170
SELECTIVE CLEAVAGE WITH ENZYMES
TI
[19]
T'°
T'cl
Val -Ala - T r p - lle - Lys -Ala - P h e - S e r - A s p - A r g - Tyr- His -Ala - Val - T h r - Gly -Arg- 2"yr- Pro- M e t
1
-T-3
r
Val-Ala-Trp-Ile - Lys I " T-2 • ] JAla - P h e - S e r - A s p - A r g ]
T-4
[ T y r - H i s -Ala - Val - T h r - G l y - A r g [ CI- 2
[
T-1
I
iTyr- Pro- M e t ~
I
cl-1----t
FzG. 3. Sequence of CNBr-III peptide from Chaloropsis lysozyme. Tryptin (T) and clostripain (Cl) cleavage sites as indicated by $. Edman degradation steps of individual tryptic peptides indicated by -->. Illustration from J. W. Shih and J. H. Hash, J. Biol. Chem. 246, 994 (1971).
pared to sequenced tryptie peptides. Clostripain has been used also as an aid in the primary structure determination of tuftsin 1: and as a tool in structure-function studies on glucagon. :~ ~2K. Kishioka, A. Constantopoulos, P. S. Satah, W. M. Mitchell, and V. A. Najjar, Biochim. Biophys. Ac~a 310, 217 (1973). 13p. W. Felts, N. E. C. Ferguson, K. A. Hagey, E. S. Stiff, and W. M. Mitchell, Diabetologia 6, 44 (1970).
[19] Specific H y d r o l y s i s b y T r y p s i n a t A l k a l i n e p H 1
By LEWIS J. GREENE and DIANA C. BARTELT Trypsin hydrolysis at pH 7-8 is extensively used in amino acid sequence determinations to hydrolyze the peptide bond at the carboxyl terminus of arginine, lysine, and S-2-aminoethylcysteine residues.: Specific hydrolysis at arginine by trypsin can be achieved by acylation of the c-and ~-amino groups of lysyl and S-2-aminoethylcysteinyl residues, respectively. When a reversible modification reaction, such as maleylation, 3 is employed, subsequent hydrolysis at both lysyl and S-2-aminoResearch carried out at Brookhaven National Laboratory under the auspices of the U.S. ERDA. By acceptance of this article, the publisher and/or recipient acknowledges the U.S. Government right to retain a nonexclusive royalty-flee license in and to any copyright covering this paper. 2D. G. Smyth, this series Vol. 11, p. 214 (1967). P. J. G. Butler and B. S. Hartley, this series Vol. 25, p. 191 (1972).
[19]
SPECIFIC HYDROLYSIS BY TRYPSIN AT ALKALINE
pH
171
ethylcysteinyl residues can be obtained by a second exposure to trypsin at pH 7-8 after removal of the acyl blocking groups. In this way, overlapping peptides may be obtained through the use of only one proteolytic enzyme. Hydrolysis at pH 11.0 is an alternative method to achieve specific cleavage by trypsin which does not require chemical modification of the substrate. A significant advantage over the acylation procedure is that hydrolysis at alkaline pH permits selective cleavage of tysyl residues in the presence of S-2-aminoethylcysteinyl peptide bonds. However, the discrimination between arginyl and lysyl bonds at alkaline pH is not as precise as that obtained by the acylation method.
Principle The procedure is based on the kinetic studies of Wang and Carpenter. 4,5 Selective hydrolysis by trypsin at alkaline pH is based on the requirement of the enzyme for a positively charged distal amino group adjacent to the site of hyd,rolysis. The differences in the acid dissociation constants (pK~) of the guanidine group of arginine (ca. 12.5), the e-amino group of lysine (10.3) and the w-amino group of S-2-aminoethylcysteine (9.4) provide the basis for the selectivity when hydrolysis is carried out at pH 10.7 to 11.0. However, the relative rates of hydrolysis of these peptide bonds also depend on the amino acid sequence of the substrate adjacent to the scissile bond, because both ionization of the distal amino groups and trypsin catalysis are influenced by adjacent amino acids. Procedure
Apparatus. Trypsin hydrolysis is carried out in a pH-stat to maintain the pH at 11.0 and to monitor the rate and extent of reaction. ~,6 An automatic burette with a small total delivery volume (0.25 ml) and a combination electrode with a narrow diameter (Radiometer GK 2322 C, or equivalent) is recommended for reaction volumes of 5-8 ml. The reaction vessel (8-10 ml total volume) is fitted with a jacket for circulating water at a constant temperature (25 °) and mixing is achieved with a plastic-coated magnetic stirring bar. A slow stream of nitrogen freed of S. S. Wang and F. H. Carpenter, Biochemistry 6, 215 (1967). S. S. Wang and F. H. Carpenter, d. Biol. Chem. 243, 3702 (1968). C. F. Jacobsen, J. L6onis, K. Linderstr0m-Lang, and M. Ottesen, Methods Biochem. Anal. 4, 171 (1957).
