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Isolation of 1-[32P]phosphohistidine from rat-liver cell sap after incubation with [32P]adenosine triphosphate
279
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25698
ISOLATION OF I - ~ 3 2 p I P H O S P H O H I S T I D I N E FROM R A T - L I V E R CELL SAP A F T E R INCUBATION W I T H E~2PIADENOSINE T R I P H O S P H A T E 0 R JAN Z E T T E R Q V I S T
Institute of Medical Chemistry, University of Uppsala, Uppsala (Sweden) (Received July 25th, I966)
SUMMARY
High molecular weight material, obtained from rat-liver cell sap by chromatography on Sephadex G-5o, was incubated for 15 sec at pH 7.4 and o ° with highly labelled t32PIATP. Incubation was terminated by addition of NaOH to a final concentration of o.I M. After removal of excess [a~pIATP on Sephadex G-5 o the a"Plabelled, high molecular weight material was precipitated with acetone and hydrolyzed in 3 M K O H at IOO° for 3 h. In addition to the 3-[a2Plphosphohistidine previously isolated, a2P-labelled material was obtained which co-migrated with synthetic I-phosphohistidine when the alkaline hydrolysate was chromatographed on Dowex I. The identity of this material with I-phosphohistidine was established by paper electrophoresis at five different pH levels, paper chromatography in two systems, rechromatography on Dowex I and a study of its rate of hydrolysis in acid molybdate.
INTRODUCTION
The Ea~p]phosphohistidine isolated from mitochondria by BOYER and coworkers, after incubation with a=Pi, has been shown to be identical with the more stable of the two isomers of monophosphohistidine l. This isomer, as well as the phosphohistidine prepared by RATHLEV AND ROSENBERG 2, has recently been characterized as 3-phosphohistidine a. Convincing evidence was also presented showing that the other isomer is I-phosphohistidine which, so far, has not been isolated from biological material. In a recent work, a2P-labelled material, identical with the phosphohistidine prepared according to RATHLEV AND ROSENBERG 2, could be isolated flom all rat-liver cell fractions after incubation with ~32PIATP (ref. 4). The time course of acid hydrolysis of the E32plphosphohistidine obtained from the cell-sap fraction might, however, indicate the presence of small amounts of 3~P-labelled material even more labile than the added, synthetic 3-phosphohistidine. A later finding (0. ZETTERQVIST, unpublished observation), i.e., that the two isomers of monophosphohistidine were poorly resolved under the conditions used in Dowex I chromatography in the previous work 4, suggested that part of the small amount of more labile material might be I-phosphohistidine. In fact, when the resolving power of the Dowex I chromatography was improved by increasing the column dimensions and decreasing the slope of the eluting Biochim. Biol)7~vs. Acta, 136 (i967) 279 285
280
O. ZETTERQV J2ST
bicarbonate gradient, I-[32P]phosphohistidine could be isolated from the high molecular weight material of rat-liver cell sap after incubation with [a2P]ATP. An account of the isolation is given in this paper. EXPERIMENTAL
Analytical methods Radioactivity, orthophosphate, histidine and phosphohistidine were deterlnined as described previously 4. Orthophosphate and phosphohistidine, Fig. 4, were assayed according to BARTLETT'S method for the determination of inorganic orthophosphate ~. Conductivity was measured in a Philips conductometer, GM 414 o, with cell P R 951o.
@,~¢theses [y-a2P]ATP was prepared according to ENGSTROM~. I-Phosphohistidine and 3phosphohistidine were prepared according to DELUcA e! al. ~, with the modification that Dowex I in the bicarbonate form was used for isolation, elution being carried out with a linear gradient containing only potassium bicarbonate. The two isomers were desalted by chromatography on Sephadex G-zo (Pharmacia, Uppsala, Sweden), Fig. I. By this procedure, I-phosphohistidine was also separated from any free
F i
O
E %
4~
1.o i~
1-Phosphohistidine
°I
×
g
0.5_
.-> 2
o
0
0 2
10
J
20 Tube numbeP
I
"O---
30
I
40
0
.C
a_
Fig. i. Sephadex G - i o c h r o m a t o g r a m of unlabelled i - p h o s p h o h i s t i d i n e after isolation b y D o w e x I c h r o m a t o g r a p h y . Column dimensions : 3 cm × 3 2 cm. E l u t i o n was performed with t r i e t h y l a m i n e c a r b o n dioxide buffer (pH 9.5), o.o5 M with respect to added triethylamine. F r a c t i o n volume: 4 ml. O - O , conductivity; O - - O , p h o s p h o h i s t i d i n e and histidine.
histidine, but only partially separated front orthophosphate, the latter resulting from hydrolysis of the I-phosphohistidine during Dowex I chromatography. The two isomers thus prepared were subjected to paper electrophoresis at pH 8.25, and their electrophoretic mobilities were in accordance with those reported by HIJLTQUIST, MOYER AND BOYER a.
