A MEMBRANE-BOUND FORM OF GLUTAMATE-DEHYDROGENASE POSSESSES AN ATP-DEPENDENT HIGH-AFFINITY MICROTUBULE-BINDING ACTIVITY

We previously identified a 50 kDa membrane protein which bound to in v itro assembled microtubles [Mithieux and Rousset (1989) J. Biol. Chem. 264. 4664-4668]. This protein exhibited the expected properties for m ediating the ATP-dependent of vesicles with microtubules [Mithieux. Au debet and Rousset (1988) Biochim. Biophys. Acta 969. 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount fr om isolated pig thyroid lysosomes/endosomes, has now been purified fro m membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein b y affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtai ned from 100 g of liver tissue. Phase partitioning in Triton X-114 ind icated that MP50 is a peripheral membrane protein. Radioiodinated live r MP50 bound to microtubules assembled in vitro. The binding was inhib ited by ATP (K(i) = 0.76 mM) and displaced by unlabelled liver or brai n MP50. Equilibrium binding studies yielded K(D) values of 1.8 x 10(-7 ) M. By N-terminal amino acid sequence analysis. MP50 was identified a s glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH a ctivity: 4-5 units/mg compared with 18 and 34 units/mg for purified bo vine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrop horesis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commer cial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of s pecific and nucleotide-sensitive interaction with microtubles. Our dat a suggest that GDH isoproteins, the number of which has been undervalu ed up to now, could have cellular functions other than that of an enzy me.