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States of amino acid residues in proteins X. Tyrosine residues in oxytocin and glucagon
338
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25559
STATES OF AMINO ACID R E S I D U E S IN P R O T E I N S X. T Y R O S I N E R E S I D U E S IN OXYTOCIN AND GLUCAGON AYAI(O MATSUSHIMA, YUJI INADA AND [K2,A~ZUOSH1BATA The Tohugawa Institute for Biological Research, Mejiromachi, Tokyo*, and Tokyo institute of Technology, Megurohu, Tokyo (Japan)
(Received November 1st, 1965)
SUMMARY The ionization characteristics and reactivity with cyanuric fluoride of the tyrosine residues in two small peptides, oxytocin and glucagon, were studied with respect to side-chain hydrogen-bonding. The tyrosine residue in the cyclic hexapeptide of oxytocin ionized rapidly with p K ~ IO.O -E o.I, the same value as obtained for free tyrosine, and reacted completely with IO mM cyanuric fluoride. This tyrosine residue was therefore inferred to be free and not involved in bonding with the glutamine or asparagine residue in its vicinity in the cyclic peptide. Each of the two tyrosine residues at positions IO and I3 in the glucagon molecule ionized normally with p K = Io.o ~ o.I, but Tyr I3 was much less reactive with cyanuric fluoride than Tyr IO. The reactivity of Tyr 13 was increased by digestion with trypsin. However, the reactivity after complete digestion was still lower than that of Tvr IO or free tyrosine. The possibility of hydrogen-bonding of T y r 13 with other residues in the single peptide chain of glucagon was suggested from these results.
INTRODUCTION Some amino acid residues in proteins are exposed and free to react with chemicals while others are embedded in the interior of the molecule or bound by hydrogen or hydrophobic bonds with other residues, these bonds serving to maintain the tertiary structure of the protein. Little is known of the pairs of amino acid residues hydrogenbonded in proteins, and three kinds of reagents have been explored in previous studies l-a for discriminating chemically between these free and bound (or exposed and buried) residues of tyrosine, histidine and tryptophan. One of these reagents, cyanuric fluoride, for tyrosine residues, was applied to insulin, and the following facts were revealed as to the states of its tyrosine residues 1, ~. (i) Two residues at positions AI 9 and BI6 out of the total four tyrosine residues in the insulin molecule are reactive with cyanuric fluoride, and the remaining AI 4 and B26 residues are not reactive. (ii) The non-reactive B26 residue ionizes rapidly with alkali and is transformed by tryptic * Postal address. Biochim. Biophys. Acta,
tzt
(t966) 338-342
TYROSINE
RESIDUES
IN O X Y T O C I N A N D
GLUCAGON
339
digestion into the reactive type. (iii) On the other hand, the non-reactive AI 4 residue ionizes slowly with an abnormally high pK value and is not transformed into the reactive and rapidly ionizing type by either tryptic or chymotryptic digestion. Intrachain hydrogen-bonding of Tyr B26 with Arg B22 and interchain hydrogen-bonding of Tyr AI 4 with Glu BI3 were suggested from these results. Glucagon is a small protein made up of a single peptide chain, and is more suitable than insulin for demonstrating intrachain hydrogen-bonding if either of the two tyrosine residues in the molecule is bonded. Another substance of interest in the present study is oxytocin which is a cyclic hexapeptide with a tripeptide branch. The possibility of hydrogen-bonding between tyrosine and asparagine residues opposite each other in the cyclic peptide may be examined with this peptide. Throughout this paper, n represents the moles of tyrosine residues ionized or reacted with cyanuric fluoride per mole of protein. EXPERIMENTAL