Preparation of high molecular weight material from ral-liver cell sap 16 g of liver tissue from two male Sprague-Dawley rats were homogenized at o ° Biochim. Biophys. Acta, 136 (1967) 279 285
I-PHOSPHOHISTIDIXE FROM RAT-LIVER CELL SAP
28I
in a Potter-Elvehjem homogenizer with 8o ml of o.25 M sucrose which contained o.ooi M EDTA (pH 7-4). The homogenate was centrifuged in a refrigerated centrifuge (International, model PR-2) at 4ooo × g for 15 rain. The supernatant (87 ml) was centrifuged at 1o5ooo x g for I h at o ° (Spinco, model L). 71 ml of the cell sap thus obtained was carefully sucked off, leaving the top layer (I1.5 ml) which contained lipid material. The cell sap was chromatographed on a Sephadex G-5o (Pharmacia, Uppsala, Sweden) column (5 cm x 3o cm) in triethanolamine-acetic acid (pH 7-4), o.oi M with respect to added triethanolamine, and containing o.oo5 M magnesium acetate. The excluded material, corresponding to the high molecular weight fraction of the cell sap, was obtained in a total volume of 119 ml.
Incubatio~, with [3~P]ATP 69 ml of the high molecular weight material was incubated at o ° with I ml of 3.5" lO-4 M [a2P~ATP, which had a specific radioactivity of 8. 7" IOa counts/min per mffmole. After 15 sec, 7.7 ml of I M NaOH was added, followed 6o sec later by 2.4 ml of IO °o sodium lauryl sulphate, to prevent precipitation of denatured protein. After a further 6o sec, the p H was adjusted to approximately 8.5 by the addition of 15.5 ml of triethanolamine-acetic acid (pH 7.4), I M with respect to added triethanolamine. Low molecular a2P-labelled compounds, mainly unreacted [a2p]ATP, were removed by chromatography on a Sephadex G-5o column (6 cm x 2o cm) equilibrated with triethanolamine-acetic acid (pH 8.5), o.o25 M with respect to added triethanolamine and containing o.25 °'o sodium lauryl sulphate. The fractions containing the a2p_ labelled, high molecular weight material were pooled, and 9 vol. of cold acetone were added. The precipitate formed was collected by centrifugation, washed three times with acetone and dried overnight in vacuo.
Isolation and identification of z-[a2P]phosphohistidi~w The dried precipitate was heated with IO ml of 3 M potassium hydroxide in a sealed test tube for 3 h on a boiling-water bath 7. After dilution with 9 vol. of water E 7° 20
E
3~
x E
.E
.-
8
2
4
1
v >.
:-2 ._>
40
60
BO Tube n u m b e r
100
120
Fig. 2. D o w e x ~ - X I o (bicarbonate) c h r o m a t o g r a m of alkaline h y d r o l y s a t e of cell-sap material (corresponding to 1.6 g of rat-liver tissue) i n c u b a t e d w i t h [a2PIATP. Column dimensions : 1.6 cm x 53 cm. E l u t i o n was p e r f o r m e d w i t h a linear gradient of o.2 to o.8 M p o t a s s i u m bicarbonate. Total elution v o l u m e IOOO ml, collected in 5.5-ml fractions. O - - O , r a d i o a c t i v i t y ; O - - O , p h o s p h o histidine a n d histidine.
Biochim. Biophys. Acta, 136 (1967) 279-285
282
O. ZETTERQVIST
and filtration, 2o ml of the diluted hydrolysate was mixed with 8.9 t~moles of unlabelled I-phosphohistidine, which contained no free histidine. The mixture was c h r o m a t o g r a p h e d on a Dowex I - X I o (bicarbonate) colunm (Fig. 2). 8o % of the material migrating as I-phosphohistidine was diluted with 3 vol. of water, and rec h r o m a t o g r a p h e d on another Dowex I column (Fig. 3). Of the material still migrating as i-phosphohistidine, 80 % was rechromatographed, after dilution, on a third b o w e x i column (Fig. 4).
o O
0.6
~30.6 1-Phosphohistidine
o
20
40 Tube number
60
]
80
Fig. 3. Dowex I - X I o (bicarbonate) c h r o m a t o g r a m of 80 % of t h e I-[32P]phosphohistidine peak in Fig. 2. Column dimensions: z.2 cm × 44 cm. Elution was performed with a linear gradient of o.2 to o.8 M potassium bicarbonate. Total elution volume 6oo nil, collected in 5-ml fractions. O - - O , radioactivity; O - - Q , phosphohistidine and histidine.
D.3~
g O_
/0.2~
o 4i
!o.2t .~
0.1 ~ ~E
1-Phosphohistidine
2U el CX~
c~
0
20
40 Tube number-
60
80
Fig. 4- Dowex I - X I o (bicarbonate) c h r o m a t o g r a m of 8o°J.,o of the I-[32P]phosphohistidine. peak in Fig. 3, Column dimensions: 1.2 cm × 43 cm. Elution was performed as in Fig. 3. © - - O , radioactivity; Q - - O , Pt and phosphohistidine.
