Research Communication

A Novel D-Glyceraldehyde-3-phosphate Binding Protein, a TruncatedAlbumin, with D-Glyceraldehyde-3-phosphate DehydrogenaseInhibitory Property

Amrita Roy, Soumen Bera, Subrata Patra, Subhankar Ray and Manju Ray*Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata, India

Summary

We have purified a novel protein from mice muscle, whichthrough N-terminal amino acid sequencing was identified as atruncated form of mouse albumin. The protein was found to bea monomer of �64 kDa and located in the cytosol. The purifiedprotein strongly crossreacted with commercial albumin anti-body. Presence of this protein was observed in different mouseorgans. Further biochemical studies as well as CD spectroscopyindicated that the protein binds D-glyceraldehyde-3-phosphatelimiting the availability of the substrate to the enzyme D-glycer-aldehyde-3-phosphate dehydrogenase, thereby inhibiting its cat-alytic activity. The implication of this protein in the control ofglycolysis has been discussed. � 2009 IUBMB

IUBMB Life, 61(10): 995–1000, 2009

Keywords D-glyceraldehyde-3-phosphate dehydrogenase inhibition;

albumin; D-glyceraldehyde-3-phosphate binding protein;

CD spectroscopy.

Abbreviations GAPDH, D-glyceraldehyde-3-phosphate dehydrogen-

ase; GAP, D-glyceraldehyde-3-phosphate; GBP, D-glyceraldehyde-3-

phosphate-binding protein; NP buffer, sodium phosphate buffer; TPI,

triosephosphate isomerase; MSA, mouse serum albumin.

INTRODUCTION

D-Glycearldehyde-3-phosphate dehydrogenase (GAPDH; EC

1.2.1.12) is a glycolytic enzyme converting D-glyceraldehyde-3-

phosphate (GAP) to 1,3-diphosphoglycerate requiring NAD and

inorganic phosphate (Pi) as cosubstrates. Apart from its glyco-

lytic function, it is involved in phosphorylating transverse

tubule, RNA transcription and catalysis, DNA replication and

repair and apoptosis. Its involvement is also indicated in various

neurological diseases and upregulation of GAPDH is also found

to be associated with development of malignancy (1). GAPDH

from tumor sources is reported to be selectively inhibited

by methylglyoxal suggesting a critical alteration in these cells

(2–5). During the study of GAPDH of normal and malignant

cells, we have identified a protein in normal mice muscle, here-

inafter referred as GAP binding protein (GBP), which exhibits a

novel property of inhibiting the enzymatic activity of GAPDH

by interacting with GAP. On characterization, this protein was

found to be a truncated albumin.

MATERIALS

All biochemicals were from Sigma-Aldrich (St. Louis, MO).

Prestained SDS–PAGE marker, all monoclonal primary and

suitable secondary antibodies and Western blotting Luminol

reagent kit were from Santa Cruz (Santa Cruz, CA). Sephadex

G-100 was from Pharmacia Fine Chemicals (Uppsala Sweden).

Centricon membrane filters were from Millipore Corporation

(Billerica, MA). All other reagents were of analytical grade and

from local manufacturers.

Calcium phosphate (CP) gel was prepared by reacting

CaCl2�2H2O (0.7 g/100 mL) and Na3PO4�12H2O (0.7 g/100

mL) and washing the precipitate several times in water (6).

Animal experiments were carried out following guidelines of

institutional ethics committee for animal experiments.

METHODS

Enzyme Assays

The final volume of all enzyme assay medium was kept

1 mL.

Assay medium of GAPDH contained 40 lM GAP, 0.5 mM

NAD, 50 mM Triethanolamine–HCl buffer pH 8.6, 0.2 mM

EDTA, and 50 mM Na2HPO4 (3). After 1 min of preincubation

reaction was started by addition of the requisite amount of

enzyme giving DA 5 0.03–0.04/min at 340 nm (7). One unit

(U) of enzyme activity is defined as the amount of enzyme that

Address correspondence to: Manju Ray, Department of Biological

Chemistry, Indian Association for the Cultivation of Science, Jadavpur,

Kolkata 700 032, India. Tel: 191-33-2499-0282. Fax: 191-33-2473-

2805. E-mail: bcmr@iacs.res.in

Received 20 May 2009; accepted 2 June 2009

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.238

IUBMB Life, 61(10): 995–1000, October 2009

can convert 1 lmol of substrate into 1 lmol of product per min

under standard assay conditions.

