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Crystalline phosphoglycerate kinase from human erythrocytes
SHORT COMMUNICATIONS
355
rature, were obtained 1~ for a system o.I M in CaC12 and thus are not directly comparable with those obtained in systems containing only univalent ions. Ca 2+ ion is known la to have a specific and still indeterminate effect on the kinetic constants. N-Acetyl-L-leucine methyl ester was prepared essentially as described by APPLEWHITE et al. 8. From 13.1 g of L-leucine there was obtained lO.4 g of a syrup which was twice recrystallized from a mixture of ethyl ether and pentane to give 5.9 g (32%) of acetyl-L-leucine methyl ester, large colorless cubes, m.p. 46-47 °, ~aji) 25 - 56.8 ± I.O ° (C, 3 % in water). Found: C, 57.8; H, 9.1; N, 7.4- Calcd. for CgH17NOa: C, 57-7; H, 9.2; N, 7.5%. KARRER, ESCHER AND WlDMER 15 report a m.p. of 74-75 ° and an [a]i¢ " - 17.2 °. However, it appears that the preparation obtained by these investigators was substantially racemized. The product obtained in the present studies was comparable to that obtained earlier s. The procedure employed for the kinetic studies was identical with that described previouslyS, a". All experiments were conducted in aqueous solutions at 25.o °, p H 7.9 o 4- o.Io and o.Io or o.2o M in NaC1. The enzyme preparation was bovine, crystalline, salt-free a-chymotrypsin obtained from Armour and Co., Lot No. T-972o 7. The pertinent experimental parameters are summarized in Table I. The primary data were analyzed using the Datatron 22o digital-computer program described recently 16, As for similar substrates, the data fit the rate equation d[Pl/dt = k0[E i [Si/ (K 0 + iSi). The values obtained for the constants k o and K 0 are given in Table I. This investigation was supported in part by a grant from the National Institutes of Health, U.S. Public Health Service.
Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, Calif. (U.S.A.)
GEORGE E. HEIN J. BRYAN JONES CARL NIEMANN
1 M. BRENNER, t~. R. M~ILLER AND I{. W. PFISTER, Helv. Chim. Acla, 33 (195o) 568. 2 }-[. GUTFREUND, in P. D. BOYER, I-l. LARDY AND I(. MYRBACK, Vol. I, A c a d e m i c Press, Inc., N e w Y o r k , t959, P. 2333 G. E. HEIN AND C. NIEMANN, Proc. Natl. Acad. Sci., 47 (1961) 1341. 4 j . Am. Chem. Soc., in t h e press. 5 M. i . BENDER AND W. L. GLASSON, J. Am. Chem. Soc., 82 (196o) 3336. 6 ]:{. R. WAITE AND C. NIEMANN, Biochem., I (1962) 250. J. P. WOLF, l l I , P h . D . Thesis, Calif. I n s t . Techn., P a s a d e n a (1959). a T. I-I. APPLEWHITE, }:[. WAITE AND C. NIEMANN, J. Ar~l. Chem. Soc., 80 (1958) 1465. 9 ] . B. JONES, u n p u b l i s h e d results. 10 I~. R. JENNINGS AND C. NIEMANN, J. Am, Chem. Soc., 75 (1953) 4687 • 11 S. A. BERNHARD AND H. GUTFREUND, Progr. in Biophys. and Biophys. Chem., i o (196o) l i 6 . 12 L. V. CUNNINGHAM AND C. S. BROWN, J. Biol. Chem., 22i (1956) 287. •a R. B. MARTIN .aND C. NIEMANN, J. Am. Chem. Soe., 80 (1958) 1481. 14 p. KARRER, K. ESCHER AND t~. WIDMER, Helv. Chim. Acta, 9 (1926) 322. t5 T. 1-l. APPLE'~VHITE, R. B. MARTIN AND C. NIEMANN, J. Am. Chem. Soc., 80 (1958) I457. in H. 1. ABRASH, A. N. KURTZ A~D C. NIEMANN, Biochim. Biophys. Acta, 45 (196o) 387 .
