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S L Yun, A E Aust and C H Suelter kinase.(Saccharomyces cerevisiae) pyruvate A revised preparation of yeast:
1976, 251:124-128.J. Biol. Chem.
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THE JOURNAL OF BIOUXXAL CH~MISIW Vol. 251, No. 1, Issue of January 10, pp. 124-128, 1976
Binted in USA.
A Revised Preparation of Yeast (Saccharomyces cerevisiae) Pyruvate Kinase*
(Received for publication, April 9, 1975)
SHYUN-LONG YUN, ANN E. AUST, AND CLARENCE H. SUELTER$
From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
A revised preparation of pyruvate kinase from Sacchuromyces cereuisiue is reported. By purifying this cold-labile enzyme at room temperature, an improved recovery and specific activity was obtained. More than 350 mg of pure enzyme with a specific activity of 350 to 400 units/mg at 30” were obtained from a pound of fresh yeast. The last step of the preparation, passage of the enzyme over Sephadex G-100, was required to remove a contaminating protease. The molecular parameters of the new preparation are: molecular weight, 209,000; four subunits of identical size; E,,, ,,,,,, 0.51; p1 6.6; and pH optimum, 6.28. Kinetic parameters are: K, for P-enolpyruvate and ADP, 0.09 and 0.18 mM in the presence of saturating Fru-1,6-P,, and 1.8 and 0.34 mM in the absence of Fru-1,6-P,; K, for Fru-1,6-P,, 0.014 mM. No free NH,-terminal amino acid could be detected. Amino acid composition was determined and compared with other pyruvate kinase preparations.
The original preparation of pyruvate kinase from Sac- churomyces cereuisiae which was homogeneous by ultra- centrifugal (1) and gel electrophoretic techniques had a native molecular weight of 168,000 (2) and subunit molecular weight of 42,000 (2). The revised preparation described in this paper yields enzyme with a molecular weight of 209,000. A prepara- tion by Fell et al. (3) also has a higher molecular weight. Por- tions of this study were reported previously (4).
EXPERIMENTAL PROCEDURE
The “Experimental Procedure” including materials and methods and the revised preparation of yeast pyruvate kinase is described in the adjacent miniprint. I
RESULTS
Table I summarizes the results of a typical preparation outlined in detail in the minisection. We wish to emphasize three points important in obtaining a stable preparation with
*The investigation was supported in part by Research Grant GB 25116 from the National Science Foundation, Training Grant GB 01091.11 from the National Institutes of Health, and the Michigan State Agricultural Experiment Station, Michigan State Journal No. 7160.
$ Recipient of Research Career Development Award 1 K3 GM 9726. ‘Some of‘ the data are presented as a miniprint supplement
immediately following this paper. Material published in miniprint form can be easily read with the aid of a large field reading glass of a type readily available at most opticians, For the convenience of those who prefer to obtain supplementary material in the form of full size photocopies, these same data are available as JBC Document No. 75M-489. Orders for supplementary material should specify the title, authors, and reference to this paper and the JBC Document Number, and the number of copies desired. Orders should be addressed to The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014, and must be accompanied by a remittance to the order of the Journal in the amount of $2.70.
the higher molecular weight and specific activity: First, lysis of yeast was adapted after consideration of several methods (for summary see Table IS, in the Supplement); second, the preparation was completed at room temperature; and third, the last step, passage of enzyme over Sephadex G-100, was re- quired to remove a contaminating protease. *
ENZYME PROPERTIES
Homogeneity-The purified enzyme was homogeneous by two independent criteria; (a) a single protein band either for dissociated subunits (Fig. 1BS) or for native enzyme (Fig. 2s) was obtained by analytical polyacrylamide gel electrophoresis, (6) a plot of log net fringe uersus square of radius of equilibrium sedimentation was linear (Fig. 3s). The enzyme sedimented as a single peak during velocity sedimenta- tion (data not shown).
Stability-The preparation exhibits the same cold lability (1) and susceptibility to inactivation by Fru-1,6-P, (2) as that reported for the original preparation. The purified enzyme, at 3.8 mg ml-‘, was stable at room temperature in 0.1 M sodium phosphate, pH 6.5, in the presence of 25% glycerol, 5 mM EDTA, and 5 mM mercaptoethanol for a minimum period of 31 days (Table IL’S). The enzyme may be stored at 0” as a suspension in 90% saturated ammonium sulfate.
