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Destruction of reticulocyte ribosomes by deoxycholate
500
SHORT COMMUNICATIONS
BBA 93252 Destruction of reticulocyte ribosomes by deoxycholote Ribosomes which are prepared from the microsome fraction of cells by treatment with deoxyeholate appear to maintain their structural integrity and to function adequately in protein synthesis1, 2. Although anionic detergents, such as dodecyl sulfate, readily attack ribosomes by dissociating them into an RNA fraction and a protein detergent complex fraction a, deoxyeholate has been thought to lack this property 2. It remains unclear why this bile acid does not attack ribosomal particles on the same basis as do other anionic detergents. Recent evidence has indicated that a significant proportion of reticulocyte RNA and ribosomes are bound to the cell membrane 4. In the course of these studies it was noted that treatment of reticulocyte hemolysates with increasing concentrations of deoxycholate was associated with a decrease in recovery of free ribosomes. Since there have been few investigations of the action of deoxycholate on ribosomes existing in a free, as opposed to a membrane-bound, form, the effect of deoxycholate on free ribosome recovery has been investigated. The evidence indicates that sodium deoxycholate causes a concentration-dependent breakdown of ribosomes which are not bound to cell membrane constituents, and thus attacks ribosomes in a manner similar to that of other anionic detergents. Ribosomes were prepared from the peripheral blood of rabbits with a phenylhydrazine-induced reticulocytosis, as described previously 5. The cells were washed in isotonic NaC1 made 1.5" IO 3 M in MgC12, and then lysed in 4 vol. of a solution of MgC12, 1.5"1o 3 M, and Tris-HC1 buffer, I. lO -3 M, pH 7.5. The cell membranes were separated from the whole lysates by centrifugation at 17 ooo ×g for 15 rain and a sufficient volume of IO °,o sodium deoxyeholate was added to aliquots of the membrane-free hemolysates to bring them to the desired concentration of bile acid. The hemolysates were again centrifuged at 17 ooo ×g for 15 rain and the ribosomes then separated from the supernate by centrifugation at 165 ooo × g for 6o rain. The pelleted ribosomes were rinsed three times in the lysing solution and suspended in a small volume of this solution by gentle agitation. Ribosome recovery and purity were quantitated by absorbance at 260 and 280 m/~, determination of protein by the method of LOWRY~ and determination of RNA v. Ribosomal RNA was hydrolysed with hot 5 % trichloroacetic acid after washing the ribosomes four times in cold 5 % trichloroacetic acid and the orcinol reaction was used to quantitate RNA-ribose. A factor of 4.31 was used to convert the amount of purine-bound ribose to RNA s. Fig. I shows the recovery of RNA and protein from ribosomes isolated from aliquots of membrane-free hemolysates treated with increasing concentrations of deoxyeholate prior to pelleting. The results indicate that treatment of free reticulocyte ribosomes with deoxycholate has two effects. At concentrations of deoxycholate below i % there is a loss of protein in excess of RNA, as indicated by the protein: RNA ratios in Table I. This is consistent with freeing of the ribosomes from attached proteins which are not integral to ribosome structure. At concentrations of deoxycholate above o.I % there is a decreased recovery of ribosomal RNA. The slight inBiochim. Biophys. Acta, 145 (1967) 506-508
2001
5o7
SHORT COMMUNICATIONS
8 ~soo~ ~r ,~ o
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DEOXYCHOLATE Fig. I. R e c o v e r y of R N A ( • ) and p r o t e i n ( O ) f r o m r i b o s o m e s isolated from aliquots of m e m b r a n e free h e m o l y s a t e exposed to v a r i o u s c o n c e n t r a t i o n s of s o d i u m deoxycholate. Analytic m e t h o d s are described in the text. The ratio of p r o t e i n to R N A r e m a i n s c o n s t a n t in s a m p l e s exposed to d e o x y c h o l a t e c o n c e n t r a t i o n s g r e a t e r t h a n o. 5 %. TABLE I RECOVERY OF RIBOSOMES AND RIBOSOMAL CONSTITUENTS HEMOLYSATE FOLLOWING TREATMENT WITH DEOXYCHOLATE
Deoxycholate concentration (%)
o o,i o,2 o, 5 I.O 2,o 5,o
FROM
ALIQUOTS
OF MEMBRANE-FREE
Total ribosome recovery (units)
Protein (#g )
RNA (l~g)
Protein RN A
23.3 2o.8 19.8 17. 5 15.4 13.2 9.0
228o 176o 132o lO4O 7 °0 58o 322
776 8o2 784 672 595 483 275
2.9 2.2 1. 7 1. 5 1.2 1.2 1.2
A 260 my
R N A loss* (%)
-o 2.3 16.2 25.8 39-7 65.8
* % of h i g h e s t value ( O , I O/o deoxycholate).