Received June i2th, 1962
Biochim. Biophys. Acta, 65 (I962) 353-355
SC I I O 2 0
Crystalline phosphoglycerate kinase from human erythrocytes It is well-known that the main phosphorus compound in most mammalian erythrocytes is 2,3-diphosphoglycerate. Although RAPOPORT AND LUEBERING1 have suggested that the first step in the enzymic formation of 2,3-diphosphoglycerate in erythrocyte Biochim. Biophys. Acta, 65 (1962) 355 357
356
SttORT COMMUNICATIONS
is the formation of 1,3-diphosphoglycerate, they have not studied the phosphoglycerate kinase (ATP: D-3-phosphoglycerate I-phosphotransferase, EC 2.7.2.4) of these cells. The role of the enzyme in the phosphoglycerate cycle 2 is a interesting problem for erythrocyte glycolysis. This note describes a procedure for the crystallization of the enzyme from human erythrocytes. 20o ml of washed human erythrocytes (fresh or ACD blood stored for up to 6 weeks) were hemolyzed by the addition of 2 vol. of deionized water in an ice-bath. The hemolysate was brought to p H 7.2 ± o.I with o. 5 N KOH. To the hemolysate at o °, 21o ml of ethanol chloroform mixture (2 : I) were added with stirring and kept for 2o-30 min in the cold with occasional stirring. The denatured hemoglobin
Fig. I. Photograph of recrystallized phosphoglycerate kinase ( × 400). Small dense particles converted to fine rods which were gradually grown to long-rectangular plates by warming and cooling.
was removed by centrifugation. The complete removal of hemoglobin at this stage was necessary to obtain pure preparation of the crystalline enzyme. The supernatant will be called Fraction I. The enzyme was precipitated from the supernatant fluid by the addition of 2.5 vol. of ice-cold ethanol, followed by centrifugation. The precipitate was dissolved in about IOO ml of ice-cold water and the insoluble material was removed to give Fraction 2. Fraction 2 was brought to p H 6.0 ± o.I with 0.2 M sodium acetate buffer (pH 5.8) and to it was added 2o ml of calcium phosphate-gel suspension (25 mg/ml (ref. 3) The mixture was left standing for Io min with occasional stirring; it was centrifuged and the precipitate was washed with o.o2 M sodium acetate (pH 5.8). The enzyme was eluted from the gel with 15 ml of o.oi M Na4P207. The turbid eluate (Fraction 3) was directly passed through the column of DEAEcellulose (5 X 3 cm, previously bufferized with o.oo5 M NaaP~07). To the effluent and washings containing enzyme (Fraction 4), solid (NHa)2SO 4 was added to give Biochim. Biophys. Acta, 65 (t962) 355-357
357
SHORT COMMUNICATIONS
0.40 saturation. The precipitate was centrifuged off and the clear supernatant was obtained. A saturated (NH4)~SO 4 solution was carefully added to the supernatant to give 0.75 saturation. Dense fine crystals, as evidenced by the characteristic silky sheen appearing upon swirling the tube, were formed within IO rain. These grew to to long-rectangular plates b y warming to room temperature for I or 2 h and again cooling to 0-5 °. Recrystallization was carried out in the following manner. The suspension was centrifuged and the supernatant fluid discarded. The crystal pellet was dissolved in a minimum quantity of o.oi M Na4P~O7 and was brought to 0.6o saturation with (NH4)~SO~. Insoluble material containing hemoproteins was removed, and a saturated (NH4)2SO 4 solution was added to the resulted clear solution to give 0.75 saturation. The recrystallized preparation was sometimes contaminated with traces ofhemoprotein which was removed by the further recrystallization. The purification procedure is summarized in Table I. TABLE I SUMMARY OF PUR[FICATION PROCEDURE
A t e s t t u b e w h i c h c o n t a i n e d t h e following c o m p o n e n t s e x p r e s s e d as ttmoles in a t o t a l vol. of 0.6 ml was i n c u b a t e d for IO m i n a t 37°: N H 2 O H - N a O H (pH 7.4), 250; p o t a s s i u m D - 3 - p h o s p h o g l y c e r a t e , 6.0; AT P, 2.o; MgC12, i. 5 a n d e n z y m e . The r e a c t i o n was s t o p p e d b y t h e a d d i t i o n of 0.75 ml of FeCl 3 r e a g e n t a n d 0.2o ml of 95 % e t h a n o l as d e s c r i b e d b y AXELROD AND BANDURSKI 4. Fraction
Hemolysate Fraction I Fraction 2 Fraction 3 Fraction 4 I St c r y s t a l l i n e p r e p a r a t i o n Mother liquid 2nd c r y s t a l l i n e p r e p a r a t i o n
Vol. of solution (ml)
Total* activity (uuits)
600 500 IOO 15
500 400 260 240 240 18o 5° 144
Total pr.tein (rag)
52 800 74 ° 450 142 82 54 27 42
Specific* activity
o,oo95 o,54
o,55 1.78 2.92 3-29 t .85 3.42
Yield (%)
too 8o 52 49 48
3 li 29
* An e n z y m e u n i t of t h e k i n a s e is defined as t h a t a m o u n t of e n z y m e w h i c h c a us e s a n increase in a b s o r b a n c y of o . I o o per m i n u n d e r t h e c o n d i t i o n s of t h e assay. Specific a c t i v i t y is defined as u n i t / r a g e n z y m e p r o t e i n w h i c h is d e t e r m i n e d b y t h e m e t h o d of LOWRY el al?.
