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Use of pulse labeling technique in protein structure determination: ordering of the cyanogen bromide peptides from porcine pancreatic α-amylase
222
BBA
B I O C H I M I C A E T B I O P H Y S I C A ACTA
36022
USE OF P U L S E L A B E L I N G T E C H N I Q U E IN P R O T E I N S T R U C T U R E D E T E R M I N A T I O N : O R D E R I N G OF T H E CYANOGEN B R O M I D E P E P T I D E S FROM P O R C I N E PANCREATIC a-AMYLASE
P. COZZONE AND G. MARCHIS-MOUREN
Institut de Chimie Biologique, Place Victor Hugo-z3, Marseille (3 °) (France) (Received September 6th, 1971)
SUMMARY
The nine peptides isolated after cleavage of the pancreatic amylase polypeptidic chain by cyanogen bromide, have been ordered by the use of an isotopic technique based upon pulse labeling. Porcine pancreatic slices were incubated with radioactive valine for 3 rain. Differential labeling of the peptides was obtained. As shown by comparison of the relative specific radioactivity of valine in homologous peptides, the results are reproducible. The peptides have then been lined up unambiguously from the amino terminal to the carboxyl terminal end in the following order V I I I , II, IV, VII, I, VI, V, IX, I I I . The position of the terminal peptides ( V I I I and I I I ) agrees with structural determination. As a control the relative specific radioactivity of the nine peptides was determined under two different labeling conditions. In the first control experiment, excess unlabeled valine was added after incubating for 3 rain with [3H]valine and the incubation carried on for 4 h. In the second control experiment [aH]valine was replaced by a 14C-labeled algae protein hydrolyzate. In both cases, although the slope of the radioactivity gradient was quite different from before, the same order was obtained.
INTRODUCTION Porcine pancreatic a-amylase consists of a single polypeptide chain of about 47 ° amino acid residues 1. Determination of the primary structure of such a long peptide chain is a rather formidable task. Because its amino terminal end is blocked by an acetyl group 2, the amino acid sequence at this end cannot be determined by the E d m a n procedure using a sequencer. Classical methods based on comparison of overlapping peptides are time consuming, especially in the case of a long peptide chain, since a large number of peptides have to be isolated and analyzed. This is the reason why in the case of the 47 ° residues long amylase chain an isotopic technique has been employed for ordering the peptides. Biochim. Biophys. dcta, 257 (1972) 222-229
C Y A N O G E N B R O M I D E P E P T I D E S FROM or-AMYLASE
223
This method is based upon the findings of DINTZIS3 in the study of globin biosynthesis: after incubating reticulocytes with radioactive amino acid for a short period of time, differential labeling of tryptic peptides from globin was obtained. Since the primary structure of rabbit a-globin was already known, DINTZIS~ could thus demonstrate that the globin chain is growing by sequential addition of amino acids from the N-terminal end to the C-terminal position. The same polarity in the elongation process was also found in the biosynthesis of other polypeptide chains of known sequence such as ribonuclease 4 and lysozyme 5. It is now admitted that this mechanism is universal. More recently the pulse labeling technique was applied by FLEISCHMAN~ to study the biosynthesis of v-globulin heavy chain and by VUUST AND PIEZ~ in the case of a-chains of collagen. In view of the determination of the primary structure of a-amylase this technique was presently used for ordering the nine peptides resulting from the cleavage of the amylase chain by cyanogen bromide. These peptides have been previously purified and characterized by molecular weight determination, end groups and amino acid analyses 8. Porcine pancreatic slices were incubated for short period of time with labeled amino acid. Radioactive amylase was then isolated and submitted to CNBr treatment. Unequal labeling of peptides was obtained. I t was then possible to line them up on the basis of the specific radioactivity which increases from the N-terminal to the C-terminal peptides. MATERIALS AND METHODS
Porcine pancreas was cut in small pieces (5-8 g) and incubated at 25 ° in 75 ml of Krebs I I I medium 9. For the 3-min pulse experiments the incubation medium contained 250/~C of L-[aHlvaline (25 C/mmole, CEA, Saclay, France) or 125 #C of 1*C-labeled algae protein hydrolyzate (0. 5 mC/mg, CEA, Saclay). Incorporations were stopped by cooling. In the chase experiment a lO 4 molar excess of unlabeled valine was added and the incubation continued under 02 in a Dubnoff metabolic shaking incubator. At the end of the labeling period, the medium was poured off and the tissue was thoroughly rinsed with 250 ml of cold 0.9% NaC1. All subsequent operations were performed at 2-4 °. The tissue was homogenized in presence of D F P (IO 3 M) in o.I M sodium phosphate buffer p H 8.0 with a Potter-Elvehjem homogenizer. The homogenate was sonicated 3 times at io-sec intervals for 30 sec (8 A audiofrequency current), then centrifuged for 2 h at lO5 ooo × g. The clear supernatant was lyophilized after overnight dialysis against water. To the radioactive lyophilized powder (5-700 mg), unlabeled lyophilizate powder (I.8-2 g) was added as a carrier. Amylase was then purified 1°. Pure labeled amylase (3/,moles) was cleaved by cyanogen bromide n and the CNBr peptides were isolated by gel filtration as already described 8. Acid hydrolyzates from amylase and CNBr peptides were analyzed for amino acid content 12. Total protein from tissue homogenate or lO5 ooo × g supernatant were precipitated by hot 5% trichloroacetic acid then dissolved in o.I M NaOH, and assayed by the method of LOWRY et al. 13. Amylase content was measured spectrophotometrically (El°/% 280 nm = 25) and by amino acid analysis. Radioactive samples were counted in a liquid scintillation spectrometer using BRAY'S fluid 14. Biochim. Biophys. Acta, 257 (1972) 222-229
-q
.q
g~
r~
E3H]Val, 3 min pulse I s t experiment 2nd experiment ? H ] V a l , 3 rain pulse + 4 h chase 14C-labeled Chlorella algae protein h y d r o lyzate 3 min pulse 342 380 2OlO
390
3881
939
22.50
57.00
25.57 28.oo
Total Amylase protein in supernatant
1 lO 4 lO5O
Total protein in homogenate
6.30
19.18
8.75 9.12
I
2.56
6.05
1.8o 2.2o
II
C N B r peptides
The figures represent total radioactivity i n c o r p o r a t e d in c o u n t s / m i n × lO-3.
