Aldolase B Protein, Human (GST, solution)

Aldolase B protein catalyzes fructose 1,6-bisphosphate to trisaccharide phosphate during glycolysis and performs reverse aldol addition during gluconeogenesis. Aldolase B Protein, Human (GST, solution) is the recombinant human-derived Aldolase B protein, expressed by E. coli , with N-GST labeled tag.

Aldolase B protein serves a pivotal role in glycolysis by catalyzing the aldol cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, as well as the reverse stereospecific aldol addition reaction in gluconeogenesis. Additionally, in fructolysis, it metabolizes fructose 1-phosphate, derived from the phosphorylation of dietary fructose by fructokinase, into dihydroxyacetone phosphate and D-glyceraldehyde. Beyond its enzymatic function, Aldolase B acts as an adapter and functions as a tumor suppressor by stabilizing a ternary complex with G6PD and TP53, inhibiting G6PD activity. This regulatory mechanism helps maintain control over oxidative pentose phosphate metabolism, contributing to the overall balance of cellular metabolic pathways.

Materials
Substrate: Accurately weigh 16.24 mg of substrate powder and dissolve in 1 mL of deionized water to achieve a concentration of 40 mM.
Aldolase B Protein, Human (GST, solution) (HY-P74419)
NADH: Accurately weigh 5.672 mg of solid NADH powder and dissolve in 1 mL of deionized water to achieve a concentration of 8 mM.

Procedure
1. Serially dilute NADH to the following concentrations: 0, 0.0150625, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 mM. Add 200 μL per well to a 96-well plate with three replicates.
2. Measure the absorbance at 340 nm using a microplate reader. Plot a standard curve with the amount of NADH as the abscissa and OD value as the ordinate.
3. Dilute the substrate to 40 mM and set aside.
4. Mix 50 μL of 40 mM substrate with 50 μL of 1 mM NADH, add to the 96-well plate in advance, and set three replicates.
5. Dilute Human Aldolase B to 0.01 mg/mL. Initiate the reaction by adding 100 μL per well.
6. Zero the microplate reader using the OD value of the blank group, and read the absorbance at 340 nm for 10 min in kinetic mode.
7. Define one enzyme activity unit (U) as the amount that consumes 1 μmol of NADH per minute. Calculate the specific enzyme activity:

Human

E. coli

N-GST

P05062 (A2-Y364)

229  [NCBI]

Full Length of Mature Protein

Approximately 60 KDa

• 
  Greater than 90% as determined by reducing SDS-PAGE.

<1 EU/μg, determined by LAL method.