Effects of Growth Hormone and Tri-Iodothyronine on Amino Acid Incorporation by Microsomal Subfractions from Rat Liver

Transcription

1 European J. Biochem. 2 (1967) Effects of Growth Hormone and Tri-Iodothyronine on Amino Acid Incorporation by Microsomal Subfractions from Rat Liver J. R. TATA and H. G. WILLIAMS-ASHMAN National Institute for Medical Research, Mill Hill, London, and The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, and the Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland (Received June 1, 1967) A two-step procedure without the use of detergents for the separation of smooth and rough endoplasmic reticulum of rat-liver and the fractionation of the latter into light rough membranes, heavy rough membranes and Yree polysomed is described. The distribution of RNA, protein and phospholipid in these fractions as well as their amino acid incorporation characteristics are also described. The heavy rough membrane fraction incorporated 14C-labelled phenylalanine, isoleucine and lysine into protein at a rate 3-5 times greater than that exhibited by the light rough membranes and free polysomes. Stimulation of the incorporation of [14C]phenylalanine and [14C]- isoleucine by poly U and poly UA showed that, per mg ribosomal RNA, the heavy rough membrane fraction was also more active in its response to synthetic messenger RNAs than the other two fractions. Hypophysectomy caused a marked reduction in the amount of ribosomes and phospholipids, the amino acid incorporation capacity and the response to poly U and poly UA in all three rough microsomal subfractions. These effects were reversed in 3-4 days by a combined treatment of hypophysectomized rats with human growth hormone and 3,3,5-tri-iodo-~-thyronine. Although the amino acid incorporation activity in the presence of endogenous and synthetic template RNA was affected in all the subfractions, the effect of hormone deprivation and replacement was most marked in the heavy rough membrane fraction. The relative stimulation of the incorporation of [14C]phenylalanine and [14C]isoleucine by poly U and poly UA, respectively, by free and membrane-bound polysomes was only slightly lowered in a few cases after hormonal treatment of hypophysectomized rats. However, per mg ribosomal RNA, hormone treatment led to a 2-4-fold enhancement of the incorporation of phenylalanine and isoleucine in response to the synthetic polyribonucleotides. Treatment of heavy rough membranes with sodium deoxycholate showed that much of the hormonal effect on amino acid incorporation was manifested in the ribosomes but that the maximal hormonal effect was detected when these were associated with the membranes. It is concluded that growth hormone and thyroid hormone together control both the amount and amino acid incorporation activity of free and membrane-associated polyribosomes of ratliver. It has now been well established that the stimulation of protein synthesis during growth hormoneand thyroid hormone-induced growth of the liver is mainly at the level of ribosomes [ i 31. The stimulation caused by the administration of these hormones to hypophysectomized or thyroidectomized rats is largely due to : (a) an increase in the relative amount - Non-Standard Abbreviations. Messenger RNA, m-rna; 3,3,5-tri-iodo-~-thyronine, T3; human growth hormone, HGH; polyuridylic acid, poly U; copolymer of polyuridylic and polyadenylic acids, poly UA; sodium deoxycholate, DOC. Enzymes. F yruvate kinase or ATP : phosphorylase phosphotransferaae (EC ). of cytoplasmic m-rna per ribosome [3] and: (b) an increase in the amount of newly formed ribosomes (and polysomes) accompanying the enhancement of amino acid incorporation [4,5]. At the same time, hormone administration was found to enhance the attachment of ribosomes to membranes of the endoplasmic reticulum [5,6]. Later studies also showed that the rate of proliferation of cytoplasmic membranes was co-ordinated with the accumulation of ribosomes [6,7]. Although the characteristics of protein synthesis in vitro by liver microsomes, and of ribosomes derived