A purified preparation was used to observe the back-ward reaction coupled with crystalline muscle glyceraldehyde phosphate dehydrogenase. The turnover number of the erythrocyte enzyme was of the same order as that of the yeast enzyme 6.
Department of Physiological Chemistry and Nutrition, Faculty of Medicine, The University of Tokyo, Tokyo (Japan)
TAKASHI HASHIMOTO H A R U H IS A YOSHIKAWA
J" S. RAPOPORT AND J. LUEBERING, J. Biol. (:hem., 183 (195o) 507. 2 S. GRISOLIA, Ann. N . Y . Acad. Sci., 72 (I959) 462. 3 D. I~EILIN AND E. F. HARTREE, Proc. Roy. Soc. (London), B i 24 (i938) 397. 4 B. AXELROD AND R. S. BANDURSKI. J. Biol. Chem., 204 (1953) 939. '50. H. LowRY, N . J . ROSEBROUGH, A. L. FARRAND R. J. RANDALL, J. Biol. Chem., 193 (195 L) z65. 6 T. BUCHER, Biochim. Biophys. Acta, 1 (1947) 292.
Received June 29th, 1962 Biochim. Biophys. ,4cla, 65 (19()2) 355-357
355
rature, were obtained 1~ for a system o.I M in CaC12 and thus are not directly comparable with those obtained in systems containing only univalent ions. Ca 2+ ion is known la to have a specific and still indeterminate effect on the kinetic constants. N-Acetyl-L-leucine methyl ester was prepared essentially as described by APPLEWHITE et al. 8. From 13.1 g of L-leucine there was obtained lO.4 g of a syrup which was twice recrystallized from a mixture of ethyl ether and pentane to give 5.9 g (32%) of acetyl-L-leucine methyl ester, large colorless cubes, m.p. 46-47 °, ~aji) 25 - 56.8 ± I.O ° (C, 3 % in water). Found: C, 57.8; H, 9.1; N, 7.4- Calcd. for CgH17NOa: C, 57-7; H, 9.2; N, 7.5%. KARRER, ESCHER AND WlDMER 15 report a m.p. of 74-75 ° and an [a]i¢ " - 17.2 °. However, it appears that the preparation obtained by these investigators was substantially racemized. The product obtained in the present studies was comparable to that obtained earlier s. The procedure employed for the kinetic studies was identical with that described previouslyS, a". All experiments were conducted in aqueous solutions at 25.o °, p H 7.9 o 4- o.Io and o.Io or o.2o M in NaC1. The enzyme preparation was bovine, crystalline, salt-free a-chymotrypsin obtained from Armour and Co., Lot No. T-972o 7. The pertinent experimental parameters are summarized in Table I. The primary data were analyzed using the Datatron 22o digital-computer program described recently 16, As for similar substrates, the data fit the rate equation d[Pl/dt = k0[E i [Si/ (K 0 + iSi). The values obtained for the constants k o and K 0 are given in Table I. This investigation was supported in part by a grant from the National Institutes of Health, U.S. Public Health Service.
Gates and Crellin Laboratories of Chemistry, California Institute of Technology, Pasadena, Calif. (U.S.A.)
