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Significance of the endoplasmic reticulum membranes for protein biosynthesis in rat liver and Yoshida hepatoma ascites cells
E$perimental
Cell Research 30, 193-199
SIGNIFICANCE MEMBRANES
(1963)
OF THE ENDOPLASMIC
FOR PROTEIN
AND YOSHIDA
RETICULUM
BIOSYNTHESIS
HEPATOMA
G. P. BARBIERI Farmitalia,
193
ASCITES
and A. DI
IN RAT LIVER CELLS
MARCO
Laboratori Ricerche Microbiologia e Chemioterapia, Istituto Nazionale Tumori, Milano, Italy
and
Received July 2, 1962
little, up to now, is known about the role of the microsomal membrane structures in the biosynthesis of proteins. Although in liver the endoplasmic reticulum consists of both particle-covered membranes and free particles in various proportions, we know that the former disappear almost completely in other cells, such as, for instance, hepatoma ascites cells. Ribosomes are today considered to be an important, if not the main site of amino acid incorporation into cell protein. The process of protein synthesis, however, should not necessarily require the membranous moiety of the microsomes, fortified, also incorporate since isolated ribosomal particles, if properly amino acids. On the other hand, it cannot be excluded that in certain cases, the membrane structures may be intimately concerned with the protein biosynthesis. Kuff [6] suggested that all the ribosomal particles in a certain step of the protein synthesis must be bound to the membrane structures. Without arriving at such a general admission, Butler, Godson and Hunter [l] found that the membrane structures enhanced the amino acid incorporation into ribosomal proteins; on the other hand, however, Campbell [3] showed that their presence reduced in vitro the incorporation processes. Since in certain in vitro systems it was suggested by Emmelot [4] that the high ATPase activity of the microsomal membrane structures might produce depletion of the .energy supplied for the amino acid incorporation, then such contradictory results become comprehensible. It has been suggested by Howatson and Ham [5] that the E.R. membranes play some role in the transfer of the proteins to be used extracellularly, and, in any case, Siekewitz and Palade [8] showed that there are some differences in the amino acid incorporation activity from membrane-bound and free ribosomal particles. Results of our in vitro and, in particular, in vivo experiments fortify, in our opinion, these latter hypotheses put forward by Howatson and Ham.
VERY
13 - 631809
Esperimental
Cell Research
30
G. P. Barbieri and A. di Marco METHODS Albino rats of about 150 g were used in all the experiments. The ascitic fluid was drawn from rats 5-6 days after inoculation of Yoshida hepatoma ascites (AH130). In the experiments that we improperly called in vivo, hepatoma ascites cells were labeled by incubating the ascitic fluid out of the animal body (10 min incubation of 100 ml of freshly extracted ascitic fluid at 37°C with 50 ,UCalgal protein hydrolysate1% as suggested by Littlefield-Keller [7]); liver, on the other hand, was normally labeled by i.v. injecting animals with 0.12 ,uc/g b.w. 30 min before sacrifice. Livers and hepatoma ascites fluids of 20 rats were pooled in each of the in vitro experiments. Pooled livers from six rats injected with algal protein hydrolysateJ% and pooled ascitic fluid from IO rats supplied the material for each of the in vivo experiments. After incubation labeled ascitic fluid was rapidly cooled, centrifuged at 2000 rpm and the packed cells washed three times with a cold solution of inert protein hydrolysate in 0.04 M glucose and 0.02 tris buffer at pH 7.8. In the in vitro experiments, samples consisted of: microsome suspension (5 mg of protein); cytoplasmic sap (10 mg of protein) and ATP (1 ,uM)-GTP (0.5 PM)-Phosphenol-pyruvate (10 yM) -PEP kinase 0.025 mg-Algal protein hydrolysate/% 15 ,ug (2 PC), in a final volume of 1 ml of Webster medium [9].’ Preparation of microsomal fractions.-The washed hepatoma packed cells were suspended in 3 vols cold distilled water; after 5 min at OX, 0.3 vols of a tenfold concentrated Webster medium was added and the suspension was homogenized in a glass teflon Potter homogenizer for 4 min at 0°C. Liver was minced and directly suspended in 3.3 vols Webster medium. The suspension was homogenized for 20 set at 0°C. Liver and hepatoma cell homogenates were centrifuged IO min at 5000 g. Sediments were discarded and supernatants centrifuged 15 min at 15,000 g. Sediments were discarded and supernatants, that represent the “postmitochondrial homogenates”, were centrifuged 15 min at 36,000 g. The sediments of this latter centrifugation and the following ones, that constituted the microsomal fractions, presented two distinct layers: an overhanging one, that appeared woolly and light, and a lower one, vitreous and heavy. “Light” and “heavy” microsomes were pooled for analysis and incubation purposes, in the in vitro experiments, but in the in vivo experiments they were separated by a gentle washing with Webster medium and separately analyzed. Supernatants from the centrifugation at 36,000 g were again centrifuged at 68,000 g for 15 min. Microsomal fractions were isolated and supernatants cent.rifuged at 95,000 g for 15 min. Microsomal fractions were isolated and supernatants centrifuged again at 144,000 g for 2 hr. Microsomal fractions were isolated and supernatants that constituted the “cytoplasmic sap” supplied the required enzymatic systems to the microsomal samples in the in vitro experiments. All the samples of the in vitro and in vivo experiments and aliquots from postmitochondrial homogenates and from cytoplasmic saps, after protein determination (biuret), were submitted to the following analytical procedure: 1 Tris-HCl
buffer
0.05
AI pH
0.008 M.
Experimental Cell Research 30
7.8, sucrose
0.175
M, KC1 0.03
M, KHC03
0.024
M, MgC12
Protein biosynthesis in liver and hepatoma microsomes
195
(1) Precipitation of the acid-insoluble material in 0.5 N HCIO, at 0°C. The insoluble material was washed twice with cold 0.5 N HClO,. (2) Extraction of the phospholipid fraction: the insoluble material was washed twice with cold ethanol-ether (3/l), then extracted twice with hot ethanol-ether and twice with ethanol-ether-CHCl, (2/2/l). Finally, it was washed twice with ether. Extracts and washings were collected and the phosphorus content of this fraction was determined (lipid phosphorus). (3) Extraction of RNA from the residual nucleoprotein was obtained by boiling the material from extraction (2) I hr in 2 N NaCl. Residual protein was washed twice with 2 N NaCl and the washings were added to the extract. RNA was precipitated from the saline solution with 2 vols ethanol. RNA was collected, dissolved in distilled water and determined either spectrophotometrically or by orcinol and phosphorus reactions. (4) Residual proteins were dissolved in 0.4 N NaOH containing inert protein hydrolysate, at 40X, and precipitated in 5 per cent TCA. Dissolutions and precipitations were repeated three times. Protein samples were then boiled in 5 per cent TCA for 15 min and finally dried in ethanol-ether. The dry samples were transferred to polyethylene discs, weighed and placed under a thin mica window G.M. tube for the radioactivity determination. RESULTS
It is a well-known finding that normal and tumoral cells generally show a clear difference in their ergastoplasmic composition. The former are rich in particle-covered membranes (Phospholipo-proteins), and the latter in free particles (RNA-proteins). The existence of these constitutive discrepancies might reasonably account for the differences in the electrophoretic patterns of liver and hepatoma microsomal components recently observed by Di Marco et al. [a]. The aim of our present experiments was to determine whether the abovementioned changes in the ergastoplasmic structures from normal to tumoral cells were accompanied by variations in the protein biosynthetic activity of the structures themselves. Therefore we assayed in vitro and in vim the amino acid incorporation activity of several microsomal fractions isolated from liver and hepatoma by differential centrifugations. The patterns usually showed variations from one group of animals to another, but the overall trend was always the same. Therefore the figures of only one experiment are presented in each case. These results of in vitro and in vivo experiments (Fig. 1 a, b and Fig. 2 a, b) are as follows. (1) The ribosomal fractions sedimented at the highest centrifugal speed (144,000 CJper 2 hr) generally contained less lipid phosphorus (membrane Experimental
Cell Research 30
196
G. P. Rarbieri and A. di Marco
structures) and more RNA than all the others, both in liver and in hepatoma ascites cells. They generally presented a reduced amino acid incorporation activity. ,411 the microsomal fractions isolated from hepatoma ascites cells always presented a remarkably higher RNA/protein ratio than the corresponding ones isolated from liver.
Fig. 1 a.
Fig. la.-Rat liver microsomal fractions incubated with algal protein plasmic sap. mg RNA/mg prot = 0.04; y Plip/v RNA = 0.09.
hydrolysate-%.
Cyto-
Fig. 1 b.-Microsomal fractions from AH130 ascites hepatoma cells incubated with algal protein hydrolysate-W. Cytoplasmic sap. mg RNA/mg prot = 0.15; y Plip/r RNA = 0.0008. Experimental
Cell Research 30
Protein biosynthesis in liver and hepatoma microsomes
197
(2) In the amino acid incorporation activity of the “light” portion of the microsomal fractions, a clear difference between liver and hepatoma ascites cells is evident. The light microsomes isolated from liver in in uiuo experi,2.20
"$
0.15
: F 60 300' 0.4 A
01
0.3.
0.2
200
q 0
100
OD5 0.1
15DOOP
36Jmog
69DOOg
95ooog
144Doog
rract
ion8s
Fig. 2 a.
-‘ .oo
; : 9 L 100"
ZOO
,100
Fig. 2a.-Liver microsomal fractions isolated from lysate-W. In viuo rat hepatoma ascites microsomal Fig. 2b. Microsomal fractions isolated from AH130 fluid with algal protein hydrolysate-“C. In uivo rat
animals injected with algal protein hydrofractions. ascites hepatoma cells incubated in ascitic liver microsomal fractions. Experimental
Cell Research 30
198
G. P. Barbieri and A. di Marco
ments always showed a higher protein specific activity than the corresponding heavy microsomes; on the other hand, the light microsomes isolated from hepatoma ascites cells always showed a lower protein specific activity than the corresponding heavy ones. Light microsomes always contained more lipid phosphorus (membrane structures) than the corresponding heavy microsomes in both liver and hepat0ma.l The decrease of amino acid incorporation activity observed in the highspeed sedimented microsomes in in vivo experiments is to be considered a real metabolic characteristic of this fraction and not the result of mechanical or enzymatic damages that might have occurred during the preparation. Such a possibility, however, could not be excluded in the in vitro experiments. As for the different behavior of the light microsomes in liver and in hepatoma, revealed by our in vivo experiments, we assume that the membrane structures which sedimented preferentially together with the light microsomes in both liver and hepatoma (see the high lipid phosphorus content of these fractions) play a role in protein biosynthesis, a role which is more important in liver than in ascites cells. It should also be noted, however, that in these latter cells the most active fractions are always the heavy ones; in these heavy fractions the ribosomal free particles are most probably predominant. Since no biosynthetic pattern of a particular protein was studied here, our results are themselves not immediately amenable to rigorous interpretation. However, bearing in mind the fact that protein export processes are much more displayed by liver than by hepatoma cells, it may be thought that the increase in protein specific activity of the membrane-rich particles (light microsomes) of liver is related to the greater amount of extracellular proteins synthesized by this tissue (Fig. 2 a). On the other hand, in the hepatoma cells, where the “export protein” production is most probably reduced, it seems that the main role in the protein synthesis is played by the heavy microsomes (Fig. 2 b).
SUMMARY
The amino acid incorporation activity of several microsomal fractions, obtained either from rat liver or from rat hepatoma ascites cells, was determined. The overall trend was always the same for both liver and hepatoma, _____ 1 The specific activity of proteins of heavy microsomes isolated from hepatoma ascites cell suspensions previously incubated with 14C algal protein hydrolysate, either for 30 min or for 60 min, was always higher than that of light ones. Experimental
Cell Research 30
199
Protein biosynthesis in liver and hepatoma microsomes
with no significant variation of activity between the fractions, save for the highest speed sedimented fractions that generally showed the lowest activity. A light and a heavy portion were separated from each microsomal fraction.
The light one presented a much higher content of lipid phosphorus (membrane structure) than the heavy one; this latter, on the other hand, presented a much higher content of RNA (ribosomal particles) than the former. In liver the light microsomes always incorporated amino acids into proteins more actively than the heavy ones. In hepatoma, however, the former ones always presented a lower amino acid incorporation activity than the latter. Such results give prominence to the different significance of the E.R. membranes for protein synthetic processes in liver and in hepatoma. REFERENCES G. C., GODSON, G. N. and HUNTER, G. D., Protein Biosynthesis, Symposium, 1961. DI MARCO, A., CALENDI, E. and GAETANI, M., Tumori 47, 439 (1961). CAMPBELL, P. N. and GREENGARD, O., Biochem. J. 71,148 (1959). EMMELOT, P., Exptl. Cell Res. 13, 601 (1957). HOWATSON, A. F. and HAM, A. W., Proc. Can. Cancer Res. Con/. 2, 17 (1957). KUFF, E. I.. and ZEIGEL, R. F., J. Biophys. Biochem. Cytol. 7, 405 (1960). LITTLEFIELD, J. W. and KELLER, E. B., J. Biol. Chem. 224, 345 (1957). SIEKEWITZ, P. and PALADE, G. E., J. Biophys. Biochem. Cytol. 4, 557 (1958). WEBSTER, G. C., Arch. Biochem. Biophys. 85, 159 (1959).
1. BUTLER,
2. 3. 4. 5. 6. 7. 8.
9.
Experimental
p. 349,
Cell Research
30
Cell Research 30, 193-199
SIGNIFICANCE MEMBRANES
(1963)
OF THE ENDOPLASMIC
FOR PROTEIN
AND YOSHIDA
RETICULUM
BIOSYNTHESIS
HEPATOMA
G. P. BARBIERI Farmitalia,
193
ASCITES
and A. DI
IN RAT LIVER CELLS
MARCO
Laboratori Ricerche Microbiologia e Chemioterapia, Istituto Nazionale Tumori, Milano, Italy
and
Received July 2, 1962
little, up to now, is known about the role of the microsomal membrane structures in the biosynthesis of proteins. Although in liver the endoplasmic reticulum consists of both particle-covered membranes and free particles in various proportions, we know that the former disappear almost completely in other cells, such as, for instance, hepatoma ascites cells. Ribosomes are today considered to be an important, if not the main site of amino acid incorporation into cell protein. The process of protein synthesis, however, should not necessarily require the membranous moiety of the microsomes, fortified, also incorporate since isolated ribosomal particles, if properly amino acids. On the other hand, it cannot be excluded that in certain cases, the membrane structures may be intimately concerned with the protein biosynthesis. Kuff [6] suggested that all the ribosomal particles in a certain step of the protein synthesis must be bound to the membrane structures. Without arriving at such a general admission, Butler, Godson and Hunter [l] found that the membrane structures enhanced the amino acid incorporation into ribosomal proteins; on the other hand, however, Campbell [3] showed that their presence reduced in vitro the incorporation processes. Since in certain in vitro systems it was suggested by Emmelot [4] that the high ATPase activity of the microsomal membrane structures might produce depletion of the .energy supplied for the amino acid incorporation, then such contradictory results become comprehensible. It has been suggested by Howatson and Ham [5] that the E.R. membranes play some role in the transfer of the proteins to be used extracellularly, and, in any case, Siekewitz and Palade [8] showed that there are some differences in the amino acid incorporation activity from membrane-bound and free ribosomal particles. Results of our in vitro and, in particular, in vivo experiments fortify, in our opinion, these latter hypotheses put forward by Howatson and Ham.
VERY
13 - 631809
Esperimental
Cell Research
30
G. P. Barbieri and A. di Marco METHODS Albino rats of about 150 g were used in all the experiments. The ascitic fluid was drawn from rats 5-6 days after inoculation of Yoshida hepatoma ascites (AH130). In the experiments that we improperly called in vivo, hepatoma ascites cells were labeled by incubating the ascitic fluid out of the animal body (10 min incubation of 100 ml of freshly extracted ascitic fluid at 37°C with 50 ,UCalgal protein hydrolysate1% as suggested by Littlefield-Keller [7]); liver, on the other hand, was normally labeled by i.v. injecting animals with 0.12 ,uc/g b.w. 30 min before sacrifice. Livers and hepatoma ascites fluids of 20 rats were pooled in each of the in vitro experiments. Pooled livers from six rats injected with algal protein hydrolysateJ% and pooled ascitic fluid from IO rats supplied the material for each of the in vivo experiments. After incubation labeled ascitic fluid was rapidly cooled, centrifuged at 2000 rpm and the packed cells washed three times with a cold solution of inert protein hydrolysate in 0.04 M glucose and 0.02 tris buffer at pH 7.8. In the in vitro experiments, samples consisted of: microsome suspension (5 mg of protein); cytoplasmic sap (10 mg of protein) and ATP (1 ,uM)-GTP (0.5 PM)-Phosphenol-pyruvate (10 yM) -PEP kinase 0.025 mg-Algal protein hydrolysate/% 15 ,ug (2 PC), in a final volume of 1 ml of Webster medium [9].’ Preparation of microsomal fractions.-The washed hepatoma packed cells were suspended in 3 vols cold distilled water; after 5 min at OX, 0.3 vols of a tenfold concentrated Webster medium was added and the suspension was homogenized in a glass teflon Potter homogenizer for 4 min at 0°C. Liver was minced and directly suspended in 3.3 vols Webster medium. The suspension was homogenized for 20 set at 0°C. Liver and hepatoma cell homogenates were centrifuged IO min at 5000 g. Sediments were discarded and supernatants centrifuged 15 min at 15,000 g. Sediments were discarded and supernatants, that represent the “postmitochondrial homogenates”, were centrifuged 15 min at 36,000 g. The sediments of this latter centrifugation and the following ones, that constituted the microsomal fractions, presented two distinct layers: an overhanging one, that appeared woolly and light, and a lower one, vitreous and heavy. “Light” and “heavy” microsomes were pooled for analysis and incubation purposes, in the in vitro experiments, but in the in vivo experiments they were separated by a gentle washing with Webster medium and separately analyzed. Supernatants from the centrifugation at 36,000 g were again centrifuged at 68,000 g for 15 min. Microsomal fractions were isolated and supernatants cent.rifuged at 95,000 g for 15 min. Microsomal fractions were isolated and supernatants centrifuged again at 144,000 g for 2 hr. Microsomal fractions were isolated and supernatants that constituted the “cytoplasmic sap” supplied the required enzymatic systems to the microsomal samples in the in vitro experiments. All the samples of the in vitro and in vivo experiments and aliquots from postmitochondrial homogenates and from cytoplasmic saps, after protein determination (biuret), were submitted to the following analytical procedure: 1 Tris-HCl
buffer
0.05
AI pH
0.008 M.
Experimental Cell Research 30
7.8, sucrose
0.175
M, KC1 0.03
M, KHC03
0.024
M, MgC12
Protein biosynthesis in liver and hepatoma microsomes
195
(1) Precipitation of the acid-insoluble material in 0.5 N HCIO, at 0°C. The insoluble material was washed twice with cold 0.5 N HClO,. (2) Extraction of the phospholipid fraction: the insoluble material was washed twice with cold ethanol-ether (3/l), then extracted twice with hot ethanol-ether and twice with ethanol-ether-CHCl, (2/2/l). Finally, it was washed twice with ether. Extracts and washings were collected and the phosphorus content of this fraction was determined (lipid phosphorus). (3) Extraction of RNA from the residual nucleoprotein was obtained by boiling the material from extraction (2) I hr in 2 N NaCl. Residual protein was washed twice with 2 N NaCl and the washings were added to the extract. RNA was precipitated from the saline solution with 2 vols ethanol. RNA was collected, dissolved in distilled water and determined either spectrophotometrically or by orcinol and phosphorus reactions. (4) Residual proteins were dissolved in 0.4 N NaOH containing inert protein hydrolysate, at 40X, and precipitated in 5 per cent TCA. Dissolutions and precipitations were repeated three times. Protein samples were then boiled in 5 per cent TCA for 15 min and finally dried in ethanol-ether. The dry samples were transferred to polyethylene discs, weighed and placed under a thin mica window G.M. tube for the radioactivity determination. RESULTS
It is a well-known finding that normal and tumoral cells generally show a clear difference in their ergastoplasmic composition. The former are rich in particle-covered membranes (Phospholipo-proteins), and the latter in free particles (RNA-proteins). The existence of these constitutive discrepancies might reasonably account for the differences in the electrophoretic patterns of liver and hepatoma microsomal components recently observed by Di Marco et al. [a]. The aim of our present experiments was to determine whether the abovementioned changes in the ergastoplasmic structures from normal to tumoral cells were accompanied by variations in the protein biosynthetic activity of the structures themselves. Therefore we assayed in vitro and in vim the amino acid incorporation activity of several microsomal fractions isolated from liver and hepatoma by differential centrifugations. The patterns usually showed variations from one group of animals to another, but the overall trend was always the same. Therefore the figures of only one experiment are presented in each case. These results of in vitro and in vivo experiments (Fig. 1 a, b and Fig. 2 a, b) are as follows. (1) The ribosomal fractions sedimented at the highest centrifugal speed (144,000 CJper 2 hr) generally contained less lipid phosphorus (membrane Experimental
Cell Research 30
196
G. P. Rarbieri and A. di Marco
structures) and more RNA than all the others, both in liver and in hepatoma ascites cells. They generally presented a reduced amino acid incorporation activity. ,411 the microsomal fractions isolated from hepatoma ascites cells always presented a remarkably higher RNA/protein ratio than the corresponding ones isolated from liver.
Fig. 1 a.
Fig. la.-Rat liver microsomal fractions incubated with algal protein plasmic sap. mg RNA/mg prot = 0.04; y Plip/v RNA = 0.09.
hydrolysate-%.
Cyto-
Fig. 1 b.-Microsomal fractions from AH130 ascites hepatoma cells incubated with algal protein hydrolysate-W. Cytoplasmic sap. mg RNA/mg prot = 0.15; y Plip/r RNA = 0.0008. Experimental
Cell Research 30
Protein biosynthesis in liver and hepatoma microsomes
197
(2) In the amino acid incorporation activity of the “light” portion of the microsomal fractions, a clear difference between liver and hepatoma ascites cells is evident. The light microsomes isolated from liver in in uiuo experi,2.20
"$
0.15
: F 60 300' 0.4 A
01
0.3.
0.2
200
q 0
100
OD5 0.1
15DOOP
36Jmog
69DOOg
95ooog
144Doog
rract
ion8s
Fig. 2 a.
-‘ .oo
; : 9 L 100"
ZOO
,100
Fig. 2a.-Liver microsomal fractions isolated from lysate-W. In viuo rat hepatoma ascites microsomal Fig. 2b. Microsomal fractions isolated from AH130 fluid with algal protein hydrolysate-“C. In uivo rat
animals injected with algal protein hydrofractions. ascites hepatoma cells incubated in ascitic liver microsomal fractions. Experimental
Cell Research 30
198
G. P. Barbieri and A. di Marco
ments always showed a higher protein specific activity than the corresponding heavy microsomes; on the other hand, the light microsomes isolated from hepatoma ascites cells always showed a lower protein specific activity than the corresponding heavy ones. Light microsomes always contained more lipid phosphorus (membrane structures) than the corresponding heavy microsomes in both liver and hepat0ma.l The decrease of amino acid incorporation activity observed in the highspeed sedimented microsomes in in vivo experiments is to be considered a real metabolic characteristic of this fraction and not the result of mechanical or enzymatic damages that might have occurred during the preparation. Such a possibility, however, could not be excluded in the in vitro experiments. As for the different behavior of the light microsomes in liver and in hepatoma, revealed by our in vivo experiments, we assume that the membrane structures which sedimented preferentially together with the light microsomes in both liver and hepatoma (see the high lipid phosphorus content of these fractions) play a role in protein biosynthesis, a role which is more important in liver than in ascites cells. It should also be noted, however, that in these latter cells the most active fractions are always the heavy ones; in these heavy fractions the ribosomal free particles are most probably predominant. Since no biosynthetic pattern of a particular protein was studied here, our results are themselves not immediately amenable to rigorous interpretation. However, bearing in mind the fact that protein export processes are much more displayed by liver than by hepatoma cells, it may be thought that the increase in protein specific activity of the membrane-rich particles (light microsomes) of liver is related to the greater amount of extracellular proteins synthesized by this tissue (Fig. 2 a). On the other hand, in the hepatoma cells, where the “export protein” production is most probably reduced, it seems that the main role in the protein synthesis is played by the heavy microsomes (Fig. 2 b).
SUMMARY
The amino acid incorporation activity of several microsomal fractions, obtained either from rat liver or from rat hepatoma ascites cells, was determined. The overall trend was always the same for both liver and hepatoma, _____ 1 The specific activity of proteins of heavy microsomes isolated from hepatoma ascites cell suspensions previously incubated with 14C algal protein hydrolysate, either for 30 min or for 60 min, was always higher than that of light ones. Experimental
Cell Research 30
199
Protein biosynthesis in liver and hepatoma microsomes
with no significant variation of activity between the fractions, save for the highest speed sedimented fractions that generally showed the lowest activity. A light and a heavy portion were separated from each microsomal fraction.
The light one presented a much higher content of lipid phosphorus (membrane structure) than the heavy one; this latter, on the other hand, presented a much higher content of RNA (ribosomal particles) than the former. In liver the light microsomes always incorporated amino acids into proteins more actively than the heavy ones. In hepatoma, however, the former ones always presented a lower amino acid incorporation activity than the latter. Such results give prominence to the different significance of the E.R. membranes for protein synthetic processes in liver and in hepatoma. REFERENCES G. C., GODSON, G. N. and HUNTER, G. D., Protein Biosynthesis, Symposium, 1961. DI MARCO, A., CALENDI, E. and GAETANI, M., Tumori 47, 439 (1961). CAMPBELL, P. N. and GREENGARD, O., Biochem. J. 71,148 (1959). EMMELOT, P., Exptl. Cell Res. 13, 601 (1957). HOWATSON, A. F. and HAM, A. W., Proc. Can. Cancer Res. Con/. 2, 17 (1957). KUFF, E. I.. and ZEIGEL, R. F., J. Biophys. Biochem. Cytol. 7, 405 (1960). LITTLEFIELD, J. W. and KELLER, E. B., J. Biol. Chem. 224, 345 (1957). SIEKEWITZ, P. and PALADE, G. E., J. Biophys. Biochem. Cytol. 4, 557 (1958). WEBSTER, G. C., Arch. Biochem. Biophys. 85, 159 (1959).
1. BUTLER,
2. 3. 4. 5. 6. 7. 8.
9.
Experimental
p. 349,
Cell Research
30