3.21
7.o5
5.09 6.45
III
0.98
3.87
1.4o 1.56
IV
3.12
3.46
1.9o 2.o 5
V
2.02
4.98
2.4o 2.75
VI
1.5o
5-95
2.40 2.56
VII
TOTAL RADIOACTIVITYINCORPORATED INTO THE VARIOUS PROTEIN FRACTIONS AND THE C N B r PEPTIDES FROM AMYLASE
TABLE I
o.39
1.9 °
0.36 0.48
VIII
1.76
2.oo
1.32 1.55
IX
21.84
54.44
25.42 28.72
S u m of peptides
to to
CYANOGEN BROMIDE PEPTIDES FROM (Z-AMYLASE
225
RESULTS AND DISCUSSION
Pulse labeling with valine Valine was first used as a tracer because this stable amino acid is present in all CNBr peptides. Pancreas slices were incubated at the slaughterhouse as soon as possible after the animal was killed, otherwise the system rapidly inactivates even when kept at o °. The incubation was carried out as described in MATERIALS AND METHODS and the tissue was homogenized; the total protein of the homogenate and the soluble protein (supernatant) were counted after hot trichloroacetic acid precipitation (Table I). After a 3-rain pulse only 1/3 of the radioactivity incorporated into polypeptidic material was soluble. Further sonication did not improve the extraction of labeled protein*. The total radioactivity incorporated into purified amylase (3/*moles) and CNBr peptides (3/*moles) is also given in Table I. Extracted radioactive amylase makes about 7.5% of the label present in soluble protein; all the CNBr peptides are labeled. Quantitative recovery of the radioactivity incorporated in the whole amylase molecule can be obtained by summation of the values of the CNBr peptides (Table I, last column). Since the amount of radioactivity incorporated into the peptides depends on the valine content, each peptide was hydrolyzed and analyzed. The specific radioactivity of valine in each peptide was calculated by dividing the total radioactivity incorporated into 3 /,moles of the peptide by the number of valine residues present. The specific radioactivity of valine in the nine peptides is given in Table II. As one can see, differential labeling is obtained. The absolute value of specific radioactivity for a given peptide does, of course, depend on the incubation conditions. However, under our conditions, small changes from one experiment to another were observed (Tables I and II, Experiments I and 2). These variations are not due to random experimental errors in the analysis of valine content nor in the counting of incorporated radioactivity, the accuracy of which is satisfactory, but more likely due to the preparation of tissue slices. Actually, the relative specific radioactivity of the homologous peptides calculated by dividing the specific radioactivity of valine in the peptides by the specific radioactivity of valine in the corresponding amylase (Table III) is much more constant within two experiments. In each experiment no overlapping of these values is apparent and the nine peptides TABLE II SPECIFIC RADIOACTIVITY IN THE C N B r PEPTIDES AND IN AMYLASE AFTER VARIOUS INCUBATIONS A n a l y s e s were p e r f o r m e d on 3 /zmoles of each p e p t i d e .
