- Email: [email protected]
Isoleucyl-tRNA synthetase inactivation and the extent of aminoacylation of tRNAIle from Escherichia coli
84
Biochimica et Biophysica Acta, 477 (1977) 84--88 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 91446
I S O L E U C Y L - t R N A S Y N T H E T A S E INACTIVATION AND THE EXTENT OF A M I N O A C Y L A T I O N OF t R N A Ile F R O M ESCHERICHIA COLI
F A R H A D MARASHI and CHARLES L. HARRIS
Department of Biochemistry, West Virginia University School of Medicine, Morgantown, W. Va. 26506 (U.S.A.) (Received March 14th, 1977)
Summary A difference in isoleucine acceptance between normal and sulfur-deficient t R N A from Escherichia coli C6 (rel-, met-, cys-) was eliminated when more isoleucyl-tRNA synthetase was added at the reaction plateau. Enzymatic deacylation was similar for b o t h tRNAs. These results suggest that enzyme inactivation caused a premature reaction plateau which was n o t predicted by the rates of acylation and deacylation.
Besides its role in protein biosynthesis, aminoacyl-tRNA has been found to serve as d o n o r of amino acids in the biosynthesis of aminoacyl-phosphatidylglycerol [1,2], glycyllipopolysaccharide [3], and cell wall biosynthesis [4]. Aminoacyl-tRNA also participates in b o t h the regulation of R N A synthesis [5], and of the synthesis of certain enzymes [6]. Because of its importance in metabolism, methods of measuring aminoacyl-tRNA levels in vivo [ 7,8] and in vitro [9] have been developed. The aminoacylation capacity of a t R N A preparation must be determined under optimal assay conditions, and is generally estimated when the plateau level of the reaction has been reached. The aminoacylation plateau for t R N A Val [ 1 0 ] and t R N A Phe [11] from Escherichia coli was reported to be a balance b e t w e e n the rates of acylation and deacylation reactions. Contrary to enzyme kinetics theory, b o t h these studies reported that this plateau was a function of the enzyme concentration. The effect was n o t due to enzyme inactivation, as aminoacylation increased when t R N A was added at the plateau level [10]. We n o w report that the plateau of the aminoacylation of t R N A Ile from E. coli is also affected b y the a m o u n t of isoleucyl-tRNA synthetase present. This was observed when comparing the isoleucine acceptances of normal and sulfur-deficient t R N A from E. coli C6 (HfrC, tel-, k, met-, cys-), with a con-
85 sistently lower plateau seen for normal tRNA. The lower aminoacylation plateau is shown to be caused by inactivation of isoleucyl-tRNA synthetase during the assays. Transfer R N A was isolated from E. coli C6 by the method of von Ehrenstein [12]. Normal t R N A was obtained from cellsgrown in medium containing 20/~g/ml of cysteine and methionine, while sulfur-deficientt R N A was obtained after 6 h of cysteine starvation of the same organism [13]. The mixture of aminoacyl-tRNA synthetases was prepared from E. coli Q13 by the method of Kelmers et al. [14]. This preparation contained 4 rag/ ml protein as measured by the method of Lowry et al. [15]. Purified isoleucylt R N A synthetase was obtained from E. coli K12 according to the method of Baldwin and Berg [16], except that the Alumina C7 Gel step was omitted. An overall 680-fold purification was obtained through this procedure and the preparation contained 0.74 mg/ml protein. Reaction mixture A (0.61 ml) contained 61.5 m M Tris-HCl (pH 7.3), 9.83 m M magnesium acetate, 1.64 m M ATP, 5.25 # M L-[14C]isoleucine (312.5 Ci/mol, N e w England Nuclear), 1 A260 unit of either normal or sulfur-deficientt R N A and 0.11 m g of the aminoacyl-tRNA synthetase mixture. Reaction mixture B was a modification of the method of Nishimura et al. [17] and contained 20 m M Tris-HCI (pH 7.5), 10 m M magnesium acetate, 10 mM potassium chloride, 2.0 mM ATP, 6.4 #M L-[14Clisoleucine (312.5 Ci/mol, New England Nuclear), 1 A260 unit of each of normal or sulfurdeficient tRNA and 0.74/~g of pure isoleucyl-tRNA synthetase in total volume of 0.5 ml. All assays were carried out at 37°C. Normal and sulfur-deficient [ ~4C]isoleucyl-tRNA was isolated from reaction mixture A by phenol extraction and ethanol precipitation. Enzymatic IO0,
E-
80
0
W 60
<~80 W z 60
o
U
40
v1
7
20
M 2o
0 I[ I:l
o
~
~
TIME (re,n)
