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  • Title: [Effect of erythrocyte membranes and tubulin on the activity of NAD-dependent dehydrogenases].
    Author: Shcherbatova NA, Nagradova NK, Muronets VI.
    Journal: Biokhimiia; 1996 Aug; 61(8):1512-25. PubMed ID: 8962925.
    Abstract:
    Interactions of NAD-dependent dehydrogenases (glyceraldehyde-3-phosphate dehydrogenase, GAPDH, and lactate dehydrogenase, LDH) with band 3 erythrocyte membrane protein and tubulin were characterized. At low ionic strength and un-saturating substrate concentrations, LDH tightly binds to tubulin and is thus inactivated. The Kd of the LDH-tubulin complex was calculated in inhibition and direct binding experiments (15.0 and 13.6 nM, respectively); the stoichiometry of the complex was 1.66 moles of tubulin dimer bound per mole of LDH tetramer. In the presence of 0.15 M NaCl, LDH does not bind to tubulin and tubulin-dependent inhibition of LDH activity is not detected. At low ionic strength, erythrocyte membranes affect both dehydrogenases similarly. GAPDH activity is completely inhibited by excess of erythrocyte membranes (or by excess of cytoplasmic fragment of band 3 protein). Under similar conditions, LDH activity was inhibited by 70% by erythrocyte membranes. In these experiments, 14.8.10(6) GAPDH tetramers or 25.6.10(6) LDH tetramers bound to one erythrocyte ghost (Kd is 0.13 and 0.6 microM, respectively). Increase in ionic strength (0.15 m NaCl) completely abolished the membrane-dependent inhibition of dehydrogenases; however, membranes still bound GAPDH and LDH. Under these conditions, the Kd for GAPDH was increased (up to 4.43 microM), whereas the number of membrane-bound enzyme molecules has not been significantly affected (0.75 nmoles of tetramer per 100 micrograms membrane protein). The Kd for LDH was not changed (0.76 microM), whereas the number of membrane-bound enzyme molecules was decreased (down to 0.48 nmoles of tetramer per 100 micrograms membrane protein). It is suggested that at low ionic strength, the "acidic tails" of band 3 protein and tubulin can interact with positively charged NAD-binding domains of both dehydrogenases thus inhibiting their activity. Increase in ionic strength reduces these interactions, decreasing the binding and inhibition of enzyme activities. At "physiological" ionic strength, catalytically active GAPDH and LDH can possibly bind to various sites of the erythrocyte membrane. This can be important in regulation of the transfer of the common cofactor (NAD/NADH) between their active sites.
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