The radioactive material to be studied by paper electrophoresis and paper c h r o m a t o g r a p h y was obtained in the following way. Diluted hydrolysate of 3~p_ labelled cell-sap material was chromatographed as described in Fig. 2, except t h a t no I3iochim. t3iophys. ,'fcta, i36 (1967) 279-285
I-PHOSPHOHISTIDINE FROM RAT-LIVER CELL SAP
283
unlabelled I-phosphohistidine was added. The radioactive material, which was eluted in the position of I-phosphohistidine, was rechromatographed as described in Fig. 3. The radioactive material which still migrated as i-phosphohistidine was concentrated i n vacuo at 2o ° by rotary evaporation to 1/5 of its volume, and chromatographed on a Sephadex G-io column as described in Fig. I, except that the buffer used was o.oo2 M with respect to added triethylamine. The radioactive material was thus obtained free from salt, and was concentrated to o.5 ml by rotary evaporation. Aliquots of 2o/zl of this concentrate were mixed with 25 m~moles of desalted, unlabelled I-phosphohistidine and subjected to paper chromatography and highvoltage paper electrophoresis. Two paper-chromatographic systems were used ~. Electrophoresis was carried out at o ° and 4 ° V/cm (using a Pherograph Original Frankfurt D.B.G.M No. 1713858, L. Hormuth, Heidelberg-Wiesloch, Germany). The following buffers were used: (a) 4 M acetic acid (pH 1.9), (b) I M acetic acid (pH 2.3), (c) triethylamine-acetic acid (pH 5.5), o.o5 M with respect to added acetic acid, (d) triethylamine-carbon dioxide (pH 8.5), o.o5 M with respect to added triethylamine, (e) triethylamine-carbon dioxide (pH 9.5), o.o5 M with respect to added triethylamine. After drying at IOO°, the chromatograms and electrophoretograms were sprayed with ninhydrin s, followed b y molybdate reagent 9 to detect unlabelled Iphosphohistidine and orthophosphate. Autoradiography of the papers was carried out with Ilford Ilfex X-ray film (exposure time 5 days). RESULTS
The Dowex i chromatogram of an alkaline hydrolysate of a2p-labelled, high molecular weight material from rat-liver cell sap shows that part of the radioactive material was eluted with added, unlabelled, I-phosphohistidine (Fig. 2). On rechromatography, about 15 °/o of the i-phosphohistidine-containing radioactive material still migrated as unlabelled I-phosphohistidine, the remainder appearing as a2PL (Fig. 3)- Only part of this a2Pi could, however, have been derived from I-[32P~phosphohistidine, since about 5o % of the rechromatographed, unlabelled, I-phosphohistidine remained intact. The alkaline hydrolysate m a y thus have contained a2P-labelled material which was even more labile than I-phosphohistidine on Dowex I chromatography. When rechromatographed once more, the i-phosphohistidine-containing material proved to contain two radioactive components, the first migrating as orthophosphate, the second as I-phosphohistidine (Fig. 4). Both peaks showed approximately the same specific radioactivity. The second peak was examined as described in Table I. Most of the radioactive material had the same lability as synthetic I-phosphohistidine. Such extreme lability in acid molybdate has recently been reported for I-phosphohistidine by HULTQU~IST, MOYER AND BOYER3. The specific radioactivity of the labile material was the same as that of the I-phosphohistidine-containing peak in Fig. 3- Analysis of the radioactive material eluted in the position of i-phosphohistidine b y paper electrophoresis and paper chromatography, as described under EXPERIMENTAL,revealed two radioactive components of approximately equal intensity, one migrating as I-phosphohistidine, the other as orthophosphate. The aforementioned results establish the identity of the main part of the radioBiochim. Biophys. Acta, I30 (19()7) 279 285
284
i5. ZETTEI,~QVIST
TABLE i SPECIFIC HISTIDINE
RADIOACTIVITY
OF THE
CONTAINING
MATERIAL
ORTHOPHOSPHATE AFTER
LIBERATED
TREATMENT
FROM PURIFIED
\VITH ACID AND
I-PHOSPHO-
ACID MOLYBDATE
The three peak fractions of the l-phosphohistidine-containing peak in 1;ig. 4 were pooled, and I.O-nll aliquots were used for the following analyses. A. The aliquots were diluted with 1.o ml of water, 2.5 ml of a benzene isobutanol mixture and o. 5 ml of acid ammonium molybdate reagent ~2 were added, and the phosphomolybdic acid rapidly extracted as previously described 4. ]3. Tile aliquots were diluted with o. 5 inl of water and o. 5 ml of 2 M H 2 S O 4. After 3o min at 2o '', 2.5 ml of benzene-isobutanol mixture and o. 5 ml of neutral ammoniunl molybdate reagent 12 were added, and extraction carried out as before. Radioactivity4 and phosphomolybdic acid 12 were measured on I.o-ni1 aliquots of the organic phase. The values given are means of 4 analyses.