The sample of crystalline glucagon was donated by Eli Lilly Co. (Lot No. 258234B-I67-I), and the sample of oxytocin was a synthetic product of Hoechst. Ionization curves of tyrosine residues in these proteins were determined by the same procedure as described previously 5, from the increment, AA 295m~, in absorbance at 295 m~ due to the ionization of the phenolic hydroxyl group. The reaction of tyrosine residues with cyanuric fluoride was conducted at pH 9.1, and the degree of reaction was estimated from the AA 995m~ value for the ionization of intact tyrosine not reacted with cyanuric fluoride. The details of procedure were the same as in the previous experiment 1. AA295mp values were reduced to moles of tyrosine residues ionized or not reacted with cyanuric fluoride per mole of protein with the aid of Ae295m# ~ - 2305 M-l"cm-L Protein concentrations were determined assuming the e2s0m/~ values of tyrosine and tryptophan in these proteins to be 1375 and 5500 M-1. cm -1, respectively. As will be shown later, one of the two tyrosine residues in the glucagon molecule is much less reactive with cyanuric fluoride than the other. These two types of tyrosine residues were located in the amino acid sequence by the following procedure. Glucagon was treated with 13 mM cyanuric fluoride at pH 9. I to modify only the more reactive tyrosine residue. Cyanuric fluoride-modified glucagon was then precipitated and washed with 0.5 M trichloroacetic acid and with ether. The precipitate was dissolved in o.I M ammonium carbonate-bicarbonate mixture (pH 9.5) and was subjected to gel filtration on a Sephadex G-25 column. A protein fraction was eluted and lyophilized. Cyanuric fluoride-modified glucagon thus isolated was dissolved in o.I M ammonium carbonate, and was digested with trypsin (substrate: enzyme ---- 3 o, by weight) at 37 ° and pH 8. 7 for 18 h. Water was added to the lyophilized digest (final pH ---- 5.0) to dissolve free arginine and two acid-soluble peptides ~, and the insoluble peptides were discarded. The solution was applied on Toyo No. 50 paper to obtain a peptide map. Electrophoresis was conducted for 1.5 h with pyridine-acetic acid-water (8: I:iOO, by vol.) at pH 6.1 and a potential gradient of 30 V/cm, and chromatography was performed for 15 h with the upper layer of butanol-acetic acid-water (4: I : 5, by vol.). The amino acid compositions of the three spots in the peptide map were determined by two-dimensional paper chromatography 4 with Toyo No. 51 paper and by the SAKAGUCHI reaction with 8-oxyquinoline and N-bromosuccinimide 7-9. Biochim. Biophys. Acta, 121 (1966) 3 3 8 - 3 4 2
34 °
a . MATSUSHIMA, Y. INADA, K. SHIBATA
RESULTS AND DISCUSSION
Oxytocin The ionization curve obtained for oxytocin is shown by Curve A in Fig. I, which indicates the ionization of the tyrosine residue with pK ~ IO.O ± o. I, the same value as obtained for free tyrosine 5. The ionization took place instantaneously on mixing with alkali. As shown by Curve A in Fig. 2, the tyrosine residue reacted with cyanurie fluoride and the reaction was complete at IO raM, the same cyanuric fluoride concentration as required for the reaction to the free tyrosine residues in insulin. This residue does not therefore seem to be hydrogen-bonded with a residue in its vicinity such as glutamine at position 4 or asparagine at position 5 on the opposite side of the hexapeptide ring. The lack of side-chain bonding between tyrosine and asparagine residues was previously demonstrated with three angiotensin analogues by PAIVAAND PAIVA 10.
I
i
i
i
i
n = 1.0
0.6
A
•
A _ ~ •
:2-
EO.4 LO O~ OJ
B
.
n = 2.0 .
.
.
i/
"KO.2 <:3
I
9
I0
II
12
13
pH
0
O0
I0
20
30
Cyanuric fluoride (raM)
Fig. I. Ionization curves obtained for tyrosine residues in oxytocin (276 ffM, Curve A) and those in glucagon (97 ffM, Curve B) ; ionic strength = o.56. Fig. 2. Reaction curves (the degree of reaction, in zl-d29Sm/*, plotted against cyanuric fluoride concentration) obtained for oxytocin (I78/iM, Curve A) and for glucagon (Io3 if.M, Curve I3) at p H 9.I.