172
SELECTIVE CLEAVAGE WITH ENZYMES
[19]
C Q by passage ~hrough KOH is directed over the surface of the reaction solution during hydrolysis to reduce the adsorption of CO~ + NH3 from air. Trypsin. Trypsin (bovine, crystalline, salt-free) is treated with ~-(1-tosylamido-2-phenyl)ethyl ehloromethyl ketone (TPCK) as described by Carpenter 7 in order to inactivate chymotrypsin, which is a frequent contaminant of commercial bovine trypsin preparations. Stock solutions of trypsin (1 mg/ml) are prepared in 1 mM HC1 and stored at 5 °. The concentration of TPCK-treated trypsin is given in terms of the weight of the lyophilized powder. These preparations usually contain only 50-60% active trypsin. Trypsin is sufficiently active and stable at pH 10.7-11.0 to be used at concentrations of 1-4% w/w relative to the substrate for periods of 1-2 hr. For example, a 4% w/w ratio corresponds to a molar ratio of 1:600 for active trypsin: substrate molecular weight 2000. There is, however, a significant decrease in trypsin activity when going from pH to 10.7 to 11.0. This is due to an ionizable group {pK, 10.4) in trypsin which must be protonated in order to have a fully active enzyme2 TPCK-treated trypsin (100 t~g/ml) at pH 11.0, 25% for 1 hr loses 15 and 25% of its activity, respectively, in the presence and in the absence of CaCl2. Trypsin Hydrolysis at pH 11.0. The substrate (1-5 ~mol) dissolved in 4 ml of 0.1 M KC1 is transferred to the reaction vessel of the pH-stat and equilibrated at 25 ° under a stream of nitrogen. The pH of the solution is manually titrated to 10.5 with 0.1 N NaOH and then automatically to pH 11.0 with the autoburette containing 0.033 N NaOH. The reaction is started by the addition of 0.1-0.3 ml of trypsin. Since the trypsin solution is acidic, it is necessary to determine the amount of base required to neutralize the trypsin solution before hydrolysis of the peptide or protein is carried out, so that the value for total base uptake can be corrected. The reaction is stopped by adjusting the pH to 2.0 with normal HC1 when base uptake has ceased, when the theoretical number of bonds has been hydrolyzed, or after a predetermined amount of time which is based on a preliminary experiment. Examples
1. Selective Hydrolysis at Lysine in the Presence of S-2-Aminoethylcysteine. Residues 6 through 44 of S-2-aminoethylcysteinyl porcine pancreatic secretory inhibitor I has arginine at the carboxyl terminus and 7 F.
I-I. Carpenter, this series, Vol. 11, p. 237 (1967).
