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The isolation and identification of polyribosomes from cellular slime molds
HORT COMMUNICATIONS
26
ion of polyribosomes from cell
molds
~l to the question of the generality c nthetic his brief report is to describe an ize the in the cellular slime molds. Recently it has been shown in mammalian cells that proteins ized on sters of ribosomes held together b y RNA, rather than m on single In the iculocyte 1-4 it has been shown that these ribosomal cl clusters, ca osomes ?olysomes, contain about five or six ribosomes. The reticulocyt re mufacmany es a single protein, hemoglobin. However, in a mammalian mal teins, such as liver 5, or H e L a cells s-s there is a broad bro; distrit lysome :s. In the case of H e L a cells, this extends up to 3o oor 40 ribo distri:ion of polysome sizes is believed to be related to the distribution dis' r-RNA gths which hold the polysomes togethere,~, 1°. In order to investigate the generality of the polyribc rribosome sy~ anism, looked for their presence in cellular slime molds. Slime Sli mold unique ~ediate /logenetic position in that they are regarded as ha having a sl celled ween plants and animals. They can be grown in suspension, su oeba-like form but under appropriate conditions these the, cells can also organize to Iormm a more complex multicellular organism. In these experiments, e~ Polyspondylium palladum ladum was grown serially in liquid medium as a singlele-membered culture and was labelled elled for various time periods with 14C-labelled amino acids, as described in Fig. I. A varlet /ariety of methods was examined to discover a way of lysing the cells without sub,jecting them to strong sheer gradients. The most successful procedure involved su the addition of sodium deoxycholate to a final concentr ~ncentration of 0.5 %. The chilled SUSpension could be shaken with this detergent and the cells lysed directly. I)eoxycholate )late has a potential disadvantage in that it also lyses the nuclear membrane, and rele~ases DNA. However., since mce there is little DNA in slime slb molds, it was not found necessary to add I)NAase (EC 3.1.4.5) before sucrose-gradient centrifugation. The results of an amin~o acid labelling experiment of this type are shown in Fig. 1. Fig. Ia shows the absorbanc~ racy and radioactivity profile after IO min of incubation with the materials sedimentin lting to the left. The large peak in absorbancy consists of in be seen that in addition, there is a rapidly sedimenting single 8o-S ribosomes. I t can aximum absorbancy peak with a maximum u m near 200 S. The absorbancy curve has a series of small contours which have been identified as single ribosomal increments in a polyribosomal series 1. It can be seen that the broad rapidly sedimenting peak of absorbancy also has a broad peak off radioactivity associated with it. The polysome peak has in the region of polysomes containing 5-6 ribosomes and an absorbancy m a x i m u m m the radioactivity maximuma is in the same region. After 2o min incubation (Fig. Ib) it can be seen that there is only a slight increase in the radioactivity associated with the polysomes. However, the absorbancy profile is substantially the same as that seen in Fig. Ia. An identical ical aliquot of the 2o-min incubation was subjected to .7.16) for 30 min at 4 °, and the absorbancy measured. I t can I #g/ml of RNAase (EC 2.7.7.16) Biochim. Biophys. 14cta, 80 (1964) 5 o 8 - 5 I o
FIORT COMMUNICATIONS
20rnin INCUBATION ~ t
EFFECT O,
(b)
(c)
l~
;"%
',L'
ii! 0.2
0.1~ < 0¢
%
'
I
~
'
,
20
,.,
30
,
0
FRACTION N U M B E R
