Isolation of saline’s bacteria:

The isolated bacteria from salt works, were cultivated in medium M2: NaCl, 98 g; KCl, 2 g; MgSO₄, 7H₂O, 1 g; CaCl₂, 2H₂O, 0.36 g; NaHCO₃, 0.06 g; NaBr, 0.24 g; FeCl₃, 6H₂O, 1 g; Bactotryptone (Difco), 10 g; glucose, 1 g; agar, 20 g (per liter).

The pH was adjusted to 7.4. The plates were incubated at 37˚C aerobically in a salt saturated atmosphere. The isolated bacterial strains were cultivated and identified.

In the present study the purification of the GAPDH was done with the Idiomarina loihiensis which solubilizes the inorganic phosphate in the NBRIP medium. Idiomarina loihiensis was cultivated in medium LuriaBertani (LB) with NaCl and the enzyme was purified using traditional procedure [10,11] modified according to our conditions. All purification, steps were carried out at 4˚C.

Liquid culture cells were harvested by centrifugation at 15,000× g for 20 min at 4˚C. Cell pellets were washed twice in 25 mM Tris-HCl (pH 7.5) and resuspended in the same buffer supplemented with 2 mM EDTA and 10 mM β-mercaptoethanol). Cells were then disrupted by ultrasonic treatment in a chilled water bath using a Branson 25U sonifier at medium strength. The resulting suspension was centrifuged at 15,000× g for 45 min to obtain the cell-free extract.

Phosphorylating NAD⁺-dependent GAPDH activity was measured as described by Heina and Freimuller [12]. The reaction was started by the addition of 10 μg of cell-free extract to an assay mixture containing 50 mM Tricine buffer (pH 8.5), 1 mM NAD⁺, inorganic phosphate or arsenate (10 mM) in the reaction solution and 2 mM D-glyceraldehyde-3-phosphate at 25˚C. Absorbance at 340 nm was measured in a spectrophotometer (model BioMate 3, Thermo Electron Corporation, Madison WI 53711, USA).

The crude extract was subjected to protein precipitation in the 60% - 88% (W/V) saturation range of ammonium sulphate. The final pellet was dissolved in a minimal volume of 25 mM Tris-HCl buffer (pH 7.5), containing 2 mM EDTA and 10 mM 2β-mercaptoethanol (buffer A). The protein solution was dialyzed twice against 2 l of the same buffer overnight.

The dialyzed enzyme preparation was applied to a Blue Sepharose CL-6B column (1 cm × 10 cm) equilibrated with 100 ml buffer A (approximately 10 times bed volumes).

The column was washed with 10 bed volumes of buffer A and 5 bed volumes of the same buffer adjusted to pH 9.5 (buffer B).

The enzyme was eluted with buffer B containing 10 mM NAD⁺ at a flow rate of 10 ml/h. Active fractions were collected, pooled and preserved in 50% glycerol (V/V) at −20˚C.

During enzyme purification, a coupled assay in which aldolase (1 U/ml) produced the stoichiometric breakage of D-fructose 1 - 6 biphosphate (2 mM) to dihydroxyacetone-phosphate and D-G3P the actual substrate of the oxidative reaction [13], was used.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli [14] on 12% polyacrylamide slab gels containing 0.1% SDS. Gels were run on a miniature vertical slab gel unit (Hoefer Scientific Instruments, San Francisco, USA). After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 at 0.2% (W/V) in the mixture of methanol/acetic acid/water (4:1:5, V/V/V) for 30 min at room temperature.

The apparent subunit molecular weight was determined by measuring and comparing relative mobility of the following pre-stained SDS-PAGE molecular weight standards (Broad Range Protein Molecular Weight Markers; Promega).

Determination of native molecular weight was carried out by electrophoresis on non-denaturing polyacrylamide slab gels (Bio-Rad, Hercules, USA) using the following protein standards: Apoferritin (443 kDa); Amylase (200 kDa); Alcohol dehydrogenase (150 kDa); Monomeric BSA (66 kDa); Carbonic anhydrase (29 kDa); and Lactalbumin (14.2 kDa). As described by the method of Hedrick and Smith [15], a calibration curve can be calculated from the relative mobility of standard proteins on non-denaturing polyacrylamide gels with different acrylamide concentrations (6%, 8%, 10%, and 12%, W/V).

By constructing the Ferguson plot [Log(Rf × 100) versus the concentration of polyacrylamide gels (%)], the resulting slopes versus the known molecular weights of standard native proteins allowed the determination of the native molecular weight of purified GAPDH.

Polyclonal antiserum was raised in New Zealand White rabbits to GAPDH that had been purified from Idiomarina loihiensis. The purified protein (approximately 0.5 mg) was mixed 1:1 with Freund’s complete adjuvant. After 21 days, a sample of blood was collected, and a second dose of 500 µg of the protein was injected into rabbits in multiple places, as described by Vaitukaitis [16]. After one week, 50 ml of rabbit blood was collected and serum was separated after its coagulation overnight at 4˚C then its centrifugation. The obtained serum, containing monospecific anti-GAPDH polyclonal antibodies, was sampled and stored at −20˚C.