o
30
60
90
120
TIME (rain)
Fig. I . K i n e t i c s o f i s o l e u c y l - t R N A f o r m a t i o n w i t h n o r m a l and s u l f u r - d e f i c i e n t t R N A . T h e r e a c t i o n w a s mitiated b y t h e a d d i t i o n o f u n f r a e t i o n a t e d a m i n o a c y l - t R N A s y n t h e t a s e s to t h e r e a c t i o n m i x t u r e A . o . .o, n o r m a l t R N A ; o o, s u l f u r - d e f i c i e n t t R N A . Fig. 2. E n z y m a t i c h y d r o l y s i s o f i s o l e u c y l - t R N A i n t h e absence o f AMP a n d PPi" 1 A260 u n / t each o f either n o r m a l (-~) o r s u l f u r - d e f i c i e n t (e _-) [i 4 0 ] l s o l e u e y l . t R N A w e r e i n c u b a t e d in a s s a y m i x t u r e A c o n t a i n i n g 0 . 1 1 m g o f s y n t h e t a s e m i x t u r e , b u t l a c k i n g A T P and i s o l e u c i n e . C o n t r o l s (o o, n o r m a l ; o o, s u l f u r - d e f i c i e n t ) had n o s y n t h e t a s e added.
86
I00
'< BO hi Z W J
0 U.I _J
0
2O
36
6b TIME (rain)
Fig. 3. K i n e t i c s o f i s o l e u c y l - t R N A f o r m a t i o n w i t h n o r m a l and s u l f u r - d e f i c i e n t t R N A . This s t u d y w a s carried o u t w i t h 0 . 7 4 ~g o f p u r i f i e d i s o l e u c y l - t R N A s y n t h e t a s e , using assay m i x t u r e B. 7 . 4 ~g o f addit i o n a l e n z y m e w e r e a d d e d at t h e p o i n t s i n d i c a t e d b y a r r o w s , o o, n o r m a l t R N A ; o o, sulfurdeficient tRNA.
hydrolysis of 1 A260 unit each of either normal or sulfur-deficient isoleucylt R N A was performed in the same assay mixture, b u t lacking ATP and isoleucine. The filter disc m e t h o d [13] was used to determine the a m o u n t of acid precipitable radioactivity in 0.05 ml samples taken at various times in all assays. Fig. 1 shows the typical kinetics observed for enzymatic acylation of normal and sulfur-deficient E. coli C6 t R N A with [ 14C] isoleucine in reaction mixture A as described above. The plateau level of acylation is lower for normal tRNA. This difference was n o t due to misacylation or lack of integrity of the -CCA end in normal t R N A , and all reaction c o m p o n e n t s were present in optimal amounts [18]. It had been reported that such differences in plateau levels could be the result of enzymatic deacylation [10,11]. To investigate this possibility, normal and sulfur-deficient [14C]isoleucyl-tRNA was isolated, and deacylation was measured with and w i t h o u t added synthetase as described above. Fig. 2 shows that the rates of enzymatic deacylation (in the absence of AMP and PPi) were similar for normal and sulfur-deficient tRNA. Incomplete aminoacylation of E. coli t R N A with isoleucine had been previously reported to be due to an undetermined artifact of the crude in vitro system [19]. Therefore, we prepared purified isoleucyl-tRNA synthetase and tested the acylation of these tRNAs, the results being shown in Fig. 3. With 0.74/~g of synthetase, the acylation patterns for the t w o tRNAs were similar to those seen with unfractionated enzyme (Fig. 1). However, when additional enzyme was added at the reaction plateau (30 min), the extent o f normal isoleucyl-tRNA formation was increased. After the second synthetase addition at 75 min, the extent of isoleucylation of normal t R N A rose to that seen for sulfur-deficient tRNA. A similar b u t less pronounced response was observed with reaction mixture A when more crude enzyme was added at the plateau level. Other experiments showed that values of 82 pmol isoleucine per A260 unit were obtained for either t R N A when the reaction was initiated with 7.4 ~g of synthetase. Under these conditions further enzyme additions
