295
Biochimica et Biophysica Acta, 476 (1977) 295--302 © Elsevier/North-Holland Biomedical Press
BBA 98942
PURIFICATION AND PROPERTIES OF SOYBEAN LEGHEMOGLOBIN MESSENGER RNA
ROSE SIDLOI-LUMBROSO and HERBERT M. SCHULMAN Lady Davis Institute for Medical Research of the Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal, Quebec, H3T 1E2 (Canada) (Received January 24th, 1977)
Summary Poly(A)-containing leghemoglobin mRNA from soybean root nodules has been purified 84-fold, as judged by its ability to direct the in vitro synthesis of leghemoglobin in a wheat germ system. It has a poly(A) content of 8.6% and a molecular weight, estimated by formamide gel electrophoresis, of 260 000. mRNA with a molecular weight of around 143 000 would be sufficient to code for leghemoglobin. Thus, with respect to both its poly(A) content and its unexpectedly high molecular weight, leghemoglobin mRNA is similar to mRNAs isolated from animal tissues.
Introduction Leghemoglobin is the major soluble protein found in legume root nodules which form after legume roots are invaded by soil bacteria of the genus Rhizobium. Although the presence of leghemoglobin is confined to root nodules, there is considerable evidence that the protein is a product of plant genes [1--3] and as such represents a major contribution of the legume genome to the expression of rhizobial nitrogenase within the nodule tissue. Soybean leghemoglobin is composed of two major and two minor components [4], which together comprise about 40% of the total soluble protein of mature nodules [5]. Because of differences in their amino acid compositions the two major soybean leghemoglobin components are thought to be the products of different genes [4]. It has been proposed that leghemoglobin functions by facilitating oxygen diffusion in nodules, delivering oxygen at a high flux but low partial pressure to sites of oxidative metabolism while protecting nitrogenase from inactivation by oxygen [6]. It has been shown that leghemoglobin mRNA with a sedimentation coefficient of around 10 S is present ih the poly(A)-containing fraction of soybean root nodule polysomal RNA [7]. As a step toward elucidating the mechanism
296 responsible for the selective expression of leghemoglobin genes during legume symbiotic nitrogen fixation we have undertaken the purification and characterization of poly(A)-containing leghemoglobin m R N A from soybean r o o t nodules. Materials and Methods Soybean plants (Glycine max vat. Kanrich) were grown from inoculated seeds (Atlee Burpee Seed Co., Riverside, Calif.) as described previously [5]. Frozen root nodules from 35-day-old plants were ground to a powder under liquid nitrogen with a porcelain mortar and pestle. The p o w d e r was mixed with four volumes of buffer composed of 0.15 M Tris/acetate; 0.05 M KC1; 0.02 M magnesium acetate; 0.2 M sucrose; 0.005 M 2-mercaptoethanol; 0.125% diethylpyrocarbonate and adjusted to a final pH of 8.5 at 4 ° C. After mixing in the cold for 5--10 min the slurry was centrifuged at 4°C in a SorvaU S-34 rotor at 11 500 rev./min for 10 min. Polysomes were isolated from the post-mitochondrial supernatant by centrifugation through t w o volumes of 1.5 M sucrose prepared in 0.01 M magnesium acetate; 0.04 M KC1 and 0.05 M Tris/acetate, pH 8.5. Centrifugation was at 50 000 rev./min in a Beckman Ti-50 rotor for 75 min, at 4 ° C. R N A was extracted from resuspended polysomes by centrifugation in CsC1 as described by Glisin et al. [8]. Poly(A)-containing R N A was isolated from polysomal R N A by t w o passages on oligo(dT)-cellulose (Collaborative Research) according to Aviv and Leder [9] except that poly(A)-containing R N A was eluted with distilled water. 10-S leghemoglobin m R N A was prepared b y centrifuging poly(A)-containing R N A on 10--35% linear sucrose gradients [10] in a Beckman SW-56 rotor at 50 000 rev./min for 4 h at 21°C. A2s4nm was monitored by pumping the contents of the tubes through an Isco ultraviolet analyzer. Fractions under the 10 S peak were pooled and precipitated several times a t - - 2 0 ° C with 2.5 volumes of ethanol in the presence of 0.2 M potassium acetate, pH 5.6. The precipitated R N A was vacuum dried, dissolved in H20 and stored at --70 ° C. Leghemoglobin mRNA-directed protein synthesis was measured with a wheat germ system prepared from non-toasted wheat germ (General Mills). The composition of the system was as described [11] except that optimum magnesium and potassium concentrations of 3 and 65 mM, respectively, were used. Samples containing non-saturating levels of R N A and 5 pCi of [3H]leucine (50 Ci/ mmol) in a final volume of 100/~1 were incubated for 90 min at 30°C. For the determination of total protein synthesis, carrier bovine serum albumin was added to the ribosome-free supernatants which where then precipitated with t w o volumes of 10% trichloroacetic acid (containing 0.1% leucine) at 0°C for 60 min. Precipitates were collected b y centrifugation, extracted with 5% trichloroacetic acid at 100°C for 10 min, washed twice with 5% trichloroacetic acid at 0°C and dissolved in NCS (Amersham/Searle). Radioactivity was measured b y liquid scintillation counting at an efficiency of a b o u t 35%. To measure leghemoglobin synthesis 1 pg of carrier leghemoglobin and rabbit antibody (IgG fraction) at equivalence was added to each ribosome-free supernatant. The samples were incubated at 37°C for 40 min and overnight at 4°C. The immuno-
