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Stable messenger RNA in the synthesis of contractile protein in human platelets
IS~
SHORT COMMUNICATIONS
BBA 93243 Stoble messenger R N A in the synthesis of controctUe protein in human
platelets Platelets incubated with zaC-labelled amino acids have the ability to incorporate radioactivity into hot trichloroaeetic acid-insoluble material. This process can be inhibited by puromycin. Labelled contractile protein can be isolated from platelets incubated with 14C-labelled amino acids b y extraction with Weber-Edsall solution (o.6 M KC1, o.oi M Na2CO 3, o.o 4 M NaHCOa). Contractile protein can be precipitated and purified b y decreasing the ionic strength of the KC1 to o.o5 according to the procedure of BETTEX-GALLANDAND LUSCHER3. Electrophoresis and extensive dialysis did not remove the radioactivity from the isolated contractile protein. Fingerprinting of the partially acid hydrolyzed protein gave fifteen ninhydrin-positive spots which all contained radioactivity. From these data we conclude that contractile protein is synthesized de novo in platelets. We have repolted on the protein synthesizing ability of platelets z. Platelets are enucleated cells and according to the diphenylamine reaction 2, no DNA could be detected in the cytoplasm of the cell. This would then imply that the messenger RNA (mRNA) directing the synthesis of the contractile protein would either have to be (a) stable or (b) transcribed from a "piece" of DNA which was so small that it could not be detected by the diphenylamine reaction. In an attempt to elucidate this problem, the amino acid incorporating ability of platelets was studied in the presence of actinomycin D. Platelets used for actinomycin D studies were isolated from blood in which E D T A was used as anticoagulant. Freshly isolated platelets were incubated for 9 ° min at 37 ° in 0. 9 °Jo NaC1, I ° o glucose, a 14C-labelled amino acid mixture and increasing levels of actinomycin D. Radioactivity incorporated into hot trichloroacetic acid-insoluble material was similar to that of the controls which did not contain actinomycin D. Since the incorporation process was insensitive to actinomycin D and no incorporation of ~14C~orotic acid of ~aH~uridine was observed, the amino acid-incorporation was considered to be independent of synthesis of new mRNA. It m a y be noted that both Ia4C~orotic acid and ~3H~uridine could be detected in tbe cytoplasm within 5-1o min after the labelled compounds had been added. The stability of the presumed m R N A directing the synthesis of contractile protein was studied under in vitro conditions. Platelets (9 g wet weight cells isolated from 14 units of fresh blood) were isolated and erythrocytes removed by differential lyses. The cells were washed in isotonic saline and placed in sterilized polyethylene containers. Each fraction contained I-1. 5 g wet weight cells. Preincubations were carried out in a bicarbonate buffer at 37 ° under a gas phase of 02 plus CO 2 (95 "5). The bicarbonate buffer (pH 7,45) contained 0. 5 mM KH2PO ,, 4.5 mM NaHC() 3, 0. 3 mM Na2HPO 4, 5 mM KC1, o.14 M NaC1, I o,,o glucose, IO O-o human plasma, penicillin (1oo maits/ml) and streptomycin (IOO mg/ml), Incubation mixtures consisted of 5 ml of bicarbonate buffer, I ml amino acid mixture (containing I m g casein hydrolysate plus 20/,g cysteine, adjusted to p H 7.5 with NaOH). Abbreviation: mRNA, messenger RNA. Biochim. l~iophys, Acta, i45 (1967) t88 z9o
189
SHORT COMMUNICATIONS
Incubation mixtures were changed every 8 h. Samples were removed at I2-h intervals, centrifuged at 2500 ×g and resuspended in 6 ml bicarbonate buffer containing a mixture of 13 L-[14C]amino acids (Schwarz BioResearch, Inc., IOo/~C/ml) plus a mixture of 6 L-Ea2CJamino acids (cysteine, glutamine, glycine, histidine, methionine, and tryptophan). Final concentration of a4C label per sample was 2/~C/ 6 ml. Sterility was maintained throughout the entire period of preincubation. Preincubated platelets were then incubated for an additional 4 h in the presence of the 14C-labelled amino acids. In each case the 14C-labelled contractile protein was isolated and reprecipitated twice as described previously 3. Specific activities of labelled contractile protein isolated after various preincubation periods are expressed in Table I.