[19]
SPECIFICHYDROLYSIS BY TRYPSIN AT ALKALINE p H
173
contains three residues of lysine and five residues of S-2-aminoethylcysteine per molecule2 The peptide (0.3 ~mol/ml) was hydrolyzed with 4.5%, w/w, TPCK-treated trypsin at pH 11.0, 25 ° for 20 min. Glu-Ala-Thr-Cys (Ae) - Thr- Ser-Glu-Val-Ser-Gly-Cys (Ae)-Pro-Lys-Ile- Tyr-Asn-Pro6
10
15
18
20
Val-Cys(Ae)-Gly-Thr-Asp-Gly- Ile-Thr-Tyr-Ser-Asn-Glu-Cys(Ae)-Val-Leu-Cys(Ae)25
30
35
Ser-Glu-Asn-Lys-Lys-Arg 40 42 %
Glu 16
42
43
44
Lys
Ile
18
45 %
19 44 %
Glu
68 %
Lys
Lys - -
Arg
42
43
44
Lys
6
42
7%
Ile
Arg
19
44
The partially hydrolyzed products, residues 6 through 42 and residues 19 through 44, were redigested under the same conditions for 40 and 50 min, respectively. The overall recoveries of the products relative to the amount of the starting peptide, residues 6 through 44, were: residues 6 through 18, 80%; residues 19 through 42, 89%, and residues 43 and 44, 74%. No hydrolysis at any of the five residues of S-2-aminoethyleysteine was detected. Hydrolysis at the Lys~2-Lys~3 peptide bond occurred more rapidly than at the Lysls-Ile~9 peptide bond by a factor of at least 2. 2. Selective Hydrolysis at Arginine in the Presence of Lysine. Residues 40 through 56 of performic acid-oxidized bovine pancreatic secretory trypsin inhibitor contain one arginine and two lysine residues. 9 The peptide (0.5 tLm/ml) was hydrolyzed with 4%, w/w, TPCK-treated Glu-Asn-Lys-Glu-Arg-Gln-Thr-Pro-Val-Leu-Ile-Gln-Lys-Ser-Gly-Pro-CySOaH 40
Glu 4O
42
76%
44
50
Arg Gin 44
52
56
60%
CySO~H
45
Gln 45
56 24%
25%
Lys Ser . . . . . . 52
53
CySO~H 56
trypsin at pH 11.0, 25°C. The reaction was terminated after 7 rain when the initial rapid release of acid had stopped (cf. Fig. 3 of Greene and Bartelt. 9 s D. C. Bartelt and L. J. Greene, J. Biol. Chem. 246, 2218 (1971). L. J. Greene and D. C. Bartelt, J. Biol. Chem. 244, 2646 (1969).
174
SELECTIVE CLEAVAGE WITH ENZYMES
[19]
The ratio of the relative rates of hydrolysis of Arg-Gln compared with Lys-Ser is at leas~ 4:1. Selectivity was reduced when the duration of hydrolysis was extended. After 13 rain of hydrolysis, the following recoveries were obtained: residues 40 through 44, 82%; residues 45 through 52, 75%; residues 53 through 56, 68%; and residues 44 through 56, 19%. The Lys-Glu peptide bond (residues 42 and 43) was not hydrolyzed in either experiment. Comments
1. The effect of neighboring amino acids on the relative rates of hydrolysis is illustrated by the more rapid hydrolysis of the Lys-Lys bond than the Lys-Ile bond in Example 1 and by the hydrolysis of the Lys-Ser bond, but not the Lys-Glu bond in Example 2. The experimental conditions given in the examples provide some guidelines, but it is generally necessary to carry out some preliminary experiments before applying the procedure. In Example 2 the duration of hydrolysis was selected on the basis of a preliminary experiment in which the kinetics of the total hydrolysis at pH 112 were monitored in a pH-stat (cf. Fig. 3 of Greene and Bartelt2 2. The relative rates of hydrolysis of lysyl and S-2-aminoethylcysteinyl bonds by trypsin at alkaline pH are sufficiently different to permit complete hydrolysis of lysyl bonds before S-2-aminoethylcysteinyl bonds are attacked. At pH 11.0 this may require 60-80 rain for some peptides. Since exposure of peptides to this pH may lead to deamidation (especially at Asn-Gly bonds), it may be advisable to attempt to carry out the reaction at pH 10.7, where trypsin is more active, thereby reducing the incubation time. 3. The discrimination between arginyl and lysyl bonds at alkaline pH is not as precise as obtained with the acylation method. The use of short incubation periods to limit the extent of hydrolysis increases the effective selectivity, provides additional overlap information, but also reduces the yields of products and makes their purification more complex. In view of this limitation, it may be advisable in some cases to apply both methods: acylation and hydrolysis at alkaline pH to obtain selective trypsin hydrolysis in three stages. 8,1° The reduced aminoethylated protein may first be maleylated to obtain specific cleavage at arginine and then, after fragments are separated and demaleylated, trypsin hydrolysis at alkaline pH is employed to obtain specific hydrolysis at lysine. Finally, the products are treated at pH 7-8 with trypsin to hydrolyze to S-2-aminoethylcysteine residues. ~°D. C. Bartelt, R. Shapanka, and L. J. Greene, Arch. Biochem. Biophys. 179, 189199 (1977).