297 precipitates were collected by centrifugation and washed with phosphate-buffered saline. Carrier bovine serum albumin was added and the samples were processed as above. The presence of poly(A) in RNA fractions was determined using 3H-labeled poly(U) (Schwarz/Mann) hybridization to RNA [12]. The poly(A) content of RNA was calculated by reference to a standard curve of 3H-labeled poly(U) hybridization to increasing amounts of poly(A) (Schwarz/Mann) with excess 3H-labeled poly(U). Mouse globin mRNA was a gift from Dr. L. Kleiman. Results and Discussion
In trying to optimize our procedures for isolating mRNA from nodule tissue we found that while omitting non-ionic detergent [7] from the buffer used to extract the tissue resulted in a polysome yield of only 64% of that obtained when the detergent was present, the polysomes isolated in the absence of detergent were about twice as active in supporting protein synthesis in the wheat germ system as those obtained with the use of detergent. Possibly the detergent solubilizes some ribonucleases which partially degrade polysomal mRNA [13]. We also found that, although phenol was used previously for extracting RNA from nodule polysomes [7], extraction of polysomal RNA by centrifugation through CsC1 [8] yielded RNA which had about 35% more protein-synthesizing activity in the wheat germ system. When these procedures were employed, sucrose gradient centrifugation of poly(A)-containing RNA resulted in a 10 S fraction (Fig. 1B) with an 84-fold purification of leghemoglobin mRNA activity (Table I). The poly(A) content of this leghemoglobin mRNA of 8.6% (Table I) is close to that reported for mammalian globin mRNAs [14]. Slightly further purification could be achieved by pooling a few fractions from a subsequent sucrose gradient centrifugation of this 10 S RNA (Fig. 1C), however, the yield was extremely low. Our estimates for the purity of the leghemoglobin mRNA shown in Table I were made utilizing the technique of immunoprecipitation by antibodies to leghemoglobin of the RNA-directed products of the wheat germ system and are close to those reported for the purification of ovalbumin mRNA [15]. Since both ovalbumin and leghemoglobin account for about 50% of the protein synthesis directed by the total poly(A)-containing RNA extracted from chick oviduct and root nodules, respectively, our purification results are in close agreement. In order to estimate the functional purity of the leghemogiobin mRNA by techniques not involving immunoprecipitation, all the proteins synthesized by the wheat germ system incubated with leghemoglobin mRNA were analyzed by SDS and disc gel electrophoresis. Since the molecular weights of the two main soybean leghemoglobin components are very similar [4] they are not separated by SDS gel electrophoresis. As shown in Fig. 2 the bulk of the radioactive products which entered such a gel (over 70%) corresponded electrophoretically to the carrier leghemoglobin. The major portion of the remaining radioactivity migrated ahead of leghemoglobin, indicating that it was contained in material having molecular weights lower than 16 000. Since the most of the soluble proteins, other than leghemoglobin, in nodules from 35
Biochimica et Biophysica Acta, 476 (1977) 295--302 © Elsevier/North-Holland Biomedical Press
BBA 98942
PURIFICATION AND PROPERTIES OF SOYBEAN LEGHEMOGLOBIN MESSENGER RNA
ROSE SIDLOI-LUMBROSO and HERBERT M. SCHULMAN Lady Davis Institute for Medical Research of the Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal, Quebec, H3T 1E2 (Canada) (Received January 24th, 1977)
Summary Poly(A)-containing leghemoglobin mRNA from soybean root nodules has been purified 84-fold, as judged by its ability to direct the in vitro synthesis of leghemoglobin in a wheat germ system. It has a poly(A) content of 8.6% and a molecular weight, estimated by formamide gel electrophoresis, of 260 000. mRNA with a molecular weight of around 143 000 would be sufficient to code for leghemoglobin. Thus, with respect to both its poly(A) content and its unexpectedly high molecular weight, leghemoglobin mRNA is similar to mRNAs isolated from animal tissues.