Sample
:~'2p~ t'adioactivily (com~ls/mi~)
:~.,.p~ (/tmoles)
Specific ~'adioactivit)'
A
I ~4
o.028
41oo
]3
127
o.03 ~
4too
oj
a2P i
cot~llts/mi~ per/*~Jzole
active m a t e r i a l in the second radioactive peak, Fig. 3, as x-phosphohistidine. Analysis for phosphohistidine a n d histidine in the first a n d second Dowex I chromatograms (Figs. 2 a n d 3) revealed two peaks, the first of which represented histidine and the second i-phosphohistidine. No measurable a m o u n t s of phosphohistidine could be d e m o n s t r a t e d unless unlabelled x-phosphohistidine was added to the hydrolysate before the first Dowex I chromatography. Since the added, unlabelled I-phost)hohistidine c o n t a i n e d no detectable a m o u n t s of free histidine, a n d only a b o u t 3o o; of the histidine in Fig. 2 derived from the cell-sap material, it can be concluded t h a t Iphosphohistidine was hydrolyzed to a considerable degree during Dowex I chromatography. Consequently, the original a m o u n t of I-[32Pi]phosphohistidine in the alkaline hydrolysate of 32P-labelled cell-sap m a t e r i a l has to be calculated from the a m o u n t of added, unlabelled, I-phosphohistidine (S. 9 /~moles) a n d the specific r a d i o a c t i v i t y of the isolated I-~a~Plphosphohistidine, which was 41oo c o u n t s / m i n per ~mole, according to Table I. P r o v i d e d t h a t the specific r a d i o a c t i v i t y of the I-[a2P)phosphohistidine in the alkaline hydrolysate before addition of unlabelled i-phosphohistidine was the same as t h a t of the [3zP]ATP, it can be calculated t h a t a b o u t o.o3 m~moles of I[32P]phosphohistidine could be isolated from each gram of rat-liver tissue. If N a O H was added to the cell-sap material prior to addition of ['~2P]ATP the only radioactive material detected in a Dowex I c h r o m a t o g r a m was a m i n u t e a m o u n t of 32Pi. I t is thus obvious t h a t the cell-sap material m u s t be u n d e n a t u r e d for formation of the a2P-labelled m a t e r i a l from which i-[a2P]phosphohistidine can be derived. The first peak after I-[a2P]phosphohistidine in Fig. 2 co-migrated with unlabelled 3-phosphohistidine on r e c h r o m a t o g r a p h y on Dowex I. This obviously represented the [aeP]phosphohistidine studied earlier ~, a n d a m o u n t e d to a p p r o x i m a t e l y o.2 m~moles per gram of rat-liver tissue. The peak s u b s e q u e n t to 3-[32P~phosp hohistidine in Fig. 2 has not been further studied. However, it is likely t h a t it c o n t a i n e d a2P-labelled phosphohistidine peptides, in accordance with previous work 4. The i d e n t i t y of the radioactive material in the first peak after 32P1 in Fig. 2 is u n d e r investigation.
Biochim. Biophys. Acta, i36 (~967) 279-285
I-PHOSPHOHISTIDINE FROM RAT-LIVER CELL SAP
285
DISCUSSION
This is evidently the first isolation of i-phosphohistidine from biological material. One possible origin of this I-phosphohistidine, as well as of the 3-phosphohistidine previously isolated from rat-liver cell sap*, seems to be active sites of phosphorylenzymes, as in the case of 3-phosphohistidine in mitochondria 1°. However, this possibility awaits verification. The amount of I-E32P~phosphohistidine isolated from rat-liver cell sap was considerably smaller than that of 3-E32PJphosphohistidine isolated from the same source and by the same methods. With the present data it is difficult to know whether any interrelation exists between the formation of the two isomers. The isomers may, for example, be derived from different proteins, or from the same or different histidine residues in the same protein. With respect to the last possibility, it can be pointed out that the reactivity of the two imidazole nitrogens may differ in different histidine residues of the same protein, as has been shown to apply in the alkylation of ribonuclease n. The possibility of phosphoryl-group migration must also be considered in this context. It is, however, obvious that no definite conclusions can be drawn concerning the formation of the two isomers of monophosphohistidine, until the cell-sap material from which the isomers are isolated has been purified and characterized. ACKNOWLEDGEMENTS
I wish to thank Dr. L. ENGSTRIJM for invaluable discussions throughout the work. I am also indebted to Miss I. SVEXSSONand Miss M. LINDQUIST for their skilful assistance. The work was supported by grants to Dr. L. ENGSTROM from the Swedish Medical Research Council (Project No. I3X-5o-o2), and by grants to the author from the Medical Faculty of the University of Uppsala, and Svenska S/~llskapet f6r Medicinsk Forskning. REFERENCES t M. DELucA, I¢. E. EBNER, D. E. HULTQUIST, G. KREIL, J. B. PETER, R. W. MOYER AND l ). D. BOYER, Biochem. Z., 338 (1963) 512. 2 T. RATHLEV AND T. ROSENBERG, Arch. Biochem. Biophys., 65 (1956) 319. 3 D. E. HULTQUIST,R_. \V. MOYER AND P. El. BOYER, Biochemistry, 5 (1966) 322. 4 0. ZETTERQVIST AND L. ENGSTR6M, Biochim. Biophys. Acta, 113 (1966) 520. 5 G. 1~. BARTLETT, J. Biol. Chem., 234 (1959) 466. 6 L. ENGSTR6M, Arkiv Kemi, 19 (1962) 129. 7 P. D. ]3OYER, M. DELUCA, K. E. EBNER, D. E. HULTQUIST AND J. ]3. PETER, J. Biol. Chem., 237 (1962) PC 3306. 8 A. L. LEVY AND D. CHUNG, Anal. Chem., :'5 (I953) 396. 9 R. S. ]AANDURSKI AND ]3. AXELROD, J. Biol. Chem., 193 (1951) 405 • IO ]~_. A. MITCHELL, L. G. BUTLER AND P. D. ]3OYER, Biochem. Biophys. Res. Commun., 16 (1964) 545. I I A. M. CRESTFIELD, \V. H. STEIN AND S. M~OORE, J. Biol. Chem., 238 (1963) 2413. 12 J. ]3. MARTIN AND D. M. DOTY, Anal. Chem., 21 (1949) 965.
