Resealing of Protein Tyrosine Kinase Substrates into Human Erythrocytes by Rapid Freezing and Thawing in Liquid Nitrogen
We have proposed a hypothesis that the rate of glycolysis in erythrocytes can be controlled in part by the phosphorylation-dependent interaction of several glycolytic enzymes with the N-terminus of the cytoplasmic domain of band 3.1-3 Briefly, as shown by Steck and coworkers4-7, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and phosphofructokinase bind to an acidic sequence within the first 40 amino acids of the anion transport protein, band 3. Because the enzymes are inhibited upon association with the cytoplasmic terminus of band 3, glycolysis is retarded under conditions that promote enzyme binding. However, when the enzymes are released from their inhibitory site, substrates flow more freely down the pathway and glycolysis accelerates. Based on in vitro studies, three potentially physiological transitions can lead to displacement of the glycolytic enzymes from band 3: i) elevation of pH8,9, ii) an increase in the concentrations of certain glycolytic substrates8-10, and iii) phosphorylation of tyrosine 8 or 21 within the enzyme binding site on band 3.1,2 Because little evidence was available to support any of the above regulatory mechanisms in vivo, we sought to develop a method that would allow quantitative assessment of the phosphorylation of band 3 on tyrosine 8 in vivo.
KeywordsSucrose Tyrosine Chloroform Serine Polypeptide
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