Interactions of Recombinant Mouse Erythrocyte Transglutaminase with Membrane Skeletal Proteins

Interactions of Recombinant Mouse Erythrocyte Transglutaminase with Membrane Skeletal Proteins Transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent γ-glutamyl-ε-lysine crosslinks between glutamine (Q) acyl-donors and lysine (K) acyl-acceptors. Here, we report the cDNA cloning of a TG from mouse reticulocytes, its 4.6-kb message size and high-yield synthesis of recombinant TG in yeast cultures. Its activity was assayed by crosslinking the amine of monodansylcadaverine (DC) onto casein and inside-out vesicles of erythrocytes. The latter contain TG substrates including the anion ion exchanger (AE1) or band 3, and the crosslinking activity was the highest at physiological [GTP] and [ATP] of erythrocytes. To study individually how TG interacts with band 3 and what role P4.2, a pseudo-TG that is normally associated with band 3, may play in their interaction, recombinant cytoplasmic domain of band 3 (cdb3) and P4.2 were also cloned by polymerase chain reaction from mouse reticulocytes, expressed and affinity-purified from Escherichia coli. Enzyme-linked immunosorbent assay and Western blot analysis revealed that increasing [CaCl2] enhanced TG-mediated crosslinking of DC to cdb3 but decreased TG binding to cdb3. P4.2 inhibited the TG-mediated crosslinking of cdb3 but stabilized the binding of TG to cdb3 in the presence of calcium. This in vitro study suggests a relationship among TG, cdb3 and P4.2 in erythrocyte membrane during calcium influx. The Journal of Membrane Biology Springer Journals

Interactions of Recombinant Mouse Erythrocyte Transglutaminase with Membrane Skeletal Proteins

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Copyright © 2007 by Springer Science+Business Media, LLC
Life Sciences; Human Physiology ; Biochemistry, general
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