described n w i t h palladum w a s g r o w n u n d e r c o n d i t i o n s p r e v i o u s l y descri lecithin i ~o b a c t o p e p t o n e as a p r o t e i n source. Cells w e r e ggrowin~ r o w i n g logarithn with a eration t i m e of 8 h. A t t h e b e g i n n i n g of t h e e x p e r i m e n t s tthe h e cells were d o w n at Approx. × g for 5 min, and t h e n resuspended in t h e s a m e salt m e d i u m w i t h o u t b o s cells were resuspended in i o o m l w i t h o.o3 m l of p4C]AI~gal h y d r o l 3 0.05 m C C). A l i q u o t s were r e m o v e d at i o and 2o min, chilled b y p o u r i n g the cell sus crushed and t h e n centrifuged in the cold for 5 rain at 5o0 × g. The cells cell were res washed in in t h e s t a n d a r d salt m e d i u m and t h e n centrifuged as before. befor* The pellq reded in 1 o.oo15 M MgC12, 0.o 5 IV[ KC1, o . o i M Tris-HC1 buffer ( p H 7.2) and ¢cholate t h e n a d d e d t o a final c o n c e n t r a t i o n of 0. 5 % to lyse the cells. o A low . f uvg a t i o n m i n a t 50o × g) was used to r e m o v e u n l y s e d cells and m membranes. embr The s u p e r n a t a n t w a s t h e n anted and layered on a I 5 - 3 o % sucrose gradient c o n t a i n i n g t h e s a m e buffer. This w a s centrifuu g e d for 9o rain at 24 ooo r e v . ] m i n in a Spinco m o d e l L ultracentrifuge u using the S W 25 rotor2, ) ~r2,s. A t t h e end of t h e spin, t h e b o t t o m of the tube was punctm 3unctured and the a b s o r b a n c y ( W a S read c o n t i n u o u s l y at 2 6 o o / ~ in a Gilford s p e c t r o m e t e r . Fractions Fr were collected and precipitated tared as described previously*, s to c o u n t the r a d i o a c t i v i t y ( O - - O ) . (a) R a d i o a c t i v i t y and absorban~ ~rbancy a t i o m i n ; (b) r a d i o a c t i v i t y and a b s o r b a n c y at 2o 20 min; m (c) an a l i q u o t of t h e 2 o - m i n s a m Lple was k e p t at 4 ° in t h e presence of i / z g R N A a s e per ,dient 9er m l befor before placing on the sucrose gradient and the a b s o r b a n c y was t h e n recorded. recor I. P .
seen m i n Fig. F i g . IC r e that t h a t the t h e .~ polyribosomes r m l v r i b o ~ o r n e ~ have h a v e disappeared di~anne; be seen :ional entirely and additional absorbancy is now found in the 8o-S peak. Thus, the polysomal material has the characteristics associated wit] vith polyribosomes from mammalian sources l-s, in that it takes up short pulses of radioacti, ctivit, showing that it is the site of nascent protein tdioactivity synthesis and, in addition, small amounts of RNAase will degrade the polysomes into single ribosomal units. It is of interest to com pare the distribution of polyribosome size seen in the slime mold with that found in other rich ler cellsL The distribution is somewhat similar to that which obtains in mammalian cells in that there is a maximum in the region of polysomes containing 5 or 6 ribosomes and nd there is a spread of larger polysomes extending out to structures which we estime tte to contain 20-30 ribosomes. In view of the mechanism of protein synthesis on pol 37ribosomesT, s this is consistent with the manufacture of a wide variety of proteins b y the slime mold. Polyribosomes have now low been described from several mammalian sources, a variety of chick-embryo tissues, ~sues, bacterial cells, plant cells as well as protozoa 1°,1sa4. The addition of this report concerning cellular slime molds further suggests that poly-
Biochim. Biophys. Acta, 80 (1964) 5 o 8 - 5 1 o
fORT COMMUNICATIONS
r distribution, and points out the
of the
d b y grants from the U.S. Publ lation.
ervice
,ology, and
WILt ALEX RAQI
Natl. Acad. Sci. A. MARKS, E. R. BURKA AND D. SCHLESSINGER, Proc. Natl.. R. WARNER, k . RICH AND E. E. HALL, Science, 138 (1962) 1399. R. WARNER, P. M. KNOPF AND A. RICH, Bloc. Natl. Acad. 5 Sci. U.S., 4 • GIERER, J. Mol. Biol., 6 (1963) 148. . O. WETTSTEIN, T. STAEHELIN AND H. NOLL, Nature• 197 (I! t963) 43 o. ,c. Natl. Acad 1PENMAN, K. SCHERRER, Y. BECKER AND J. DARNELL, Proc.. ~4. ?. GILBERT, J. Mol. Biol., 6 (1963) 374. • GOODMAN AND A. RICH, Nature, 199 (I963) 318. • RICH, S• PENMAN, Y. BECKER, j. DARNELL AND C. E. HALL, HAL Science, Syrup . RICH, J. R. WARNER AND H. M. GOODMAN, Cold Spring; Harbor Ha ?ess. :. SUSSMAN, Science, 139 (1963) 338• • STAEHELIN, C. C. BRINTON, F. O. WETTSTEIN AND H. NOLL~,, Nature, I • SCHAECHTER, J. Mol. Biol., 7 (1963) 561. • SCHLESSINGER, J. Mol. Biol., 7 (1963) 569-
LLIPS* H ;MAN
) 2163.
