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Insect ribosomes: Effect of monovalent cation deficiency
276
SHORT COMMUNICATIONS
BBA 93557
Insect ribosomes:
Effect of monovolent cotion deficiency
The importance of magnesium to the structural integrity of ribosomes was recognized in the early attempts at ribosome preparation 1,2. The role of monovalent cations, such as potassium, has been visualized as relatively nonspecific, contributing to the ionic strength of the medium as well as exerting a modifying influence on the binding of Mg 2+ (ref. 3). However, recent studies have suggested a more specific requirement for K + (refs. 4, 5). Potassium deficiency appears to have a drastic effect on the ability of ribosomes to utilize poly U as an artificial template, and has far reaching effects on conformation of mammalian ribosomes 5 and in promoting dissociation of some invertebrate ribosomes 4. House cricket ribosomes 6, as well as some other systems (refs. 7, 8), appear to have no requirement for K + in protein synthesis in vitro, and on this basis would appear to have no stringent K + dependency. Such ribosomes should provide an appropriate material to test the generality of a specific role for K ÷ in ribosome structure and activity. At least for cricket ribosomes, such tests were found to be desirable in order to resolve the apparently contradictory observations of a lack of K + requirement for protein synthesis in vitro~, and decreased endogenous incorporation and poly U-utilizing ability after brief dialysis of ribosomes against K+-free buffers (M. S. KAULENAS, unpublished data). Ribosomes were isolated 9 from adult crickets, Acheta domesticus, and were either used immediately, or, after filtration through Sephadex G-Ioo (equilibrated with the appropriate buffer), dialysed against buffers of ionic composition given in Fig. I and Table I. Dialysis was carried out with stirring for 6 h (4 °) against IOOOvol. of the appropriate medium, which was changed twice. Endogenous and poly U-directed incorporation of L3H]phenylalanine into protein was performed as previously described 6. Sucrose density gradients were analysed by passage through a flow cell of a Gilford recording spectrophotometer. The effects of K ÷ deficiency were analysed by comparing the sedimentation patterns of control ribosomes and of ribosomes dialysed against K+-free buffer (Fig.I). Control ribosomes sedimented into a gradient containing 5 mM MgZ+-I5 o mM K ÷ consist predominantly of monomers and dimers. In the presence of K +, lowering the Mg ~+ concentration to I mM results in a partial dissociation of the monomer peak, while decreasing the Mg 2+ concentration to o.I mM completely dissociates the ribosomes into subunits. Not unexpectedly 3, centrifugation of normal ribosomes into gradients containing 11o K ÷ retards dissociation at lower Mg 2+ concentrations; no subunits are formed in I mM Mg ~÷, while at o.I mM Mg 2+ partial dissociation results, accompanied by changes in the sedimentation coefficients of the monomers and dimers. Sedimentation patterns of ribosomes dialysed against K+-free buffer are drastically altered. Partial dissociation of ribosomes, with changed S values for monomers and subunits, is observed in gradients containing no K +. Lowering the Mg 2+ concentration in K+-free gradients increases the amount of dissociation and further decreases the sedimentation coefficients. The dissociation into subunits brought about by dialysis against K÷-free buffer is not reversible under the conditions tested, with subunits present even in gradients containing K + and 5 mM Mg2+. Complete dissociation of the dialysed ribosomes can be brought about by decreasing Mg~+ to o.I mM in Biochim. Biophys. Acta, 224 (197o) 276-278
SHORT COMMUNICATIONS
2"77
NORMAL
C
tO
20
30
40
20
30
40
SEDIMENTATION DISTANCE (MM)
Fig. I. S e d i m e n t a t i o n p a t t e r n s of n o r m a l cricket r i b o s o m e s a n d of r i b o s o m e s dialysed a g a i n s t K+-free buffer. Dialysis of t h e r i b o s o m e s was carried o u t as described in t h e t e x t , a g a i n s t a buffer c o n t a i n i n g 20 m M Tris, p H 7.8 (at 25°}, I m M MgSO 4. Controls dialysed a g a i n s t 20 m M Tris, p H 7.8, I m M MgSO4, 5 m M KC1 gave identical s e d i m e n t a t i o n p a t t e r n s to t h o s e of freshly prep a r e d (normal) ribosomes. G r a d i e n t s of 8-30 °/o sucrose, c o n t a i n i n g 20 m M Tris, p H 7.8, a n d (A) o.I m M MgSO4, (B) I m M MgSO4, (C) 5 m M MgSO a, (D) o.I m M M g S O t - i 5 o m M KC1, (E) i m M M g S O , - I 5 O m M KC1, (F) 5 m M MgSO4-I5o m M KCI. C e n t r i f u g a t i o n w a s for 75 m i n in t h e Spinco S W - 5 o r o t o r at 50 ooo r e v . / m i n at 4 °. A p p r o x i m a t e l y I A260 nm u n i t of n o r m a l or of dialysed ribosomes, in a v o l u m e of 0.05 ml, was loaded on each gradient. T h e direction of s e d i m e n t a t i o n is f r o m left to right.