C N B r Peptides I [~H]Val, 3 m i n pulse ( c o u n t s / m i n per Val residue) 1st e x p e r i m e n t 73 ° 2nd e x p e r i m e n t 76o [~H]Val, 3 m i n pulse + 4 h Chase ( c o u n t s / m i n p er Val residue) 16oo 1~C-labeled Chlorella a l g a e p r o t e i n h y d r o l y z a t e 3 m i n pulse ( c o u n t s / r a i n p e r a m i n o acid residue) 47
II
A mylase
III
IV
V
VI
VII
VIII
IX
3oo 37 °
I695 215o
47 ° 52o
95 ° lO25
81o 915
6oo 64o
18o 24o
132o 155o
71o 775
iOlO
2350
129 °
173o
166o
149o
95 °
2000
158o
3°
80
35
6o
51
43
15
68
48
Biochim. Biophys. Acta, 257 (1972) 222-229
226
P. COZZONE, G. MARCHIS-MOUREN
TABLE III RELATIVE SPECIFIC RADIOACTIVITIESOF THE NINE CNBr PEPTIDES The relative specific radioactivity is calculated assigning a value of i for the specific radioactivity of valine (or amino acid residue) in the whole amylase.
C N B r peptides Amylase
L-[3H] Val, 3 rain pulse Ist experiment 2nd e x p e r i m e n t L-Jail] Val, 3 rain pulse + 4 h chase ~4C-labeled protein hydrolyzate 3 min pulse
VIII
II
IV
VII
I
VI
V
IX
[I[
0.25 o.31 0.60
0.42 0.47 0.63
0.66 0.67 o.81
0.84 0.82 0.94
1.o2 0.98 I.OI
1.14 1.18 1.o 5
1.33 1.32 1.o9
1.86 2.00 1.26
2.39 2.77 1.48
i i i
o.31
0.62
0.72
0.89
0.97
1.o6
1.25
1.41
1.66
i
can be ordered unambigouusly (Fig. I). The relative specific radioactivity increases gradually (dotted line) from peptide VIII (o.25-o.31) to peptide III (2.39-2.77). The increase is linear from peptide VIII to peptide V, the higher values are measured in the case of the C-terminal peptides IX and III. This break point in the radioactivity gradient (dotted line) may be explained by the position of the valine residues
3-
• .[3.] ,-3min
pulse
>.
P_ _>
®
,//
2. _o
,/
Q:
,-r a. u~
®
uJ
®
uJ n,
N.terminal
100
J
2 I0 0
300 RESIDUE NUMBER
400
C_terminal
Fig. i. Ordering of the nine CNBr peptides from anlylase. The abcissa is proportional to the length of the peptides. The specific radioactivities, 3H c o u n t s per valine, are normalized with a value of i assigned to the specific radioactivity of amylase. Two e x p e r i m e n t s are given and the differences between the specific radioactivity of homologous peptides is made a p p a r e n t b y the hatching. The specific radioactivity gradient was tentatively d r a w n ( - - - - ) . * The radioactivity in the pellet is likely to consist either of growing peptide chains attached to ribosomes or of newly synthesized proteins still b o u n d to m e m b r a n e s .
Biochim. Biophys. Acta, 257 (1972) 222-229
CYANOGEN BROMIDE PEPTIDES FROM ~I-AMYLASE
227
in the peptides. The specific radioactivity of valine in amylase is an average value similar to the one in peptide I (localized in the central part of the amylase chain). Confirmation of the proper ordering of the peptides is obtained by comparing the N- and C-terminal peptides (VIII and III) previously determined by structural analysis. Actually, peptide VIII is also found to be the N-terminal peptide, since in this peptide valine has the lowest specific radioactivity value (18o) ; at the other end of the chain, the specific radioactivity of valine in peptide III is at the highest value (1695). However, differential labeling of the internal peptides might, at least in part, result from various valyl-tRNA pools with unequal sizes and radioactivities. To ascertain the ordering of the internal peptides two control experiments were carried out.
The chase experiment An attempt to demonstrate that the specific radioactivity of incorporated valine is the same in all growing peptide chains, at a given time, was accomplished by chasing. The growing radioactive peptide chains were then allowed to terminate in unlabeled medium. After a short pulse followed by a prolonged chase, uniform labeling was expected. As in the first experiment pancreas slices were incubated for 3 rain with radioactive valine, then excess of cold valine was added and the incubation was carried on for 4 h. Amylase was then purified and the CNBr peptides prepared. The incorporation of radioactivity into the total polypeptidic material from the homogenate is greatly enhanced (Table I, first column). This clearly indicates that the chase was not efficient. However, more radioactivity is extracted. The specific radioactivity of valine in amylase and in the peptides was determined (Table II). The value for amylase is twice higher than in the preceeding experiment. The slope of the radioactivity gradient in the peptides is much reduced (Table III). Therefore, the results thus obtained are more indicative of a prolonged labeling rather than of a chase experiment. Nevertheless, the main interest of these results obtained under labeling conditions which are quite different from those used in the first set of experiments is that the order of the peptides is found to be the same.