Proteins were separated by SDS-PAGE as described previously. Separated protein bands were electrophoretically transferred from the slab gel to a nitrocellulose membrane (Schleicher and Schuell, Keene, USA) using a BioRad Trans-Blot system. The transferred proteins were then visualized by pre-staining in 0.2% (W/V) Ponceau red in trichloroacetic acid.

The nitrocellulose membrane was then incubated for 1 h in blocking solution containing 5% (W/V) non-fat dry milk, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.01% (W/V) NaN₃, and 0.05% (V/V) Tween-20, followed by incubation with the anti-GAPDH antiserum (1:1000 dilution) as a primary antibody. Western blots were visualized by coupled immunoreaction with peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:1000; Boehringer Mannheim, Hamburg, Germany) as a secondary antibody using 4-chloro-1-naphthol as a chromogenic substrate.

For kinetic studies, initial velocities of the enzymatic reaction were carried out by varying the concentration of the substrates, D-G3P (from 0.04 to 10 mM) or NAD⁺ (from 0.02 to 2 mM). Values of the Michaelis constants (Km), dissociation constants (KD), and maximal velocity (V_max) were calculated according to the method of Cleland [17]. One unit of enzyme activity was defined as the amount of enzyme that catalyses the formation of 1 μmol NADH/min under the conditions used. Protein concentrations were estimated by the method of Bradford [18] using bovine serum albumin (BSA) as a standard. Activity levels in cell-free extracts were expressed as specific activity (U/mg protein).

To determine optimal pH, enzymatic activity was measured over a wide range of pH values (4 - 10), using a mixture of different buffers with different pKa (Tris, MES, HEPES, potassium phosphate at 50 mM, and sodium acetate at 180 mM) adjusted to the same ionic strength than the standard reaction mixture. Temperature effects were characterized by activation and denaturation processes. For activation, the tricine-NaOH buffer (50 mM; pH 8.5) was incubated for 10 min at temperatures from 20˚C to 80˚C using a thermostated cuvette holder connected to a refrigerated bath circulator. Then 2 mM NAD⁺, 200 mM sodium arsenate, and 10 μg purified GAPDH were added to the mixture. The reaction was started immediately by addition of 10 mM D-G3P.

For the denaturation, 10 μg of the purified GAPDH were incubated at temperatures from 20˚C to 80˚C for 10 min in the 50 mM tricine-NaOH buffer. Then 2 mM NAD⁺ and 200 mM sodium arsenate were added. The enzymatic activities were measured after 2 min incubation at 25˚C immediately started by addition of 10 mM D-G3P.

GAPDH was purified from a soluble protein fraction of the saline strain Idiomarina loihiensis crude extract, using a simple procedure involving only one chromatography step, namely dye affinity chromatography on Blue Sepharose. Table 1 summarizes a representative purification protocol. Approximately 1.8 U/mg proteins were obtained for the specific activity of the purified enzyme with a yield of 32.5% and an approximately 6-fold increase of purification.

SDS-PAGE analysis of the different fractions obtained during the purification procedure showed a progressive enrichment of a 36 kDa protein [Figure 1]. Only this protein band, with the same size as the GAPDH subunit

Table 1. Purification of GAPDH from Idiomarina loihiensis.

monomer, was evidenced in the electrophoretically homogeneous final enzyme preparation [Figure 1(a), Lane 4].

Non-denaturing PAGE showed that the native molecular weight of the isolated protein was approximately of 147 kDa [Figure 2].

Values of the Michaelis constants (Km), dissociation constants (KD) and maximal velocity for the oxidation of G3P and the reduction of NAD⁺ by the GAPDH were calculated. The KmNAD⁺, KmG3P and KDNAD⁺ values were estimated and showed a significant difference (Table 2). The V_max of the purified protein was estimated to be 2.06 U/mg (Table 2). In comparison with the GAPDH from human erythrocytes, the KmNAD⁺, KmG3P and V_max values were 17.8 mM, 205 mM and 4.3 U/mg respectively [19] and for Tetrahymena pyriformis, they were 5 mM, 150 mM and 5.6 U/mg respectively [20] and for Pleurodeles waltl, they were 60 mM, 27 mM and 33.2 U/mg [21].

Pre-incubation of Idiomarina GAPDH for 10 min at

Table 2. Kinetic constants (Km and V_max) for oxidation reaction of GAPDH from Idiomarina loihiensis.

temperatures varying between 15˚C and 45˚C did not irreversibly affect the enzyme activity. However, thermal inactivation occurred above 50˚C and even at 80˚C, but no total activity loss was noted [Figure 3(a)]. Studies on the effect of temperature on enzyme activity revealed an optimal value around 45˚C.

The pH activity profile of purified GAPDH was determined in pH range from 4 to 10; the maximum of relative enzymatic activity is observed between pH 8 and 8.5 (Figure 3(b)).