Fructose 1, 6 Bisphosphate Aldolase from Gestational Diabetic Human Placenta: Purification, Identification, and Investigation of Kinetic Properties

Neşe Hayat Aksoy,Pakize Dogan

doi:10.13189/adm.2015.030302

Neşe Hayat Aksoy, Pakize Dogan

Open Access

https://doi.org/10.13189/adm.2015.030302

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Abstract

Gestational diabetes mellitus is described as glucose intolerance at various degrees that is first detected during pregnancy. In diabetic complications, there are changes in placental function, particularly with respect to intake, transmit, and utilization of glucose, and also in glycolysis and glycolytic enzymes. The placenta possibly plays a critical role in protecting the fetus from adverse effects caused by the maternal diabetic conditions. Fructose 1,6-bisphosphate aldolase, a main glycolytic enzyme, catalyses the cleavage of fructose 1,6-bisphosphate, resulting in two three-carbon products in many cells. In this study, we principally have investigated the presence of aldolase in diabetic human placenta and then purified the enzyme. We also determined the optimum conditions of enzyme assay measurements. With this procedure, we determined the specific activity of placental aldolase as 590, 94 mU/mg protein, and aldolase was purified about 63,0 fold from gestational diabetic human placenta. The molecular weight of human placental aldolase was found as 160 kDa. In present study, substrate kinetics were also investigated, and two different Km and Vmax values at high and low concentrations of substrate Fructose 1,6-bisphosphate were observed. High substrate concentration range is determined as the linear substrate concentration zone. Therefore, advanced kinetic studies had been performed at this linear zone. Enzymatic assays were carried out, and substrate kinetic properties were determined. According to this determination, Vmax value of gestational diabetic placental fructose 1,6-bisphosphate aldolase was found as 939,548±60,869 U/mg and Km as 24,304±2,948 mM.

Highlights

Fructose–1,6-bisphosphate aldolase (DFru–1,6-bisphosphate D glyceraldehyde–3-P-lyase; FBPA;E.C. 4.1.2.13) catalyses the reversible cleavage of fructose–1,6-bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde–3-phosphate (GAP)in the glycolytic pathway of prokaryotic and eukaryotic organisms
Non-linear regression analysis module of Systat 11 statistic program was used to calculate of kinetic parameters. With this modified method [32], the enzyme was purified about 63,0 fold from gestational diabetic human placenta
Elution graphic from phosphocellulose column of gestational diabetic human placental aldolase is provided at Fig. 1

Summary

Introduction

Fructose–1,6-bisphosphate aldolase (DFru–1,6-bisphosphate D glyceraldehyde–3-P-lyase; FBPA;E.C. 4.1.2.13) catalyses the reversible cleavage of fructose–1,6-bisphosphate (FBP) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde–3-phosphate (GAP)in the glycolytic pathway of prokaryotic and eukaryotic organisms. FBPA is an essential enzyme for the reversible gluconeogenesis and the fructose metabolic pathways [1,2,3,4,5,6,7,8,9,10]. Aldolases are classified as Class I and Class II aldolases depending on their requirement of divalent ions in catalysis. For their catalytic activity, Class I Aldolases do not depend on cations, and they found in higher animals and plants. Class II aldolases are homodimers (80 kDa), found in bacteria and fungi, and they require a metal cofactor such as Zn+2 for catalysis [1,2,3,7,8,9,10,11,12,13]

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