Introduction Leghemoglobin is the major soluble protein found in legume root nodules which form after legume roots are invaded by soil bacteria of the genus Rhizobium. Although the presence of leghemoglobin is confined to root nodules, there is considerable evidence that the protein is a product of plant genes [1--3] and as such represents a major contribution of the legume genome to the expression of rhizobial nitrogenase within the nodule tissue. Soybean leghemoglobin is composed of two major and two minor components [4], which together comprise about 40% of the total soluble protein of mature nodules [5]. Because of differences in their amino acid compositions the two major soybean leghemoglobin components are thought to be the products of different genes [4]. It has been proposed that leghemoglobin functions by facilitating oxygen diffusion in nodules, delivering oxygen at a high flux but low partial pressure to sites of oxidative metabolism while protecting nitrogenase from inactivation by oxygen [6]. It has been shown that leghemoglobin mRNA with a sedimentation coefficient of around 10 S is present ih the poly(A)-containing fraction of soybean root nodule polysomal RNA [7]. As a step toward elucidating the mechanism
296 responsible for the selective expression of leghemoglobin genes during legume symbiotic nitrogen fixation we have undertaken the purification and characterization of poly(A)-containing leghemoglobin m R N A from soybean r o o t nodules. Materials and Methods Soybean plants (Glycine max vat. Kanrich) were grown from inoculated seeds (Atlee Burpee Seed Co., Riverside, Calif.) as described previously [5]. Frozen root nodules from 35-day-old plants were ground to a powder under liquid nitrogen with a porcelain mortar and pestle. The p o w d e r was mixed with four volumes of buffer composed of 0.15 M Tris/acetate; 0.05 M KC1; 0.02 M magnesium acetate; 0.2 M sucrose; 0.005 M 2-mercaptoethanol; 0.125% diethylpyrocarbonate and adjusted to a final pH of 8.5 at 4 ° C. After mixing in the cold for 5--10 min the slurry was centrifuged at 4°C in a SorvaU S-34 rotor at 11 500 rev./min for 10 min. Polysomes were isolated from the post-mitochondrial supernatant by centrifugation through t w o volumes of 1.5 M sucrose prepared in 0.01 M magnesium acetate; 0.04 M KC1 and 0.05 M Tris/acetate, pH 8.5. Centrifugation was at 50 000 rev./min in a Beckman Ti-50 rotor for 75 min, at 4 ° C. R N A was extracted from resuspended polysomes by centrifugation in CsC1 as described by Glisin et al. [8]. Poly(A)-containing R N A was isolated from polysomal R N A by t w o passages on oligo(dT)-cellulose (Collaborative Research) according to Aviv and Leder [9] except that poly(A)-containing R N A was eluted with distilled water. 10-S leghemoglobin m R N A was prepared b y centrifuging poly(A)-containing R N A on 10--35% linear sucrose gradients [10] in a Beckman SW-56 rotor at 50 000 rev./min for 4 h at 21°C. A2s4nm was monitored by pumping the contents of the tubes through an Isco ultraviolet analyzer. Fractions under the 10 S peak were pooled and precipitated several times a t - - 2 0 ° C with 2.5 volumes of ethanol in the presence of 0.2 M potassium acetate, pH 5.6. The precipitated R N A was vacuum dried, dissolved in H20 and stored at --70 ° C. Leghemoglobin mRNA-directed protein synthesis was measured with a wheat germ system prepared from non-toasted wheat germ (General Mills). The composition of the system was as described [11] except that optimum magnesium and potassium concentrations of 3 and 65 mM, respectively, were used. Samples containing non-saturating levels of R N A and 5 pCi of [3H]leucine (50 Ci/ mmol) in a final volume of 100/~1 were incubated for 90 min at 30°C. For the determination of total protein synthesis, carrier bovine serum albumin was added to the ribosome-free supernatants which