Prior Dephosphorylation Research Articles

Regulation of vitamin B6 metabolism in human red cells

The effects of pyridoxine and pyridoxal 5-phosphate (PLP) administration on pyridoxine kinase (PnK) and asparate aminotransferase (EGOT), a PLP-dependent enzyme, were studied in human red cells separated into young and old populations by density centrifugation. After a delay of 48 hr, both pyridoxine and PLP increase EGOT activity in mature red cells by activating preformed GOT apoenzyme. In addition, in young erythroid cells, pyridoxine therapy induces synthesis of PnK, while both pyridoxine and PLP induce synthesis of GOT apoprotein. Thus, PLP stimulates EGOT induction without a change in PnK activity, suggesting that PLP enters erythroid precursor cells without prior dephosphorylation. However, with both pyridoxine and PLP, the full induction of enzyme activities reflects the gradual replacement of circulating red cells by newly formed cells with higher enzyme levels. Therefore, the use of EGOT as a measure of vitamin B6 nutritional status requires recognition of the complexities of intracellular enzyme regulation.

Properties of the 5′-terminus of tRNA His: Kinetics of polynucleotide kinase catalyzed exchange and effect of dephosphorylation on the aminoacylation reaction

Bacteriophage T 4 induced polynucleotide kinase was found to be ineffective in transferring 32P from [γ- 32P]ATP to the 5′-terminus of 5′-phosphorylated E. coli tRNA His using the ADP mediated exchange reaction. However, prior dephosphorylation with alkaline phosphatase allowed polynucleotide kinase catalyzed phosphorylation of tRNA His. Contrary to reports for other tRNA species, alkaline phosphatase catalyzed 5′-terminus dephosphorylation destroys the amino acid accepting ability of tRNA His. Aminoacylation competency of the tRNA His is restored after phosphorylation with polynucleotide kinase.

End group labelling of RNA and double stranded DNA by phosphate exchange catalyzed by bacteriophage T 4 induced polynucleotide kinase

End group labelling of sheared double-stranded DNA, and tRNA has been effected without prior dephosphorylation, utilizing the reversal of T 4 polynucleotide kinase activity. Incubation of DNA with polynucleotide kinase in the presence and absence of a phosphate acceptor (ADP) allowed the determination of the relative ratio of 5′ hydroxyl and 5′ phosphoryl terminii in the polynucleotide. This method of analysis has demonstrated a high preference in the formation of 5′ vs 3′ phosphomonoesters during high pressure shearing of double stranded DNA.