87 were without effect. The extent of aminoacylation of sulfur~leficient tRNA was not significantly altered by enzyme addition presumably because it was fully acylated during the initial reaction. We recently reported that the rate of i8oleucylation of this tRNA was greater than that of fully-thiolated tRNA [18]. This higher rate could account for the complete acylation of sulfurdeficient tRNA prior to enzyme inactivation. Addition of either tRNA to reaction mixtures which had reached the plateau level failed to increase the extent of the reaction, substantiating the conclusion that enzyme inactivation had occurred. Addition of 10 mM 2-mercaptoethanol, 10 mM glutathione, or 1 mg/ml of bovine serum albumin to the reaction mixture B failed to prevent the inactivation of synthetase. Preincubation of the synthetase for 6 min with either normal or sulfur-deficient tRNA resulted in a 50% loss of activity. The same loss was observed when tRNA was absent during preincubation. Hence, these results suggest that tRNA does not play a role in the inactivation of the enzyme. Our results suggest that isoleucyl-tRNA 8ynthetase inactivation causes the attainment of a premature plateau level for normal tRNA. This effect can be overcome by the use of higher concentrations of purified synthetase. We determined that all reaction components were at optimal concentrations [18], but these conditions were not sufficient to protect the enzyme against inactivation. Our data do not confirm the hypothesis of Bonnet and Ebel [9l, that the plateau level of an aminoacylation reaction is a function of the rates of acylation and deacylation. For isoleucyl-tRNA synthetase, and perhaps for other synthetases, enzyme inactivation may cause a premature reaction plateau which may not be predicted using the above theory. These findings demonstrate the importance of defining all reaction parameters when comparing aminoacylation capacities of two tRNA preparations. If assays are carried out at low synthetase levels, the plateau level may indicate the amount of a given tRNA present only if enzyme inactivation is ruled out. Conversely, rate differences may be overlooked if the assays are carried out at high enzyme levels. Finally, a system optimized for complete in vitro aminoacylation of a tRNA preparation with one amino acid may not be suitable for aminoacylation using other amino acids. Supported in part by NCI grant CA-16567. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Nesbitt, III, J.A. and Lennarz, W.J. (1968) J. Biol. Chem. 243, 3088--3095 Gould, R.M., Thornton, M.P., Liepkalns, V. and Lennarz, W.J. (1968) J. Biol. Chem. 243, 3096--3104 Genter, N. and Berg~ P. (1971) Fed. Proc. 30, 1218 (Abstr.) Momose, K. and Kaji, A. (1966) J. Biol. Chem. 241, 3294--3307 Ryan, A.M. and Borek, E. (1971) Prog. Nucleic Acid Res. Mol. Biol. 11,193--228 Brenchley, J.E. and Williams, L.S. (1975) Ann. Rev. Microbiol. 29, 251--274 Yegian, C.D., Stent, G.S. and Martin, E.M. (1966) Proe. Natl. Acad. Sci. U.S. 55, 839--846 Butler, M., Derbre, A. and Arnstein, H.R.V. (1975) Biochem. J. 150, 419--432 $51/, D. and Sehimmel, P.R. (1974) in The Enzymes (Boyer, P.D., ed.), Vol. 10, pp. 469--588, A c a d e m i c Press, N e w York Bonnet, J. and Ebel, J.P. (1972) Eur. J. Bioehem. 3 1 , 3 3 5 - - 3 4 4 Renaud, M., Bollack, C. and Ebel, J.P. (1974) Btoehimie 56, 1203--1209 yon Ehrenstein, G. (1967) Methods Enzymol. 12A, 588--596 Harris, C.L., Titchener, E.B. and Cline, A.L. (1969) J. Bacteriol. 100, 1322--1327 Kelmers, A.D., Novelli, G.D. and Stulberg, M.P. (1965) J. Biol. Chem. 240, 3979--3983
88
15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - 275 16 Baldwin, A.N. and Berg, P. (1966) J. Biol. Chem. 2 4 1 , 8 3 1 - - 8 3 8 17 Nishimu~a, S., Harada, F., Narushima, U, and Seno, T, (1967) Biochim. Biophys. Acta 142, 133--148 18 Harris, C.L., Marashi, F. and Titehener, E.B. (1976) Nucleic Acid Res. 3, 2 1 2 9 - - 2 1 4 2 19 Yegian, C.D. and Stent, G.S. (1969) J. Mol. Biol. 39, 59--71
Biochimica et Biophysica Acta, 477 (1977) 84--88 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 91446
I S O L E U C Y L - t R N A S Y N T H E T A S E INACTIVATION AND THE EXTENT OF A M I N O A C Y L A T I O N OF t R N A Ile F R O M ESCHERICHIA COLI
F A R H A D MARASHI and CHARLES L. HARRIS
Department of Biochemistry, West Virginia University School of Medicine, Morgantown, W. Va. 26506 (U.S.A.) (Received March 14th, 1977)
Summary A difference in isoleucine acceptance between normal and sulfur-deficient t R N A from Escherichia coli C6 (rel-, met-, cys-) was eliminated when more isoleucyl-tRNA synthetase was added at the reaction plateau. Enzymatic deacylation was similar for b o t h tRNAs. These results suggest that enzyme inactivation caused a premature reaction plateau which was n o t predicted by the rates of acylation and deacylation.