For the assay of GBP, the protein was preincubated in a

standard assay medium of GAPDH and after 1 min, the reaction

was initiated by addition of enzyme.

The esterase activity was assayed in 250 mM sodium phos-

phate (NP) buffer pH 7.4 with 50 lM of p-nitrophenyl acetate.

Formation of p-nitrophenol was monitored at 400 nm. Specific

esterase activity was calculated using EmM 5 14.9 3 103 (8).

The triosephosphate isomerase activity was assayed in 50

mM Triethanolamine-HCl buffer pH 8.6 containing 0.2 mM

EDTA and 50 mM Na2HPO4 with 40 lM GAP 100 lM NADH

and a-glycerophosphate dehydrogenase (giving DA 5 0.15/min

at 340 nm). The reaction was monitored at 340 nm (9).

Purification of GBP

Skeletal muscles of Swiss albino mice were collected,

washed thoroughly with 20 mM NP buffer pH 7.4 (hereinafter

buffer A), minced and centrifuged in 5 volumes of buffer A at

100 g for 5 min to remove any trace of blood. The pellet was

homogenized with 6 volumes of prechilled acetone in Omni

GLH homogenizer, sucked dry in Buckner funnel and resulting

protein precipitate was air dried overnight to remove excess ac-

etone. Dried acetone powder was stirred in 4 volumes of buffer

A for 90 min and centrifuged at 23000 g for 20 min. The super-

natant or ‘‘crude extract’’ was subjected to ammonium sulfate

fractionation. Protein precipitated at 50–90% saturation of am-

monium sulfate was subjected to gel filtration on Sephadex G-

100 column previously equilibrated with buffer A. The most

active fractions without traces of GAPDH were pooled and con-

centrated by ultra-filtration through Centricon-30 membrane fil-

ter. Concentrated GBP was applied to CP gel (1 mg protein/44

mg dry weight of CP gel). Adsorbed GBP was eluted by wash-

ing the gel with 25 mM NP buffer pH 7.4 and was again sub-

jected to CP gel treatment in a similar manner, 25 mM NP

buffer washed fraction after second CP gel contained purified

GBP.

The homogeneity of the protein preparation was checked by

PAGE (7.5% matrix) according to the method of Davis (10).

Molecular Weight Determination

Molecular weight was determined by gel filtration in a

Sephadex G-100 column using BSA, Ovalbumin, and Cyto-

chrome c as reference molecular weight markers. Subunit mo-

lecular weight was determined by SDS–PAGE (10% matrix)

according to the method of Laemmli (11).

Effect of GAP, NAD, and Pi on GAPDH Inhibition

The effect of GAP, NAD, and Pi on the inhibition was deter-

mined by increasing their concentration in the assay medium.

Partial N-Terminal Amino Acid Sequencing of GBP

Amino acid sequencing of purified GBP was determined by

using Model 491Procise protein/peptide sequencer from Applied

Biosystems.

Immunoblotting

Samples were subjected to 10% SDS–PAGE followed by

immunoblot analysis. Dilutions of primary and secondary anti-

bodies were 1:500 and 1:1000 respectively, and blots were

developed using Western blotting Luminol reagent kit.

Subcellular Localization and Organ Distribution

Subcellular fractions were prepared according to the method

of Cox and Emili (12). Crude extract of mice liver, brain, kid-

ney, and muscle was prepared by homogenizing the tissue in

Omni GLH homogenizer in 4 volumes of buffer A, centrifuging

at 23000 g for 10 min, and collecting the clear supernatant.