2 Vo1.2, N0.3,1967 J. R. TATA and H. G. WILLIAMS-ASHMAN 367 from them by detergent treatment, have been compared in different situations of hormone-dependent growth [1,4,8], the behaviour of free and membranebound ribosomes prepared without detergent treatment has not been studied. Such an investigation was important both because of the hormone-induced alterations of ribosome-membrane attachment and formation and because of the suggestion that virtually all hepatic protein synthesis in vivo may occur on polyribosomes that are firmly attached to endoplasmic reticulum membranes [9]. For this purpose we have devised a method for fractionation of microsomes without detergents which is a combination of Dallner s [lo] separation of the smooth and rough endoplasmic reticulum membranes and the procedure of Bloemendal et al. [11] to separate free and bound polysomes. We were thus able to obtain from the rough endoplasmic reticulum a light and a heavy membrane fraction, as well as a free polysome fraction, all of which are active in incorporating amino acids in the presence of endogenous template RNAs and also capable of responding to synthetic polyribonucleotides. Using this separation procedure, we have examined the effects of hypophysectomy and the additive growth-promotion caused by human growth hormone and tri-iodotyronine on the amino-acid incorporation activity of the different submicrosomal fractions, in the presence of endogenous and exogenous template RNAs. It will be shown that the LCheavy ) membrane fraction is the most active fraction in. vitro and that a substantial effect of hormone deprivation and replacement is manifested in the activity of this as well as the free polysome component. MATERIALS AND METHODS Animals Male rats of the Sprague-Dawley strain were used in all experiments. Hypophysectomized animals were obtained either from the Hormone Assay Laboratories, Chicago, Illinois, U.S.A. (Table 4) or from the National Institutes of Health, Bethesda, Maryland, U.S.A. (Tables 2, 3, 5), by courtesy of Dr Robert Bates. Human growth hormone (from the National Institutes of Health, Public Health Service, Endocrinology Section, No. HS 600, Lot 1064) and 3,3,5- tri-iodo-l-thyronine were injected subcutaneously. The doses and the frequency with which they were injected are shown in the protocols. Normal rats were fed a diet of rat cake. The diet of the hypophysectomized animals was supplemented with Quick Rear Flakes. All animals received water ad libitum. The normal animals weighed between 250 and 350 g. The hypophysectomized rats of approximately the same age weighed about 120 g (Table 4) or 150 g (Tables 2, 3, 5). Isolation of Xubcellular Fractions The animals were killed by a blow on the head, the livers were removed immediately and placed in ice-cold Medium A containing sucrose (0.35 M), KC1 (25 mm), MgC1, (10 mm) and tris-hc1 buffer of ph 7.6 (50 mm). All subsequent operations were conducted at 0-2. Separation of various subcellular fractions was patterned on a combination of the procedures of Dallner [lo] and of Bloemendal, Bont and Benedetti [Ill. The tissue was chopped with scissors and washed twice with about 10 volumes of MediumA before homogenization with 3.5 volumes of MediumA using a glass homogenizer equipped with a Teflon pestle. The homogenate was centrifuged at 9,500 xg for 15 min and aliquots of the supernatant fluid were layered over 0.47 volume of a medium containing sucrose (1.3 M) and MgC1, (10 mm) in Spinco No. 40 rotor tubes. The material was centrifuged for 90 min at 40,000 rev./min. The resulting supernatant fluid, an opaque intermediate layer at the junction of the two sucrose solutions (consisting largely of the smooth membrane fraction of the endoplasmic reticulum), and the lower dense sucrose solution were removed with the aid of a Pasteur pipette from the pellet ( rough membrane fraction of the endoplasmic reticulum). The pellets of rough membranes were rinsed gently with Medium A and then suspended in Medium A by mild homogenization by hand in an all-glass apparatus. Aliquots (20 ml) of this suspension were layered gently in Spinco SW25-2 rotor tubes over a discontinuous sucrose density gradient comprised of a lower layer (19 ml) of sucrose (2 M) and MgC1, (I0 mm) and an upper layer (21 ml) of sucrose (1.5 M) and MgC1, (I0 mm). After centrifugation for h at 25,000 rev./min, the rough membrane fraction of the endoplasmic reticulum was effectively separated into three particulate fractions : (a) an upper layer termed light membranes, present just below the reddish-brown supernatant fluid ; (b) an intermediate heavy membrane layer; and (c) a pellet containing free ribosomes and polyribosomes at the bottom of the tube. The two membrane layers were removed with a Pasteur pipette, and gently homogenized by hand in an all-glass apparatus. The pellet at the bottom of the tube was then rinsed twice with Medium A and finally suspended by gentle homogenization in the latter medium. The fractionation procedure is summarized in the Scheme. Soluble Liver Eztract As a source of amino acid-activating enzymes and transfer ribonucleic acids, soluble extracts of rat liver were prepared as follows. Fresh rat liver, or rat liver frozen for 1-2 weeks at -20 and subsequently thawed at room temperature, was homogenized in a Waring blender operating at one-half maximum