Pulse labeling with algae protein hydrolyzate This control experiment was carried out in an attempt to eliminate any interference due to the location of valine residues (or of any single labeled residue) in the CNBr peptides and also to possible differences in the radioactivity of various valyltRNA pools. In the incubation mixture, valine was replaced by the radioactive amino acid mixture from a protein algae hydrolyzate. The amount of radioactivity incorporated into amylase and into the CNBr peptides is of the same order of magnitude as the value obtained when valine was used as a tracer (Table I). The specific radioactivity was calculated by dividing the incorporation value by the number of amino acid residues present in the peptide (Table II). Unequal labeling is still obtained; the specific radioactivity ratio between the terminal peptides is I to 5.5. The CNBr peptides can be arranged without any ambiguity, the radioactivity gradient is linear (dotted line) and the order is the same as above. When discussing these results, it should be noticed that the specific radioactivity Biochim. Biophys. Acta, 257 (1972) 222-229
228
P. COZZONE, G. MARCHIS-MOUREN 139
112
174
140
®
M+,I
I+l \
I s I
175
464 455
i rl.,, i0,,Is.r ,. IV.II,,. i L,1 S I
coon SH
®
24
®
..~399 / S
SH 347
346
I
I rl''l ®
s
307
306
I+1
lJ
/
N. AC ETYL PORCINE PANCREATIC AMYLASE
Fig. 2. The a-amylase chain. This figure summarizes our present knowledge of the amylase prim a r y structure. The length of the peptide is proportional to the n u m b e r of residues, except for the C-terminal peptide which is longer due to presentation of the C-terminal sequence. The amino acid composition of each peptide has been previously reported 8. Black bars indicate the CNBr cleavage points in the chain. Since the precise location of the 2 SH-groups and of the 4 disulfide bridges inside the peptide is not yet known, their position on the corresponding peptides is only indicative.
of the various amino acids in the protein algae hydrolyzate certainly differs from one amino acid to another; hence differences in the specific radioactivity of the various aminoacyl-tRNA in the cell are expected. Therefore the amount of radioactivity incorporated into the peptides depends at least partially on their amino acid composition. In this type of experiment the differential labeling observed might then be misleading with respect to the proper ordering. In the case of amylase due to a rather even distribution of the amino acids in the CNBr peptides such difficulty is not encountered and the results are consistent with the use of a single amino acid. In general the difficulty discussed above could be overcome by successively comparing the specific radioactivity of individual amino acids among peptides. This would allow a large number of cross-checks on the order. CONCLUSIONS
The pulse labeling technique has been applied to the elucidation of the proper Biochim. Biophys. Acta, 257 (1972) 222-229
CYANOGEN BROMIDE PEPTIDES FROM a-AMYLASE
229
alignment of the nine peptides obtained by CNBr cleavage of the single polypeptide chain of porcine pancreatic a-amylase. We propose this method for other structural determination and ordering of any type of peptides. Although a valuable tool in the case of rather small peptide chains, this technique might be much more helpful when applied to long peptide chains, since sequence determination by classical methods is getting more difficult with increasing length of the chain. However, its most obvious limitation is the biosynthetic capacity of the cell and the rate of synthesis of the protein which is studied. In the case of porcine pancreatic slices, although the tissue rapidly inactivates, the rate of synthesis of amylase is high enough at the beginning of the incubation periods to allow significant labeling of the amylase molecule and CNBr peptides. It was then possible to order the nine peptides which account for the entire amylase chain. From the above reported experiments carried out under different conditions a unique sequence of the peptides was obtained. It is noteworthy that the peptides assigned to the N- and C-terminal position of the amylase chain were found to correspond to those determined independently in previous structural studies. The ordering of the 9 CNBr peptides of porcine pancreatic a-amylase is a contribution to the future elucidation of the primary structure of this enzyme. ACKNOWLEDGMENTS
The expert technical assistance of Miss Ch. Teissier is gratefully acknowledged. We thank Dr. R. Monier for helpful advice in the preparation of the manuscript. We thank the "Commissariat /t l'Energie Atomique" for financial help in the supply of radioisotopes. The work described was supported by a grant from the C.N.R.S. (ERA No. 173 ). REFERENCES I P. COZZONE, L. PASERO, B. BEAUPOIL 'AND G. MARCHIS-MOUREN, Biochim. Biophys. Acta, 207 (197 ° ) 490. 2 P. COZZONE AND G. MARCHIS-MOUREN, F E B S Lett., 9 (197 o) 342. 3 H. M. DINTZlS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 247. 4 D. N. L u c k AND J. M. BARRY, J. Mol. Biol., 9 (1964) 186. 5 R. E. CANFIELD AND C. B. ANFINSEN, Biochemistry, 2 (1963) lO73. , 6 J. B. FLEISCHMAN, Biochemistry, 6 (1967) 1312. 7 J. V u o s T AND K. A. PIEZ, J . Biol. Chem., 245 (197o) 62Ol. 8 P. COZZONE, L. PASERO, g . BEAUPOIL AND G. MARCHIS-MOUREN, Biochimie, 54 (I972), in t h e press. 9 H. A. KREBS, Biochim. Biophys. Acta, 4 (195 °) 256. IO G. MARCHIS-MOUREN AND L. PASERO, Biochim. Biophys. Acta, 14o (1967) 6366. I I E. GRoss AND B. WlTKOP, J. Biol; Chem., 237 (1962) 1856. 12 S. MOORE AND W. 1~,~. STEIN, Methods Enzymol., 6 (1963) 819. 13 O. H. LowRY, A. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 14 G. A. BRAY, Anal. Biochem., I (196o) 279.