Biochim. Biophys. Acta, 136 (1967) 279 285
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25698
ISOLATION OF I - ~ 3 2 p I P H O S P H O H I S T I D I N E FROM R A T - L I V E R CELL SAP A F T E R INCUBATION W I T H E~2PIADENOSINE T R I P H O S P H A T E 0 R JAN Z E T T E R Q V I S T
Institute of Medical Chemistry, University of Uppsala, Uppsala (Sweden) (Received July 25th, I966)
SUMMARY
High molecular weight material, obtained from rat-liver cell sap by chromatography on Sephadex G-5o, was incubated for 15 sec at pH 7.4 and o ° with highly labelled t32PIATP. Incubation was terminated by addition of NaOH to a final concentration of o.I M. After removal of excess [a~pIATP on Sephadex G-5 o the a"Plabelled, high molecular weight material was precipitated with acetone and hydrolyzed in 3 M K O H at IOO° for 3 h. In addition to the 3-[a2Plphosphohistidine previously isolated, a2P-labelled material was obtained which co-migrated with synthetic I-phosphohistidine when the alkaline hydrolysate was chromatographed on Dowex I. The identity of this material with I-phosphohistidine was established by paper electrophoresis at five different pH levels, paper chromatography in two systems, rechromatography on Dowex I and a study of its rate of hydrolysis in acid molybdate.
INTRODUCTION
The Ea~p]phosphohistidine isolated from mitochondria by BOYER and coworkers, after incubation with a=Pi, has been shown to be identical with the more stable of the two isomers of monophosphohistidine l. This isomer, as well as the phosphohistidine prepared by RATHLEV AND ROSENBERG 2, has recently been characterized as 3-phosphohistidine a. Convincing evidence was also presented showing that the other isomer is I-phosphohistidine which, so far, has not been isolated from biological material. In a recent work, a2P-labelled material, identical with the phosphohistidine prepared according to RATHLEV AND ROSENBERG 2, could be isolated flom all rat-liver cell fractions after incubation with ~32PIATP (ref. 4). The time course of acid hydrolysis of the E32plphosphohistidine obtained from the cell-sap fraction might, however, indicate the presence of small amounts of 3~P-labelled material even more labile than the added, synthetic 3-phosphohistidine. A later finding (0. ZETTERQVIST, unpublished observation), i.e., that the two isomers of monophosphohistidine were poorly resolved under the conditions used in Dowex I chromatography in the previous work 4, suggested that part of the small amount of more labile material might be I-phosphohistidine. In fact, when the resolving power of the Dowex I chromatography was improved by increasing the column dimensions and decreasing the slope of the eluting Biochim. Biol)7~vs. Acta, 136 (i967) 279 285
280
O. ZETTERQV J2ST
bicarbonate gradient, I-[32P]phosphohistidine could be isolated from the high molecular weight material of rat-liver cell sap after incubation with [a2P]ATP. An account of the isolation is given in this paper. EXPERIMENTAL
Analytical methods Radioactivity, orthophosphate, histidine and phosphohistidine were deterlnined as described previously 4. Orthophosphate and phosphohistidine, Fig. 4, were assayed according to BARTLETT'S method for the determination of inorganic orthophosphate ~. Conductivity was measured in a Philips conductometer, GM 414 o, with cell P R 951o.
@,~¢theses [y-a2P]ATP was prepared according to ENGSTROM~. I-Phosphohistidine and 3phosphohistidine were prepared according to DELUcA e! al. ~, with the modification that Dowex I in the bicarbonate form was used for isolation, elution being carried out with a linear gradient containing only potassium bicarbonate. The two isomers were desalted by chromatography on Sephadex G-zo (Pharmacia, Uppsala, Sweden), Fig. I. By this procedure, I-phosphohistidine was also separated from any free
F i
O
E %
4~
1.o i~
1-Phosphohistidine
°I
×
g
0.5_
.-> 2
o
0
0 2
10
J
20 Tube numbeP
I
"O---
30
I
40
0
.C
a_
Fig. i. Sephadex G - i o c h r o m a t o g r a m of unlabelled i - p h o s p h o h i s t i d i n e after isolation b y D o w e x I c h r o m a t o g r a p h y . Column dimensions : 3 cm × 3 2 cm. E l u t i o n was performed with t r i e t h y l a m i n e c a r b o n dioxide buffer (pH 9.5), o.o5 M with respect to added triethylamine. F r a c t i o n volume: 4 ml. O - O , conductivity; O - - O , p h o s p h o h i s t i d i n e and histidine.
histidine, but only partially separated front orthophosphate, the latter resulting from hydrolysis of the I-phosphohistidine during Dowex I chromatography. The two isomers thus prepared were subjected to paper electrophoresis at pH 8.25, and their electrophoretic mobilities were in accordance with those reported by HIJLTQUIST, MOYER AND BOYER a.