GEORGE E. HEIN J. BRYAN JONES CARL NIEMANN
1 M. BRENNER, t~. R. M~ILLER AND I{. W. PFISTER, Helv. Chim. Acla, 33 (195o) 568. 2 }-[. GUTFREUND, in P. D. BOYER, I-l. LARDY AND I(. MYRBACK, Vol. I, A c a d e m i c Press, Inc., N e w Y o r k , t959, P. 2333 G. E. HEIN AND C. NIEMANN, Proc. Natl. Acad. Sci., 47 (1961) 1341. 4 j . Am. Chem. Soc., in t h e press. 5 M. i . BENDER AND W. L. GLASSON, J. Am. Chem. Soc., 82 (196o) 3336. 6 ]:{. R. WAITE AND C. NIEMANN, Biochem., I (1962) 250. J. P. WOLF, l l I , P h . D . Thesis, Calif. I n s t . Techn., P a s a d e n a (1959). a T. I-I. APPLEWHITE, }:[. WAITE AND C. NIEMANN, J. Ar~l. Chem. Soc., 80 (1958) 1465. 9 ] . B. JONES, u n p u b l i s h e d results. 10 I~. R. JENNINGS AND C. NIEMANN, J. Am, Chem. Soc., 75 (1953) 4687 • 11 S. A. BERNHARD AND H. GUTFREUND, Progr. in Biophys. and Biophys. Chem., i o (196o) l i 6 . 12 L. V. CUNNINGHAM AND C. S. BROWN, J. Biol. Chem., 22i (1956) 287. •a R. B. MARTIN .aND C. NIEMANN, J. Am. Chem. Soe., 80 (1958) 1481. 14 p. KARRER, K. ESCHER AND t~. WIDMER, Helv. Chim. Acta, 9 (1926) 322. t5 T. 1-l. APPLE'~VHITE, R. B. MARTIN AND C. NIEMANN, J. Am. Chem. Soc., 80 (1958) I457. in H. 1. ABRASH, A. N. KURTZ A~D C. NIEMANN, Biochim. Biophys. Acta, 45 (196o) 387 .
Received June i2th, 1962
Biochim. Biophys. Acta, 65 (I962) 353-355
SC I I O 2 0
Crystalline phosphoglycerate kinase from human erythrocytes It is well-known that the main phosphorus compound in most mammalian erythrocytes is 2,3-diphosphoglycerate. Although RAPOPORT AND LUEBERING1 have suggested that the first step in the enzymic formation of 2,3-diphosphoglycerate in erythrocyte Biochim. Biophys. Acta, 65 (1962) 355 357
356
SttORT COMMUNICATIONS
is the formation of 1,3-diphosphoglycerate, they have not studied the phosphoglycerate kinase (ATP: D-3-phosphoglycerate I-phosphotransferase, EC 2.7.2.4) of these cells. The role of the enzyme in the phosphoglycerate cycle 2 is a interesting problem for erythrocyte glycolysis. This note describes a procedure for the crystallization of the enzyme from human erythrocytes. 20o ml of washed human erythrocytes (fresh or ACD blood stored for up to 6 weeks) were hemolyzed by the addition of 2 vol. of deionized water in an ice-bath. The hemolysate was brought to p H 7.2 ± o.I with o. 5 N KOH. To the hemolysate at o °, 21o ml of ethanol chloroform mixture (2 : I) were added with stirring and kept for 2o-30 min in the cold with occasional stirring. The denatured hemoglobin
Fig. I. Photograph of recrystallized phosphoglycerate kinase ( × 400). Small dense particles converted to fine rods which were gradually grown to long-rectangular plates by warming and cooling.