3 368 Amino Acid Incorporation by Microsomal Subfractions European J. Biochem. Centrifuge at 105,000xg for 90 min over 1.3 M sucrose + 10 mm Mgtf Cell sap Rough membranes Smooth membranes I Suspend in Medium A Layer over medium containing 1.5 M and 2 M sucrose I Centrifuge at 50,000 x g for 16 hrs J st Interface band 2nd Interface band Pellet Light rough membranes Heavy rough membranes polysomes Scheme. Summarizing sub-microsomal fractionation speed with approximately 3 volumes of Medium A for 1-2 min at 2". Subsequent operations were carried out as close to 0" as possible. The homogenate was centrifuged at 10,000 xg for 15 min and the supernatant fluid was centrifuged for 2 hr at 105,000 xg. A superficial layer of fat was removed; t,he clear supernatant fluid, separated from the sediment, was passed through a column of Sephadex G-25 (coarse, particle size p) that had been previously equilibrated with Medium A in order to remove as many free amino acids and other low molecular weight substances as possible. The liver "105,000 xg supernatant fluids" that had been treated with Sephadex G-25 contained mg of protein per ml. They were frozen at -20" in small batches and used only once after thawing. Determination of Amino Acid Incorporation into Protein The reactions were carried out in conical centrifuge tubes at 37". Each vessel contained tris-hc1 buffer of ph 7.8 (50 pmoles) ; 2-mercaptoethanol (I0 pmoles) ; ATP (3 pmoles) ; GTP (I pmole) ; phosphoenolpyruvate (5 pmoles) ; MgC1, (12 pmoles) ; KC1 (30 pmoles); crystalline pyruvate kinase (10 pg) ; 0.1 pmole of each of the 19 L-12C-amino acids complemental to the radioactive amino acid employed ; the particular 14C-labelled amino acid (0.5-1 pc) ; rat liver Sephadex G-25-treated supernatant fluid (approximately 5 mg of protein). The total volume of the reaction mixtures was in the vicinity of 1 ml; the precise volume used in each experiment is shown in the protocols. In most instances the Sephadex G-25-treated soluble rat liver extract was added after all other components except the ribosome-containing fractions. After 5 min incubation at 37", the aminoacyl incorporations were initiated by addition of the ribosome preparations, and allowed to proceed for the stated time intervals (usually I0 min). The reactions were terminated by addition of 5 ml of 501, trichloroacetic acid (w/v) which was made O.IO/,with respect to the particular DL-W-amino acid equivalent to the particular ~PC-amino acid employed as a precursor. The contents of the tubes were immediately mixed and heated for 10min. After cooling on ice, the suspension was centrifuged, the sediment ciissolved in I ml of NaOH (0.05 N) and re-precipitated with 5 ml of 50/, trichloracetic acid (w/v). The cycle of acid-base washing was repeated. The acid-insoluble precipitate was then washed with 5ml of ethanoldiethyl ether (3:1, v/v), and finally suspended in 0.4ml of 9701, formic acid. This was mixed with 15 ml of scintillation counting fluid, for 14C determination. Incorporation of radioactivity into such protein fractions was corrected for background counts (about 50 countslmin) and also for the very small incorporation (about 40 counts/min) catalyzed by the Sephadex G-25-treated soluble rat liver extract in the absence of any of the ribosome fractions. Measurement of Radioactivity 14C was determined with a scintillation counter from Nuclear Instruments of Chicago, Inc., under conditions of an efficiency of about 70 /, at room temperature (about 25"). Measurements of at least 1,000 counts were made on all samples. The scintillation counting fluid contained 1,000 ml of xylene, 1,000ml of dioxane, 1,125ml of ethanol, 240g of naphthalene, 1.5 g of 2,5dphenyloxazole, and 0.15 g of 1,4-bis-(5-phenyl-osazole-2-yl)-benzene. Materials All solutions were prepared in deionized water, and were adjusted to ph 7.4 with NaOH or NaHCO,. Deoxycholic acid (sodium salt), ATP, GTP a.nd tri-

