Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver

Abstract

Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The K m value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with K i value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

Biochemical Characterization of Buffalo Liver Glucose-6-Phosphate Dehydrogenase Isoforms

“…The purification scheme started with precipitation step using ammonium sulfate, followed by DEAE-cellulose column (ion exchange), then, Sephacryl S-300 column (gel filtration) and finally, 2 0 ,5 0 ADP Sepharose 4B column (affinity). Similar purification procedures of G6PDs were reported from; mouse liver [23], bovine lens [10], human placental [2], dog liver [3], human erythrocyte [29], sheep kidney cortex [5], rat kidney [30] and camel liver [31]. The elution pattern from the DEAE-cellulose column resolved the buffalo G6PD isoenzymes.…”

“…G6PD purified and characterized from different sources including; rabbit liver lumenal endoplasmic reticulum [18], mouse liver [23], rat brain [24], nematodes [25], Schizosaccharomyces pombe [26], guinea pig small intestine [27], bovine lense [10], human placenta [2], dog liver [3], the hyperthermophilic bacterium Thermotoga maritima [28], buffalo erythrocyte [16], human erythrocyte [29], sheep kidney cortex [5], rat kidney [30] and from camel liver [31].…”

Overexpression, purification and enzymatic characterization of a recombinant Arabian camel Camelus dromedarius glucose-6-phosphate dehydrogenase

“…The isoelectric point of the enzyme was determined by chromatofocusing (Fig. 1D) as 6.4 and this value is in agreement with the native Arabian camel G6PD isolated and purified from the liver [27]. Lower isoelectric point value was reported for G6PD from bovine lens at 5.14 [16].…”

“…3B). It was reported that the camel liver native G6PD displayed optimum pH at 7.8 [27]. Similarly, the pH optimum for human placenta G6PD was determined to be 7.8 [28], lamb kidney cortex was 7.8 [29] and for sheep kidney cortex was 7.4 [30].…”

“…One of the most important housekeeping genes is the G6PD which plays a significant role in maintaining the levels of NADPH for fatty acid synthesis, generation of pentose sugars for nucleotide synthesis and elimination of the generated toxic ROS resulted from different metabolic pathways and environmental insults [4]. G6PD has been extensively studied in recent years in many different species but the information about the Arabian camel G6PD is scarce [2,27]. The elucidation of the Arabian camel G6PD function and structure relies on the availability of highly purified protein in sufficient quantities for detailed biochemical and biophysical characterization.…”