Table 1. Purification of rabbit liver glucose 6-phosphatase. One unit of activity represents 1.Opmol of P, formed/min. The yield and relative purification data are ...
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Table 1. Purificationof rabbit liver glucose 6-phosphatase One unit of activity represents 1.Opmol of P, formed/min. The yield and relative purification data are expressed with reference to the solubilized microsomal fraction in the presence of 1% sodium cholate, as the increase in activity on the addition of detergent does not effect a purification of the enzyme. Preparation Microsomal fraction in 0.25 M-sucrose Solubiliied microsomal fraction Resuspended poly(ethy1ene glycol) pellet DEAE+xUuIose pooled fractions Resuspended pellet from pooled fractions
protein (mg) 900 870 217 70 20
enzyme activity. The recovery of protein and enzyme activity from this step is given in Table 1. The protein concentration of the poly(ethy1ene glycol) suspension was adjusted to lOmg/ml by dilution with buffer B (0.1 M-Tridacetate buffer, pH 7.0, containing 2096 glycerol, I.OmM-EDTA and 0.5 mhldithiothreitol) before it was applied to a DEAE-cellulose column (20mm diameter x 70mm long). The column was equilibrated with buffer B before the poly(ethy1ene glycol) suspension (100mg) was loaded on the column. The column was eluted with buffer B until the flow-through protein was eliminated from the column. The buffer was then changed to buffer B containing 0.25~-KCl and the fractions exhibiting glucose 6-phosphatase activity were collected and pooled. The cloudy suspension of pooled fractions was then centrifuged at 105000g for 1.Oh at 4OC, and the resulting pellet was then solubiliied in a minimum volume of buffer B. The recovery of enzyme activity and protein from this step is given in Table 1. Glucose 6-phosphatase activity was assayed at 35OC in a 0.5 ml reaction mixture containing 50 mhl-sodium cacodylate, 30mM-gh1CO~e6-phosphate and ~ ~ M - E D TatA pH6.5. The reaction was started with enzyme and was stopped by removing a sample (0.05ml) of the reaction mixture and adding it to 0.25ml of 1096 (w/v) sodium dodecyl sulphate solution. The
Specific activity (unitdmg)
Relative purification
0.09
1 3.4 6.9 16.8
0.03
0.3 1 0.62 1.51
-
Yield
(%I 34 100 86 55 38
resulting solution was assayed for P, by the method of Ames (1966). Protein was measured by the method of Bradford (1978). The results obtained with the above preliminary purification procedures are collected in Table 1. The specific activity of the enzyme has increased 16.8-fold from 0.09 unit/mg in the solubilized microsomal fraction to 1.51 units/mg in the solubilized DEAE-cellulose eluate pellet. The above procedures have effectively decreased the amount of the microsomal protein to a low value while still maintaining a satisfactory yield of enzyme activity. We believe that the process is now ready for the introduction of a suitable dnity-chromatography technique to increase the purification substantially. We are grateful to Miss V. Cameron for excellent technical assistance. This work was supported by a grant from the Medical Research Council. Ames, B. N. (1966) Methods Enzymol. 8, 115-1 18 Bradford, M.M.(1976) Anal. Biochem. 72,248-254 Burchell, B. (1977) Biochem. J. 161,543-549 Nordlie, R. C. (1971) Enzymes, 3rd Ed. 4 , 5 4 3 6 1 0
The purification of isocitrate dehydrogenase from Escherichia coli using immobilized dyes H. G. NIMMO and W. H. HOLMS Department of Biochemistry, University of Glasgow, Glasgow GI2 8QQ,Scotland, U.K. Isocitrate lyase (EC 4.1.3. I), the first enzyme of the glyoxylate bypass, competes with the Krebs cycle enzyme isocitrate dehydrogenase (EC 1.1.1.42) for their common substrate. In Escherichia coli the glyoxylate bypass is an anaplerotic pathway essential for growth on acetate (Kornberg, 1966) and its operation is favoured by partial inactivation of the isocitrate dehydrogenase, an NADP-linked enzyme (Holms & Bennett, 1971; Bennett & Holms, 1975). The isocitrate dehydrogenase is inactivated only when the bypass actually operates (Holms & Bennett, 1971) by a process that does not depend on low-molecular-weighteffectors (Bennett & Holms, 1975). If the bypass is made redundant (e.g. by adding pyruvate to a culture grown on acetate) the ismitrate dehydrogenase is rapidly re-activated by a process independent of protein synthesis (Bennett & Holms, 1975). Recent evidence suggested that phosphorylation of isocitrate dehydrogenase may be involved (Garnak & Reeves, 1979a) and it was therefore of interest to purify the protein so that its phosphorylation could be studied in vitro. In this communication we report on some of the properties of the enzyme isolated by chromatography on immobilized dyes. Reeves’ group have recently reported the use of immobilized