4 ~ VOl.2, No.3, 196i J. R. TATA and H. G. WILLISNS-ASHMAN 369 sodium phosphoenolpyruvate, and pyruvate kinase (Type 11, crystalline, from rabbit skeletal muscle) were purchased from the Sigma Chemical Company, St. Louis, Mo., U.S.A. Synthetic polyribonucleotides, prepared by the action of polynucleotide phosphorylase on the appropriate ribonucleoside diphosphates, were obtained from the Miles Chemical Company, Elkhart, Ind., U.S.A. Polyuridylic acid (Lot No ) was obtained as the ammonium salt. Copolymer of polyuridylic and polyadenylic acid (poly UA) (Lot No. 182) was prepared using an input UDP/ADP ratio of 5 :I. Radioactive amino acids were purchased from Schwarz BioResearch, Inc., Orangcburg, N.P., U.S.A. The samples were of the following specific radioactivities : ~-[1~C]phenylalanine (360 nic/ mmole) ; ~-[ ~C]isoleucine (160 mc/mmole) ; L-[~~C]- lysine (240 mc/mmole). Rat liver soluble RNA was determined by the method of Ceriotti [I41 with yeast RNA as the standard. Phospholipid was extracted from a 0.4 M HClO,-precipitate (washed 3 times with the acid) with chloroform-methanol (2: I, v/v) (two extractions at 20 and two at 45 ). After back-washin$ with 0.1 N HC1, the extracts were evaporated to dryness and the phospholipid was cstimated as 25x phosphorus as determined by the method of Fiske and Subbarow [15]. RESULTS AND DISCUSSIOK Isolation and Composition of the Different Liver Polyribosomnl Fractions In the initial separations of the endoplasmic reticulum into rough and smooth membrane components, the post-mitochondria1 supernat,ant fluids Table 1. Protein, RNA and phospholipid content of subfractions of t?le rough membrane endoplasmic reticulum fraction of livers from normal, hypophysectomized and hormone-treated hypophysectomized rats Values are expressed as mean i S.D. from all experiments. Hormone treatment consisted of 3 injections of 75 pg of human growth hormone (HGH) and 25 pg of tri-iodothyronine (T,) over a period of 3-4 days (see Table 2). After separation from the smooth membranes, the rough membrane fraction of the cndoplasmic reticulum was further fractionated into the light nnd heavy menibranes and free polyribosome fractions as described in the text Rats Polysome fractions mg proteinig In cr.t ENA/protcin PhosphoIipid,/protein Normal Hypophysectomized Hypophysectomized $- HGH + T, ~~ Light membrane Heavy membrane Light membrane Heavy membrane Light membrane Heavy membrane i & i i e & & & * & & & : & ~& & f a Tllrsc values limy be /o below the actual protein content as no sttempt \?a% made to obtain quantitative recoveries of thc different fractions. prepared by treatment with an equal volume of goo/,, phenol of the supernatant fluid resulting from centrifugation for 1 hr at 105,000 xg of homogenates prepared in Medium A. The aqueous phase resulting from the phenol treatment was extracted with peroxide-free diethyl ether at room temperature. Excess ether was removed from the aqueous solution by a stream of nitrogen. After addition of 0.1 vol. of potassium acetate (2M), the soluble RNA was precipitated by addition of 3 vols of cold ethanol, the solution being allowed to stand at 0 overnight. The precipitate was washed with cold ethanol, and finally dissolved in and exhaustively dialyzed against cold 0.15 M NaC1. Chemicul Determinations Protein was estimated either by the Biuret method [I21 or by the procedure of Lowry, Rosebrough, Farr and Randall [I31 using crystalline bovine plasma albumin as the standard. RNA was 26 European J. Biochem., Vo1.2 were processed directly without the prior separation of microsomes. This expedient was employed to minimize the degradation of polyribosomes by ribonucleases associated with the endoplasmic reticulum. The action of such ribonucleases is believed to be depressed by ribonuclease inhibitors present in the soluble portion of the post-mitochondria1 supernatant [16,17]. At the first fractionation step, a substantial amount of microsomal phospholipid and protein was recovered in the smooth membrane fraction. This fraction, which contained little RNA, exhibited only feeble or no amino acid incorporating activity and was routinely discarded. Fo this reason only results obtained with the subfractions of the rough endoplasmic reticulum will be discussed below. Table 1 summarizes the recovery of protein, RNA and phospholipid in the three subfractions obtained from the rough endoplasmic reticulum fraction of livers from normal and hypophysectomized rats as well as hypophysectomized rats treated with human growth hormane and tri-iodothyronine.