Besides its role in protein biosynthesis, aminoacyl-tRNA has been found to serve as d o n o r of amino acids in the biosynthesis of aminoacyl-phosphatidylglycerol [1,2], glycyllipopolysaccharide [3], and cell wall biosynthesis [4]. Aminoacyl-tRNA also participates in b o t h the regulation of R N A synthesis [5], and of the synthesis of certain enzymes [6]. Because of its importance in metabolism, methods of measuring aminoacyl-tRNA levels in vivo [ 7,8] and in vitro [9] have been developed. The aminoacylation capacity of a t R N A preparation must be determined under optimal assay conditions, and is generally estimated when the plateau level of the reaction has been reached. The aminoacylation plateau for t R N A Val [ 1 0 ] and t R N A Phe [11] from Escherichia coli was reported to be a balance b e t w e e n the rates of acylation and deacylation reactions. Contrary to enzyme kinetics theory, b o t h these studies reported that this plateau was a function of the enzyme concentration. The effect was n o t due to enzyme inactivation, as aminoacylation increased when t R N A was added at the plateau level [10]. We n o w report that the plateau of the aminoacylation of t R N A Ile from E. coli is also affected b y the a m o u n t of isoleucyl-tRNA synthetase present. This was observed when comparing the isoleucine acceptances of normal and sulfur-deficient t R N A from E. coli C6 (HfrC, tel-, k, met-, cys-), with a con-
85 sistently lower plateau seen for normal tRNA. The lower aminoacylation plateau is shown to be caused by inactivation of isoleucyl-tRNA synthetase during the assays. Transfer R N A was isolated from E. coli C6 by the method of von Ehrenstein [12]. Normal t R N A was obtained from cellsgrown in medium containing 20/~g/ml of cysteine and methionine, while sulfur-deficientt R N A was obtained after 6 h of cysteine starvation of the same organism [13]. The mixture of aminoacyl-tRNA synthetases was prepared from E. coli Q13 by the method of Kelmers et al. [14]. This preparation contained 4 rag/ ml protein as measured by the method of Lowry et al. [15]. Purified isoleucylt R N A synthetase was obtained from E. coli K12 according to the method of Baldwin and Berg [16], except that the Alumina C7 Gel step was omitted. An overall 680-fold purification was obtained through this procedure and the preparation contained 0.74 mg/ml protein. Reaction mixture A (0.61 ml) contained 61.5 m M Tris-HCl (pH 7.3), 9.83 m M magnesium acetate, 1.64 m M ATP, 5.25 # M L-[14C]isoleucine (312.5 Ci/mol, N e w England Nuclear), 1 A260 unit of either normal or sulfur-deficientt R N A and 0.11 m g of the aminoacyl-tRNA synthetase mixture. Reaction mixture B was a modification of the method of Nishimura et al. [17] and contained 20 m M Tris-HCI (pH 7.5), 10 m M magnesium acetate, 10 mM potassium chloride, 2.0 mM ATP, 6.4 #M L-[14Clisoleucine (312.5 Ci/mol, New England Nuclear), 1 A260 unit of each of normal or sulfurdeficient tRNA and 0.74/~g of pure isoleucyl-tRNA synthetase in total volume of 0.5 ml. All assays were carried out at 37°C. Normal and sulfur-deficient [ ~4C]isoleucyl-tRNA was isolated from reaction mixture A by phenol extraction and ethanol precipitation. Enzymatic IO0,
E-
80
0
W 60
<~80 W z 60
o
U
40
v1
7
20
M 2o
0 I[ I:l
o
~
~
TIME (re,n)
o
30
60
90
120
TIME (rain)