TABLE I S P E C I F I C A C T I V I T I E S ( C O U N T S / m i n P E R m g CONTRACTILE P R O T E I N ) OF 14C-LABELLED CONTRACTILE P R O T E I N I S O L A T E D A F T E R VARIOUS P E R I O D S OF P R E I N C U B A T I O N
Preincubation period
(h)
o (control) 12 24 36 48 6o 72
Speci/ic activity o/contractile protein Expt. •
Expt. 2
lO63 12oo 1163 lO98 121o 1243 1182
ilOO 1176 12Ol 115o 1185 12oo 1132
The ability of human platelets to incorporate xaC-labelled amino acids into contractile protein after 72 h of preincubation, indicates that the incorporating system has a very long life and that the mRNA directing this incorporation process must have a minimum life span of at least 72 h. It is of interest to note that this value falls within the life span values of 3-8 days reported for human platelets4, 5. 15 % of the platelet protein consists of contractile protein a, in addition we have recently obtained evidence for the membrane location of this protein (manuscript in preparation). Autoradiographic results of platelets which have been pulse-labelled and chased for various periods of time, verify these findings. The life span of the mRNA directing the synthesis of contractile protein may, therefore, actually determine the life span of the platelets in vivo. These investigations were supported in part by grants from the U. S. Public Health Service (HE 07565) and the Chicago Heart Association.
Department o~ Biochemistry, Presbyterian-St. Luke's Hospital and University o~ Illinois College o~ Medicine, Chicago, Ill. (U.S.A.)
FRANCOIS M. BOOYSE MAX E. RAFELSON, J R .
Biochim. Biophys. Acta, 145 (1967) 188-19o
190 I 2 3 4 5
SHORT COMMUNICATIONS
F. M. BooYsE AND M. E. }{AFELSON,Nature, in the press. •. BURTON, Biochem. J,, 62 (I956) 315 • M. BETTEX-GALLAND AND E. F. Li)SCHER, Biochim. Biophys. Acta, 49 (I96I) 536. C. ~V. H. LEEKSMA AND J. A. COHEN, J . Clin. Invest., 35 (1956) 964, E. H. REISNER, R. P. ]{EATING, C. FRIESEN AND E. LOEFI~'LER, in A. R. JONES, -Proc. 6th Intern. Congr. Intern. Soc. Hernatol. Boston, Mass., 1956, Grune and S t r a t t o n , New York, 1958 , p. 292.
Received March 23rd, 1967 Revised manuscript received May I6th, 1967 Biochim. Biophys. Acta, 145 (1967) 188-19o
BBA 93244
Differentiol Iobeling of 28-S R N A in free ond membrane-bound ribosomes of kidney In earlier work we showed that there was little difference between the kinetics of Ii*Cluridine labeling of free (i.e., non-membrane-bound) and membrane-bound kidney ribosomes in both control and in uninephrectomized mice 1. The results were similar to those obtained from rat liverS, a, but different from what might now be predicted from recent work on H e L a cells*, 5. In H e L a cells incorporation of the 45-S subunit containing fleshly-made 18-S ribosomal RNA (rRNA) into a finished ribosome occurs in the polyribosome; the ribosome then passes from the polysome free into the cytoplasm. If most kidney ribosomes active in protein synthesis were principally membrane-bound as in liver~,L and if the mechanism of their synthesis were analogous to the assembly of polyribosomes in H e L a cells, membrane-bound ribosomes in kidney should therefore have been labeled before the free ribosomes rather than at a similar rate. Since it is now known that rapidly-labeled messenger RNA (mRNA) cam be associated with the rapidly-labeled 45-S ribosomal particle s-n, a possible source of error could have arisen in previous work from inclusion of radioactive m R N A with radioactivity of the ribosomes themselves. Further experiments were therefore done to investigate the kinetics of labeling of renal 28-S RNA, which is derived exclusively from the 6o-S ribosomal particle free from m R N A n. The experiments showed that in 28-S RNA as in intact ribosomes the kinetics of labeling of free and membrane-bound ribosomes of mouse kidney were largely parallel, but label appeared a little faster in the free ribosomes. To be sure (a) that microsomal rRNA of different labeling characteristics was not being lost in the mitochondrial pellet, (b) that free ribosomes were not contaminating the microsomal pellet, and (c) that microsomes were not contaminating the ribosomal pellet, additional experiments were done in which 28-S RNA was also prepared from the mitochondrial pellet and from a "barfer pellet" resulting from centrifugation at 65 ooo rev./min for 3 to 5 min after the microsomes had been deposited and before the free ribosomes were deposited. Fig. l a shows a typical result, in which labeling of all four pellets was mostly parallel except that the specific activity of the free ribosomes was again highest until the eighth hour. Confirmatory results Abbreviations: r R N A , ribosomal R N A ; m R N A , messenger RNA.