crease in RNA recovery in the aliquot exposed to o.i % deoxycholate was consistently observed, and is most likely due to release of ribosomes from a small amount of membrane remaining in the hemolysates following centrifugation at 17 ooo ×g. The equal loss of ribosomal RNA and protein at concentrations of deoxycholate above o. 5 % indicates a true loss of ribosomes. This loss varied from as little as 2.3 % at a concentration of 0.2 % deoxycholate to as much as 65.8 % at a concentration of 5 % deoxycholate (Table I). The data presented here demonstrate that free ribosomes, unattached to cell membranes, are broken down by deoxycholate, similar to the action of other anionic Biochim. Biophys. Acta, 145 (1967) 506-508
508
SHORT COMMUNICATIONS
detergents. The concentrations of deoxycholate usually used for freeing ribosomes from microsomal particles range from 0.3 to 1.2 °/o (ref. 2). Significant breakdown of free ribosomes was observed in this study at these concentrations. This does not indicate, however, that under like conditions membrane-bound ribosomes would be similarly broken down. Since steroids can be bound to serum proteins °, it is possible that the protein constituents of cell membranes m a y influence the action of deoxycholate. In addition, the data indicate that free reticulocyte ribosomes prepared in the absence of deoxycholate have a considerable amount of attached non-ribosomal protein. It is evident that even at low concentrations of deoxycholate solubilization of n o n r i b o s o m a l protein is accompanied b y loss of ribosomal RNA. The optimal concentration of deoxycholate for preparing free reticulocyte ribosomes without significant loss appears to be 0.2 °/o, a concentration also usaally sufficient to dissolve the membrane of the endoplasmic reticulam 1. The findings indicate that caution should be exercised in drawing conclusions concerning the quantity of ribosomes within cells when the ribosomes have been prepared b y treatment of cell fractions with deoxycholate. This work has been supported b y U.S. Public Health Service grants H E 10473 and H E 06374 .
Cardeza Foundation/or Hematologic Research, Jefferso1¢ Medical College, Philadelphia, Pa. (U.S.A.)
EDWARD R. B U R K A
I G. E. PALADE AND P. SIEKEVITZ, J . Biophys. Biochem. Cytol., 2 (1956) 171. 2 P. O. P. T s ' o , Ann. Rev. Plant Physiol., 13 (1962) 45. 3 B. D. HALL AND P. J. DOTY, J. Mol. Biol., i (I959) i i i . 4 E. R. BURKA, W. SCHREML AND C. J. KICK, Biochem. Biophys. Res. Commun., 26 (1967) 3345 E. R. BURKA AND P. A. MARKS, jr. Mol. Biol., 9 (1964) 439. 6 0 . H. LOWRY, N. J. ]~OSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 7 E. R. BURKA,J. Lab. Clin. Med., 68 (1966) 833. 8 E. ]{. BURKA, W. GCHREML AND C. J. KICK, Biochemistry, in t h e press. 9 F. BISCHOFF AND H. R. PILHORM, J. Biol. Chem., 176 (1948) 663.
Received J a n e 9th, 1967 Biochim. Biophys. deta, 145 (1967) 506-508
BBA 93256
On the functional activity of conventionally prepared chloramphenicol particles It is known that Escherichia coli cells accumulate protein-deficient subribosomal particles when chloramphenicol is added to the culture medium z. These abnormal particles, referred to as chloramphenicol particles in this paper, consist of two major Abbreviations: rRNA, ribosomal RNA; tRNA, transfer RNA; mRNA, messenger RNA; p o l y (U), p o l y u r i d y l i c acid.
