Native gel Sds Page

Figure 2. Purification procedures used for isolating Asp-NAT from rat brain.

2.1 Anion-exchange chromatography

Bio-Rad macro-prep DEAE cellulose resin (~100 ml in bed volume) was useful as an anion exchange matrix to separate the Asp-NAT from the bulk of the proteins. Asp-NAT was bound to the resin on mixing the resin with the solubilized enzyme, and chromatography was performed as follows. The column was washed sequentially in two steps: 1) with 10 column volumes of equilibrating buffer (EB) (0.15 M NaCl, 10 mM sodium phosphate buffer pH 7.1, 1 mM CHAPS, 1 mM DTT and 10 % glycerol), and 2) with 5 column volumes of EB with 0.25 M NaCl. The enzyme activity was eluted with the EB having 0.5 M NaCl in about 6 fractions of 45 ml each.

DEAE cellulose facilitated binding of ~80% of the enzyme activity and yielded ~90% of the bound enzyme activity on elution. This method enriched the enzyme activity by about 4-5 fold (activity increased from 5-6 nmol/h/mg protein to 24-26 nmol/h/mg protein). Fractions 2 and 3 containing the bulk of the enzyme activity were pooled, concentrated and applied to the native gel electrophoresis system.

2.2 Native gel electrophoresis

The Bio-Rad Model 491 Prep Cell was the apparatus of choice for gel electrophoresis purification of Asp-NAT after DEAE ion exchange chromatography, and was carried out by continuous flow electrophoresis under native conditions. Continuous buffer of Tris-Glycine-HCl of pH 8.0—8.5 was used in both the upper and lower chamber. Concentrations of Tris and Glycine were in the range of 6—25 mM and 60—192 mM, respectively. Asp-NAT activity was consistently detected in eluates after 21 hours when the electrophoresis buffer pH was 8.0. In order to retain the enzyme activity this buffer also consisted of 6% glycerol, 1 mM CHAPS and 1 mM DTT. Elution of Asp-NAT was carried out with a buffer consisting of PBS, 10% glycerol, 1 mM CHAPS, 1 mM DTT, pH adjusted to 7.2. Resolving gel (6%) of 3 cm height and stacking gel (3%) of 3 cm height were cast overnight according to the manufacturer's instructions using 30% acrylamide/Bis, 37.5:1 (2.6% C) stock solutions. Adding 10% glycerol, 1 mM CHAPS and Tris-Glycine-HCl buffer of pH 8.0 in the casting gel solution protected the enzyme activity. The entire electro-elution was carried out in a cold room (2-4 oC) at a constant power of 5W. About 2 hours of pre-electrophoresis was carried out before loading the protein sample, to remove the unreacted catalysts from the polyacrylamide gel. Sample protein (~6-8 ml) was dialysed against 2 liters of a medium containing 10 mM sodium phosphate pH 7.2, 10% glycerol, 1 mM CHAPS and 1 mM DTT before loading on to the native gel column, which retained the activity of the enzyme at the same level as opposed to dialysing in the electrophoresis buffer (Tris-HCl-Glycine, pH 8.0), which resulted in a loss of activity by ~ 20-25%. At the elution rate of 0.3 ml/min, about 250 fractions of 5 ml were collected. Every fourth fraction was assayed using the radiometric method ([14C] L-Asp, 100 mCi/mmol) after concentrating 2 ml into ~0.25 ml (Centricon YM 10 concentrator).

Asp-NAT was also purified from the supernatant obtained after separating the crude mitochondrial pellet (26,000 x g, 1h). When a large number of rat brains (>100) are homogenized using domestic blenders there is an almost equal distribution of Asp-NAT activity in the supernatant and in the crude mitochondrial pellet. Interestingly, Asp-NAT in the soluble fraction (high-speed supernatant fraction) was found to be identical in substrate specificity and molecular weight to that obtained from solubilization of the crude mitochondrial pellet. A possible explanation would be that some of the Asp-NAT that is loosely held inside the mitochondrial membranes will be released into the soluble fraction on homogenization due to partial disruption of mitochondria. This explanation is consistent with the observation that most of the Asp-NAT activity is associated with the crude mitochondrial pellet on gentle homogenization that is used to prepare tightly coupled mitochondria.12 However, a recent report suggests occurrence of Asp-NAT in the microsomal fraction as well.33 Although, identical steps of purification were followed to purify Asp-NAT from the supernatant fraction and the CHAPS solubilized fraction of the crude mitochondrial pellet, some adjustments were made in the native gel electrophoresis to achieve maximal purity and recovery of enzyme activity. A comparison of the salient features of native gel electrophoresis is given in Table 1.

Table 1. Comparison of the purification of Asp-NAT from the crude mitochondrial pellet vs. the supernatant fraction after high-speed centrifugation.

Feature

Asp-NAT from pellet

Asp-NAT supernatant

(a) Source of Asp-NAT

Crude mitochondrial pellet (26,000 g), solubilized with 10 mM CHAPS

Supernatant after high-speed centrifugation (26,000 g) without CHAPS solubilization

(b) CHAPS

1mM CHAPS included in all buffers

CHAPS excluded

(c) DEAE anion exchange chromatography

Elutes with 0.5 M NaCl buffer

Elutes with 0.25 M NaCl and 0.5 M NaCl buffers

(d) Native gel electro-elution

(i) Electrophoresis buffer

Tris-glycine (25mM-192mM), pH 8.0

Tris-glycine (6mM-60 mM), pH 8.5

(ii)Stacking and resolving gel

3 cm (3%) stacking and 3 cm (6%) resolving gel

2 cm (4%) stacking and 2 cm (6%) resolving gel

(iii)Dialysis medium

10 mM sodium phosphate buffer pH 7.0

Tris-Glycine (6mM-60mM), pH adjusted to 7.0

(iv) Elution rate and fraction size

0.3 ml/min; 5ml

0.6ml/min; 8 ml

(v) Elution time

24-48 h

6-9 h

(vi) Electrophoresis setting

5 watt constant; beginning Voltage ~280 and current ~17 mA with gradual decrease in V (up to 100 V) and increase in current (up to 47 mA).

5 watt constant; beginning Voltage ~490 and current ~6 mA with gradual decrease in V (up to 280 V) and increase in current (up to 20 mA)

(e) Size exclusion chromatography

Analytical grade with column volume of~15ml

Semi-preparative grade with total column volume of ~100 ml

(f) Peak activity

Single peak; ~670 kD

Single peak; ~670 kD

One important point of difference was that CHAPS was not required for activity or stability of the Asp-NAT preparation obtained from the supernatant fraction. The final enzyme preparation after HPLC (with the column exclusion limit of ~800 kD) showed a single peak on size exclusion chromatography and a single band on native gel electrophoresis (Fig. 3). However, multiple bands were detectable on SDS-PAGE as was the case with the mitochondria-solubilized enzyme preparation.12 Although all these protein bands may not be part of Asp-NAT, the indication was that of an enzyme complex containing multiple enzymes or subunits, which is a characteristic of a variety of mitochondrial enzymes.

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