sequence analysis indicated that the cloned PIPK gene had a high degree of homology with other mammalian PIPKs at the DNA and protein level. The fusion protein was purified with GST-affinity chromatography. Western blot analysis showed that the fusion protein had the similar immunoreactivity to a known Type II PIPK characterized previously in our laboratory. Enzymatic assay of the fusion protein using PI-4-P and PI-5-P as substrates suggested that PI-5-P was the preferable substrate for the GST-PIPK protein. Conclusion: We have cloned and expressed an enzymatically active PIPK from rat retina.
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