Glucagon The two tyrosine residues in glucagon are at positions IO and 13 according to the amino acid sequence determined by BROMER et al. 6,11-14. As shown by Curve B in Fig. i, these residues ionized uniformly with pK--~ IO.O ~_ o.I, and the ionization took place instantaneously. However, one of these residues was much less reactive with cyanuric fluoride than the other (Curve B in Fig. 2) ; the reaction with the less reactive residue proceeded only to 35 % at 34 mM cyanuric fluoride while the reaction with the more reactive residue was complete at io raM. Glucagon was treated with 13 mM cyanuric fluoride to modify only the more reactive residue, and the cyanuric fluoride-modified glucagon was digested with tliochim. Biophys. Acta, 121 (1966) 338 342
TYROSINE RESIDUES IN OXYTOCIN AND GLUCAGON
341
trypsin in order to locate the two residues in the amino acid sequence, one modified and the other not modified with cyanuric fluoride, by the procedure described under EXPERIMENTAL.Three spots were identified in the peptide map of the tryptic digest by the ninhydrin spray, and one of them contained free arginine. The peptide in one of the remaining two spots contained all of the amino acids from His I to Lys 12 except tyrosine, and the peptide in the other spot contained tyrosine and the other four amino acids from positions 13 to 17. It is evident from this result that Tyr IO is the residue modified with cyanuric fluoride and Tyr 13 is the one which did not react with 13 mM cyanuric fluoride.
Set - A
~
,o
r
g
- Arg- A I o - G l u N H
t
It
Tr
Tr
2 - -
Tr
Native glucagon was digested with trypsin to investigate the effect of digestion on the reactivity of Tyr 13. According to the result shown by Curve B in Fig. 2, the tyrosine residue which reacted with 13 mM cyanuric fluoride in the native state represented 1.o6 mole per mole of protein, and the remaining 0.94 mole of tyrosine residue was not reactive. The number of moles of residue not reactive with this concentration of cyanuric fluoride was reduced to o.31 and 0.23 mole by 2.25 and 50 h of digestion, respectively. This implies that the reaction curve for Tyr 13 was shifted by the digestion toward lower cyanuric fluoride concentrations such that Tyr 13 reacted appreciably with cyanuric fluoride. The reactivity of Tyr 13 after the digestion was thus found to be greater than that in the native state, but was still lower than that of free tyrosine or Tyr IO which reacted completely with 13 mM cyanuric fluoride. The tryptic digestion cleaves the peptide bond between Lys 12 and Tyr 13. The increase of reactivity by the digestion suggests that Tyr 13 in the native state is hydrogen-bonded with a residue in the dodecapeptide (His I to Lys 12) released by the digestion. One a-helical turn brings Asp 9 in the proximity of Tyr 13, so that Asp 9 or tile neighboring Lys 12 may be the counterpart of Tyr 13 in the side-chain bonding. On the other hand, the reactivity being lower than free tyrosine after the digestion suggests that Tyr 13 is hydrogenbonded with a residue in the pentapeptide (Tyr 13 to Arg 17) formed by the digestion. Ser 16 or Arg 17 may be the counterpart in this bonding if the pentapeptide takes the a-helical structure. The bondings of this single tyrosine residue with two residues (Asp 9 or Lys 12 and Ser 16 or Arg 17) in native glucagon is conceivable because the phenolic hydroxyl group could be bifunctional in hydrogen-bonding. These possibilities are shown above in relation to the bonds hydrolyzed with trypsin (indicated by an arrow and Tr). ACKNOWLEDGEMENTS
The authors wish to express their deep gratitude to Drs. W. W. BROMER, J. M. McGumE and O. K. BEHRENS at the Lilly Laboratories for their generous gift of crystalline glucagon.