(1963)
658 . • in t h e
Received eived November I8th, 1963 Del. (U.S.A.). ' P e r m a n e n t address: E. I. du P o n t de N e m o u r s and Co., Wilmington, Wil
Biochim. Biophys. Acta, 80 (1964) 5o8-51o
SC 7130
-carboxylic acid by a partially purified enzyme from box Activation of azetidine-2
that azetidine 2-carboxylic acid is incorporated chia coli and mungbean seedlings in the absence of exointo the protein of Escherichia ~lace genous proline. In their s~ystem, azetidine 2-carboxylic acid appeared to replace ;sized protein, and its incorporation was prevented by the proline in the newly-synthesized presence of proline. More recently, azetidine 2-carboxylic acid has been found to compete with proline in theLe activation reaction catalyzed b y plant extracts ~. Inas,mlno en reported to be competitive with proline in the amino much as no analog has been mammalian tissue, it was considered of interest to deteracid activation reaction of mine if azetidine 2-carboxy4ic acid is activated b y extracts of mammalian tissue iinn a rnle al amino manner similar to the natural amin nino acids, and whether the proline-activating enzyme ~fP)) is involved. (L-proline :sRNA ligase (AMP)) was Lzyme-fraction enriched in proline-activating activity was In these studies, an enz 51o-514 Biochim. Biophys. Acta, 80 (1964) 510-514
26
ion of polyribosomes from cell
molds
~l to the question of the generality c nthetic his brief report is to describe an ize the in the cellular slime molds. Recently it has been shown in mammalian cells that proteins ized on sters of ribosomes held together b y RNA, rather than m on single In the iculocyte 1-4 it has been shown that these ribosomal cl clusters, ca osomes ?olysomes, contain about five or six ribosomes. The reticulocyt re mufacmany es a single protein, hemoglobin. However, in a mammalian mal teins, such as liver 5, or H e L a cells s-s there is a broad bro; distrit lysome :s. In the case of H e L a cells, this extends up to 3o oor 40 ribo distri:ion of polysome sizes is believed to be related to the distribution dis' r-RNA gths which hold the polysomes togethere,~, 1°. In order to investigate the generality of the polyribc rribosome sy~ anism, looked for their presence in cellular slime molds. Slime Sli mold unique ~ediate /logenetic position in that they are regarded as ha having a sl celled ween plants and animals. They can be grown in suspension, su oeba-like form but under appropriate conditions these the, cells can also organize to Iormm a more complex multicellular organism. In these experiments, e~ Polyspondylium palladum ladum was grown serially in liquid medium as a singlele-membered culture and was labelled elled for various time periods with 14C-labelled amino acids, as described in Fig. I. A varlet /ariety of methods was examined to discover a way of lysing the cells without sub,jecting them to strong sheer gradients. The most successful procedure involved su the addition of sodium deoxycholate to a final concentr ~ncentration of 0.5 %. The chilled SUSpension could be shaken with this detergent and the cells lysed directly. I)eoxycholate )late has a potential disadvantage in that it also lyses the nuclear membrane, and rele~ases DNA. However., since mce there is little DNA in slime slb molds, it was not found necessary to add I)NAase (EC 3.1.4.5) before sucrose-gradient centrifugation. The results of an amin~o acid labelling experiment of this