5 370 Amino Acid Incorporation by Microsomal Subfractions European J. Biochem. The major characteristics of the three fractions were (a) that the upper light membrane layer contained little RNA but was relatively rich in phospholipids ; (b) that the intermediate heavy membrane layer comprised more than 50 /, of the protein, RNA and phospholipid of the rough membrane fraction but contained relatively more RNA and less phospholipid than the light membrane fraction ; (c) that the pellet of free polyribosomes at the bottom of the tube contained virtually no phospholipid (30/0 of that present in rough membrane fraction), whereas the RNA/protein ratio of was much higher than that of the two membrane fractions. The free polysomes appeared therefore to be essentially free of endoplasmic reticulum membranes. The above findings were supported by electron microscopy performed by Dr. J. A. Armstrong at the National Institute for MedicalResearch, and the ultrastructural characteristics were similar to those recently described by Bloemendal et al. [23]. Electron microsopical analysis also showed that the difference between the heavy and light membrane fractions was a difference of the density of ribosomes present per unit surface of the membrane. Thin layer chromatography of the phospholipids of light and heavy membrane fractions gave identical patterns of distribution of the different constituents which were the same as the average composition of rough microsomal membranes from rat-liver described by other workers [18,19]. From their chemical analyses, their sedimentation characteristics and their amino acid incorporation properties (see below), it is concluded that the heavy membrane fraction is rich in both microsomal membranes and ribosomes (or polysomes), the light fraction being rich in membranes but poor in ribosomes and the free polysome fraction virtually free from membranes. Table 1 also shows that hypophysectomy markedly reduces the amount of RNA, protein and phospholipids in all three microsomal subfractions, but to different extents in each fraction. Treatment of hypophysectomized rats with HGH and T3 reversed this effect, being more marked in the heavy membrane and free polysome fractions than in light membranes. This enhanced proliferation of microsomes produced by hormonal stimulation of hepatic growth is consistent with the co-ordinated formation of membrane phospholipid and microsomal RNA and protein following the administration of HGH and T3 [6,7]. Characteristics of Amino Acid Incorporation by the Various Polysome-containing Preparations A series of preliminary experiments established conditions under which incorporation of labelled amino acids into protein-like material by the various polysome-containing fractions was proportional to both the time of incubation at 37 and the quantity of ribosomal material (as determined by RNA content) added to the reaction mixtures. This necessitated employment of incubation periods of not greater than 10 min and addition of an excess (2 mg protein) of Sephadex G-25-treated cell-sap preparations as a source of amino acid activating enzymes, transfer RNAs, and soluble transfer enzymes. (Gel filtration of the liver cell-sap preparations enha.nced by 2-3 fold their ability to support the incorporation of amino acids into proteins on addition of the polysome fractions ; presumably this is a result of removal of 12C-amino acids in the crude soluble liver extracts that decreased entry of labelled amino acids into the protein fractions by isotope dilution.) That the transfer RNA content of the Sephadex G-25-treated cell-sap preparations was not rate-limiting to the aminoacyl transfers was evident from the fact that addition of further refined rat liver soluble RNA preparations (0.5 mg) to the reaction mixtures did not enhance the amino acid incorporations. The 14C-labelled L-amino acids used were phenylalanine, isoleucine, and lysine. With any particular I4C-labelled L-amino acid as labelled substrate, an excess of the 19 corresponding other L-amino acids (final concentration approximately 0.1 mm) was added to the incubation mixtures. In many experiments, incorporation of radioisotope from L-[~*C]- phenylalanine into the protein fraction was measured in the presence and absence of poly U. With various free and membrans-bound ) polysome-containing fractions, maximal stimulation of phenylalanine incorporation was observed when the final concentration of poly U was 0.2 mg per ml (higher levels of poly U did not depress the phenylalanine incorporations). The relative stimulations of phenylalanine by poly U were of the same order as that frequently reported for mammalian polysomes [20] but were considerably lower than those reported for rat-liver by Bloemendal and his associates [ll, 16,211, presumably because these workers pre-incubated their preparations