ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
166, 683-684 (1975)
COMMUNICATION Multiple
Forms
of Elongation
Elongation factor 1 (EFl), which catalyzes the GTP-dependent binding of aminoacyl-tRNA (AAtRNA) to the A site on the ribosome, has been reported to be present as multiple molecular-weight forms in a variety of eukaryotic cells (l-6). Data from several sources (2. 3. 6. 7) indicate that the heavy form of the enzyme I EFl H), having molecular weights that can be over 1 x 106, are aggregates of a light species (EFl,,) having a molecular weight 50-60,000. There is evidence (8) that the heavy forms of the enzyme contain lipids which may be involved in maintaining the integrity of the aggregates. The role of various forms of EFl is not known; however, there are several differences between EFl,, and EF,,. Lego’cki et al. (9) have reported that EFl,. from calf brain is about five times more effective than EFl,, in forming a stable ternary complex with GTP and AA-tRNA, although both forms appear to be equally efficient in catalyzing the binding of PhetRNA to ribosomes. It has been suggested that the heavy forms of EFl may represent storage forms of the enzyme or that the intercoversion of EFl,, and EFl,. may have a regulatory function. In an attempt to understand the physiological role of the multiple forms of this protein. we have investigated the presence of the various species of EFl in rat tissue. The present report shows that while EFl can be found as a mixture of heavy and light forms in rat liver, kidney. and spleen, the brain contains almost exclusively the light form. In addition, the same molecular-weight pattern in each tissue is maintained from 2 mo to 24 mo of age. Male CD strain Sprague-Dawley rats of different ages were obtained from Charles River Breeding Laboratories. Meridan, MA. These animals had been raised by the breeder under carefully controlled environmental conditions. The animals were decapitated, and brain. liver, kidney, and spleen removed. The tissues were rinsed in ice-cold 0.9% NaCl and homogenized with a glass homogenizer with a motor-driven Teflon pestle. The homogenate was centrifuged for 30 min at 30,OOOg and then for 1.5 hr at lOO.OOOg.The resulting supernatant solution was assayed for EF! activity as described by Weissbach et al. (3). Sucrose gradient centrifugation to separate the various forms of EFl was done according to the procedure of Moon et al. (6). In general. loo-~1 aliquots of the 100,OOOgsupernatant solution containing between 40 and 60 units of enzyme were layered on Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Factor
1 in Various
Rat Tissues
4.2 ml of a 5-20s linear sucrose gradient in 50 mM Tris (pH 7.4), 5 mM DTT, and centrifuged for 2 hr at 50,000 rpm in a Spinco SW56 rotor. Eight-drop fractions were collected from the bottom of the tube and assayed for enzymatic acitivity (6). EFl,. appeared in fractions 28-30, whereas the heavy forms of the enzyme were seen in fractions 15-25. Chemicals and other materials were obtained from commercial sources or prepared as previously described (3). Figure 1 shows the sucrose gradient profiles of EFl activity in the supernatant fractions from brain, liver, kidney, and spleen of 2.mo-old rats. It can be seen that the EFl pattern in the brain is significantly different from that of the other tissues. Thus, only the light form (M, 50-70,000) of this enzyme is found in the brain, while in other tissues, although EFl, is present, a large percentage of the activity is seen as high molecular-weight aggregates (2 x 105-1.2 x 106). Table I shows that the ratio of EFl./EFl,, ranges from about 2-3 in extracts of liver, kidney, and spleen, while in the brain, virtually 100% of the enzyme is
FRACTION
NUMBER
1. Sucrose gradient profiles of EFl in supernatants from rat tissues at 2 mo of age. For details see text and Ref. 6. FIG.