gradients containing 15o mM K +, although it should be noted that such subunits sediment more slowly than controls. As shown in Table I, dialysis of ribosomes against K+-free buffer not only reduces endogenous incorporation, but strongly impairs the ability of the ribosomes to utilize poly U. The presence of I mM K + during dialysis is sufficient to protect against the loss of activity, although noticeable inactivation is found at o. I mM K +. Ammonium ion is as effective as K + in its protective role; Na + does not maintain the ability of ribosomes to respond to poly U, although endogenous activity is unaffected. Neither Li+ nor 2-mercaptoethanol can substitute for K + as protective agents. Potassium-deficient cricket ribosomes undergo partial irreversible dissociation and apparent alteration in conformation, with attendant lowering of endogenous incorporation as well as loss of their ability to utilize poly U as template. The presence Biochim. Biophys. Acta, 224 (197 o) 276-278
278
SHORT C O M M U N I C A T I O N S
TABLE I INCORPORATION OF [~H]PHENYLALANINE INTO PROTEIN BY DIALYSED CRICKET RIBOSOMES I(ibosomes were dialysed for 6 h a g a i n s t buffers c o n t a i n i n g 20 m M Tris, p H 7.8 a n d additional c o m p o n e n t s as i n d i c a t e d in t h e Table. T h e c o m p o s i t i o n of t h e i n c u b a t i o n m i x t u r e a n d t h e procedure for t h e detection of incorporation were as previously described".
Dialysis bullet (raM)
E3H]phenylalanine incorporation (disint./min per mg RNA )
I~+
NH4+Na+
5 i o.i . . 5 . -
. . .
-
-
. . .
. .
. . .
. . . 5 i --
2-mercapto- Mg 2+ ethanol
. . .
. . -
.
Li +
i i i i 2 i i i i i I
. . 5 5
. I
i
Endogenous +po~ U
--
5080 5629 4417 1758 1557 8333 1893 5774 4803 5246 2200
of l o w l e v e l s o f K + o r N H 4 + , b u t n o t e q u i v a l e n t
15079 13994 6087 2798 2513 27680 2615 16164 8313 5377 3328
additional
amounts
o f M g ~+, p r e v e n t
these changes, arguing for a specific role for this type of monovalent some structure;
the absence of a requirement
by cricket ribosomes may mean that sufficient K + remains satisfy structural
needs. The exact role of K÷ remains
other eukaryote
r i b o s o m e s 4,5 t h e s t r u c t u r a l
extensive
the
transferase
than
K+-dependent
bound
to the particles to
obscure, and with cricket and
effects of K + deficiency appear to be more
conformational
alterations
around
the peptidyl
s i t e 1° d e s c r i b e d f o r b a c t e r i a l r i b o s o m e s . C l a r i f i c a t i o n o f t h e s p e c i f i c r o l e o f
K + may be important
in studies on eukaryote
This work was supported
ribosome
structure.
in part by NSF grant GB-8450.
Department o/Zoology, University o/Massachusetts, Amherst, Mass. ozo02 (U.S.A.) I 2 3 4 5 6 7 8 9 IO
cation in ribo-
f o r a d d e d K + i n p r o t e i n s y n t h e s i s in vitro
M.S.
KAULENAS
F.-C. CHAO, Arch. Biochem. Biophys., 7 ° (1957) 426. M. G. HAMILTON AND M. L. PETERMANN, J. Biol. Chem., 234 (1959) 1441. A. S. SPIRIN AND L. P. GAVRILOVA, "The Ribosome", Springer-Verlag, N e w York, 1969, p. 29. T. HULTIN, P. H. NASLUND AND M. O. NILSSON, Exptl. Cell Res., 55 (1969) 269. P. H. NASLUND AND T. HULTIN, Biochim. Biophys. Acta, 204 (197 o) 237. M. S. KAULENAS, J. Insect Physiol., in t h e press. A. R. MEANS AND C. A. BAKER, Biochim. Biophys. Acta, 182 (1969) 461. F. O. WETTSTEIN, T. STAEHELIN AND H. ~N~OLL,Nature, 197 (1963) 43 o. M. S. KAULENAS, J. Insect Physiol., 16 (197o) 813. R. MISKIN, A. ZAMIR AND D. ELSON, Biochem. Biophys. Res. Commun., 33 (1968) 551.