before the addition of the synthetic messenger, whereas we did not. In some instances, copolymers of polyuridylic and polyadenylic acids (poly UA of input UDP/ADP ratio of 1. : 5) were used as a synthetic template when isoleucine incorporations were studied. Small but reproducible stimulations by poly UA of entry of radioisotope from isoleucine into the protein fraction were observed routinely (cf. Tables 3 and 4). Since equivalent levels of poly U actually inhibited isoleucine incorporation, it appeared that the stimulatory effect of poly UA was due to its content of codons for isoleucine [20] rather than to any ambiguous coding properties with respect to isoleucine of any polyuridylic acid that may have been present in the poly UA preparations. Control experiments showed that the levels of ATP, GTP, and 2-mercaptoethanol in the various amino acid

6 ~~ Vol.2, N0.3, 1967 J. R. TATA and H. G. WILLIAMS-BSHMAN 371 incorporation reaction mixtures were not ratelimiting. A high-energy phosphate generating system (phosphoenolpyruvate and pyruvate kinase) was invariably added in order to maintain the levels of ATP and GTP. Tala1 and Exum [22] have reported that incorporation of phenylalanine into proteins by membrane-bound polysome preparations from rat spleen were inhibited by the in vitro addition of chloramphenicol ( pm), whereas this antibiotic did not inhibit the incorporations promoted by free polyribosomes from the same tissue. We were unable to demonstrate any large (> 20 /,,) effects of chloramphenicol (46p.M) on the incorporation into the protein fraction of either phenylalanine (measured in absence or presence of poly U) or isoleucine (measured in presence or absence of poly UA) with either the free or heavy membrane-bound liver polyribosome preparations described in this paper. Effect of Administration of Growth Hormone and Triiodothyronine to Hypophysectomixed Rats on Amino Acid Incorporation by Hepatic Sub-Microsomal Practions The three microsomal subfractions incorporated 14C-labelled phenylalanine, isoleucine and lysine at quite different rates, the heavy membrane fraction being 3-6 times as active as the light membranes and free polysomes (see Tables 2,3 and 4). When one considers that more than half the ribosomes were recovered in the heavy membrane fraction (see Table I), this finding of amino acid incorporation in vitro is in general agreement with the concept that it is the membrane-bound ribosomes that account for the majority of the protein synthesis in vivo 191. Our present results, however, differ from a recent report in which similar incorporation activities for the membrane-bound and free polysomes from ratliver were reported [23]. The reason for this diver- Table 2. Incorporation of [14C]phenylalanine into protein by free polysomes and polysomes in light and heavy rough membrane fractions of liver microsomes from normal and hypophysectomized rats, with or without hormonal treatment: Response to poly U Hormonal treatment consisted of 3 injections of 75 pg of HGH at 21, 47 and 74 h before killing and 2 injections of 25 [*g of T, at 47 and 74 h. Each value is the average of two determinations on material obtained from livers of 5 rats per group. Ribosomal RNA used for incubation: polysomes: pg; light rough membranes: pg; heavy rough membranes: pg. Total volume of incubation mixture = 1.05 ml Eats Polgsome fraction [14ClPhenylalanine incorporated in 10 min - Polv IT + POlV u Poly U cffecta Normal Hypophysectomized + HGH + T3 Hypophysectomized Light membranes Heavy membranes Light membranes Heavy membranes Light membranes Heavy membranes counts/min/mg RNA 13,590 38,090 18,620 41,840 58, ,400 6,770 30,520 14,550 44,250 40, ,900 26,700 86,300 22, ,130 66, ,000 ~ ~~ counts/min/mg RXA 24,500 23,220 69,600 23,750 30,700 62,800 59,600 78,500 78, Increase in incorporation produced by the addition of 200 Wg of poly U. Table 3. Incorporation of [14C]isoleucine (with and without poly UA) and [14C]lysine into protein by free and membrane-bound polysomes, of liver from normal or hypophysectomized rats, with or without hormonal treatment The same polysome preparations used in Table 2 were used in this experiment and all conditions were identical Rats Polysorne fraction [ 4C]Isoleucine incorporat,ed in 10 min - poi7 UA + Poly UA poly UA erect& [L4ClLysine incorporated in 10 rnin counts/min/mg RNA countslminlmg RNA counts/min/mg RNA Normal 5,320 6,383 1,063 14,600 Light membranes 3,590 5,748 2,158 12,200 Heavy membranes 23,800 29,980 6,180 59,800 Hypophysectomized 3,120 6,150 3,030 9,500 Light membranes 3,560 5,036 1,476 8,500 Heavy membranes 18,550 21,190 2,640 47,800 Hypophysectomized 10,900 20,720 9,820 31,300 + HGH + T3 Light membranes 6,980 11,590 4,610 16,400 Heavy membranes 32,100 45,700 13,600 66, a Increase in incorporation produced upon the addition of 200 vg of poly UA.