Presence of GBP in different samples was detected by immuno-

blotting with monoclonal mouse anti-albumin IgG.

CD Spectroscopy

Ten microgram GBP (90% pure after 1st CP treatment) and

120 lM GAP were incubated in 20 mM NP buffer pH 7.8 and

scanned after 0, 5, 10, 15, 30, 45, and 60 min without disturb-

ing the system in a Jasco CD Spectrophotometer J815. To as-

certain the specificity of the interaction, GBP was selectively

denatured after formation of GBP–GAP complex. For this, GBP

and GAP were incubated and after attaining initial saturation

level at 20 min, the system was gradually heated using a Peltier

setup from initial temperature of 25–90 8C over a period of

90 min. Control set was kept at 25 8C for the same time span.

Interaction between 10 lg MSA and 120 lM GAP was studied

similarly.

Retrieval of GAP

GAP (350 lM) was incubated with GBP in 50 mM NP

buffer pH 8.0 at room temperature for 20 min and the complex

was then denatured by adding 1 mL of diethyl ether. After

evaporating the ether, the retrieved GAP was enzymatically esti-

mated and calculated with EmM 5 6.22 (7). The GBP fraction

used contained no TPI activity and yielded �50% inhibition in

standard assays.

Determination of Kd

To determine the Kd �1 lg of GBP with no TPI contamina-

tion and yielding �50% inhibition in a standard assay was incu-

bated for 20 min with increasing concentration of GAP in 50

mM NP buffer pH 8.0. The bound and unbound GAP was then

separated by ultrafiltration followed by enzymatic estimation of

the free GAP. The Kd of MSA–GAP interaction was similarly

determined with �2 lg of MSA. The negative X-axis intercept

of [RL]/[L] versus [RL] plot gave the value of 21/Kd where

996 ROY ET AL.

[RL] is the amount of GBP 3 the concentration of bound GAP

and [L] is the concentration of the free GAP (13).

pH Optima

Inhibitory activity of GBP as a function of pH was investi-

gated in TEA-HCl buffer pH 7.5–9.0.

Protein Estimation

Using BSA as standard protein was estimated by methods of

either Lowry et al. or Warburg and Christian as outlined by

Layne (14).

RESULTS

Purification

The purification process was reproducible in several batches

of mice muscle. Both PAGE and SDS–PAGE showed a single

band of protein (Fig. 1). Thus, GBP was found to be a �64,000

(determined by gel filtration chromatography) monomer (by

SDS–PAGE).

Because of the presence of high GAPDH and TPI activity in

crude and ammonium sulfate fraction quantification of GBP

was not possible. Inactivating the GAPDH activity of those

fractions by p-HMB or iodoacetate (15) and thereby quantifying

GBP remained unsuccessful as GAPDH required for assaying

GBP was also inhibited. As TPI could only be removed after

the second CP gel treatment, it was difficult to precisely deter-

mine the amount of inhibition rendered by GBP and hence a

typical purification table could not be designed.

Inhibition of GAPDH by GBP

GBP was found to strongly inhibit GAPDH activity. After

final purification step, �0.02 lg of GBP was found to elicit

almost 35% inhibition of GAPDH in a standard assay medium

(Fig. 2A). The GBP preparation was free from TPI contamina-

tion, which could limit GAP availability to GAPDH thus sup-

pressing its catalytic activity.

Effect of GAP, NAD, and Pi on the Inhibition

The inhibitory activity of GBP was found to be dependent

on the concentration of GAP as well as on the preincubation

time of the assay system. The inhibition was reduced to �20%

as the concentration of GAP was increased to 640 lM, while it

increased from �35 to 100% as the preincubation time

increased from 0 to 12 min (Fig. 2B). NAD and Pi had no

Figure 1. Purified GBP in PAGE (A) and SDS–PAGE (B). In

both (A) and (B) a-standard marker [in (A) BSA; (B) prestained

markers, numbers indicating molecular mass in kDa], b-GBP

(10 lg) after Sepahdex G-100, and c-purified GBP (5 lg).