Fig. I . K i n e t i c s o f i s o l e u c y l - t R N A f o r m a t i o n w i t h n o r m a l and s u l f u r - d e f i c i e n t t R N A . T h e r e a c t i o n w a s mitiated b y t h e a d d i t i o n o f u n f r a e t i o n a t e d a m i n o a c y l - t R N A s y n t h e t a s e s to t h e r e a c t i o n m i x t u r e A . o . .o, n o r m a l t R N A ; o o, s u l f u r - d e f i c i e n t t R N A . Fig. 2. E n z y m a t i c h y d r o l y s i s o f i s o l e u c y l - t R N A i n t h e absence o f AMP a n d PPi" 1 A260 u n / t each o f either n o r m a l (-~) o r s u l f u r - d e f i c i e n t (e _-) [i 4 0 ] l s o l e u e y l . t R N A w e r e i n c u b a t e d in a s s a y m i x t u r e A c o n t a i n i n g 0 . 1 1 m g o f s y n t h e t a s e m i x t u r e , b u t l a c k i n g A T P and i s o l e u c i n e . C o n t r o l s (o o, n o r m a l ; o o, s u l f u r - d e f i c i e n t ) had n o s y n t h e t a s e added.
86
I00
'< BO hi Z W J
0 U.I _J
0
2O
36
6b TIME (rain)
Fig. 3. K i n e t i c s o f i s o l e u c y l - t R N A f o r m a t i o n w i t h n o r m a l and s u l f u r - d e f i c i e n t t R N A . This s t u d y w a s carried o u t w i t h 0 . 7 4 ~g o f p u r i f i e d i s o l e u c y l - t R N A s y n t h e t a s e , using assay m i x t u r e B. 7 . 4 ~g o f addit i o n a l e n z y m e w e r e a d d e d at t h e p o i n t s i n d i c a t e d b y a r r o w s , o o, n o r m a l t R N A ; o o, sulfurdeficient tRNA.
hydrolysis of 1 A260 unit each of either normal or sulfur-deficient isoleucylt R N A was performed in the same assay mixture, b u t lacking ATP and isoleucine. The filter disc m e t h o d [13] was used to determine the a m o u n t of acid precipitable radioactivity in 0.05 ml samples taken at various times in all assays. Fig. 1 shows the typical kinetics observed for enzymatic acylation of normal and sulfur-deficient E. coli C6 t R N A with [ 14C] isoleucine in reaction mixture A as described above. The plateau level of acylation is lower for normal tRNA. This difference was n o t due to misacylation or lack of integrity of the -CCA end in normal t R N A , and all reaction c o m p o n e n t s were present in optimal amounts [18]. It had been reported that such differences in plateau levels could be the result of enzymatic deacylation [10,11]. To investigate this possibility, normal and sulfur-deficient [14C]isoleucyl-tRNA was isolated, and deacylation was measured with and w i t h o u t added synthetase as described above. Fig. 2 shows that the rates of enzymatic deacylation (in the absence of AMP and PPi) were similar for normal and sulfur-deficient tRNA. Incomplete aminoacylation of E. coli t R N A with isoleucine had been previously reported to be due to an undetermined artifact of the crude in vitro system [19]. Therefore, we prepared purified isoleucyl-tRNA synthetase and tested the acylation of these tRNAs, the results being shown in Fig. 3. With 0.74/~g of synthetase, the acylation patterns for the t w o tRNAs were similar to those seen with unfractionated enzyme (Fig. 1). However, when additional enzyme was added at the reaction plateau (30 min), the extent o f normal isoleucyl-tRNA formation was increased. After the second synthetase addition at 75 min, the extent of isoleucylation of normal t R N A rose to that seen for sulfur-deficient tRNA. A similar b u t less pronounced response was observed with reaction mixture A when more crude enzyme was added at the plateau level. Other experiments showed that values of 82 pmol isoleucine per A260 unit were obtained for either t R N A when the reaction was initiated with 7.4 ~g of synthetase. Under these conditions further enzyme additions