7 372 Amino Acid Incorporation by illicrosomal Subfractions European J. Biochem. Table 4. Incorporation of 14G-labeEEed amino acids into protein by free and membrane-bound polysomes from normal and hypophysectomized rats with or without hormonal treatment In this experiment hormonal treatment consisted of 3 injections of 100 pg of human growth hormone (HGH) at 26, 53, and 72 h before killing and 2 injections of 30 pg tri-iodothyronine at 53 and 72 h before killing. Each value is the average of two determinations on fractions derived from livers pooled from 5 rats; pg ribosomal RNA were used per incubation (final volume of 1.06 ml) Isoleucine Rats Polj smies Phen) lalanine ~- ~ - Poly u + Poly Ub C-Labelled amino acid incorporated in 10 min Lysine counts/min/mg RNA counts/min/mg RNA counts/min/mg RNA Normal 31, ,600 10,800 24,750 Membrane-bo und a 102, ,600 36,200 92,700 Hypophysectomized 8,720 33,120 1,070 5,320 Membrane-bound 27, ,500 9,270 22,300 Hypophysectomized 40, ,200 15,750 45,400 + HGH + T, Membrane-bound 103, ,000 41,300 94,000 a The membrane-bound fraction refers to the hem y membrane fraction. b 200 wg of poly IJ. gence may lie in the differences in techniques since we did not starve our animals, or repeatedly wash the preparation, or pre-incubate the particles before assay. The capacity of all the microsomal sub-fractions to incorporate the three amino acids was markedly depressed in hypophysectomized rats as compared to the normal animals. This deficiency was more than corrected in the stimulation obtained by the combined treatment with growth hormone and triiodothyronine. (It should be noted that the particular sequence and the amounts in which the two hormones were given were based on the fact that such a combination of hormonal treatment produces an additive increase in the rate of RNA synthesis in hypophysectomized rat-liver [24]). These findings of the effects of hormone deprivation and replacement on protein synthesis are consistent with earlier observations on the effects of hypophysectomy and thyroidectomy or the administration of growth hormone and thyroid hormone on amino acid incorporation in the presence of endogenous messenger [3-5, 25,261. In general the effect of hormone deprivation or replacement was of a greater magnitude in the heavy membrane and free polysome fractions than in the light membrane fraction. (A hypophysectomized rat is also functionally thyroidectomized due to the absence of thyrotrophic hormone.) In another experiment, not reported here, in which hormonal treatment was for a day less than that described in the experiments in Tables 2, 3 and 4, the stimulatory effect of hormone administration was more marked in the free polysome than in the heavy membrane fraction. Tables 2, 3 and 4 also show that the response of ribosomes from the different groups of animals to poly U and poly UA in incorporating [14C]phcnyl- alanine and [14C]isoleucine, respectively. The response of both the membrane-associated and free polysomes to synthetic polyribonucleotides paralleled their activity in the presence of endogenous messenger. In other words, hypophysectomy depressed, and hormone administration stimulated, the capacity of all classes of polysomes to utilize messenger RNA. It seems therefore that the major effect of hormone deprivation and replacement in modifying the amino acid incorporating ability of the ribosome preparations could not be explained solely on the basis of alterations in the amount of m-rnas associated with polysomes. In considering the possible ways in which growth hormone and thyroid hormone may modulate protein synthesis, it is important to mention that this stimulation of protein synthetic activity coincides with the appearance in the cytoplasm of additional newly-formed ribosomes following hormone administration [2,5,7,27]. This raises the possibility that the effect of each hormone on protein synthesis may be restricted to a small population of newly generated ribosomes rather than modification of the activity of all the ribosomes in the cytoplasm. Similar findings to ours on the response of hepatic ribosomes to poly U after hypophysectomy and thyroidectomy have now been observed in different laboratories [5,25,26]. They disagree with Korner s [3] suggestion, based on the higher response to poly U by ribosomes from hypophysectomized rats than from normal or growth hormone-treated animals, that growth hormone regulated protein synthesis on the basis of mrna levels in the cytoplasm. However, recently Earl and Korner [as] have shown that cardiac ribosomes were less able after hypophysect,omy to respond to poly U and that growth hormone administration enhanced protein synthesis with a concomitantly accelerated production of ribosomes.