Biochim. Biophys. Acta, 145 (1967) z9o-I92
SHORT COMMUNICATIONS
BBA 93243 Stoble messenger R N A in the synthesis of controctUe protein in human
platelets Platelets incubated with zaC-labelled amino acids have the ability to incorporate radioactivity into hot trichloroaeetic acid-insoluble material. This process can be inhibited by puromycin. Labelled contractile protein can be isolated from platelets incubated with 14C-labelled amino acids b y extraction with Weber-Edsall solution (o.6 M KC1, o.oi M Na2CO 3, o.o 4 M NaHCOa). Contractile protein can be precipitated and purified b y decreasing the ionic strength of the KC1 to o.o5 according to the procedure of BETTEX-GALLANDAND LUSCHER3. Electrophoresis and extensive dialysis did not remove the radioactivity from the isolated contractile protein. Fingerprinting of the partially acid hydrolyzed protein gave fifteen ninhydrin-positive spots which all contained radioactivity. From these data we conclude that contractile protein is synthesized de novo in platelets. We have repolted on the protein synthesizing ability of platelets z. Platelets are enucleated cells and according to the diphenylamine reaction 2, no DNA could be detected in the cytoplasm of the cell. This would then imply that the messenger RNA (mRNA) directing the synthesis of the contractile protein would either have to be (a) stable or (b) transcribed from a "piece" of DNA which was so small that it could not be detected by the diphenylamine reaction. In an attempt to elucidate this problem, the amino acid incorporating ability of platelets was studied in the presence of actinomycin D. Platelets used for actinomycin D studies were isolated from blood in which E D T A was used as anticoagulant. Freshly isolated platelets were incubated for 9 ° min at 37 ° in 0. 9 °Jo NaC1, I ° o glucose, a 14C-labelled amino acid mixture and increasing levels of actinomycin D. Radioactivity incorporated into hot trichloroacetic acid-insoluble material was similar to that of the controls which did not contain actinomycin D. Since the incorporation process was insensitive to actinomycin D and no incorporation of ~14C~orotic acid of ~aH~uridine was observed, the amino acid-incorporation was considered to be independent of synthesis of new mRNA. It m a y be noted that both Ia4C~orotic acid and ~3H~uridine could be detected in tbe cytoplasm within 5-1o min after the labelled compounds had been added. The stability of the presumed m R N A directing the synthesis of contractile protein was studied under in vitro conditions. Platelets (9 g wet weight cells isolated from 14 units of fresh blood) were isolated and erythrocytes removed by differential lyses. The cells were washed in isotonic saline and placed in sterilized polyethylene containers. Each fraction contained I-1. 5 g wet weight cells. Preincubations were carried out in a bicarbonate buffer at 37 ° under a gas phase of 02 plus CO 2 (95 "5). The bicarbonate buffer (pH 7,45) contained 0. 5 mM KH2PO ,, 4.5 mM NaHC() 3, 0. 3 mM Na2HPO 4, 5 mM KC1, o.14 M NaC1, I o,,o glucose, IO O-o human plasma, penicillin (1oo maits/ml) and streptomycin (IOO mg/ml), Incubation mixtures consisted of 5 ml of bicarbonate buffer, I ml amino acid mixture (containing I m g casein hydrolysate plus 20/,g cysteine, adjusted to p H 7.5 with NaOH). Abbreviation: mRNA, messenger RNA. Biochim. l~iophys, Acta, i45 (1967) t88 z9o
189
SHORT COMMUNICATIONS
Incubation mixtures were changed every 8 h. Samples were removed at I2-h intervals, centrifuged at 2500 ×g and resuspended in 6 ml bicarbonate buffer containing a mixture of 13 L-[14C]amino acids (Schwarz BioResearch, Inc., IOo/~C/ml) plus a mixture of 6 L-Ea2CJamino acids (cysteine, glutamine, glycine, histidine, methionine, and tryptophan). Final concentration of a4C label per sample was 2/~C/ 6 ml. Sterility was maintained throughout the entire period of preincubation. Preincubated platelets were then incubated for an additional 4 h in the presence of the 14C-labelled amino acids. In each case the 14C-labelled contractile protein was isolated and reprecipitated twice as described previously 3. Specific activities of labelled contractile protein isolated after various preincubation periods are expressed in Table I.
