Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD + /NADH redox method.

Autor: Syngkli S; Biological Chemistry Laboratory, Department of Zoology, North-Eastern Hill University, Shillong, 793022, India., Das B; Biological Chemistry Laboratory, Department of Zoology, North-Eastern Hill University, Shillong, 793022, India. Electronic address: dasbidyadhar@nehu.ac.in.
Jazyk: angličtina
Zdroj: Biochimie [Biochimie] 2023 Nov; Vol. 214 (Pt B), pp. 199-215. Date of Electronic Publication: 2023 Jul 20.
DOI: 10.1016/j.biochi.2023.07.015
Abstrakt: Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD + /NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)
Databáze: MEDLINE
Externí odkaz: