Distribution of Ribosomes in Hypophysectomized Rat Liver. An Improved Method for the Determination of Free and Membrane-bound Ribosomes

Transcription

1 Agr. Biol. Chem., 37 (2), 219 `224, 1973 Distribution of Ribosomes in Hypophysectomized Rat Liver An Improved Method for the Determination of Free and Membrane-bound Ribosomes Masamichi TAKAGI, Tadashi INOUE and Yasukiyo UMEMURA* Laboratory of Radiation Genetics, Faculty of Agriculture, University of Tokyo, and *National Institute of Animal Health, Kodaira, Tokyo Received June 27, 1972 Post-mitochondrial fraction of rat liver was submitted directly onto the ultracentrifugation with newly improved discontinuous sucrose gradient system, which consisted of high con centration of sucrose. By this method, free ribosomes, membrane-bound ribosomes and smooth-membranes were fractionated, and the ratio of amounts of free ribosomes to membrane bound ribosomes was calculated. The ratio of polysomes to monosomes could also be determined by application of the same procedure on DOC-treated post-mitochondrial frac tion. In hypophysectomized rat liver, it was revealed that the ratio of free ribosomes to membrane-bound ribosomes was not changed as compared with that in control rat liver, while that of polysomes to monosomes was 56% smaller. Most of large polysomes were degraded into smaller polysomes by the operation. As a consequence of hypophysectomy, vascular albumin mass was decreased1 `3) following to the impairing of metabolic activi ties which include biosynthesis of albumin.4) The lowering activity of albumin synthesis in operated rat is considered to be a reflect of the changes of ribosomal distribution in liver. It is well known that there are two kinds of types in ribosomes; free ribosomes and membrane-bound ribosomes, and since the study of Siekevitz and Palade5) biochemical evidences have been accumulated showing functional differences between these two types of ribosomes in animal cells.6,7) Together with the results obtained in electron-micro scopic studies,8) it is suggested that free ribosomes synthesize proteins for intracellular purposes and membrane-bound ribosomes synthesize those to be secreted. It is import ant, therefore, to determine the amounts of these two types of ribosomes for evaluation of the function of various kinds of cells. The present study is an attempt to determine the quantitative ratio of free ribosomes to mem brane-bound ribosomes as well as that of polysomes to monosomes in hypophysecto mized rat liver. It is intended to know whether the change in the distribution of ribosomes is caused by the operation. The methods so far applied for this purpose, however, are indirect and intricated ones.9,10) They include at least two steps; the first step is to separate free and membrane-bound ribosomes, and the second is to analyze each of them. In this paper, a direct and simple method is also presented, in which only one step of ultracentrifugation with a discontinu ous sucrose gradient system is involved. "Ribosomes" in this paper includes both monosomes and polysomes. MATERIALS AND METHODS Chemicals. Sucrose (ribonuclease-free) was pur chased from Nakarai Chem. Co. Ltd., Kyoto, and sodium deoxychorate (DOC) from Difco Laboratories, Detroit, Michigan. Animals. Female rats of Wistar Imamichi strain, about 100g, were hypophysectomized, and 40days after the operation they were used for experiments.

2 220 M. TAKAGI, T. INOUE and Y. UMEMURA Fractionation of rat liver homogenate. After overnight starvation, rats were sacrificed by decapita tion and livers were quickly removed and chilled in ice-cold 0.25 M-sucrose in buffer A (5mm MgCl2, 25mm KCl, 50mm Tris-HCI, ph7.6). They were homogenized using a glass homogenizer with a motor driven Teflon pestle in 2.5 volumes of a same sucrosebuffer. The post-mitochondria) fraction was obtained as a supernatant by centrifuging the homogenate at 7000 ~g for 10min. The supernatant was centrifuged at 40,000rpm for 1.5hr in a Spinco Type 65 rotor. A brown pellet, microsomal fraction, was suspended in the same sucrose-buffer of an equal volume of the original supernatant. DOC-treatment. Treatment of the post-mito chondrial and the microsomal fraction with DOC was carried out by the addition of one-tenth volume of 13% DOC, which was freshly dissolved in 0.25 M sucrose-buffer A. Discontinuous sucrose density gradient centrifugation. Each solution of sucrose-buffer A was layered succes sively in a tube for a Spinco SW 41 rotor as shown in Fig. 1 and the gradients were stabilized for several hours at 4 Ž. A subcellular fraction of 0.5ml to be analyzed was slowly put onto the gradient and spun at 41,000rpm for 6hr at 4'C. After the centrifuga tion, each 0.27ml fraction was collected through a needle pierced into the bottom of the tube. Determination of RNA. Quantitative analysis of RNA in each fraction collected from the tube was done by a modified method of Fleck and Munro.11) To each fraction, 2ml of 0.34N KOH was added (the final concentration of KOH was 0.3N) and it was incubated for 1hr at 37 Ž to hydrolyze RNA. Then, 0.22ml of 60% (9.25N) perchloric acid was added to remove protein with the precipitate of potassium perchlorate. OD260mƒÊ of the supernatant was deter mined for each fraction after standing for at least 24hr at 0 Ž. RESULTS AND DISCUSSION Improved method for a direct and simple analysis of free and membrane-bound ribosomes Ribosomes of mammalian cells consist of about an equal amount of RNA and protein.12) The bouyant density of free ribosomes in sucrose solution is more than 1.5, while that of bound ones, having protein and lipid as components of membrane, is less than 1.3. The sedimentation coefficients of free ribo somes range from 80 S to several hundreds S FIG. 1. Discontinuous Sucrose Gradient System. Each sucrose solution contained the same constituents as those of buffer A was layered in the tube for a Spinco SW 41 rotor. On the top of the gradient, 0.5ml of sample to be analyzed was overlayered. or more. In a usually used sucrose density gradient centrifugation, in which sucrose concentration is less than 1M (d=1.33), free ribosomes with large S values and membranebound ribosomes can never be well separated.13) In principle, however, separation of these two kinds of ribosomes might be possible if the density of sucrose solution is adjusted smaller than that of free robosomes and higher than that of membrane-bound ribosomes. Several kinds of such a type of sucrose gradient were examined under various centrifugational con ditions. Among these, the system shown in Fig. 1 were found to be the best for the separa tion of two kinds of ribosomes. In Fig. 2a and 2b a sedimentation profile of the post mitochondrial fraction in the gradient system of Fig. I is shown. Two main peaks appeared clearly; peaks 1 and 2, which were observed as a white turbid zone and a brown turbid one, respectively, in tube after centrifugation (Fig. 2a). A small peak, peak 1 L, also existed between two main peaks, which was observed as a brown zone in the tube. The

3 Distribution of Ribosomes in Hypophysectomized Rat Liver 221 FIG. 2. Sedimentation Profile of Post-mitochondrial Fraction. Post-mitochondrial fraction was overlayered on the discontinuous sucrose gradient system and spun for 6hr at 41,000rpm. (a) Optical observation of the tube was shown schematically. (b) After collection of 0.27 ml-fraction from the tube, RNA in each fraction was determined as described in the text. (c) Post-mitochondria) fraction of hypophysectomized rat liver was analyzed by the same pro cedure as that of (b). Peak 1, peak 1 L, peak 2 and "S" were found to contain free polysomes, free monosomes, membrane-bound ribosomes and endoplasmic reticulum, respectively. Details are given in the text. FIG. 3. Sedimentation Profile of DOC-treated Post-Mitochondrial Fraction. DOC-treated post-mitochondria) fraction was analyzed as described in the legend to Fig. 2b. (a) Control, (b) Hypophysectomized. DOC-treated post-mitochondrial fraction was also examined in the same gradient system. Membranous components were solubilized by DOC-treatment and ribosomes released from membrane,14) so one could expect that mem brane-bound ribosomes in DOC-treated post mitochondrial fraction will become to sediment at the same position as that of free ribosomes. In a tube after centrifugation, though two peaks were also observed, brown turbid zone was not detected as shown in Fig. 3a. Peaks 1 and 1 L of Fig. 3a were sedimented at ap proximately the same position as those of peaks 1 and 1 L of Fig. 2b, respectively. The ultraviolet absorption spectra of peaks 1 and 2 in Fig. 2b were shown in Fig. 4. The

4 222 M. TAKAGI, T. INOUE and Y. UMEMURA former had the ribosome-like spectrum, and the latter had no such a characteristic spectrum. These results may well be explained if it is ultraviolet spectrum of this peak (Fig. 4c) supported the conclusion. assumed that peak 1 contains free ribosomes and peak 2 membrane-bound ribosomes. Besides the two peaks, a fluffy white band ("S" in Fig. 2a), which was located beneath the boundary of 1.0M sucrose layer in the tube, was obtained. This band was presumed to be smooth-membrane, since the absorption spectrum of it had no peak at 260mƒÊ (not shown) and it could not be detected in the RNA-determination procedure as shown in Fig. 2b. FIG. 5. Sedimentation Profile of DOC-Treated Microsomal Fraction. DOC-treated microsomal fraction was analyzed as described in the legend to Fig. 2b. We would like to emphasize again that the bouyant density of free ribosomes is larger than that of membrane-bound ones, although the sedimentation coefficient of the former is FIG. 4. Ultraviolet Absorption Spectra of the Peaks. Each fraction was diluted with water and the ultra violet absorption spectrum was measured by the Hitachi 124 spectrophotometer connected with QPD- 73 recorder. (a) Fraction No. 15 (peak 1) in Fig. 2b. (b) Fraction No. 28 (peak 2) in Fig. 2b. (c) Fraction No. 23 (peak 1 L) in Fig. 5. It is well known that if the microsomal fraction is treated with DOC, membrane bound ribosomes are released from membrane and, concomitantly, because of the absence of ribonuclease inhibitor, most of all polysomes are degraded into monosomes by the action of ribonuclease which is released from the membrane.15) The result of analysis of DOC-treated microsomal fraction is shown in Fig. 5. In this case, only one peak was ob served, which was sedimented at the same position as that of peak 1 L in both Fig. 2 and Fig. 3. It can be concluded, therefore, that peak 1 L contains free monosomes. The smaller than that of the latter. In the system shown in Fig. 1, both types of ribosomes in the post-mitochondrial fraction can be sepa rated clearly, showing membrane-bound ribo somes cannot penetrate 1.5M sucrose layer. Distribution of monosomes and polysomes in the post-mitochondrial fraction can also be known when the DOC-treated post-mitochon drial fraction is analyzed by the same system, in which monosomes stay in the lower part of 1.5M sucrose layer and polysomes distribute in the layers of 1.6 `2.0M sucrose. The cusion of 2.4M sucrose, layered at the bottom of the tube, is essential to prevent for large free polysomes to precipitate. Among the various centrifugational con ditions, centrifuging at a large forces for a shorter term (i.e., 41,000rpm, 6hr) seems to be necessary to obtain a clear sedimentation profile. In summary, ribosomes can be separated into free polysomes, free monosomes and

5 Distribution of Ribosomes in Hypophysectomi zed Rat Liver 223 membrane-bound ribosomes in the discontinu ous sucrose density gradient centrifugation system. And by analyzing the post-mitochon drial fraction, the ratio of the amounts of the free to the membrane-bound ribosomes can be determined. The ratio of the polysomes to the monosomes can also be obtained from the result of analysis of the DOC-treated post mitochondrial fraction. It should be mentioned, however, that all of ribosomes in the cell are not recovered in the post-mitochondrial fraction, but some parts of membrane-bound ribosomes are lost in the mitochondrial fraction, so that the ratio of the amounts of both types of ribosomes de scribed in this paper does not represent the ratio in situ. According to the system of Fig. I, the attempts to analyze the cell homo genate or post-nuclear fraction, without further fractionation could not succeeded because of their complicated sedimentation profiles after ultracentrifugation. Blobel and Potter,10) analyzing membrane-bound ribosomes in the mitochondrial and post-mitochondrial frac tions of rat liver, showed that the amount of membrane-bound ribosomes in post-mitochon drial fraction was not changed between prepa rations when the constant conditions for homogenization and centrifugation were em ployed. Taking it into consideration, the method described here could be usefully ap plied to various kinds of cells to know the distribution of ribosomes. Distribution of ribosomes in hypophysectomized rat liver The post-mitochondrial fraction and DOG treated post-mitochondrial fraction of liver from hypophysectomized rat were submitted to the above-mentioned discontinuous sucrose gradient centrifugation system. As shown in Fig. 2c, the operated rat liver contained a slightly less amount of membrane-bound ribosomes and a greater amount of free mono somes than the control rat liver. It is also shown in Fig. 2c that the large free polysomes in the operated rat liver were degraded ; the peak of the fraction No. 5 was negligible, and peak 1, which was located at fraction No. 19 in the control, was shifted to fraction No by the operation. It is clear that fraction No. 21 contains smaller polysomes than fraction No The degree of polymerization of membranebound ribosomes could be speculated by comparing the profiles shown in Fig. 3 (DOCtreated) with those in Fig. 2 (non-treated). The increases in optical density of all free ribosomal fractions (fraction No. 1 `No. 25 in Fig. 3) caused by DOC-treatment are con sidered to come from the ribosomes bound to membranes. Whereas marked increases in optical density were observed mainly in the large polysomal region in the control rat liver (fraction No. 2 `No.19 in Fig. 3a), such increases were observed exclusively in the smaller polysomal region in the operated rat liver (fraction No. 15 `No.21 in Fig. 3b). The amount of membrane-bound monosome seemed to be unchanged by the operation. From these profiles, it is concluded that membrane-bound ribosomes are consisted of smaller polysomes and that free large poly somes degraded into smaller polysomes or monosomes in hypophysectomized rat liver. The amounts of these ribosomes are calculated from the profiles and summarized in Table I. TABLE 1. RELATIVE AMOUNT OF RIBOSOMES IN HYPOPHYSECTOMIZED RAT LIVER Amounts of ribosomes are expressed as percentages of that of free ribosomes in non-treated post-mito ehondrial fraction of control rat liver. The ratio of free ribosomes to membrane bound ribosomes, and the amount of me

6 224 M. TAKAGI, T. INOUE and Y. UMEMURA brane-bound ribosomes were the same in the operated rat liver as those in the control rat liver, however, the ratio of total polysomes to total monosomes in the former was 56 % smaller than that in the latter. Although the primary effect is not clear, it is known that hypophysectomy results in a considerable decrease in both albumin syn thesis and metabolic activities in rat liver cells.4) The degradation of polysomes in both types of free and membrane-bound might be the cause to reduce these activities. Studies on the effects of regulatory agents, i.e., hormones, antibiotics, and other chemi cals on the distribution of ribosomes in animal cells are under investigation by using the new method described here. Acknowledgement. We are greatly indebted to Dr. A. Ogasa of the National Institute of Animal Health for performing hypophysectomies. REFERENCES 1) L. Levin and J. H. Leathem, Am. J. Physiol., 136, 306 (1942). 2) C. H. Li and W. O. Reinhardt, J. Biol. Chem., 167, 487 (1947). 3) L. Enerback, P. M. Lundin and J. Mellgren, Acta Endocr. Copnh., 32, 552 (1959). 4) L. M. Kernoff, B. L. Pimstone, J. Solomon and J. F. Brock, Biochem., J. 124, 592 (1971). 5) P. Siekevitz and G. E. Palade, J. Biophys, Biochem. Cyt., 4, 557 (1958). 6) C. R. Redman, J. Biol. Chem., 244, 4308 (1969). 7) M. Takagi, T. Tanaka and K. Ogata, Biochim. Biophys. Acta, 217, 148 (1970). 8) M. S. C. Birbeck and E. M. Mercer, Nature, 189, 558 (1961). 9) T. E. Webb, G. Blobel, V. R. Potter and H. P. Morris, Cancer Res., 25, 1291 (1965). 10) G. Blobel and V. R. Potter, J. Mol. Biol., 26, 279 (1967). 11) A. Fleck and H. N. Munro, Biochim. Biophys. Acta, 55, 571 (1962). 12) M. G. Hamilton, L. F. Cavalieri and M. L. Petermann, J. Biol. Chem., 237, 1155 (1962). 13) N. Tatal and E. D. Exum, Proc. Natl. Acad. Sci. U. S., 55, 1288 (1966). 14) H. Sugano, I. Watanabe and K. Ogata, J. Bio chem., Tokyo, 61, 778 (1967). 15) R. Morais and G. D. Lamirande, Biochim. Biophys. Acta, 95, 40 (1965).