Received
June
30th, 1970
Biochim. Biophys. Acta, 224 (197 o) 276-278
SHORT COMMUNICATIONS
BBA 93557
Insect ribosomes:
Effect of monovolent cotion deficiency
The importance of magnesium to the structural integrity of ribosomes was recognized in the early attempts at ribosome preparation 1,2. The role of monovalent cations, such as potassium, has been visualized as relatively nonspecific, contributing to the ionic strength of the medium as well as exerting a modifying influence on the binding of Mg 2+ (ref. 3). However, recent studies have suggested a more specific requirement for K + (refs. 4, 5). Potassium deficiency appears to have a drastic effect on the ability of ribosomes to utilize poly U as an artificial template, and has far reaching effects on conformation of mammalian ribosomes 5 and in promoting dissociation of some invertebrate ribosomes 4. House cricket ribosomes 6, as well as some other systems (refs. 7, 8), appear to have no requirement for K + in protein synthesis in vitro, and on this basis would appear to have no stringent K + dependency. Such ribosomes should provide an appropriate material to test the generality of a specific role for K ÷ in ribosome structure and activity. At least for cricket ribosomes, such tests were found to be desirable in order to resolve the apparently contradictory observations of a lack of K + requirement for protein synthesis in vitro~, and decreased endogenous incorporation and poly U-utilizing ability after brief dialysis of ribosomes against K+-free buffers (M. S. KAULENAS, unpublished data). Ribosomes were isolated 9 from adult crickets, Acheta domesticus, and were either used immediately, or, after filtration through Sephadex G-Ioo (equilibrated with the appropriate buffer), dialysed against buffers of ionic composition given in Fig. I and Table I. Dialysis was carried out with stirring for 6 h (4 °) against IOOOvol. of the appropriate medium, which was changed twice. Endogenous and poly U-directed incorporation of L3H]phenylalanine into protein was performed as previously described 6. Sucrose density gradients were analysed by passage through a flow cell of a Gilford recording spectrophotometer. The effects of K ÷ deficiency were analysed by comparing the sedimentation patterns of control ribosomes and of ribosomes dialysed against K+-free buffer (Fig.I). Control ribosomes sedimented into a gradient containing 5 mM MgZ+-I5 o mM K ÷ consist predominantly of monomers and dimers. In the presence of K +, lowering the Mg ~+ concentration to I mM results in a partial dissociation of the monomer peak, while decreasing the Mg 2+ concentration to o.I mM completely dissociates the ribosomes into subunits. Not unexpectedly 3, centrifugation of normal ribosomes into gradients containing 11o K ÷ retards dissociation at lower Mg 2+ concentrations; no subunits are formed in I mM Mg ~÷, while at o.I mM Mg 2+ partial dissociation results, accompanied by changes in the sedimentation coefficients of the monomers and dimers. Sedimentation patterns of ribosomes dialysed against K+-free buffer are drastically altered. Partial dissociation of ribosomes, with changed S values for monomers and subunits, is observed in gradients containing no K +. Lowering the Mg 2+ concentration in K+-free gradients increases the amount of dissociation and further decreases the sedimentation coefficients. The dissociation into subunits brought about by dialysis against K÷-free buffer is not reversible under the conditions tested, with subunits present even in gradients containing K + and 5 mM Mg2+. Complete dissociation of the dialysed ribosomes can be brought about by decreasing Mg~+ to o.I mM in Biochim. Biophys. Acta, 224 (197o) 276-278
SHORT COMMUNICATIONS
2"77
NORMAL
C
tO
20
30
40
20
30
40
SEDIMENTATION DISTANCE (MM)
Fig. I. S e d i m e n t a t i o n p a t t e r n s of n o r m a l cricket r i b o s o m e s a n d of r i b o s o m e s dialysed a g a i n s t K+-free buffer. Dialysis of t h e r i b o s o m e s was carried o u t as described in t h e t e x t , a g a i n s t a buffer c o n t a i n i n g 20 m M Tris, p H 7.8 (at 25°}, I m M MgSO 4. Controls dialysed a g a i n s t 20 m M Tris, p H 7.8, I m M MgSO4, 5 m M KC1 gave identical s e d i m e n t a t i o n p a t t e r n s to t h o s e of freshly prep a r e d (normal) ribosomes. G r a d i e n t s of 8-30 °/o sucrose, c o n t a i n i n g 20 m M Tris, p H 7.8, a n d (A) o.I m M MgSO4, (B) I m M MgSO4, (C) 5 m M MgSO a, (D) o.I m M M g S O t - i 5 o m M KC1, (E) i m M M g S O , - I 5 O m M KC1, (F) 5 m M MgSO4-I5o m M KCI. C e n t r i f u g a t i o n w a s for 75 m i n in t h e Spinco S W - 5 o r o t o r at 50 ooo r e v . / m i n at 4 °. A p p r o x i m a t e l y I A260 nm u n i t of n o r m a l or of dialysed ribosomes, in a v o l u m e of 0.05 ml, was loaded on each gradient. T h e direction of s e d i m e n t a t i o n is f r o m left to right.