Biochim. Biophys. Acta, 145 (1967) 508--511
SHORT COMMUNICATIONS
BBA 93252 Destruction of reticulocyte ribosomes by deoxycholote Ribosomes which are prepared from the microsome fraction of cells by treatment with deoxyeholate appear to maintain their structural integrity and to function adequately in protein synthesis1, 2. Although anionic detergents, such as dodecyl sulfate, readily attack ribosomes by dissociating them into an RNA fraction and a protein detergent complex fraction a, deoxyeholate has been thought to lack this property 2. It remains unclear why this bile acid does not attack ribosomal particles on the same basis as do other anionic detergents. Recent evidence has indicated that a significant proportion of reticulocyte RNA and ribosomes are bound to the cell membrane 4. In the course of these studies it was noted that treatment of reticulocyte hemolysates with increasing concentrations of deoxycholate was associated with a decrease in recovery of free ribosomes. Since there have been few investigations of the action of deoxycholate on ribosomes existing in a free, as opposed to a membrane-bound, form, the effect of deoxycholate on free ribosome recovery has been investigated. The evidence indicates that sodium deoxycholate causes a concentration-dependent breakdown of ribosomes which are not bound to cell membrane constituents, and thus attacks ribosomes in a manner similar to that of other anionic detergents. Ribosomes were prepared from the peripheral blood of rabbits with a phenylhydrazine-induced reticulocytosis, as described previously 5. The cells were washed in isotonic NaC1 made 1.5" IO 3 M in MgC12, and then lysed in 4 vol. of a solution of MgC12, 1.5"1o 3 M, and Tris-HC1 buffer, I. lO -3 M, pH 7.5. The cell membranes were separated from the whole lysates by centrifugation at 17 ooo ×g for 15 rain and a sufficient volume of IO °,o sodium deoxyeholate was added to aliquots of the membrane-free hemolysates to bring them to the desired concentration of bile acid. The hemolysates were again centrifuged at 17 ooo ×g for 15 rain and the ribosomes then separated from the supernate by centrifugation at 165 ooo × g for 6o rain. The pelleted ribosomes were rinsed three times in the lysing solution and suspended in a small volume of this solution by gentle agitation. Ribosome recovery and purity were quantitated by absorbance at 260 and 280 m/~, determination of protein by the method of LOWRY~ and determination of RNA v. Ribosomal RNA was hydrolysed with hot 5 % trichloroacetic acid after washing the ribosomes four times in cold 5 % trichloroacetic acid and the orcinol reaction was used to quantitate RNA-ribose. A factor of 4.31 was used to convert the amount of purine-bound ribose to RNA s. Fig. I shows the recovery of RNA and protein from ribosomes isolated from aliquots of membrane-free hemolysates treated with increasing concentrations of deoxyeholate prior to pelleting. The results indicate that treatment of free reticulocyte ribosomes with deoxycholate has two effects. At concentrations of deoxycholate below i % there is a loss of protein in excess of RNA, as indicated by the protein: RNA ratios in Table I. This is consistent with freeing of the ribosomes from attached proteins which are not integral to ribosome structure. At concentrations of deoxycholate above o.I % there is a decreased recovery of ribosomal RNA. The slight inBiochim. Biophys. Acta, 145 (1967) 506-508
2001
5o7
SHORT COMMUNICATIONS
8 ~soo~ ~r ,~ o
I000
Pp~TEIN
'.2_
50(
I I
I 2
I 3
I 4
I 5
DEOXYCHOLATE Fig. I. R e c o v e r y of R N A ( • ) and p r o t e i n ( O ) f r o m r i b o s o m e s isolated from aliquots of m e m b r a n e free h e m o l y s a t e exposed to v a r i o u s c o n c e n t r a t i o n s of s o d i u m deoxycholate. Analytic m e t h o d s are described in the text. The ratio of p r o t e i n to R N A r e m a i n s c o n s t a n t in s a m p l e s exposed to d e o x y c h o l a t e c o n c e n t r a t i o n s g r e a t e r t h a n o. 5 %. TABLE I RECOVERY OF RIBOSOMES AND RIBOSOMAL CONSTITUENTS HEMOLYSATE FOLLOWING TREATMENT WITH DEOXYCHOLATE
Deoxycholate concentration (%)
o o,i o,2 o, 5 I.O 2,o 5,o
FROM
ALIQUOTS
OF MEMBRANE-FREE
Total ribosome recovery (units)
Protein (#g )
RNA (l~g)
Protein RN A
23.3 2o.8 19.8 17. 5 15.4 13.2 9.0
228o 176o 132o lO4O 7 °0 58o 322
776 8o2 784 672 595 483 275
2.9 2.2 1. 7 1. 5 1.2 1.2 1.2
A 260 my
R N A loss* (%)
-o 2.3 16.2 25.8 39-7 65.8
* % of h i g h e s t value ( O , I O/o deoxycholate).