Biochim. Biophys. Acta, 121 (1966) 338-342
342
A. MATSUSHIMA, Y. INADA, K. SHIBATA
REFERENCES I K. KURIHARA, H. HORINISHI AND •. SHIBATA, Biochim. Biophys. Acta, 74 (1963) 678" 2 H. HORINISHI, Y. HACHIMORI, K. ]~URIHARA AND K. SHIBATA, Biochim. Biophys. Aeta, 86 (1964) 4773 Y. ]-IACHIMORI, H. HORINISHI, K. KURIHARA AND K. SHIBATA, Biochim. Biophys. Acta, 93 (1964) 346. 4 M. AOYAMA, K. KURIHARA AND K. SHIBATA, Biochim. Biophys. Acta, lo 7 (1965) 257. 5 Y" 1NADA, J. Biochem. Tokyo, 49 (1961) 217. 6 \ ¥ . W. BROMER, A. STAUB, L. G. SINN AND O. K. BEHRENS, J. Am. Chem. Soe., 79 (1957) 28Ol. 7 S. SAKAGUCHI, J. Biochem. Tokyo, 5 (1925) 25" 8 S. SAKAGUCHI, J. Biochem. Tokyo, 37 (195o) 231. 9 l. SZlLOC-YI AND I. SZABO, Nature, 181 (1958) 52 . IO A. C..'~{. PAIVA AND T. B. PAIVA, Bioehim. Biophys. Acla, 56 (1962) 339. I I \V. W. ~BROMER, A. STAUI3, E. [{. DILLER, H. L. BIRS, L. G. SINN AND (). ~[(. ]~EHRENS, J..4~l. Chem. Soc., 79 (1957) 2794. 12 \V. W. t3ROMER, L. G. SINN AND O. K. BEHRENS, J. Am. Chem. Soe., 79 (1957) 279813 L. G. SINN, 0 . K. BEHRENS AND \ ¥ . W. BROMER, J. Am. Chem. Soc., 79 (t957) 2805. 14 "%~. ~V. ]3ROMER, L. G. SINN AND (). K. BEHRENS, J. Am. Chem. Soc., 79 (I957) 2807.
Biochim. Biophys. Acta, i 2 i (1960) 338-342
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25559
STATES OF AMINO ACID R E S I D U E S IN P R O T E I N S X. T Y R O S I N E R E S I D U E S IN OXYTOCIN AND GLUCAGON AYAI(O MATSUSHIMA, YUJI INADA AND [K2,A~ZUOSH1BATA The Tohugawa Institute for Biological Research, Mejiromachi, Tokyo*, and Tokyo institute of Technology, Megurohu, Tokyo (Japan)
(Received November 1st, 1965)
SUMMARY The ionization characteristics and reactivity with cyanuric fluoride of the tyrosine residues in two small peptides, oxytocin and glucagon, were studied with respect to side-chain hydrogen-bonding. The tyrosine residue in the cyclic hexapeptide of oxytocin ionized rapidly with p K ~ IO.O -E o.I, the same value as obtained for free tyrosine, and reacted completely with IO mM cyanuric fluoride. This tyrosine residue was therefore inferred to be free and not involved in bonding with the glutamine or asparagine residue in its vicinity in the cyclic peptide. Each of the two tyrosine residues at positions IO and I3 in the glucagon molecule ionized normally with p K = Io.o ~ o.I, but Tyr I3 was much less reactive with cyanuric fluoride than Tyr IO. The reactivity of Tyr 13 was increased by digestion with trypsin. However, the reactivity after complete digestion was still lower than that of Tvr IO or free tyrosine. The possibility of hydrogen-bonding of T y r 13 with other residues in the single peptide chain of glucagon was suggested from these results.