87 were without effect. The extent of aminoacylation of sulfur~leficient tRNA was not significantly altered by enzyme addition presumably because it was fully acylated during the initial reaction. We recently reported that the rate of i8oleucylation of this tRNA was greater than that of fully-thiolated tRNA [18]. This higher rate could account for the complete acylation of sulfurdeficient tRNA prior to enzyme inactivation. Addition of either tRNA to reaction mixtures which had reached the plateau level failed to increase the extent of the reaction, substantiating the conclusion that enzyme inactivation had occurred. Addition of 10 mM 2-mercaptoethanol, 10 mM glutathione, or 1 mg/ml of bovine serum albumin to the reaction mixture B failed to prevent the inactivation of synthetase. Preincubation of the synthetase for 6 min with either normal or sulfur-deficient tRNA resulted in a 50% loss of activity. The same loss was observed when tRNA was absent during preincubation. Hence, these results suggest that tRNA does not play a role in the inactivation of the enzyme. Our results suggest that isoleucyl-tRNA 8ynthetase inactivation causes the attainment of a premature plateau level for normal tRNA. This effect can be overcome by the use of higher concentrations of purified synthetase. We determined that all reaction components were at optimal concentrations [18], but these conditions were not sufficient to protect the enzyme against inactivation. Our data do not confirm the hypothesis of Bonnet and Ebel [9l, that the plateau level of an aminoacylation reaction is a function of the rates of acylation and deacylation. For isoleucyl-tRNA synthetase, and perhaps for other synthetases, enzyme inactivation may cause a premature reaction plateau which may not be predicted using the above theory. These findings demonstrate the importance of defining all reaction parameters when comparing aminoacylation capacities of two tRNA preparations. If assays are carried out at low synthetase levels, the plateau level may indicate the amount of a given tRNA present only if enzyme inactivation is ruled out. Conversely, rate differences may be overlooked if the assays are carried out at high enzyme levels. Finally, a system optimized for complete in vitro aminoacylation of a tRNA preparation with one amino acid may not be suitable for aminoacylation using other amino acids. Supported in part by NCI grant CA-16567. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Nesbitt, III, J.A. and Lennarz, W.J. (1968) J. Biol. Chem. 243, 3088--3095 Gould, R.M., Thornton, M.P., Liepkalns, V. and Lennarz, W.J. (1968) J. Biol. Chem. 243, 3096--3104 Genter, N. and Berg~ P. (1971) Fed. Proc. 30, 1218 (Abstr.) Momose, K. and Kaji, A. (1966) J. Biol. Chem. 241, 3294--3307 Ryan, A.M. and Borek, E. (1971) Prog. Nucleic Acid Res. Mol. Biol. 11,193--228 Brenchley, J.E. and Williams, L.S. (1975) Ann. Rev. Microbiol. 29, 251--274 Yegian, C.D., Stent, G.S. and Martin, E.M. (1966) Proe. Natl. Acad. Sci. U.S. 55, 839--846 Butler, M., Derbre, A. and Arnstein, H.R.V. (1975) Biochem. J. 150, 419--432 $51/, D. and Sehimmel, P.R. (1974) in The Enzymes (Boyer, P.D., ed.), Vol. 10, pp. 469--588, A c a d e m i c Press, N e w York Bonnet, J. and Ebel, J.P. (1972) Eur. J. Bioehem. 3 1 , 3 3 5 - - 3 4 4 Renaud, M., Bollack, C. and Ebel, J.P. (1974) Btoehimie 56, 1203--1209 yon Ehrenstein, G. (1967) Methods Enzymol. 12A, 588--596 Harris, C.L., Titchener, E.B. and Cline, A.L. (1969) J. Bacteriol. 100, 1322--1327 Kelmers, A.D., Novelli, G.D. and Stulberg, M.P. (1965) J. Biol. Chem. 240, 3979--3983
88
15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - 275 16 Baldwin, A.N. and Berg, P. (1966) J. Biol. Chem. 2 4 1 , 8 3 1 - - 8 3 8 17 Nishimu~a, S., Harada, F., Narushima, U, and Seno, T, (1967) Biochim. Biophys. Acta 142, 133--148 18 Harris, C.L., Marashi, F. and Titehener, E.B. (1976) Nucleic Acid Res. 3, 2 1 2 9 - - 2 1 4 2 19 Yegian, C.D. and Stent, G.S. (1969) J. Mol. Biol. 39, 59--71