TABLE I S P E C I F I C A C T I V I T I E S ( C O U N T S / m i n P E R m g CONTRACTILE P R O T E I N ) OF 14C-LABELLED CONTRACTILE P R O T E I N I S O L A T E D A F T E R VARIOUS P E R I O D S OF P R E I N C U B A T I O N
Preincubation period
(h)
o (control) 12 24 36 48 6o 72
Speci/ic activity o/contractile protein Expt. •
Expt. 2
lO63 12oo 1163 lO98 121o 1243 1182
ilOO 1176 12Ol 115o 1185 12oo 1132
The ability of human platelets to incorporate xaC-labelled amino acids into contractile protein after 72 h of preincubation, indicates that the incorporating system has a very long life and that the mRNA directing this incorporation process must have a minimum life span of at least 72 h. It is of interest to note that this value falls within the life span values of 3-8 days reported for human platelets4, 5. 15 % of the platelet protein consists of contractile protein a, in addition we have recently obtained evidence for the membrane location of this protein (manuscript in preparation). Autoradiographic results of platelets which have been pulse-labelled and chased for various periods of time, verify these findings. The life span of the mRNA directing the synthesis of contractile protein may, therefore, actually determine the life span of the platelets in vivo. These investigations were supported in part by grants from the U. S. Public Health Service (HE 07565) and the Chicago Heart Association.
Department o~ Biochemistry, Presbyterian-St. Luke's Hospital and University o~ Illinois College o~ Medicine, Chicago, Ill. (U.S.A.)
FRANCOIS M. BOOYSE MAX E. RAFELSON, J R .
Biochim. Biophys. Acta, 145 (1967) 188-19o
190 I 2 3 4 5
SHORT COMMUNICATIONS
F. M. BooYsE AND M. E. }{AFELSON,Nature, in the press. •. BURTON, Biochem. J,, 62 (I956) 315 • M. BETTEX-GALLAND AND E. F. Li)SCHER, Biochim. Biophys. Acta, 49 (I96I) 536. C. ~V. H. LEEKSMA AND J. A. COHEN, J . Clin. Invest., 35 (1956) 964, E. H. REISNER, R. P. ]{EATING, C. FRIESEN AND E. LOEFI~'LER, in A. R. JONES, -Proc. 6th Intern. Congr. Intern. Soc. Hernatol. Boston, Mass., 1956, Grune and S t r a t t o n , New York, 1958 , p. 292.
Received March 23rd, 1967 Revised manuscript received May I6th, 1967 Biochim. Biophys. Acta, 145 (1967) 188-19o
BBA 93244
Differentiol Iobeling of 28-S R N A in free ond membrane-bound ribosomes of kidney In earlier work we showed that there was little difference between the kinetics of Ii*Cluridine labeling of free (i.e., non-membrane-bound) and membrane-bound kidney ribosomes in both control and in uninephrectomized mice 1. The results were similar to those obtained from rat liverS, a, but different from what might now be predicted from recent work on H e L a cells*, 5. In H e L a cells incorporation of the 45-S subunit containing fleshly-made 18-S ribosomal RNA (rRNA) into a finished ribosome occurs in the polyribosome; the ribosome then passes from the polysome free into the cytoplasm. If most kidney ribosomes active in protein synthesis were principally membrane-bound as in liver~,L and if the mechanism of their synthesis were analogous to the assembly of polyribosomes in H e L a cells, membrane-bound ribosomes in kidney should therefore have been labeled before the free ribosomes rather than at a similar rate. Since it is now known that rapidly-labeled messenger RNA (mRNA) cam be associated with the rapidly-labeled 45-S ribosomal particle s-n, a possible source of error could have arisen in previous work from inclusion of radioactive m R N A with radioactivity of the ribosomes themselves. Further experiments were therefore done to investigate the kinetics of labeling of renal 28-S RNA, which is derived exclusively from the 6o-S ribosomal particle free from m R N A n. The experiments showed that in 28-S RNA as in intact ribosomes the kinetics of labeling of free and membrane-bound ribosomes of mouse kidney were largely parallel, but label appeared a little faster in the free ribosomes. To be sure (a) that microsomal rRNA of different labeling characteristics was not being lost in the mitochondrial pellet, (b) that free ribosomes were not contaminating the microsomal pellet, and (c) that microsomes were not contaminating the ribosomal pellet, additional experiments were done in which 28-S RNA was also prepared from the mitochondrial pellet and from a "barfer pellet" resulting from centrifugation at 65 ooo rev./min for 3 to 5 min after the microsomes had been deposited and before the free ribosomes were deposited. Fig. l a shows a typical result, in which labeling of all four pellets was mostly parallel except that the specific activity of the free ribosomes was again highest until the eighth hour. Confirmatory results Abbreviations: r R N A , ribosomal R N A ; m R N A , messenger RNA.
Biochim. Biophys. Acta, 145 (1967) z9o-I92