Aldolase B Protein, Human (GST)

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) 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.

The specific activity is 8.915 U/mg. One unit will convert 1.0 µmol of fructose 1,6-diphosphate to dihydroxyacetone phosphate and glyceraldehydes 3- phosphate per minute at pH 7.5 at 37°C in the presence.

Materials
Assay buffer: 50 mM Tris-HCl (containing 0.1 mM EDTA), pH 7.4
Substrate solution: 50 mM sodium fructose-1,6-bisphosphate (prepared using assay buffer)
Coenzyme solution: 10 mM β-NADH (prepared fresh with ice-cold ultrapure water)
Coupling enzyme complex: α-Glycerophosphate Dehydrogenase-Triose Phosphate Isomerase (α-GDH-TPI)
Aldolase B Protein, Human (GST) (HY-P74419A)

Procedure
1. Preparation of reaction working solution (per well, total reaction volume 172 μL; prepare an additional 10% volume to compensate for losses)
Component — Volume per well (μL) — Final concentration
Fructose-1,6-bisphosphate (50 mM)——20 μL——50 mM
NADH (10 mM) —— 4 μL —— 0.2 mM
α-GDH-TPI (100 U/mL) —— 2 μL —— 1 U/mL
Assay buffer —— 146 μL—— /
2. Add reaction working solution: Add 172 μL of reaction working solution to each detection well of the 96-well UV plate.
3. Pre-incubation: Place the 96-well UV plate in a pre-warmed microplate reader set to 37°C and incubate for 5-10 minutes to equilibrate the system temperature.
4. Add assay protein: Resuspend protein in sterile water to 200 μg/mL. Prepare 10 μg/mL Aldolase B protein in assay buffer. Quickly add 28 μL of each sample to the wells. Use assay buffer without protein for the control group.
5. Kinetic Monitoring: Immediately return the plate to the microplate reader and initiate absorbance monitoring at 340 nm. Read every 30 seconds for 10 minutes.
6. Calculate Enzyme Activity:
Enzyme activity definition: One enzyme activity unit (U) is defined as the consumption of 1 μmol of NADH per minute.
Enzyme Activity Formula:

Human

E. coli

N-GST

P05062 (A2-Y364)

229

Full Length of Mature Protein

AHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEENRRQFREILFSVDSSINQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLDQGGAPLAGTNKETTIQGLDGLSERCAQYKKDGVDFGKWRAVLRIADQCPSSLAIQENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDHHVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGMSEEDATLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMKRAMANCQAAKGQYVHTGSSGAASTQSLFTACYTY

Approximately 65 KDa

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

Lyophilized powder

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4 or PBS, pH 7.4, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.