8 ~ Vo1.2, N0.3,1967 J. R. TATA and H. G. WILLIAMS-ASEDIAN 373 Table 5. Effect of Nu deoxycholate (DOC) treatment on the incorporation of (14C]phenylalanine and [14C]isoleucine into protein by "free" and membrane-bound polysomes a from livers of I~~ypophysectomized rats, with or without hormonal treatment Hormonal treatment consisted of 3 injections to groups of 2 hypophysectomized rats of 75 pg of HGH at 21.0,46.5 and 69.5 h before killing and 2 injections of 25 pg of T3 at 46.5 and 69.5 h. To one half of mitochondria-free supernatant from the livers of each group of rats were added enough 5O/, (w/v) DOC to give a final concentration of 0.2O/, (w/v). Amounts of ribosomal RNA in each incubation were as follows: polysomes without DOC treatment: 35 pg; free polysomes after DOC: 52 to 88 pg; membrane-bound polysomes without DOC: wg; membrane-bound after DOC: 3-6 Rats Polysome fraction ['PCIPhenylalanine DOC treatment incorporated Poly U effect b in 10 min ["CIIsoleucine incorporated in 10 min Poly UA effect b counts/minjrng RNA Hypophysectomized - 22, ,700 Membrane-bound a :OOO Hypophysectomized - 42,500 + HGH + T, Membrane-bound a - 209, ,000 countslminlmg RNA 60,700 44, , , ,500 94, , ,000 counts/min/mg RNA 6,360 17,550 53, ,000 16,500 35,600 94,200 83,000.z The mcmbrsne-bound preparation comprised the heavy rough membranes. h Incrcase in incorporation of ["Clphmylalaniue or [14Clisoleucine urodnced by 200 fig of poly U or poly UA, respectively. countslminlmg RNA 840 3,400 6, ,700 7,000 24,500 It can be seen from Tables 2 and 4 that even in our experiments, poly U produced a relatively higher stimulation of phenylalanine incorporation in ribosomes from hypophysectomized than from normal or hormone-treated rats. But the absolute values for the incorporation with poly U were much lower in preparations from hypophysectomized rats. Recently, several other hormones have also been implicated in regulating translation at the level of the ribosome of the target cell [ In order to evaluate how far the attachment of ribosomes to endoplasmic reticulum membranes was essential to observe a hormonal effect on the heavy and light rough membrane fractions, we performed experiments in which an aliquot of the heavy membranes were treated with small amounts of sodium dcoxycholate. The amino acid incorporation activity of the residual membrane-attached preparations and the ribosomes thus released was then compared with preparations not treated with the detergent. As Table 5 shows, the effects of growth hormone and thyroid hormone residues both in the activity of the ribosome per se and in its association with the membrane. The polysomes released from the heavy membrane fraction by deoxycholate retained much of their high incorporation activity with both endogenous and exogenous messenger. At the same time, the activity of the residual membrane-bound polysomes not detached by deoxycholate was even higher than in the heavy membrane fraction not treated with the detergent. Similar results were obtained when incorporation of isoleucine was studied, with and without stimulation by poly UA. This finding is interesting in view of the observations that these two hormones increase the fraction of membrane-bound ribosomes that are resistant to detergent treatment [5,6,27]. The importance of the attachment of ribosomes to endoplasmic reticulum membranes was further illustrated by the finding that nearly 80 /, of nascent protein synthesized in vivo was associated with the heavy membrane fraction, isolated in the same way as reported here, whether derived from hormone-deficient or hormone-treated animals (Tata, unpublished). Recently, Campbell et al. [33] have concluded that the higher activity of microsomes in regenerating rat-liver is due to some factor in the membrane to which the ribosomes are attached. How membranes control ribosomal amino acid incorporation is not yet known but some preliminary experiments (Andrews and Tata, unpublished) suggest that growth hormone and thyroid hormone do not significantly modify the release of nascent protein into the vesicle of microsomal membranes [18,34]. These studies were supported in part by a Research Grant (HD-01453) from the United States Public Health Service. The major part of this work was carried out during a visit by one of us (J.R.T.) to the Department of Pharmacology and Experimental Therapeutics, The Johns Hoplrins University School of Medicine; he wishes to thank Prof. P. Talalay and other members of the Department for tho excellent facilities and help put at his disposal. We are grateful to Dr. R. Blizzard for the sample of human growth hormone REFERENCES Korner, A., Recent Progr. Hormone Res. 21 (1965) 205. Tata, J. R., Progr. Nucleic Acid. Res and Mol. Biol. 5 (1966) Korner, A., Biochem. J. 92 (1965) Tata, J. R., Ernster, L., Lindberg, O., Arrhenius, E., Pedersen, S., and Hedman, R., Biochem. J. 86 (1963) Tata, J. R., and Widnell, C. C., Biochem. J. 98 (1966) Tata, J. R., Nature, 213 (1967) 566.

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