SELECTIVE CLEAVAGE WITH ENZYMES
TI
[19]
T'°
T'cl
Val -Ala - T r p - lle - Lys -Ala - P h e - S e r - A s p - A r g - Tyr- His -Ala - Val - T h r - Gly -Arg- 2"yr- Pro- M e t
1
-T-3
r
Val-Ala-Trp-Ile - Lys I " T-2 • ] JAla - P h e - S e r - A s p - A r g ]
T-4
[ T y r - H i s -Ala - Val - T h r - G l y - A r g [ CI- 2
[
T-1
I
iTyr- Pro- M e t ~
I
cl-1----t
FzG. 3. Sequence of CNBr-III peptide from Chaloropsis lysozyme. Tryptin (T) and clostripain (Cl) cleavage sites as indicated by $. Edman degradation steps of individual tryptic peptides indicated by -->. Illustration from J. W. Shih and J. H. Hash, J. Biol. Chem. 246, 994 (1971).
pared to sequenced tryptie peptides. Clostripain has been used also as an aid in the primary structure determination of tuftsin 1: and as a tool in structure-function studies on glucagon. :~ ~2K. Kishioka, A. Constantopoulos, P. S. Satah, W. M. Mitchell, and V. A. Najjar, Biochim. Biophys. Ac~a 310, 217 (1973). 13p. W. Felts, N. E. C. Ferguson, K. A. Hagey, E. S. Stiff, and W. M. Mitchell, Diabetologia 6, 44 (1970).
[19] Specific H y d r o l y s i s b y T r y p s i n a t A l k a l i n e p H 1
By LEWIS J. GREENE and DIANA C. BARTELT Trypsin hydrolysis at pH 7-8 is extensively used in amino acid sequence determinations to hydrolyze the peptide bond at the carboxyl terminus of arginine, lysine, and S-2-aminoethylcysteine residues.: Specific hydrolysis at arginine by trypsin can be achieved by acylation of the c-and ~-amino groups of lysyl and S-2-aminoethylcysteinyl residues, respectively. When a reversible modification reaction, such as maleylation, 3 is employed, subsequent hydrolysis at both lysyl and S-2-aminoResearch carried out at Brookhaven National Laboratory under the auspices of the U.S. ERDA. By acceptance of this article, the publisher and/or recipient acknowledges the U.S. Government right to retain a nonexclusive royalty-flee license in and to any copyright covering this paper. 2D. G. Smyth, this series Vol. 11, p. 214 (1967). P. J. G. Butler and B. S. Hartley, this series Vol. 25, p. 191 (1972).
[19]
SPECIFIC HYDROLYSIS BY TRYPSIN AT ALKALINE
pH
171
ethylcysteinyl residues can be obtained by a second exposure to trypsin at pH 7-8 after removal of the acyl blocking groups. In this way, overlapping peptides may be obtained through the use of only one proteolytic enzyme. Hydrolysis at pH 11.0 is an alternative method to achieve specific cleavage by trypsin which does not require chemical modification of the substrate. A significant advantage over the acylation procedure is that hydrolysis at alkaline pH permits selective cleavage of tysyl residues in the presence of S-2-aminoethylcysteinyl peptide bonds. However, the discrimination between arginyl and lysyl bonds at alkaline pH is not as precise as that obtained by the acylation method.
Principle The procedure is based on the kinetic studies of Wang and Carpenter. 4,5 Selective hydrolysis by trypsin at alkaline pH is based on the requirement of the enzyme for a positively charged distal amino group adjacent to the site of hyd,rolysis. The differences in the acid dissociation constants (pK~) of the guanidine group of arginine (ca. 12.5), the e-amino group of lysine (10.3) and the w-amino group of S-2-aminoethylcysteine (9.4) provide the basis for the selectivity when hydrolysis is carried out at pH 10.7 to 11.0. However, the relative rates of hydrolysis of these peptide bonds also depend on the amino acid sequence of the substrate adjacent to the scissile bond, because both ionization of the distal amino groups and trypsin catalysis are influenced by adjacent amino acids. Procedure
Apparatus. Trypsin hydrolysis is carried out in a pH-stat to maintain the pH at 11.0 and to monitor the rate and extent of reaction. ~,6 An automatic burette with a small total delivery volume (0.25 ml) and a combination electrode with a narrow diameter (Radiometer GK 2322 C, or equivalent) is recommended for reaction volumes of 5-8 ml. The reaction vessel (8-10 ml total volume) is fitted with a jacket for circulating water at a constant temperature (25 °) and mixing is achieved with a plastic-coated magnetic stirring bar. A slow stream of nitrogen freed of S. S. Wang and F. H. Carpenter, Biochemistry 6, 215 (1967). S. S. Wang and F. H. Carpenter, d. Biol. Chem. 243, 3702 (1968). C. F. Jacobsen, J. L6onis, K. Linderstr0m-Lang, and M. Ottesen, Methods Biochem. Anal. 4, 171 (1957).