INTRODUCTION Some amino acid residues in proteins are exposed and free to react with chemicals while others are embedded in the interior of the molecule or bound by hydrogen or hydrophobic bonds with other residues, these bonds serving to maintain the tertiary structure of the protein. Little is known of the pairs of amino acid residues hydrogenbonded in proteins, and three kinds of reagents have been explored in previous studies l-a for discriminating chemically between these free and bound (or exposed and buried) residues of tyrosine, histidine and tryptophan. One of these reagents, cyanuric fluoride, for tyrosine residues, was applied to insulin, and the following facts were revealed as to the states of its tyrosine residues 1, ~. (i) Two residues at positions AI 9 and BI6 out of the total four tyrosine residues in the insulin molecule are reactive with cyanuric fluoride, and the remaining AI 4 and B26 residues are not reactive. (ii) The non-reactive B26 residue ionizes rapidly with alkali and is transformed by tryptic * Postal address. Biochim. Biophys. Acta,
tzt
(t966) 338-342
TYROSINE
RESIDUES
IN O X Y T O C I N A N D
GLUCAGON
339
digestion into the reactive type. (iii) On the other hand, the non-reactive AI 4 residue ionizes slowly with an abnormally high pK value and is not transformed into the reactive and rapidly ionizing type by either tryptic or chymotryptic digestion. Intrachain hydrogen-bonding of Tyr B26 with Arg B22 and interchain hydrogen-bonding of Tyr AI 4 with Glu BI3 were suggested from these results. Glucagon is a small protein made up of a single peptide chain, and is more suitable than insulin for demonstrating intrachain hydrogen-bonding if either of the two tyrosine residues in the molecule is bonded. Another substance of interest in the present study is oxytocin which is a cyclic hexapeptide with a tripeptide branch. The possibility of hydrogen-bonding between tyrosine and asparagine residues opposite each other in the cyclic peptide may be examined with this peptide. Throughout this paper, n represents the moles of tyrosine residues ionized or reacted with cyanuric fluoride per mole of protein. EXPERIMENTAL
The sample of crystalline glucagon was donated by Eli Lilly Co. (Lot No. 258234B-I67-I), and the sample of oxytocin was a synthetic product of Hoechst. Ionization curves of tyrosine residues in these proteins were determined by the same procedure as described previously 5, from the increment, AA 295m~, in absorbance at 295 m~ due to the ionization of the phenolic hydroxyl group. The reaction of tyrosine residues with cyanuric fluoride was conducted at pH 9.1, and the degree of reaction was estimated from the AA 995m~ value for the ionization of intact tyrosine not reacted with cyanuric fluoride. The details of procedure were the same as in the previous experiment 1. AA295mp values were reduced to moles of tyrosine residues ionized or not reacted with cyanuric fluoride per mole of protein with the aid of Ae295m# ~ - 2305 M-l"cm-L Protein concentrations were determined assuming the e2s0m/~ values of tyrosine and tryptophan in these proteins to be 1375 and 5500 M-1. cm -1, respectively. As will be shown later, one of the two tyrosine residues in the glucagon molecule is much less reactive with cyanuric fluoride than the other. These two types of tyrosine residues were located in the amino acid sequence by the following procedure. Glucagon was treated with 13 mM cyanuric fluoride at pH 9. I to modify only the more reactive tyrosine residue. Cyanuric fluoride-modified glucagon was then precipitated and washed with 0.5 M trichloroacetic acid and with ether. The precipitate was dissolved in o.I M ammonium carbonate-bicarbonate mixture (pH 9.5) and was subjected to gel filtration on a Sephadex G-25 column. A protein fraction was eluted and lyophilized. Cyanuric fluoride-modified glucagon thus isolated was dissolved in o.I M ammonium carbonate, and was digested with trypsin (substrate: enzyme ---- 3 o, by weight) at 37 ° and pH 8. 