Cibacron Blue in the purification of isocitrate dehydrogenase from E. coli strain K12 (Garnak & Reeves, 1979b; Vasquez & Reeves, 1979) and this matrix has also been used to isolate the enzyme from human heart (Seelig & Colman, 1977) and from Bacillus stearothermophilus(Nagaoka et al., 197I). We partially purified ismitrate dehydrogenase from E. coli strain ML308 using protamine sulphate treatment, ammonium sulphate fractionation and chromatography on DEAE-cellulose (H. G. Nimmo, unpublished results). The resulting material, purified 65-fold over the crude extract, was dialysed into IOmhl-potassium phosphate/5 mt,4-sodium citrate/2 mM-MgCI,/ 10% glycerol (pH6.5) at room temperature and loaded onto 1 cm x 6cm columns of either Cibacron Blue or Procion Red linked to Sepharose (gifts from Dr. L. Jervis. Department of Biology, Paisley College of Technology, Paisley, Scotland, U.K.). With the Cibacron Blue column, 42% of the applied activity (480i.u.) was considerably retarded and emerged when the column was washed with 15 bed volumes of buffer. A further 30% of the applied activity was bound by the column and was eluted by including 0.5 mM-NADP+ in the washing buffer. With the Procion Red column, over 90% of the applied activity was bound by the column and was eluted in 75% yield by the inclusion of 0.5 mM-NADP+ in the washing buffer. In each case the enzyme that was eluted by NADP+ had been purified 1980
39 1
587th MEETING, DUNDEE
0
3
6
9
Gel length (cm)
Fig. 1. Gel electrophoresis of ismitrate &hydrogenuse purified using Procion RedSepharose Non-denaturing 7% polyacrylamide gels were run at pH8.9 as described by Davis (1964). (a) Gel loaded with 2pg protein and stained for isocitrate dehydrogenase activity (Vasquez & Reeves, 1979) (6) Gel loaded with 5 pg protein and stained with Coomassie Blue. Gels were scanned at 600nm. Enzyme purified using Cibacron Blue-Sepharose gave similar results.
approximately 3-fold over the material loaded onto the columns Blue to isolate isocitrate dehydrogenase and the specific activity and had a specific activity of 160-180 units/mg at 37OC. This of their purified enzyme is lower than that recorded here. They compares favourably with the value of 56 units/mg at 25OC have not checked the purity of their preparation using reported by Vasquez & Reeves (1979) for the enzyme from E. nondenaturing gel electrophoresis at pH8.9 and accordingly their conclusions about the phosphorylation of E. colf isocitrate coli K 12. The purities of the enzyme fractions eluted by NADP+ were dehydrogenase (Garnak & Reeves, 1979a,6) may be premature. checked by gel electrophoresis. Using two different systems in the presence of sodium dodecyl sulphate (Laemmli, 1970; Bennett, P. M. & Holms, W. H. (1975) J. Gen. Mkmbiol. 87,37-5 1 Weber & Osborn, 1969) each enzyme fraction gave only a single Davis, B. J. (1964) Ann. N.Y. Acod. Sci. 121,404427 band corresponding to a subunit mo1.W. of approx. 45 OOO (not G-ak, M. & Reeves, H. C. (19790) SC&W 203.11 11-1 112 illustrated). However, using nondenaturing polyacrylarnide gels Garnak, M. & Reeves, H. C. (1979b)J. E b l . Chem. 254,7915-7920 at pH 8.9 (Davis, 1964) each fraction gave two bands, only one Holms, W. H. &Bennett, P. M. (1971)J. Gen. Mfcmbfol.6 5 , 5 7 6 8 of which stained for isocitrate dehydrogenase activity (Fig. I). Komberg. H. L. (1966) Essays Bfoclfem.2, 1-31 This pattern was not altered by pre-treatment of the enzyme Laemmli. U. K.(1970) Nature (London)227,680-685 T., Hachimori, A., Takeda, A. (Yt Samejiia, T. (1977) J . with alkaline phosphatase, suggesting that the two species were Nagaoka, Biochem. (Tokyo81,71-78 not merely phosphorylated and dephosphorylated forms of the Seclig, G. F. & Colman, R. F. (1977)J. Bfol.Chem.252,3671-3678 same protein. Vasquez, B. & Reeves. H. C. (1979) Biochfm. Bfophys. Acfa 578, Reeves and his co-workers (Vasquez & Reeves, 1979; 3 1-40 Garnak & Reeves, 19796) have also used immobilized Cibacron Weber, K.& Osborn, M. J. (1969) J. Biol. Chem. 244,44064412
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