172
SELECTIVE CLEAVAGE WITH ENZYMES
[19]
C Q by passage ~hrough KOH is directed over the surface of the reaction solution during hydrolysis to reduce the adsorption of CO~ + NH3 from air. Trypsin. Trypsin (bovine, crystalline, salt-free) is treated with ~-(1-tosylamido-2-phenyl)ethyl ehloromethyl ketone (TPCK) as described by Carpenter 7 in order to inactivate chymotrypsin, which is a frequent contaminant of commercial bovine trypsin preparations. Stock solutions of trypsin (1 mg/ml) are prepared in 1 mM HC1 and stored at 5 °. The concentration of TPCK-treated trypsin is given in terms of the weight of the lyophilized powder. These preparations usually contain only 50-60% active trypsin. Trypsin is sufficiently active and stable at pH 10.7-11.0 to be used at concentrations of 1-4% w/w relative to the substrate for periods of 1-2 hr. For example, a 4% w/w ratio corresponds to a molar ratio of 1:600 for active trypsin: substrate molecular weight 2000. There is, however, a significant decrease in trypsin activity when going from pH to 10.7 to 11.0. This is due to an ionizable group {pK, 10.4) in trypsin which must be protonated in order to have a fully active enzyme2 TPCK-treated trypsin (100 t~g/ml) at pH 11.0, 25% for 1 hr loses 15 and 25% of its activity, respectively, in the presence and in the absence of CaCl2. Trypsin Hydrolysis at pH 11.0. The substrate (1-5 ~mol) dissolved in 4 ml of 0.1 M KC1 is transferred to the reaction vessel of the pH-stat and equilibrated at 25 ° under a stream of nitrogen. The pH of the solution is manually titrated to 10.5 with 0.1 N NaOH and then automatically to pH 11.0 with the autoburette containing 0.033 N NaOH. The reaction is started by the addition of 0.1-0.3 ml of trypsin. Since the trypsin solution is acidic, it is necessary to determine the amount of base required to neutralize the trypsin solution before hydrolysis of the peptide or protein is carried out, so that the value for total base uptake can be corrected. The reaction is stopped by adjusting the pH to 2.0 with normal HC1 when base uptake has ceased, when the theoretical number of bonds has been hydrolyzed, or after a predetermined amount of time which is based on a preliminary experiment. Examples
1. Selective Hydrolysis at Lysine in the Presence of S-2-Aminoethylcysteine. Residues 6 through 44 of S-2-aminoethylcysteinyl porcine pancreatic secretory inhibitor I has arginine at the carboxyl terminus and 7 F.
I-I. Carpenter, this series, Vol. 11, p. 237 (1967).