type are shown in Fig. 1. Fig. Ia shows the absorbanc~ racy and radioactivity profile after IO min of incubation with the materials sedimentin lting to the left. The large peak in absorbancy consists of in be seen that in addition, there is a rapidly sedimenting single 8o-S ribosomes. I t can aximum absorbancy peak with a maximum u m near 200 S. The absorbancy curve has a series of small contours which have been identified as single ribosomal increments in a polyribosomal series 1. It can be seen that the broad rapidly sedimenting peak of absorbancy also has a broad peak off radioactivity associated with it. The polysome peak has in the region of polysomes containing 5-6 ribosomes and an absorbancy m a x i m u m m the radioactivity maximuma is in the same region. After 2o min incubation (Fig. Ib) it can be seen that there is only a slight increase in the radioactivity associated with the polysomes. However, the absorbancy profile is substantially the same as that seen in Fig. Ia. An identical ical aliquot of the 2o-min incubation was subjected to .7.16) for 30 min at 4 °, and the absorbancy measured. I t can I #g/ml of RNAase (EC 2.7.7.16) Biochim. Biophys. 14cta, 80 (1964) 5 o 8 - 5 I o
FIORT COMMUNICATIONS
20rnin INCUBATION ~ t
EFFECT O,
(b)
(c)
l~
;"%
',L'
ii! 0.2
0.1~ < 0¢
%
'
I
~
'
,
20
,.,
30
,
0
FRACTION N U M B E R
described n w i t h palladum w a s g r o w n u n d e r c o n d i t i o n s p r e v i o u s l y descri lecithin i ~o b a c t o p e p t o n e as a p r o t e i n source. Cells w e r e ggrowin~ r o w i n g logarithn with a eration t i m e of 8 h. A t t h e b e g i n n i n g of t h e e x p e r i m e n t s tthe h e cells were d o w n at Approx. × g for 5 min, and t h e n resuspended in t h e s a m e salt m e d i u m w i t h o u t b o s cells were resuspended in i o o m l w i t h o.o3 m l of p4C]AI~gal h y d r o l 3 0.05 m C C). A l i q u o t s were r e m o v e d at i o and 2o min, chilled b y p o u r i n g the cell sus crushed and t h e n centrifuged in the cold for 5 rain at 5o0 × g. The cells cell were res washed in in t h e s t a n d a r d salt m e d i u m and t h e n centrifuged as before. befor* The pellq reded in 1 o.oo15 M MgC12, 0.o 5 IV[ KC1, o . o i M Tris-HC1 buffer ( p H 7.2) and ¢cholate t h e n a d d e d t o a final c o n c e n t r a t i o n of 0. 5 % to lyse the cells. o A low . f uvg a t i o n m i n a t 50o × g) was used to r e m o v e u n l y s e d cells and m membranes. embr The s u p e r n a t a n t w a s t h e n anted and layered on a I 5 - 3 o % sucrose gradient c o n t a i n i n g t h e s a m e buffer. This w a s centrifuu g e d for 9o rain at 24 ooo r e v . ] m i n in a Spinco m o d e l L ultracentrifuge u using the S W 25 rotor2, ) ~r2,s. A t t h e end of t h e spin, t h e b o t t o m of the tube was punctm 3unctured and the a b s o r b a n c y ( W a S read c o n t i n u o u s l y at 2 6 o o / ~ in a Gilford s p e c t r o m e t e r . Fractions Fr were collected and precipitated tared as described previously*, s to c o u n t the r a d i o a c t i v i t y ( O - - O ) . (a) R a d i o a c t i v i t y and absorban~ ~rbancy a t i o m i n ; (b) r a d i o a c t i v i t y and a b s o r b a n c y at 2o 20 min; m (c) an a l i q u o t of t h e 2 o - m i n s a m Lple was k e p t at 4 ° in t h e presence of i / z g R N A a s e per ,dient 9er m l befor before placing on the sucrose gradient and the a b s o r b a n c y was t h e n recorded. recor I. P .