Extinction Coefficient-The extinction coefficient de- termined by dry weight gave an average value of 0.51 ml mg-’ cm-’ at 280 nm. The value obtained by the method of
*Direct proof for a protease contamination at this stage of purifica- tion is not possible. The suspected protease contamination is not sufficiently large to see on polyacrylamide gel or by analytical ultra- centrifugation; furthermore, no protease activity could be detected in an azoalbumin assay described by Tomarelli (5) or in a fluorescamine assay (6).
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Purification and Properties of Yeast Pyruvate Kinuse 125
TABLE I
Summary of yeast pyruuate kinase purification
Assay conditions are described under “Experimental Procedure.” The yields are given tar 1 pound of yea&t.
Step Volume Protein Artlvlty Spedi activity Yield
ml w units units/my 9
I. Toluolysis (37”) 475 12,400 “80.000 ‘2.6 100 II. (NH,),SO, fraction (40-X%) 102 2,500 197,000 79 70
III. Dialysate 111 :3,010 223,000 74 80 IV. DEAE-cellulose phosphate. pH 5.5 390 1,680 160,000 95 6i V. Cellulose phosphate, pH 6.5 246 390 135,000 346 48
VI. (NH,),SO,precipitation 14.6 350 126,000 360 45 VII. Sephadex G-100 Ii8 356 127 c 000 7 345 44
Scopes (7) using the ratio of A JA 2,,5 was 0.515 ml mg- 1 cm - I. The extinction coefficient of pyruvate kinase from other sources is compared in Table VS.
pH Optimum and Isoelectric Point-A pH profile of pyruvate kinase in the presence of saturating Fru-1,6-P, shows a bell-shaped curve with an optimum at pH 6.28 (data not given) in agreement with earlier data (8). Fig. 6S shows a p1 = 6.6.
Molecular Weight-High speed sedimentation equilibrium according to the method of Yphantis (9) gave an average molecular weight of 209,000 + 6,000 from three determinations (Fig. 3s). A molecular weight of 56,500 to 58,500 was obtained for the subunits by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 4s). The molecular weight determined by electrophoresis of native enzyme on polyacrylamide gels, as described by Hedrick and Smith (10) was 220,000 * 6,500 (Fig. 5s). An identical value was obtained for enzyme in crude extract. Purified pyruvate kinase mixed with crude ex- tract gave a single activity band on polyacrylamide gels of varying acrylamide concentration.
Amino Acid Composition and NH,-terminal-The prepara- tion has a typical amino acid composition (Table 111s). No free NH,-terminal amino group was detected.
Kinetic Parameters-The kinetic parameters for this prepa- ration, except for the specific activity, are not significantly
different from those reported for the original preparation (1). Initial velocity versus concentration of P-enolpyruvate under conditions specified under “Experimental Procedure” is sigmoidal with a Hill coefficient, nH = 2.3 and K,,, = 1.8 mM. The sigmoidal curve becomes hyperbolic in the presence of F~L-1,6-P,. The K, for P-enolpyruvate under the same con- dition, but with 1 mM Fru-1,6-P, is 0.09 mM. The K, for ADP is 0.34 mM without Fru-1,6-P, and 0.16 mM with Fru-1,6- P,. K, for Fru-1,6-P, is 0.014 mM at 1 mM P-enolpyruvate.
REFERENCES I. Hunsley, J. R., and Suelter, C. H. (1969) J. Biol. Chem. 244, 4815-
4818 2. Kuczenski, R. T., and Suelter, C. H. (1970) Biochemistr?/ 9,
2043-2047 3. Fell, D. A., Liddle, D. A.. Peacocke, A. R., and Dwek, R. A.
(1974) Biochem. J. 139,665-675 4. Yun. S. L., and Suelter, C. H. (1973) Abstracts of 166th National
Meeting of the American Chemical Society, No. 125 5. Tomarelli, R. M., Charney, J., and Harding, M. L. (1949) J. Lab.
Clin. Med. 34,428-433 6. Schwabe, C. (1973) Anal. Biochem. 53,484-490 7. Scopes, R. K. (1974) Anal. Biochem. 59,277-282 8. Hunsley, J. R. (1970) Ph.D. thesis, Michigan State Ilniversity,
East Lansing. Mich. 9. Yphantis, D. A.‘( 1964) Biochemistry 3, 297-317
10. Hedrick, .J. C., and Smith, A. .I. (1968) Arch. Biochem. Biophys. 126,155-164
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126 Purification and Properties of Yeast Pyruvate Kinnse
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128 Purification and Properties of Yeast Pyruvate Kinuse
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