Biochim. Biophys. Acta, 257 (1972) 222-229
BBA
B I O C H I M I C A E T B I O P H Y S I C A ACTA
36022
USE OF P U L S E L A B E L I N G T E C H N I Q U E IN P R O T E I N S T R U C T U R E D E T E R M I N A T I O N : O R D E R I N G OF T H E CYANOGEN B R O M I D E P E P T I D E S FROM P O R C I N E PANCREATIC a-AMYLASE
P. COZZONE AND G. MARCHIS-MOUREN
Institut de Chimie Biologique, Place Victor Hugo-z3, Marseille (3 °) (France) (Received September 6th, 1971)
SUMMARY
The nine peptides isolated after cleavage of the pancreatic amylase polypeptidic chain by cyanogen bromide, have been ordered by the use of an isotopic technique based upon pulse labeling. Porcine pancreatic slices were incubated with radioactive valine for 3 rain. Differential labeling of the peptides was obtained. As shown by comparison of the relative specific radioactivity of valine in homologous peptides, the results are reproducible. The peptides have then been lined up unambiguously from the amino terminal to the carboxyl terminal end in the following order V I I I , II, IV, VII, I, VI, V, IX, I I I . The position of the terminal peptides ( V I I I and I I I ) agrees with structural determination. As a control the relative specific radioactivity of the nine peptides was determined under two different labeling conditions. In the first control experiment, excess unlabeled valine was added after incubating for 3 rain with [3H]valine and the incubation carried on for 4 h. In the second control experiment [aH]valine was replaced by a 14C-labeled algae protein hydrolyzate. In both cases, although the slope of the radioactivity gradient was quite different from before, the same order was obtained.
INTRODUCTION Porcine pancreatic a-amylase consists of a single polypeptide chain of about 47 ° amino acid residues 1. Determination of the primary structure of such a long peptide chain is a rather formidable task. Because its amino terminal end is blocked by an acetyl group 2, the amino acid sequence at this end cannot be determined by the E d m a n procedure using a sequencer. Classical methods based on comparison of overlapping peptides are time consuming, especially in the case of a long peptide chain, since a large number of peptides have to be isolated and analyzed. This is the reason why in the case of the 47 ° residues long amylase chain an isotopic technique has been employed for ordering the peptides. Biochim. Biophys. dcta, 257 (1972) 222-229
C Y A N O G E N B R O M I D E P E P T I D E S FROM or-AMYLASE
223
This method is based upon the findings of DINTZIS3 in the study of globin biosynthesis: after incubating reticulocytes with radioactive amino acid for a short period of time, differential labeling of tryptic peptides from globin was obtained. Since the primary structure of rabbit a-globin was already known, DINTZIS~ could thus demonstrate that the globin chain is growing by sequential addition of amino acids from the N-terminal end to the C-terminal position. The same polarity in the elongation process was also found in the biosynthesis of other polypeptide chains of known sequence such as ribonuclease 4 and lysozyme 5. It is now admitted that this mechanism is universal. More recently the pulse labeling technique was applied by FLEISCHMAN~ to study the biosynthesis of v-globulin heavy chain and by VUUST AND PIEZ~ in the case of a-chains of collagen. In view of the determination of the primary structure of a-amylase this technique was presently used for ordering the nine peptides resulting from the cleavage of the amylase chain by cyanogen bromide. These peptides have been previously purified and characterized by molecular weight determination, end groups and amino acid analyses 8. Porcine pancreatic slices were incubated for short period of time with labeled amino acid. Radioactive amylase was then isolated and submitted to CNBr treatment. Unequal labeling of peptides was obtained. I t was then possible to line them up on the basis of the specific radioactivity which increases from the N-terminal to the C-terminal peptides. MATERIALS AND METHODS