Figure 2. GAPDH inhibition by GBP (A). Effect of GAP and preincubation time on GAPDH inhibition by GBP (B). [Color figure

can be viewed in the online issue, which is available at www.interscience.wiley.com.]

997GBP WITH GAPDH INHIBITORY PROPERTY

effects even at concentrations of 0.5 and 70 mM, respectively.

These results indicated that GBP interacted only with GAP and

reducing the availability of the metabolite brought about the in-

hibition of GAPDH.

N-Terminal Amino Acid Sequencing

The first 17 N-terminal amino acids of GBP were sequenced

as EAHKSEIAQRYNDLGEQ. The pBLAST of NCBI retrieved

�100% homology with 25th–42nd residues of mouse albumin.

From this result, GBP seems to be a truncated form of albumin

whose existence has not been previously reported.

As GBP appeared to as a truncated form of mouse albumin

it was checked for other characters of albumin such as presence

of esterase activity and was immunoblotted with monoclonal

anti-albumin IgG.

Esterase Activity of GBP

Similar to MSA, GBP was found to hydrolyze p-nitrophenyl

acetate. The specific esterase activity (units/mg) of GBP was

122.8 3 1024 (62.03) while that of MSA was 6.98 3 1024

(61.7). The relatively high specific activity of esterase by GBP

may be attributed to the presence of alterations in the GBP mol-

ecule or low specific activity of MSA in different batches of

commercial preparations.

Immunoblotting

Similar to MSA, purified GBP strongly crossreacted with

monoclonal anti-albumin IgG proving significant homology of

GBP with mouse albumin. Significant immunoreactivity was

also detected at different stages of GBP purification including

the final one (Fig. 3A).

Subcellular Localization and Organ Distribution

Because of the very high activities of both GAPDH and TPI

in the subcellular fractions and organ homogenates quantifica-

tion of GBP by its GAPDH inhibitory property was not possi-

ble. As albumin is known not to be present in intracellular loca-

tion except in some cancerous tissues (16), we could reasonably

assume that the antibody reacted with GBP and not with albu-

min. So, despite its limitations, GBP was traced by immuno-

blotting.

Presence of GBP was observed in cytosol only. a-Tubulinwas used as a positive marker for cytosolic fraction in immuno-

blot (Fig. 3B). All the mice organs tested showed the presence

of GBP (Fig. 3C).

CD Spectroscopic Determination ofGBP–GAP Interaction

The GBP–GAP interaction was visualized by CD spectros-

copy. At 250–200 nm wavelength, light interacts with the sec-

ondary structure of protein while at 300–250 nm it interacts

with the tertiary structure. In our experiments, the deflection of

the spectrum at 300–250 nm clearly indicated a strong interac-

tion between GBP and GAP involving the tertiary structure.

The interaction reached maximum within 15 min where it

remained steady for more than 1 h (data not shown). As GBP–

GAP complex was slowly heated, the intensity of interaction

slowly increased with temperature till 50 8C, which may be due

to nonspecific unfolding of the protein structure. Beyond 50 8C,secondary structure of GBP started to disintegrate telling upon

the binding of GAP and GBP. At 90 8C, the denaturation of

GBP was complete with the loss of its binding capacity indicat-

ing the interaction is due to the protein, thus confirming the

specificity of GBP–GAP interaction (Fig. 4).

Similar CD spectrophotometric study with MSA and GAP

showed no deflection of the spectrum in the region of 300–250

nm indicating that MSA did not interact with GAP while GBP

did.

Retrieval of GAP

To determine whether GBP converts GAP to other product(s)

or simply binds it, residual GAP was enzymatically estimated

following denaturation of GBP–GAP complex with ether. It was

observed after GBP denaturation 100% GAP was efficiently

retrieved whereas without diethyl ether treatment only 35% of

the added GAP could be estimated indicating that GAP bound

to GBP as its ligand and no catalytic conversion took place

upon binding.

Kd

The Kd of the GBP–GAP interaction was found to be 30 lMfor GBP while it was 2 lM for MSA.