Preparation of high molecular weight material from ral-liver cell sap 16 g of liver tissue from two male Sprague-Dawley rats were homogenized at o ° Biochim. Biophys. Acta, 136 (1967) 279 285
I-PHOSPHOHISTIDIXE FROM RAT-LIVER CELL SAP
28I
in a Potter-Elvehjem homogenizer with 8o ml of o.25 M sucrose which contained o.ooi M EDTA (pH 7-4). The homogenate was centrifuged in a refrigerated centrifuge (International, model PR-2) at 4ooo × g for 15 rain. The supernatant (87 ml) was centrifuged at 1o5ooo x g for I h at o ° (Spinco, model L). 71 ml of the cell sap thus obtained was carefully sucked off, leaving the top layer (I1.5 ml) which contained lipid material. The cell sap was chromatographed on a Sephadex G-5o (Pharmacia, Uppsala, Sweden) column (5 cm x 3o cm) in triethanolamine-acetic acid (pH 7-4), o.oi M with respect to added triethanolamine, and containing o.oo5 M magnesium acetate. The excluded material, corresponding to the high molecular weight fraction of the cell sap, was obtained in a total volume of 119 ml.
Incubatio~, with [3~P]ATP 69 ml of the high molecular weight material was incubated at o ° with I ml of 3.5" lO-4 M [a2P~ATP, which had a specific radioactivity of 8. 7" IOa counts/min per mffmole. After 15 sec, 7.7 ml of I M NaOH was added, followed 6o sec later by 2.4 ml of IO °o sodium lauryl sulphate, to prevent precipitation of denatured protein. After a further 6o sec, the p H was adjusted to approximately 8.5 by the addition of 15.5 ml of triethanolamine-acetic acid (pH 7.4), I M with respect to added triethanolamine. Low molecular a2P-labelled compounds, mainly unreacted [a2p]ATP, were removed by chromatography on a Sephadex G-5o column (6 cm x 2o cm) equilibrated with triethanolamine-acetic acid (pH 8.5), o.o25 M with respect to added triethanolamine and containing o.25 °'o sodium lauryl sulphate. The fractions containing the a2p_ labelled, high molecular weight material were pooled, and 9 vol. of cold acetone were added. The precipitate formed was collected by centrifugation, washed three times with acetone and dried overnight in vacuo.
Isolation and identification of z-[a2P]phosphohistidi~w The dried precipitate was heated with IO ml of 3 M potassium hydroxide in a sealed test tube for 3 h on a boiling-water bath 7. After dilution with 9 vol. of water E 7° 20
E
3~
x E
.E
.-
8
2
4
1
v >.
:-2 ._>
40
60
BO Tube n u m b e r
100
120
Fig. 2. D o w e x ~ - X I o (bicarbonate) c h r o m a t o g r a m of alkaline h y d r o l y s a t e of cell-sap material (corresponding to 1.6 g of rat-liver tissue) i n c u b a t e d w i t h [a2PIATP. Column dimensions : 1.6 cm x 53 cm. E l u t i o n was p e r f o r m e d w i t h a linear gradient of o.2 to o.8 M p o t a s s i u m bicarbonate. Total elution v o l u m e IOOO ml, collected in 5.5-ml fractions. O - - O , r a d i o a c t i v i t y ; O - - O , p h o s p h o histidine a n d histidine.
Biochim. Biophys. Acta, 136 (1967) 279-285
282
O. ZETTERQVIST
and filtration, 2o ml of the diluted hydrolysate was mixed with 8.9 t~moles of unlabelled I-phosphohistidine, which contained no free histidine. The mixture was c h r o m a t o g r a p h e d on a Dowex I - X I o (bicarbonate) colunm (Fig. 2). 8o % of the material migrating as I-phosphohistidine was diluted with 3 vol. of water, and rec h r o m a t o g r a p h e d on another Dowex I column (Fig. 3). Of the material still migrating as i-phosphohistidine, 80 % was rechromatographed, after dilution, on a third b o w e x i column (Fig. 4).
o O
0.6
~30.6 1-Phosphohistidine
o
20
40 Tube number
60
]
80
Fig. 3. Dowex I - X I o (bicarbonate) c h r o m a t o g r a m of 80 % of t h e I-[32P]phosphohistidine peak in Fig. 2. Column dimensions: z.2 cm × 44 cm. Elution was performed with a linear gradient of o.2 to o.8 M potassium bicarbonate. Total elution volume 6oo nil, collected in 5-ml fractions. O - - O , radioactivity; O - - Q , phosphohistidine and histidine.
D.3~
g O_
/0.2~
o 4i
!o.2t .~
0.1 ~ ~E
1-Phosphohistidine
2U el CX~
c~
0
20
40 Tube number-
60
80
Fig. 4- Dowex I - X I o (bicarbonate) c h r o m a t o g r a m of 8o°J.,o of the I-[32P]phosphohistidine. peak in Fig. 3, Column dimensions: 1.2 cm × 43 cm. Elution was performed as in Fig. 3. © - - O , radioactivity; Q - - O , Pt and phosphohistidine.