Porcine pancreas was cut in small pieces (5-8 g) and incubated at 25 ° in 75 ml of Krebs I I I medium 9. For the 3-min pulse experiments the incubation medium contained 250/~C of L-[aHlvaline (25 C/mmole, CEA, Saclay, France) or 125 #C of 1*C-labeled algae protein hydrolyzate (0. 5 mC/mg, CEA, Saclay). Incorporations were stopped by cooling. In the chase experiment a lO 4 molar excess of unlabeled valine was added and the incubation continued under 02 in a Dubnoff metabolic shaking incubator. At the end of the labeling period, the medium was poured off and the tissue was thoroughly rinsed with 250 ml of cold 0.9% NaC1. All subsequent operations were performed at 2-4 °. The tissue was homogenized in presence of D F P (IO 3 M) in o.I M sodium phosphate buffer p H 8.0 with a Potter-Elvehjem homogenizer. The homogenate was sonicated 3 times at io-sec intervals for 30 sec (8 A audiofrequency current), then centrifuged for 2 h at lO5 ooo × g. The clear supernatant was lyophilized after overnight dialysis against water. To the radioactive lyophilized powder (5-700 mg), unlabeled lyophilizate powder (I.8-2 g) was added as a carrier. Amylase was then purified 1°. Pure labeled amylase (3/,moles) was cleaved by cyanogen bromide n and the CNBr peptides were isolated by gel filtration as already described 8. Acid hydrolyzates from amylase and CNBr peptides were analyzed for amino acid content 12. Total protein from tissue homogenate or lO5 ooo × g supernatant were precipitated by hot 5% trichloroacetic acid then dissolved in o.I M NaOH, and assayed by the method of LOWRY et al. 13. Amylase content was measured spectrophotometrically (El°/% 280 nm = 25) and by amino acid analysis. Radioactive samples were counted in a liquid scintillation spectrometer using BRAY'S fluid 14. Biochim. Biophys. Acta, 257 (1972) 222-229
-q
.q
g~
r~
E3H]Val, 3 min pulse I s t experiment 2nd experiment ? H ] V a l , 3 rain pulse + 4 h chase 14C-labeled Chlorella algae protein h y d r o lyzate 3 min pulse 342 380 2OlO
390
3881
939
22.50
57.00
25.57 28.oo
Total Amylase protein in supernatant
1 lO 4 lO5O
Total protein in homogenate
6.30
19.18
8.75 9.12
I
2.56
6.05
1.8o 2.2o
II
C N B r peptides
The figures represent total radioactivity i n c o r p o r a t e d in c o u n t s / m i n × lO-3.
3.21
7.o5
5.09 6.45
III
0.98
3.87
1.4o 1.56
IV
3.12
3.46
1.9o 2.o 5
V
2.02
4.98
2.4o 2.75
VI
1.5o
5-95
2.40 2.56
VII
TOTAL RADIOACTIVITYINCORPORATED INTO THE VARIOUS PROTEIN FRACTIONS AND THE C N B r PEPTIDES FROM AMYLASE
TABLE I
o.39
1.9 °
0.36 0.48
VIII
1.76
2.oo
1.32 1.55
IX
21.84
54.44
25.42 28.72
S u m of peptides
to to
CYANOGEN BROMIDE PEPTIDES FROM (Z-AMYLASE
225
RESULTS AND DISCUSSION
Pulse labeling with valine Valine was first used as a tracer because this stable amino acid is present in all CNBr peptides. Pancreas slices were incubated at the slaughterhouse as soon as possible after the animal was killed, otherwise the system rapidly inactivates even when kept at o °. The incubation was carried out as described in MATERIALS AND METHODS and the tissue was homogenized; the total protein of the homogenate and the soluble protein (supernatant) were counted after hot trichloroacetic acid precipitation (Table I). After a 3-rain pulse only 1/3 of the radioactivity incorporated into polypeptidic material was soluble. Further sonication did not improve the extraction of labeled protein*. The total radioactivity incorporated into purified amylase (3/*moles) and CNBr peptides (3/*moles) is also given in Table I. Extracted radioactive amylase makes about 7.5% of the label present in soluble protein; all the CNBr peptides are labeled. Quantitative recovery of the radioactivity incorporated in the whole amylase molecule can be obtained by summation of the values of the CNBr peptides (Table I, last column). Since the amount of radioactivity incorporated into the peptides depends on the valine content, each peptide was hydrolyzed and analyzed. The specific radioactivity of valine in each peptide was calculated by dividing the total radioactivity incorporated into 3 /,moles of the peptide by the number of valine residues present. The specific radioactivity of valine in the nine peptides is given in Table II. As one can see, differential labeling is obtained. The absolute value of specific radioactivity for a given peptide does, of course, depend on the incubation conditions. However, under our conditions, small changes from one experiment to another were observed (Tables I and II, Experiments I and 2). These variations are not due to random experimental errors in the analysis of valine content nor in the counting of incorporated radioactivity, the accuracy of which is satisfactory, but more likely due to the preparation of tissue slices. Actually, the relative specific radioactivity of the homologous peptides calculated by dividing the specific radioactivity of valine in the peptides by the specific radioactivity of valine in the corresponding amylase (Table III) is much more constant within two experiments. In each experiment no overlapping of these values is apparent and the nine peptides TABLE II SPECIFIC RADIOACTIVITY IN THE C N B r PEPTIDES AND IN AMYLASE AFTER VARIOUS INCUBATIONS A n a l y s e s were p e r f o r m e d on 3 /zmoles of each p e p t i d e .