684
COMMUNICATION TABLE
I
RATIO OF EFl ACTIVITIES (EFl./EFl,.) IN VARIOUS RAT TISSUES AT DIFFERENT AGES’ Tissue
Age (mo) Brain 2 12 24
OF BIOCHEMISTRY
AND
BIOPHYSICS
166, 683-684 (1975)
COMMUNICATION Multiple
Forms
of Elongation
Elongation factor 1 (EFl), which catalyzes the GTP-dependent binding of aminoacyl-tRNA (AAtRNA) to the A site on the ribosome, has been reported to be present as multiple molecular-weight forms in a variety of eukaryotic cells (l-6). Data from several sources (2. 3. 6. 7) indicate that the heavy form of the enzyme I EFl H), having molecular weights that can be over 1 x 106, are aggregates of a light species (EFl,,) having a molecular weight 50-60,000. There is evidence (8) that the heavy forms of the enzyme contain lipids which may be involved in maintaining the integrity of the aggregates. The role of various forms of EFl is not known; however, there are several differences between EFl,, and EF,,. Lego’cki et al. (9) have reported that EFl,. from calf brain is about five times more effective than EFl,, in forming a stable ternary complex with GTP and AA-tRNA, although both forms appear to be equally efficient in catalyzing the binding of PhetRNA to ribosomes. It has been suggested that the heavy forms of EFl may represent storage forms of the enzyme or that the intercoversion of EFl,, and EFl,. may have a regulatory function. In an attempt to understand the physiological role of the multiple forms of this protein. we have investigated the presence of the various species of EFl in rat tissue. The present report shows that while EFl can be found as a mixture of heavy and light forms in rat liver, kidney. and spleen, the brain contains almost exclusively the light form. In addition, the same molecular-weight pattern in each tissue is maintained from 2 mo to 24 mo of age. Male CD strain Sprague-Dawley rats of different ages were obtained from Charles River Breeding Laboratories. Meridan, MA. These animals had been raised by the breeder under carefully controlled environmental conditions. The animals were decapitated, and brain. liver, kidney, and spleen removed. The tissues were rinsed in ice-cold 0.9% NaCl and homogenized with a glass homogenizer with a motor-driven Teflon pestle. The homogenate was centrifuged for 30 min at 30,OOOg and then for 1.5 hr at lOO.OOOg.The resulting supernatant solution was assayed for EF! activity as described by Weissbach et al. (3). Sucrose gradient centrifugation to separate the various forms of EFl was done according to the procedure of Moon et al. (6). In general. loo-~1 aliquots of the 100,OOOgsupernatant solution containing between 40 and 60 units of enzyme were layered on Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
Factor
1 in Various
Rat Tissues
4.2 ml of a 5-20s linear sucrose gradient in 50 mM Tris (pH 7.4), 5 mM DTT, and centrifuged for 2 hr at 50,000 rpm in a Spinco SW56 rotor. Eight-drop fractions were collected from the bottom of the tube and assayed for enzymatic acitivity (6). EFl,. appeared in fractions 28-30, whereas the heavy forms of the enzyme were seen in fractions 15-25. Chemicals and other materials were obtained from commercial sources or prepared as previously described (3). Figure 1 shows the sucrose gradient profiles of EFl activity in the supernatant fractions from brain, liver, kidney, and spleen of 2.mo-old rats. It can be seen that the EFl pattern in the brain is significantly different from that of the other tissues. Thus, only the light form (M, 50-70,000) of this enzyme is found in the brain, while in other tissues, although EFl, is present, a large percentage of the activity is seen as high molecular-weight aggregates (2 x 105-1.2 x 106). Table I shows that the ratio of EFl./EFl,, ranges from about 2-3 in extracts of liver, kidney, and spleen, while in the brain, virtually 100% of the enzyme is
FRACTION
NUMBER
1. Sucrose gradient profiles of EFl in supernatants from rat tissues at 2 mo of age. For details see text and Ref. 6. FIG.
684
COMMUNICATION TABLE
I
RATIO OF EFl ACTIVITIES (EFl./EFl,.) IN VARIOUS RAT TISSUES AT DIFFERENT AGES’ Tissue
Age (mo) Brain 2 12 24