Figure 3. Western blot analysis of GBP. (A) GBP at different

purification stages. a-Crude extract, b-Ammonium sulfate frac-

tion, c-Sephadex G-100 eluent, and d-purified GBP. (B) Subcel-

lular localization of GBP. Immunoreaction of different fractions

with (i) mouse albumin antibody, (ii) with a-tubulin antibody,

and (iii) loading control of the subcellular fractions. In all cases,

a-commercial MSA, b-whole mouse serum, c-cytosol, d-micro-

some, e-mitochondria, and f-nucleus. (C) Organ distribution of

GBP. a-liver, b-muscle, c-brain, and d-kidney.

998 ROY ET AL.

pH Optima

The optimum inhibition was found at pH 8.7 with sharp

decreases above pH 9.0 and below 8.0. GAPDH assay without

GBP at different pH acted as control.

DISCUSSION

We report here the presence of a novel protein in mice mus-

cle, a truncated form of albumin, which by binding with GAP

inhibited the catalytic activity of GAPDH. It is generally

assumed that although an important enzyme GAPDH is not a

control point of the glycolytic pathway. However, despite its

multifunctional role (1) GAPDH could not perform some of its

functions simultaneously (17). So despite occupying a signifi-

cant portion of total cellular protein, at any point of time, only

a small fraction of total GAPDH molecule remains available for

glycolysis. It had also been suggested that activity of GAPDH

in cells is controlled by various factors such as intracellular pH

and ATP concentration (3, 18). Till date, only Band 3 protein

of erythrocyte (19) and very recently another protein named

FKBP36 present in human testes (20) are reported to inhibit

GAPDH. However, to our knowledge, no protein is reported to

inhibit GAPDH activity by sequestering GAP. The present work

shows that by increasing the concentration of GAP, the inhibi-

tion could be significantly relieved. These findings suggest that

GBP could possibly control the substrate availability for

GAPDH and might play a role in controlling glycolysis. How-

ever, it would be interesting to identify precisely the possible

intracellular conditions in which GAP might get bound to or

released from GBP. Whether or not GBP has any role in control

of glycolysis, its novelty is unquestionable.

Our studies indicated that GBP shares many characteristics

with mouse albumin like electrophoretic mobility, molecular

weight, homologous amino acid sequence, epitope structure, and

esterase activity, but lacks the first 24 amino acids of albumin. An

important property of albumin is the ability to bind various

ligands like free fatty acids, steroid hormones, copper, calcium,

and so forth (21). This is reflected in the ability of GBP to bind

GAP. We also observed that both BSA and MSA could not inhibit

GAPDH. It might be that the absence of 24 amino acids from N-

terminal end altered the three-dimensional structure of albumin

rendering this gain of function. A crystallographic study might be

useful to throw some light in this regard. Besides serum albumin,

several forms of albumin like ovalalbumin and albumin of some

plant seeds act as storage proteins. This newly discovered GBP

might be another such variant of albumin.

Figure 4. CD Spectrophotometric studies of GBP–GAP and

MSA–GBP interaction. (A) Interaction 25–50 8C. (B) Interaction50–90 8C. a–GBP only, b, c, d, and e-interaction at 25 8C, 50 8C,55 8C, and 90 8C, respectively. (Inset: magnification of 300–240

nm region). (C) Interaction between MSA and GAP at 25 8C.[Color figure can be viewed in the online issue, which is avail-

able at www.interscience.wiley.com.]

999GBP WITH GAPDH INHIBITORY PROPERTY

Albumin is initially synthesized in liver as a preproprotein.

The signal peptide and a hexapeptide at the N-terminal end are

cleaved off along the secretory pathway. There are also reports

of albumin mRNA synthesis in extra hepatic tissues (22). It

would be interesting to study the mechanism of the formation

of this truncated albumin and also its species distribution.

ACKNOWLEDGEMENT

This work was sponsored by grants from Council for Scientific

and Industrial Research, New Delhi, India.

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