7 for 18 h. Water was added to the lyophilized digest (final pH ---- 5.0) to dissolve free arginine and two acid-soluble peptides ~, and the insoluble peptides were discarded. The solution was applied on Toyo No. 50 paper to obtain a peptide map. Electrophoresis was conducted for 1.5 h with pyridine-acetic acid-water (8: I:iOO, by vol.) at pH 6.1 and a potential gradient of 30 V/cm, and chromatography was performed for 15 h with the upper layer of butanol-acetic acid-water (4: I : 5, by vol.). The amino acid compositions of the three spots in the peptide map were determined by two-dimensional paper chromatography 4 with Toyo No. 51 paper and by the SAKAGUCHI reaction with 8-oxyquinoline and N-bromosuccinimide 7-9. Biochim. Biophys. Acta, 121 (1966) 3 3 8 - 3 4 2
34 °
a . MATSUSHIMA, Y. INADA, K. SHIBATA
RESULTS AND DISCUSSION
Oxytocin The ionization curve obtained for oxytocin is shown by Curve A in Fig. I, which indicates the ionization of the tyrosine residue with pK ~ IO.O ± o. I, the same value as obtained for free tyrosine 5. The ionization took place instantaneously on mixing with alkali. As shown by Curve A in Fig. 2, the tyrosine residue reacted with cyanurie fluoride and the reaction was complete at IO raM, the same cyanuric fluoride concentration as required for the reaction to the free tyrosine residues in insulin. This residue does not therefore seem to be hydrogen-bonded with a residue in its vicinity such as glutamine at position 4 or asparagine at position 5 on the opposite side of the hexapeptide ring. The lack of side-chain bonding between tyrosine and asparagine residues was previously demonstrated with three angiotensin analogues by PAIVAAND PAIVA 10.
I
i
i
i
i
n = 1.0
0.6
A
•
A _ ~ •
:2-
EO.4 LO O~ OJ
B
.
n = 2.0 .
.
.
i/
"KO.2 <:3
I
9
I0
II
12
13
pH
0
O0
I0
20
30
Cyanuric fluoride (raM)
Fig. I. Ionization curves obtained for tyrosine residues in oxytocin (276 ffM, Curve A) and those in glucagon (97 ffM, Curve B) ; ionic strength = o.56. Fig. 2. Reaction curves (the degree of reaction, in zl-d29Sm/*, plotted against cyanuric fluoride concentration) obtained for oxytocin (I78/iM, Curve A) and for glucagon (Io3 if.M, Curve I3) at p H 9.I.
Glucagon The two tyrosine residues in glucagon are at positions IO and 13 according to the amino acid sequence determined by BROMER et al. 6,11-14. As shown by Curve B in Fig. i, these residues ionized uniformly with pK--~ IO.O ~_ o.I, and the ionization took place instantaneously. However, one of these residues was much less reactive with cyanuric fluoride than the other (Curve B in Fig. 2) ; the reaction with the less reactive residue proceeded only to 35 % at 34 mM cyanuric fluoride while the reaction with the more reactive residue was complete at io raM. Glucagon was treated with 13 mM cyanuric fluoride to modify only the more reactive residue, and the cyanuric fluoride-modified glucagon was digested with tliochim. Biophys. Acta, 121 (1966) 338 342
TYROSINE RESIDUES IN OXYTOCIN AND GLUCAGON
341
trypsin in order to locate the two residues in the amino acid sequence, one modified and the other not modified with cyanuric fluoride, by the procedure described under EXPERIMENTAL.Three spots were identified in the peptide map of the tryptic digest by the ninhydrin spray, and one of them contained free arginine. The peptide in one of the remaining two spots contained all of the amino acids from His I to Lys 12 except tyrosine, and the peptide in the other spot contained tyrosine and the other four amino acids from positions 13 to 17. It is evident from this result that Tyr IO is the residue modified with cyanuric fluoride and Tyr 13 is the one which did not react with 13 mM cyanuric fluoride.