seen m i n Fig. F i g . IC r e that t h a t the t h e .~ polyribosomes r m l v r i b o ~ o r n e ~ have h a v e disappeared di~anne; be seen :ional entirely and additional absorbancy is now found in the 8o-S peak. Thus, the polysomal material has the characteristics associated wit] vith polyribosomes from mammalian sources l-s, in that it takes up short pulses of radioacti, ctivit, showing that it is the site of nascent protein tdioactivity synthesis and, in addition, small amounts of RNAase will degrade the polysomes into single ribosomal units. It is of interest to com pare the distribution of polyribosome size seen in the slime mold with that found in other rich ler cellsL The distribution is somewhat similar to that which obtains in mammalian cells in that there is a maximum in the region of polysomes containing 5 or 6 ribosomes and nd there is a spread of larger polysomes extending out to structures which we estime tte to contain 20-30 ribosomes. In view of the mechanism of protein synthesis on pol 37ribosomesT, s this is consistent with the manufacture of a wide variety of proteins b y the slime mold. Polyribosomes have now low been described from several mammalian sources, a variety of chick-embryo tissues, ~sues, bacterial cells, plant cells as well as protozoa 1°,1sa4. The addition of this report concerning cellular slime molds further suggests that poly-
Biochim. Biophys. Acta, 80 (1964) 5 o 8 - 5 1 o
fORT COMMUNICATIONS
r distribution, and points out the
of the
d b y grants from the U.S. Publ lation.
ervice
,ology, and
WILt ALEX RAQI
Natl. Acad. Sci. A. MARKS, E. R. BURKA AND D. SCHLESSINGER, Proc. Natl.. R. WARNER, k . RICH AND E. E. HALL, Science, 138 (1962) 1399. R. WARNER, P. M. KNOPF AND A. RICH, Bloc. Natl. Acad. 5 Sci. U.S., 4 • GIERER, J. Mol. Biol., 6 (1963) 148. . O. WETTSTEIN, T. STAEHELIN AND H. NOLL, Nature• 197 (I! t963) 43 o. ,c. Natl. Acad 1PENMAN, K. SCHERRER, Y. BECKER AND J. DARNELL, Proc.. ~4. ?. GILBERT, J. Mol. Biol., 6 (1963) 374. • GOODMAN AND A. RICH, Nature, 199 (I963) 318. • RICH, S• PENMAN, Y. BECKER, j. DARNELL AND C. E. HALL, HAL Science, Syrup . RICH, J. R. WARNER AND H. M. GOODMAN, Cold Spring; Harbor Ha ?ess. :. SUSSMAN, Science, 139 (1963) 338• • STAEHELIN, C. C. BRINTON, F. O. WETTSTEIN AND H. NOLL~,, Nature, I • SCHAECHTER, J. Mol. Biol., 7 (1963) 561. • SCHLESSINGER, J. Mol. Biol., 7 (1963) 569-
LLIPS* H ;MAN
) 2163.
(1963)
658 . • in t h e
Received eived November I8th, 1963 Del. (U.S.A.). ' P e r m a n e n t address: E. I. du P o n t de N e m o u r s and Co., Wilmington, Wil
Biochim. Biophys. Acta, 80 (1964) 5o8-51o
SC 7130
-carboxylic acid by a partially purified enzyme from box Activation of azetidine-2
that azetidine 2-carboxylic acid is incorporated chia coli and mungbean seedlings in the absence of exointo the protein of Escherichia ~lace genous proline. In their s~ystem, azetidine 2-carboxylic acid appeared to replace ;sized protein, and its incorporation was prevented by the proline in the newly-synthesized presence of proline. More recently, azetidine 2-carboxylic acid has been found to compete with proline in theLe activation reaction catalyzed b y plant extracts ~. Inas,mlno en reported to be competitive with proline in the amino much as no analog has been mammalian tissue, it was considered of interest to deteracid activation reaction of mine if azetidine 2-carboxy4ic acid is activated b y extracts of mammalian tissue iinn a rnle al amino manner similar to the natural amin nino acids, and whether the proline-activating enzyme ~fP)) is involved. (L-proline :sRNA ligase (AMP)) was Lzyme-fraction enriched in proline-activating activity was In these studies, an enz 51o-514 Biochim. Biophys. Acta, 80 (1964) 510-514