where then precipitated with t w o volumes of 10% trichloroacetic acid (containing 0.1% leucine) at 0°C for 60 min. Precipitates were collected b y centrifugation, extracted with 5% trichloroacetic acid at 100°C for 10 min, washed twice with 5% trichloroacetic acid at 0°C and dissolved in NCS (Amersham/Searle). Radioactivity was measured b y liquid scintillation counting at an efficiency of a b o u t 35%. To measure leghemoglobin synthesis 1 pg of carrier leghemoglobin and rabbit antibody (IgG fraction) at equivalence was added to each ribosome-free supernatant. The samples were incubated at 37°C for 40 min and overnight at 4°C. The immuno-
297 precipitates were collected by centrifugation and washed with phosphate-buffered saline. Carrier bovine serum albumin was added and the samples were processed as above. The presence of poly(A) in RNA fractions was determined using 3H-labeled poly(U) (Schwarz/Mann) hybridization to RNA [12]. The poly(A) content of RNA was calculated by reference to a standard curve of 3H-labeled poly(U) hybridization to increasing amounts of poly(A) (Schwarz/Mann) with excess 3H-labeled poly(U). Mouse globin mRNA was a gift from Dr. L. Kleiman. Results and Discussion
In trying to optimize our procedures for isolating mRNA from nodule tissue we found that while omitting non-ionic detergent [7] from the buffer used to extract the tissue resulted in a polysome yield of only 64% of that obtained when the detergent was present, the polysomes isolated in the absence of detergent were about twice as active in supporting protein synthesis in the wheat germ system as those obtained with the use of detergent. Possibly the detergent solubilizes some ribonucleases which partially degrade polysomal mRNA [13]. We also found that, although phenol was used previously for extracting RNA from nodule polysomes [7], extraction of polysomal RNA by centrifugation through CsC1 [8] yielded RNA which had about 35% more protein-synthesizing activity in the wheat germ system. When these procedures were employed, sucrose gradient centrifugation of poly(A)-containing RNA resulted in a 10 S fraction (Fig. 1B) with an 84-fold purification of leghemoglobin mRNA activity (Table I). The poly(A) content of this leghemoglobin mRNA of 8.6% (Table I) is close to that reported for mammalian globin mRNAs [14]. Slightly further purification could be achieved by pooling a few fractions from a subsequent sucrose gradient centrifugation of this 10 S RNA (Fig. 1C), however, the yield was extremely low. Our estimates for the purity of the leghemoglobin mRNA shown in Table I were made utilizing the technique of immunoprecipitation by antibodies to leghemoglobin of the RNA-directed products of the wheat germ system and are close to those reported for the purification of ovalbumin mRNA [15]. Since both ovalbumin and leghemoglobin account for about 50% of the protein synthesis directed by the total poly(A)-containing RNA extracted from chick oviduct and root nodules, respectively, our purification results are in close agreement. In order to estimate the functional purity of the leghemogiobin mRNA by techniques not involving immunoprecipitation, all the proteins synthesized by the wheat germ system incubated with leghemoglobin mRNA were analyzed by SDS and disc gel electrophoresis. Since the molecular weights of the two main soybean leghemoglobin components are very similar [4] they are not separated by SDS gel electrophoresis. As shown in Fig. 2 the bulk of the radioactive products which entered such a gel (over 70%) corresponded electrophoretically to the carrier leghemoglobin. The major portion of the remaining radioactivity migrated ahead of leghemoglobin, indicating that it was contained in material having molecular weights lower than 16 000. Since the most of the soluble proteins, other than leghemoglobin, in nodules from 35