Set - A
~
,o
r
g
- Arg- A I o - G l u N H
t
It
Tr
Tr
2 - -
Tr
Native glucagon was digested with trypsin to investigate the effect of digestion on the reactivity of Tyr 13. According to the result shown by Curve B in Fig. 2, the tyrosine residue which reacted with 13 mM cyanuric fluoride in the native state represented 1.o6 mole per mole of protein, and the remaining 0.94 mole of tyrosine residue was not reactive. The number of moles of residue not reactive with this concentration of cyanuric fluoride was reduced to o.31 and 0.23 mole by 2.25 and 50 h of digestion, respectively. This implies that the reaction curve for Tyr 13 was shifted by the digestion toward lower cyanuric fluoride concentrations such that Tyr 13 reacted appreciably with cyanuric fluoride. The reactivity of Tyr 13 after the digestion was thus found to be greater than that in the native state, but was still lower than that of free tyrosine or Tyr IO which reacted completely with 13 mM cyanuric fluoride. The tryptic digestion cleaves the peptide bond between Lys 12 and Tyr 13. The increase of reactivity by the digestion suggests that Tyr 13 in the native state is hydrogen-bonded with a residue in the dodecapeptide (His I to Lys 12) released by the digestion. One a-helical turn brings Asp 9 in the proximity of Tyr 13, so that Asp 9 or tile neighboring Lys 12 may be the counterpart of Tyr 13 in the side-chain bonding. On the other hand, the reactivity being lower than free tyrosine after the digestion suggests that Tyr 13 is hydrogenbonded with a residue in the pentapeptide (Tyr 13 to Arg 17) formed by the digestion. Ser 16 or Arg 17 may be the counterpart in this bonding if the pentapeptide takes the a-helical structure. The bondings of this single tyrosine residue with two residues (Asp 9 or Lys 12 and Ser 16 or Arg 17) in native glucagon is conceivable because the phenolic hydroxyl group could be bifunctional in hydrogen-bonding. These possibilities are shown above in relation to the bonds hydrolyzed with trypsin (indicated by an arrow and Tr). ACKNOWLEDGEMENTS
The authors wish to express their deep gratitude to Drs. W. W. BROMER, J. M. McGumE and O. K. BEHRENS at the Lilly Laboratories for their generous gift of crystalline glucagon.
Biochim. Biophys. Acta, 121 (1966) 338-342
342
A. MATSUSHIMA, Y. INADA, K. SHIBATA
REFERENCES I K. KURIHARA, H. HORINISHI AND •. SHIBATA, Biochim. Biophys. Acta, 74 (1963) 678" 2 H. HORINISHI, Y. HACHIMORI, K. ]~URIHARA AND K. SHIBATA, Biochim. Biophys. Aeta, 86 (1964) 4773 Y. ]-IACHIMORI, H. HORINISHI, K. KURIHARA AND K. SHIBATA, Biochim. Biophys. Acta, 93 (1964) 346. 4 M. AOYAMA, K. KURIHARA AND K. SHIBATA, Biochim. Biophys. Acta, lo 7 (1965) 257. 5 Y" 1NADA, J. Biochem. Tokyo, 49 (1961) 217. 6 \ ¥ . W. BROMER, A. STAUB, L. G. SINN AND O. K. BEHRENS, J. Am. Chem. Soe., 79 (1957) 28Ol. 7 S. SAKAGUCHI, J. Biochem. Tokyo, 5 (1925) 25" 8 S. SAKAGUCHI, J. Biochem. Tokyo, 37 (195o) 231. 9 l. SZlLOC-YI AND I. SZABO, Nature, 181 (1958) 52 . IO A. C..'~{. PAIVA AND T. B. PAIVA, Bioehim. Biophys. Acla, 56 (1962) 339. I I \V. W. ~BROMER, A. STAUI3, E. [{. DILLER, H. L. BIRS, L. G. SINN AND (). ~[(. ]~EHRENS, J..4~l. Chem. Soc., 79 (1957) 2794. 12 \V. W. t3ROMER, L. G. SINN AND O. K. BEHRENS, J. Am. Chem. Soe., 79 (1957) 279813 L. G. SINN, 0 . K. BEHRENS AND \ ¥ . W. BROMER, J. Am. Chem. Soc., 79 (t957) 2805. 14 "%~. ~V. ]3ROMER, L. G. SINN AND (). K. BEHRENS, J. Am. Chem. Soc., 79 (I957) 2807.
Biochim. Biophys. Acta, i 2 i (1960) 338-342