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A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding
1Institut National de la Santé et de la Recherche Médicale, Unité 369, Faculté de Médecine Alexis Carrel, Lyon, France.
Abstract:
We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine 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 electrophoresis; 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 commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme.
Insights
Researchers identified a membrane protein (MP50) that binds to microtubules. This protein was identified as glutamate dehydrogenase (GDH), suggesting a novel cellular role for GDH beyond its enzymatic function.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- A previously identified 50 kDa membrane protein (MP50) binds to microtubules.
- MP50 shows properties consistent with mediating ATP-dependent vesicle-microtubule association.
Purpose of the Study:
- To purify and identify the 50 kDa membrane protein (MP50).
- To characterize the interaction of MP50 with microtubules.
- To investigate potential non-enzymatic functions of glutamate dehydrogenase (GDH).
Main Methods:
- Purification of MP50 from pig liver and brain membranes using affinity chromatography on immobilized ATP.
- Phase partitioning in Triton X-114 to determine membrane association.
- In vitro binding assays with microtubules, including inhibition studies with ATP.
- N-terminal amino acid sequencing and V8 protease peptide mapping for protein identification.
- Enzyme activity assays and two-dimensional electrophoresis for characterizing MP50 and comparing it with purified GDH.
Main Results:
- MP50 was purified and identified as glutamate dehydrogenase (GDH).
- MP50 exhibits specific, nucleotide-sensitive binding to microtubules.
- The identified MP50 displayed lower enzymatic activity compared to soluble GDH.
- MP50 showed distinct electrophoretic properties compared to soluble GDH, with minimal microtubule-binding activity observed for soluble forms.
Conclusions:
- A membrane-bound form of GDH (MP50) has been identified with specific microtubule-binding capabilities.
- This interaction is sensitive to nucleotide presence, suggesting a regulatory mechanism.
- The findings indicate that GDH may possess cellular functions beyond its canonical enzymatic role, potentially involving cytoskeletal interactions.