was removed by centrifugation. The complete removal of hemoglobin at this stage was necessary to obtain pure preparation of the crystalline enzyme. The supernatant will be called Fraction I. The enzyme was precipitated from the supernatant fluid by the addition of 2.5 vol. of ice-cold ethanol, followed by centrifugation. The precipitate was dissolved in about IOO ml of ice-cold water and the insoluble material was removed to give Fraction 2. Fraction 2 was brought to p H 6.0 ± o.I with 0.2 M sodium acetate buffer (pH 5.8) and to it was added 2o ml of calcium phosphate-gel suspension (25 mg/ml (ref. 3) The mixture was left standing for Io min with occasional stirring; it was centrifuged and the precipitate was washed with o.o2 M sodium acetate (pH 5.8). The enzyme was eluted from the gel with 15 ml of o.oi M Na4P207. The turbid eluate (Fraction 3) was directly passed through the column of DEAEcellulose (5 X 3 cm, previously bufferized with o.oo5 M NaaP~07). To the effluent and washings containing enzyme (Fraction 4), solid (NHa)2SO 4 was added to give Biochim. Biophys. Acta, 65 (t962) 355-357
357
SHORT COMMUNICATIONS
0.40 saturation. The precipitate was centrifuged off and the clear supernatant was obtained. A saturated (NH4)~SO 4 solution was carefully added to the supernatant to give 0.75 saturation. Dense fine crystals, as evidenced by the characteristic silky sheen appearing upon swirling the tube, were formed within IO rain. These grew to to long-rectangular plates b y warming to room temperature for I or 2 h and again cooling to 0-5 °. Recrystallization was carried out in the following manner. The suspension was centrifuged and the supernatant fluid discarded. The crystal pellet was dissolved in a minimum quantity of o.oi M Na4P~O7 and was brought to 0.6o saturation with (NH4)~SO~. Insoluble material containing hemoproteins was removed, and a saturated (NH4)2SO 4 solution was added to the resulted clear solution to give 0.75 saturation. The recrystallized preparation was sometimes contaminated with traces ofhemoprotein which was removed by the further recrystallization. The purification procedure is summarized in Table I. TABLE I SUMMARY OF PUR[FICATION PROCEDURE
A t e s t t u b e w h i c h c o n t a i n e d t h e following c o m p o n e n t s e x p r e s s e d as ttmoles in a t o t a l vol. of 0.6 ml was i n c u b a t e d for IO m i n a t 37°: N H 2 O H - N a O H (pH 7.4), 250; p o t a s s i u m D - 3 - p h o s p h o g l y c e r a t e , 6.0; AT P, 2.o; MgC12, i. 5 a n d e n z y m e . The r e a c t i o n was s t o p p e d b y t h e a d d i t i o n of 0.75 ml of FeCl 3 r e a g e n t a n d 0.2o ml of 95 % e t h a n o l as d e s c r i b e d b y AXELROD AND BANDURSKI 4. Fraction
Hemolysate Fraction I Fraction 2 Fraction 3 Fraction 4 I St c r y s t a l l i n e p r e p a r a t i o n Mother liquid 2nd c r y s t a l l i n e p r e p a r a t i o n
Vol. of solution (ml)
Total* activity (uuits)
600 500 IOO 15
500 400 260 240 240 18o 5° 144
Total pr.tein (rag)
52 800 74 ° 450 142 82 54 27 42
Specific* activity
o,oo95 o,54
o,55 1.78 2.92 3-29 t .85 3.42
Yield (%)
too 8o 52 49 48
3 li 29
* An e n z y m e u n i t of t h e k i n a s e is defined as t h a t a m o u n t of e n z y m e w h i c h c a us e s a n increase in a b s o r b a n c y of o . I o o per m i n u n d e r t h e c o n d i t i o n s of t h e assay. Specific a c t i v i t y is defined as u n i t / r a g e n z y m e p r o t e i n w h i c h is d e t e r m i n e d b y t h e m e t h o d of LOWRY el al?.
A purified preparation was used to observe the back-ward reaction coupled with crystalline muscle glyceraldehyde phosphate dehydrogenase. The turnover number of the erythrocyte enzyme was of the same order as that of the yeast enzyme 6.
Department of Physiological Chemistry and Nutrition, Faculty of Medicine, The University of Tokyo, Tokyo (Japan)
TAKASHI HASHIMOTO H A R U H IS A YOSHIKAWA
J" S. RAPOPORT AND J. LUEBERING, J. Biol. (:hem., 183 (195o) 507. 2 S. GRISOLIA, Ann. N . Y . Acad. Sci., 72 (I959) 462. 3 D. I~EILIN AND E. F. HARTREE, Proc. Roy. Soc. (London), B i 24 (i938) 397. 4 B. AXELROD AND R. S. BANDURSKI. J. Biol. Chem., 204 (1953) 939. '50. H. LowRY, N . J . ROSEBROUGH, A. L. FARRAND R. J. RANDALL, J. Biol. Chem., 193 (195 L) z65. 6 T. BUCHER, Biochim. Biophys. Acta, 1 (1947) 292.
Received June 29th, 1962 Biochim. Biophys. ,4cla, 65 (19()2) 355-357