The radioactive material to be studied by paper electrophoresis and paper c h r o m a t o g r a p h y was obtained in the following way. Diluted hydrolysate of 3~p_ labelled cell-sap material was chromatographed as described in Fig. 2, except t h a t no I3iochim. t3iophys. ,'fcta, i36 (1967) 279-285
I-PHOSPHOHISTIDINE FROM RAT-LIVER CELL SAP
283
unlabelled I-phosphohistidine was added. The radioactive material, which was eluted in the position of I-phosphohistidine, was rechromatographed as described in Fig. 3. The radioactive material which still migrated as i-phosphohistidine was concentrated i n vacuo at 2o ° by rotary evaporation to 1/5 of its volume, and chromatographed on a Sephadex G-io column as described in Fig. I, except that the buffer used was o.oo2 M with respect to added triethylamine. The radioactive material was thus obtained free from salt, and was concentrated to o.5 ml by rotary evaporation. Aliquots of 2o/zl of this concentrate were mixed with 25 m~moles of desalted, unlabelled I-phosphohistidine and subjected to paper chromatography and highvoltage paper electrophoresis. Two paper-chromatographic systems were used ~. Electrophoresis was carried out at o ° and 4 ° V/cm (using a Pherograph Original Frankfurt D.B.G.M No. 1713858, L. Hormuth, Heidelberg-Wiesloch, Germany). The following buffers were used: (a) 4 M acetic acid (pH 1.9), (b) I M acetic acid (pH 2.3), (c) triethylamine-acetic acid (pH 5.5), o.o5 M with respect to added acetic acid, (d) triethylamine-carbon dioxide (pH 8.5), o.o5 M with respect to added triethylamine, (e) triethylamine-carbon dioxide (pH 9.5), o.o5 M with respect to added triethylamine. After drying at IOO°, the chromatograms and electrophoretograms were sprayed with ninhydrin s, followed b y molybdate reagent 9 to detect unlabelled Iphosphohistidine and orthophosphate. Autoradiography of the papers was carried out with Ilford Ilfex X-ray film (exposure time 5 days). RESULTS
The Dowex i chromatogram of an alkaline hydrolysate of a2p-labelled, high molecular weight material from rat-liver cell sap shows that part of the radioactive material was eluted with added, unlabelled, I-phosphohistidine (Fig. 2). On rechromatography, about 15 °/o of the i-phosphohistidine-containing radioactive material still migrated as unlabelled I-phosphohistidine, the remainder appearing as a2PL (Fig. 3)- Only part of this a2Pi could, however, have been derived from I-[32P~phosphohistidine, since about 5o % of the rechromatographed, unlabelled, I-phosphohistidine remained intact. The alkaline hydrolysate m a y thus have contained a2P-labelled material which was even more labile than I-phosphohistidine on Dowex I chromatography. When rechromatographed once more, the i-phosphohistidine-containing material proved to contain two radioactive components, the first migrating as orthophosphate, the second as I-phosphohistidine (Fig. 4). Both peaks showed approximately the same specific radioactivity. The second peak was examined as described in Table I. Most of the radioactive material had the same lability as synthetic I-phosphohistidine. Such extreme lability in acid molybdate has recently been reported for I-phosphohistidine by HULTQU~IST, MOYER AND BOYER3. The specific radioactivity of the labile material was the same as that of the I-phosphohistidine-containing peak in Fig. 3- Analysis of the radioactive material eluted in the position of i-phosphohistidine b y paper electrophoresis and paper chromatography, as described under EXPERIMENTAL,revealed two radioactive components of approximately equal intensity, one migrating as I-phosphohistidine, the other as orthophosphate. The aforementioned results establish the identity of the main part of the radioBiochim. Biophys. Acta, I30 (19()7) 279 285
284
i5. ZETTEI,~QVIST
TABLE i SPECIFIC HISTIDINE
RADIOACTIVITY
OF THE
CONTAINING
MATERIAL
ORTHOPHOSPHATE AFTER
LIBERATED
TREATMENT
FROM PURIFIED
\VITH ACID AND
I-PHOSPHO-
ACID MOLYBDATE
The three peak fractions of the l-phosphohistidine-containing peak in 1;ig. 4 were pooled, and I.O-nll aliquots were used for the following analyses. A. The aliquots were diluted with 1.o ml of water, 2.5 ml of a benzene isobutanol mixture and o. 5 ml of acid ammonium molybdate reagent ~2 were added, and the phosphomolybdic acid rapidly extracted as previously described 4. ]3. Tile aliquots were diluted with o. 5 inl of water and o. 5 ml of 2 M H 2 S O 4. After 3o min at 2o '', 2.5 ml of benzene-isobutanol mixture and o. 5 ml of neutral ammoniunl molybdate reagent 12 were added, and extraction carried out as before. Radioactivity4 and phosphomolybdic acid 12 were measured on I.o-ni1 aliquots of the organic phase. The values given are means of 4 analyses.