[19]
SPECIFICHYDROLYSIS BY TRYPSIN AT ALKALINE p H
173
contains three residues of lysine and five residues of S-2-aminoethylcysteine per molecule2 The peptide (0.3 ~mol/ml) was hydrolyzed with 4.5%, w/w, TPCK-treated trypsin at pH 11.0, 25 ° for 20 min. Glu-Ala-Thr-Cys (Ae) - Thr- Ser-Glu-Val-Ser-Gly-Cys (Ae)-Pro-Lys-Ile- Tyr-Asn-Pro6
10
15
18
20
Val-Cys(Ae)-Gly-Thr-Asp-Gly- Ile-Thr-Tyr-Ser-Asn-Glu-Cys(Ae)-Val-Leu-Cys(Ae)25
30
35
Ser-Glu-Asn-Lys-Lys-Arg 40 42 %
Glu 16
42
43
44
Lys
Ile
18
45 %
19 44 %
Glu
68 %
Lys
Lys - -
Arg
42
43
44
Lys
6
42
7%
Ile
Arg
19
44
The partially hydrolyzed products, residues 6 through 42 and residues 19 through 44, were redigested under the same conditions for 40 and 50 min, respectively. The overall recoveries of the products relative to the amount of the starting peptide, residues 6 through 44, were: residues 6 through 18, 80%; residues 19 through 42, 89%, and residues 43 and 44, 74%. No hydrolysis at any of the five residues of S-2-aminoethyleysteine was detected. Hydrolysis at the Lys~2-Lys~3 peptide bond occurred more rapidly than at the Lysls-Ile~9 peptide bond by a factor of at least 2. 2. Selective Hydrolysis at Arginine in the Presence of Lysine. Residues 40 through 56 of performic acid-oxidized bovine pancreatic secretory trypsin inhibitor contain one arginine and two lysine residues. 9 The peptide (0.5 tLm/ml) was hydrolyzed with 4%, w/w, TPCK-treated Glu-Asn-Lys-Glu-Arg-Gln-Thr-Pro-Val-Leu-Ile-Gln-Lys-Ser-Gly-Pro-CySOaH 40
Glu 4O
42
76%
44
50
Arg Gin 44
52
56
60%
CySO~H
45
Gln 45
56 24%
25%
Lys Ser . . . . . . 52
53
CySO~H 56
trypsin at pH 11.0, 25°C. The reaction was terminated after 7 rain when the initial rapid release of acid had stopped (cf. Fig. 3 of Greene and Bartelt. 9 s D. C. Bartelt and L. J. Greene, J. Biol. Chem. 246, 2218 (1971). L. J. Greene and D. C. Bartelt, J. Biol. Chem. 244, 2646 (1969).
174
SELECTIVE CLEAVAGE WITH ENZYMES
[19]
The ratio of the relative rates of hydrolysis of Arg-Gln compared with Lys-Ser is at leas~ 4:1. Selectivity was reduced when the duration of hydrolysis was extended. After 13 rain of hydrolysis, the following recoveries were obtained: residues 40 through 44, 82%; residues 45 through 52, 75%; residues 53 through 56, 68%; and residues 44 through 56, 19%. The Lys-Glu peptide bond (residues 42 and 43) was not hydrolyzed in either experiment. Comments
1. The effect of neighboring amino acids on the relative rates of hydrolysis is illustrated by the more rapid hydrolysis of the Lys-Lys bond than the Lys-Ile bond in Example 1 and by the hydrolysis of the Lys-Ser bond, but not the Lys-Glu bond in Example 2. The experimental conditions given in the examples provide some guidelines, but it is generally necessary to carry out some preliminary experiments before applying the procedure. In Example 2 the duration of hydrolysis was selected on the basis of a preliminary experiment in which the kinetics of the total hydrolysis at pH 112 were monitored in a pH-stat (cf. Fig. 3 of Greene and Bartelt2 2. The relative rates of hydrolysis of lysyl and S-2-aminoethylcysteinyl bonds by trypsin at alkaline pH are sufficiently different to permit complete hydrolysis of lysyl bonds before S-2-aminoethylcysteinyl bonds are attacked. At pH 11.0 this may require 60-80 rain for some peptides. Since exposure of peptides to this pH may lead to deamidation (especially at Asn-Gly bonds), it may be advisable to attempt to carry out the reaction at pH 10.7, where trypsin is more active, thereby reducing the incubation time. 3. The discrimination between arginyl and lysyl bonds at alkaline pH is not as precise as obtained with the acylation method. The use of short incubation periods to limit the extent of hydrolysis increases the effective selectivity, provides additional overlap information, but also reduces the yields of products and makes their purification more complex. In view of this limitation, it may be advisable in some cases to apply both methods: acylation and hydrolysis at alkaline pH to obtain selective trypsin hydrolysis in three stages. 8,1° The reduced aminoethylated protein may first be maleylated to obtain specific cleavage at arginine and then, after fragments are separated and demaleylated, trypsin hydrolysis at alkaline pH is employed to obtain specific hydrolysis at lysine. Finally, the products are treated at pH 7-8 with trypsin to hydrolyze to S-2-aminoethylcysteine residues. ~°D. C. Bartelt, R. Shapanka, and L. J. Greene, Arch. Biochem. Biophys. 179, 189199 (1977).