gradients containing 15o mM K +, although it should be noted that such subunits sediment more slowly than controls. As shown in Table I, dialysis of ribosomes against K+-free buffer not only reduces endogenous incorporation, but strongly impairs the ability of the ribosomes to utilize poly U. The presence of I mM K + during dialysis is sufficient to protect against the loss of activity, although noticeable inactivation is found at o. I mM K +. Ammonium ion is as effective as K + in its protective role; Na + does not maintain the ability of ribosomes to respond to poly U, although endogenous activity is unaffected. Neither Li+ nor 2-mercaptoethanol can substitute for K + as protective agents. Potassium-deficient cricket ribosomes undergo partial irreversible dissociation and apparent alteration in conformation, with attendant lowering of endogenous incorporation as well as loss of their ability to utilize poly U as template. The presence Biochim. Biophys. Acta, 224 (197 o) 276-278
278
SHORT C O M M U N I C A T I O N S
TABLE I INCORPORATION OF [~H]PHENYLALANINE INTO PROTEIN BY DIALYSED CRICKET RIBOSOMES I(ibosomes were dialysed for 6 h a g a i n s t buffers c o n t a i n i n g 20 m M Tris, p H 7.8 a n d additional c o m p o n e n t s as i n d i c a t e d in t h e Table. T h e c o m p o s i t i o n of t h e i n c u b a t i o n m i x t u r e a n d t h e procedure for t h e detection of incorporation were as previously described".
Dialysis bullet (raM)
E3H]phenylalanine incorporation (disint./min per mg RNA )
I~+
NH4+Na+
5 i o.i . . 5 . -
. . .
-
-
. . .
. .
. . .
. . . 5 i --
2-mercapto- Mg 2+ ethanol
. . .
. . -
.
Li +
i i i i 2 i i i i i I
. . 5 5
. I
i
Endogenous +po~ U
--
5080 5629 4417 1758 1557 8333 1893 5774 4803 5246 2200
of l o w l e v e l s o f K + o r N H 4 + , b u t n o t e q u i v a l e n t
15079 13994 6087 2798 2513 27680 2615 16164 8313 5377 3328
additional
amounts
o f M g ~+, p r e v e n t
these changes, arguing for a specific role for this type of monovalent some structure;
the absence of a requirement
by cricket ribosomes may mean that sufficient K + remains satisfy structural
needs. The exact role of K÷ remains
other eukaryote
r i b o s o m e s 4,5 t h e s t r u c t u r a l
extensive
the
transferase
than
K+-dependent
bound
to the particles to
obscure, and with cricket and
effects of K + deficiency appear to be more
conformational
alterations
around
the peptidyl
s i t e 1° d e s c r i b e d f o r b a c t e r i a l r i b o s o m e s . C l a r i f i c a t i o n o f t h e s p e c i f i c r o l e o f
K + may be important
in studies on eukaryote
This work was supported
ribosome
structure.
in part by NSF grant GB-8450.
Department o/Zoology, University o/Massachusetts, Amherst, Mass. ozo02 (U.S.A.) I 2 3 4 5 6 7 8 9 IO
cation in ribo-
f o r a d d e d K + i n p r o t e i n s y n t h e s i s in vitro
M.S.
KAULENAS
F.-C. CHAO, Arch. Biochem. Biophys., 7 ° (1957) 426. M. G. HAMILTON AND M. L. PETERMANN, J. Biol. Chem., 234 (1959) 1441. A. S. SPIRIN AND L. P. GAVRILOVA, "The Ribosome", Springer-Verlag, N e w York, 1969, p. 29. T. HULTIN, P. H. NASLUND AND M. O. NILSSON, Exptl. Cell Res., 55 (1969) 269. P. H. NASLUND AND T. HULTIN, Biochim. Biophys. Acta, 204 (197 o) 237. M. S. KAULENAS, J. Insect Physiol., in t h e press. A. R. MEANS AND C. A. BAKER, Biochim. Biophys. Acta, 182 (1969) 461. F. O. WETTSTEIN, T. STAEHELIN AND H. ~N~OLL,Nature, 197 (1963) 43 o. M. S. KAULENAS, J. Insect Physiol., 16 (197o) 813. R. MISKIN, A. ZAMIR AND D. ELSON, Biochem. Biophys. Res. Commun., 33 (1968) 551.
Received
June
30th, 1970
Biochim. Biophys. Acta, 224 (197 o) 276-278