crease in RNA recovery in the aliquot exposed to o.i % deoxycholate was consistently observed, and is most likely due to release of ribosomes from a small amount of membrane remaining in the hemolysates following centrifugation at 17 ooo ×g. The equal loss of ribosomal RNA and protein at concentrations of deoxycholate above o. 5 % indicates a true loss of ribosomes. This loss varied from as little as 2.3 % at a concentration of 0.2 % deoxycholate to as much as 65.8 % at a concentration of 5 % deoxycholate (Table I). The data presented here demonstrate that free ribosomes, unattached to cell membranes, are broken down by deoxycholate, similar to the action of other anionic Biochim. Biophys. Acta, 145 (1967) 506-508
508
SHORT COMMUNICATIONS
detergents. The concentrations of deoxycholate usually used for freeing ribosomes from microsomal particles range from 0.3 to 1.2 °/o (ref. 2). Significant breakdown of free ribosomes was observed in this study at these concentrations. This does not indicate, however, that under like conditions membrane-bound ribosomes would be similarly broken down. Since steroids can be bound to serum proteins °, it is possible that the protein constituents of cell membranes m a y influence the action of deoxycholate. In addition, the data indicate that free reticulocyte ribosomes prepared in the absence of deoxycholate have a considerable amount of attached non-ribosomal protein. It is evident that even at low concentrations of deoxycholate solubilization of n o n r i b o s o m a l protein is accompanied b y loss of ribosomal RNA. The optimal concentration of deoxycholate for preparing free reticulocyte ribosomes without significant loss appears to be 0.2 °/o, a concentration also usaally sufficient to dissolve the membrane of the endoplasmic reticulam 1. The findings indicate that caution should be exercised in drawing conclusions concerning the quantity of ribosomes within cells when the ribosomes have been prepared b y treatment of cell fractions with deoxycholate. This work has been supported b y U.S. Public Health Service grants H E 10473 and H E 06374 .
Cardeza Foundation/or Hematologic Research, Jefferso1¢ Medical College, Philadelphia, Pa. (U.S.A.)
EDWARD R. B U R K A
I G. E. PALADE AND P. SIEKEVITZ, J . Biophys. Biochem. Cytol., 2 (1956) 171. 2 P. O. P. T s ' o , Ann. Rev. Plant Physiol., 13 (1962) 45. 3 B. D. HALL AND P. J. DOTY, J. Mol. Biol., i (I959) i i i . 4 E. R. BURKA, W. SCHREML AND C. J. KICK, Biochem. Biophys. Res. Commun., 26 (1967) 3345 E. R. BURKA AND P. A. MARKS, jr. Mol. Biol., 9 (1964) 439. 6 0 . H. LOWRY, N. J. ]~OSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 7 E. R. BURKA,J. Lab. Clin. Med., 68 (1966) 833. 8 E. ]{. BURKA, W. GCHREML AND C. J. KICK, Biochemistry, in t h e press. 9 F. BISCHOFF AND H. R. PILHORM, J. Biol. Chem., 176 (1948) 663.
Received J a n e 9th, 1967 Biochim. Biophys. deta, 145 (1967) 506-508
BBA 93256
On the functional activity of conventionally prepared chloramphenicol particles It is known that Escherichia coli cells accumulate protein-deficient subribosomal particles when chloramphenicol is added to the culture medium z. These abnormal particles, referred to as chloramphenicol particles in this paper, consist of two major Abbreviations: rRNA, ribosomal RNA; tRNA, transfer RNA; mRNA, messenger RNA; p o l y (U), p o l y u r i d y l i c acid.
Biochim. Biophys. Acta, 145 (1967) 508--511