C N B r Peptides I [~H]Val, 3 m i n pulse ( c o u n t s / m i n per Val residue) 1st e x p e r i m e n t 73 ° 2nd e x p e r i m e n t 76o [~H]Val, 3 m i n pulse + 4 h Chase ( c o u n t s / m i n p er Val residue) 16oo 1~C-labeled Chlorella a l g a e p r o t e i n h y d r o l y z a t e 3 m i n pulse ( c o u n t s / r a i n p e r a m i n o acid residue) 47
II
A mylase
III
IV
V
VI
VII
VIII
IX
3oo 37 °
I695 215o
47 ° 52o
95 ° lO25
81o 915
6oo 64o
18o 24o
132o 155o
71o 775
iOlO
2350
129 °
173o
166o
149o
95 °
2000
158o
3°
80
35
6o
51
43
15
68
48
Biochim. Biophys. Acta, 257 (1972) 222-229
226
P. COZZONE, G. MARCHIS-MOUREN
TABLE III RELATIVE SPECIFIC RADIOACTIVITIESOF THE NINE CNBr PEPTIDES The relative specific radioactivity is calculated assigning a value of i for the specific radioactivity of valine (or amino acid residue) in the whole amylase.
C N B r peptides Amylase
L-[3H] Val, 3 rain pulse Ist experiment 2nd e x p e r i m e n t L-Jail] Val, 3 rain pulse + 4 h chase ~4C-labeled protein hydrolyzate 3 min pulse
VIII
II
IV
VII
I
VI
V
IX
[I[
0.25 o.31 0.60
0.42 0.47 0.63
0.66 0.67 o.81
0.84 0.82 0.94
1.o2 0.98 I.OI
1.14 1.18 1.o 5
1.33 1.32 1.o9
1.86 2.00 1.26
2.39 2.77 1.48
i i i
o.31
0.62
0.72
0.89
0.97
1.o6
1.25
1.41
1.66
i
can be ordered unambigouusly (Fig. I). The relative specific radioactivity increases gradually (dotted line) from peptide VIII (o.25-o.31) to peptide III (2.39-2.77). The increase is linear from peptide VIII to peptide V, the higher values are measured in the case of the C-terminal peptides IX and III. This break point in the radioactivity gradient (dotted line) may be explained by the position of the valine residues
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Fig. i. Ordering of the nine CNBr peptides from anlylase. The abcissa is proportional to the length of the peptides. The specific radioactivities, 3H c o u n t s per valine, are normalized with a value of i assigned to the specific radioactivity of amylase. Two e x p e r i m e n t s are given and the differences between the specific radioactivity of homologous peptides is made a p p a r e n t b y the hatching. The specific radioactivity gradient was tentatively d r a w n ( - - - - ) . * The radioactivity in the pellet is likely to consist either of growing peptide chains attached to ribosomes or of newly synthesized proteins still b o u n d to m e m b r a n e s .
Biochim. Biophys. Acta, 257 (1972) 222-229
CYANOGEN BROMIDE PEPTIDES FROM ~I-AMYLASE
227
in the peptides. The specific radioactivity of valine in amylase is an average value similar to the one in peptide I (localized in the central part of the amylase chain). Confirmation of the proper ordering of the peptides is obtained by comparing the N- and C-terminal peptides (VIII and III) previously determined by structural analysis. Actually, peptide VIII is also found to be the N-terminal peptide, since in this peptide valine has the lowest specific radioactivity value (18o) ; at the other end of the chain, the specific radioactivity of valine in peptide III is at the highest value (1695). However, differential labeling of the internal peptides might, at least in part, result from various valyl-tRNA pools with unequal sizes and radioactivities. To ascertain the ordering of the internal peptides two control experiments were carried out.
The chase experiment An attempt to demonstrate that the specific radioactivity of incorporated valine is the same in all growing peptide chains, at a given time, was accomplished by chasing. The growing radioactive peptide chains were then allowed to terminate in unlabeled medium. After a short pulse followed by a prolonged chase, uniform labeling was expected. As in the first experiment pancreas slices were incubated for 3 rain with radioactive valine, then excess of cold valine was added and the incubation was carried on for 4 h. Amylase was then purified and the CNBr peptides prepared. The incorporation of radioactivity into the total polypeptidic material from the homogenate is greatly enhanced (Table I, first column). This clearly indicates that the chase was not efficient. However, more radioactivity is extracted. The specific radioactivity of valine in amylase and in the peptides was determined (Table II). The value for amylase is twice higher than in the preceeding experiment. The slope of the radioactivity gradient in the peptides is much reduced (Table III). Therefore, the results thus obtained are more indicative of a prolonged labeling rather than of a chase experiment. Nevertheless, the main interest of these results obtained under labeling conditions which are quite different from those used in the first set of experiments is that the order of the peptides is found to be the same.