Sample
:~'2p~ t'adioactivily (com~ls/mi~)
:~.,.p~ (/tmoles)
Specific ~'adioactivit)'
A
I ~4
o.028
41oo
]3
127
o.03 ~
4too
oj
a2P i
cot~llts/mi~ per/*~Jzole
active m a t e r i a l in the second radioactive peak, Fig. 3, as x-phosphohistidine. Analysis for phosphohistidine a n d histidine in the first a n d second Dowex I chromatograms (Figs. 2 a n d 3) revealed two peaks, the first of which represented histidine and the second i-phosphohistidine. No measurable a m o u n t s of phosphohistidine could be d e m o n s t r a t e d unless unlabelled x-phosphohistidine was added to the hydrolysate before the first Dowex I chromatography. Since the added, unlabelled I-phost)hohistidine c o n t a i n e d no detectable a m o u n t s of free histidine, a n d only a b o u t 3o o; of the histidine in Fig. 2 derived from the cell-sap material, it can be concluded t h a t Iphosphohistidine was hydrolyzed to a considerable degree during Dowex I chromatography. Consequently, the original a m o u n t of I-[32Pi]phosphohistidine in the alkaline hydrolysate of 32P-labelled cell-sap m a t e r i a l has to be calculated from the a m o u n t of added, unlabelled, I-phosphohistidine (S. 9 /~moles) a n d the specific r a d i o a c t i v i t y of the isolated I-~a~Plphosphohistidine, which was 41oo c o u n t s / m i n per ~mole, according to Table I. P r o v i d e d t h a t the specific r a d i o a c t i v i t y of the I-[a2P)phosphohistidine in the alkaline hydrolysate before addition of unlabelled i-phosphohistidine was the same as t h a t of the [3zP]ATP, it can be calculated t h a t a b o u t o.o3 m~moles of I[32P]phosphohistidine could be isolated from each gram of rat-liver tissue. If N a O H was added to the cell-sap material prior to addition of ['~2P]ATP the only radioactive material detected in a Dowex I c h r o m a t o g r a m was a m i n u t e a m o u n t of 32Pi. I t is thus obvious t h a t the cell-sap material m u s t be u n d e n a t u r e d for formation of the a2P-labelled m a t e r i a l from which i-[a2P]phosphohistidine can be derived. The first peak after I-[a2P]phosphohistidine in Fig. 2 co-migrated with unlabelled 3-phosphohistidine on r e c h r o m a t o g r a p h y on Dowex I. This obviously represented the [aeP]phosphohistidine studied earlier ~, a n d a m o u n t e d to a p p r o x i m a t e l y o.2 m~moles per gram of rat-liver tissue. The peak s u b s e q u e n t to 3-[32P~phosp hohistidine in Fig. 2 has not been further studied. However, it is likely t h a t it c o n t a i n e d a2P-labelled phosphohistidine peptides, in accordance with previous work 4. The i d e n t i t y of the radioactive material in the first peak after 32P1 in Fig. 2 is u n d e r investigation.
Biochim. Biophys. Acta, i36 (~967) 279-285
I-PHOSPHOHISTIDINE FROM RAT-LIVER CELL SAP
285
DISCUSSION
This is evidently the first isolation of i-phosphohistidine from biological material. One possible origin of this I-phosphohistidine, as well as of the 3-phosphohistidine previously isolated from rat-liver cell sap*, seems to be active sites of phosphorylenzymes, as in the case of 3-phosphohistidine in mitochondria 1°. However, this possibility awaits verification. The amount of I-E32P~phosphohistidine isolated from rat-liver cell sap was considerably smaller than that of 3-E32PJphosphohistidine isolated from the same source and by the same methods. With the present data it is difficult to know whether any interrelation exists between the formation of the two isomers. The isomers may, for example, be derived from different proteins, or from the same or different histidine residues in the same protein. With respect to the last possibility, it can be pointed out that the reactivity of the two imidazole nitrogens may differ in different histidine residues of the same protein, as has been shown to apply in the alkylation of ribonuclease n. The possibility of phosphoryl-group migration must also be considered in this context. It is, however, obvious that no definite conclusions can be drawn concerning the formation of the two isomers of monophosphohistidine, until the cell-sap material from which the isomers are isolated has been purified and characterized. ACKNOWLEDGEMENTS
I wish to thank Dr. L. ENGSTRIJM for invaluable discussions throughout the work. I am also indebted to Miss I. SVEXSSONand Miss M. LINDQUIST for their skilful assistance. The work was supported by grants to Dr. L. ENGSTROM from the Swedish Medical Research Council (Project No. I3X-5o-o2), and by grants to the author from the Medical Faculty of the University of Uppsala, and Svenska S/~llskapet f6r Medicinsk Forskning. REFERENCES t M. DELucA, I¢. E. EBNER, D. E. HULTQUIST, G. KREIL, J. B. PETER, R. W. MOYER AND l ). D. BOYER, Biochem. Z., 338 (1963) 512. 2 T. RATHLEV AND T. ROSENBERG, Arch. Biochem. Biophys., 65 (1956) 319. 3 D. E. HULTQUIST,R_. \V. MOYER AND P. El. BOYER, Biochemistry, 5 (1966) 322. 4 0. ZETTERQVIST AND L. ENGSTR6M, Biochim. Biophys. Acta, 113 (1966) 520. 5 G. 1~. BARTLETT, J. Biol. Chem., 234 (1959) 466. 6 L. ENGSTR6M, Arkiv Kemi, 19 (1962) 129. 7 P. D. ]3OYER, M. DELUCA, K. E. EBNER, D. E. HULTQUIST AND J. ]3. PETER, J. Biol. Chem., 237 (1962) PC 3306. 8 A. L. LEVY AND D. CHUNG, Anal. Chem., :'5 (I953) 396. 9 R. S. ]AANDURSKI AND ]3. AXELROD, J. Biol. Chem., 193 (1951) 405 • IO ]~_. A. MITCHELL, L. G. BUTLER AND P. D. ]3OYER, Biochem. Biophys. Res. Commun., 16 (1964) 545. I I A. M. CRESTFIELD, \V. H. STEIN AND S. M~OORE, J. Biol. Chem., 238 (1963) 2413. 12 J. ]3. MARTIN AND D. M. DOTY, Anal. Chem., 21 (1949) 965.
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