Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain <i>Idiomarina loihiensis</i>

Abstract

Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) from I. loihiensis saline's bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize the I. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45˚C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD + and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

Lipid transfer protein CaMBP10 binds to glyceraldehyde-3-phosphate dehydrogenase and modulates its activity

“…NAD-linked GAPDH activity assay was performed according to the method in (Mardad et al 2013). The sample was dissolved in 0.015 M pyrophosphate buffer (pH 8.5) containing 7.5 mM NAD, 0.1 mM DTT and 0.015 M DLglyceraldehyde-3-phosphate to a 3 mL total volume.…”

“…The most common form of GAPDH is an NADHdependent enzyme located in the cytosol, mitochondria and chloroplasts. Generally, NADH-dependent activity is constant and constitutive, which is responsible for the oxidative phosphorylation of G3P and the reduction of NAD to NADH (Mardad et al 2013;Sparla et al 2005).…”

“…NADPHdependent activity is regulated by light and metabolic effectors. Meanwhile, GAPDH also displays constitutive NADdependent activity regarding the oxidative phosphorylation of G3P and the reduction of NAD to NADH (Mardad et al 2013;Sparla et al 2005). In plants chloroplast, there are two subunits of GAPDH: GAPDH-A and GAPDH-B.…”

Expresión heteróloga y caracterización bioquímica de la gliceraldehído-3-fosfato deshidrogenasa de Giardia lamblia como posible candidata para el diseño de fármacos contra la giardiasis

“…También se demostró que como dímero es más estable que como tetrámero [63]; mientras que en la proteína GAPDH de Fasciola gigantica, en una concentración 300 mM de NaCl, se estabiliza su estado tetramérico sin cambiar a la forma dímera [67]. El valor de pH registrado para GlGAPDH-I se acerca a los valores de pH de otras GAPDHs, con un rango de 7.0 y 10.0, aunque la mayoría se ha reportado entre pH 8.0 y 9.0, de los cuales podemos mencionar los siguientes: Proteus vulgaris pH 7.5 [68], Naegleria gruberi pH 8,0 [45], Homo sapiens pH 8.5 [69], Idiomarina loihiensis pH 8,0-8,5 [58], Micobacterium tuberculosis pH 9.0 [59], Trypanosoma brucei pH 9.4 [61], Trypanosoma cruzi pH 8.6 [62],…”

“…Sin embargo, este hallazgo es diferente a la temperatura óptima reportada para Idiomarina loihiensis (30-45 °C) [58], Naegleria gruberi (25 °C) [45] y Homo sapiens (43 °C) [69]. El valor T 1/2 definido como la temperatura a la que GlGAPDH-I donde la enzima pierde el 50% de su actividad original fue de 40.28 °C.…”