Pulse labeling with algae protein hydrolyzate This control experiment was carried out in an attempt to eliminate any interference due to the location of valine residues (or of any single labeled residue) in the CNBr peptides and also to possible differences in the radioactivity of various valyltRNA pools. In the incubation mixture, valine was replaced by the radioactive amino acid mixture from a protein algae hydrolyzate. The amount of radioactivity incorporated into amylase and into the CNBr peptides is of the same order of magnitude as the value obtained when valine was used as a tracer (Table I). The specific radioactivity was calculated by dividing the incorporation value by the number of amino acid residues present in the peptide (Table II). Unequal labeling is still obtained; the specific radioactivity ratio between the terminal peptides is I to 5.5. The CNBr peptides can be arranged without any ambiguity, the radioactivity gradient is linear (dotted line) and the order is the same as above. When discussing these results, it should be noticed that the specific radioactivity Biochim. Biophys. Acta, 257 (1972) 222-229
228
P. COZZONE, G. MARCHIS-MOUREN 139
112
174
140
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175
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346
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s
307
306
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N. AC ETYL PORCINE PANCREATIC AMYLASE
Fig. 2. The a-amylase chain. This figure summarizes our present knowledge of the amylase prim a r y structure. The length of the peptide is proportional to the n u m b e r of residues, except for the C-terminal peptide which is longer due to presentation of the C-terminal sequence. The amino acid composition of each peptide has been previously reported 8. Black bars indicate the CNBr cleavage points in the chain. Since the precise location of the 2 SH-groups and of the 4 disulfide bridges inside the peptide is not yet known, their position on the corresponding peptides is only indicative.
of the various amino acids in the protein algae hydrolyzate certainly differs from one amino acid to another; hence differences in the specific radioactivity of the various aminoacyl-tRNA in the cell are expected. Therefore the amount of radioactivity incorporated into the peptides depends at least partially on their amino acid composition. In this type of experiment the differential labeling observed might then be misleading with respect to the proper ordering. In the case of amylase due to a rather even distribution of the amino acids in the CNBr peptides such difficulty is not encountered and the results are consistent with the use of a single amino acid. In general the difficulty discussed above could be overcome by successively comparing the specific radioactivity of individual amino acids among peptides. This would allow a large number of cross-checks on the order. CONCLUSIONS
The pulse labeling technique has been applied to the elucidation of the proper Biochim. Biophys. Acta, 257 (1972) 222-229
CYANOGEN BROMIDE PEPTIDES FROM a-AMYLASE
229
alignment of the nine peptides obtained by CNBr cleavage of the single polypeptide chain of porcine pancreatic a-amylase. We propose this method for other structural determination and ordering of any type of peptides. Although a valuable tool in the case of rather small peptide chains, this technique might be much more helpful when applied to long peptide chains, since sequence determination by classical methods is getting more difficult with increasing length of the chain. However, its most obvious limitation is the biosynthetic capacity of the cell and the rate of synthesis of the protein which is studied. In the case of porcine pancreatic slices, although the tissue rapidly inactivates, the rate of synthesis of amylase is high enough at the beginning of the incubation periods to allow significant labeling of the amylase molecule and CNBr peptides. It was then possible to order the nine peptides which account for the entire amylase chain. From the above reported experiments carried out under different conditions a unique sequence of the peptides was obtained. It is noteworthy that the peptides assigned to the N- and C-terminal position of the amylase chain were found to correspond to those determined independently in previous structural studies. The ordering of the 9 CNBr peptides of porcine pancreatic a-amylase is a contribution to the future elucidation of the primary structure of this enzyme. ACKNOWLEDGMENTS
The expert technical assistance of Miss Ch. Teissier is gratefully acknowledged. We thank Dr. R. Monier for helpful advice in the preparation of the manuscript. We thank the "Commissariat /t l'Energie Atomique" for financial help in the supply of radioisotopes. The work described was supported by a grant from the C.N.R.S. (ERA No. 173 ). REFERENCES I P. COZZONE, L. PASERO, B. BEAUPOIL 'AND G. MARCHIS-MOUREN, Biochim. Biophys. Acta, 207 (197 ° ) 490. 2 P. COZZONE AND G. MARCHIS-MOUREN, F E B S Lett., 9 (197 o) 342. 3 H. M. DINTZlS, Proc. Natl. Acad. Sci. U.S., 47 (1961) 247. 4 D. N. L u c k AND J. M. BARRY, J. Mol. Biol., 9 (1964) 186. 5 R. E. CANFIELD AND C. B. ANFINSEN, Biochemistry, 2 (1963) lO73. , 6 J. B. FLEISCHMAN, Biochemistry, 6 (1967) 1312. 7 J. V u o s T AND K. A. PIEZ, J . Biol. Chem., 245 (197o) 62Ol. 8 P. COZZONE, L. PASERO, g . BEAUPOIL AND G. MARCHIS-MOUREN, Biochimie, 54 (I972), in t h e press. 9 H. A. KREBS, Biochim. Biophys. Acta, 4 (195 °) 256. IO G. MARCHIS-MOUREN AND L. PASERO, Biochim. Biophys. Acta, 14o (1967) 6366. I I E. GRoss AND B. WlTKOP, J. Biol; Chem., 237 (1962) 1856. 12 S. MOORE AND W. 1~,~. STEIN, Methods Enzymol., 6 (1963) 819. 13 O. H. LowRY, A. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 14 G. A. BRAY, Anal. Biochem., I (196o) 279.
Biochim. Biophys. Acta, 257 (1972) 222-229