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Isolation and properties of polysomes from human placenta
BIOCHIMICA ET BIOPHYSICA ACTA
39~
BBA 96561
ISOLATION AND P R O P E R T I E S OF POLYSOMES FROM HUMAN PLACENTA E. M. L A G A , B. S. B A L I G A AND H. N. M U N R O
Physiological Chemistry Laboratories, Department o/ Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass., o2139 (U.S.,4.) (Received M a r c h 6th, 197 o)
SUMMARY
Conditions have been investigated for obtaining free and membrane-bound polysomes from human placenta that can incorporate free amino acids into peptide bonds in a cell-free protein-synthesizing system. Optimum recoveries of polysomes were obtained when ribonuclease activity was inhibited by addition of 0. 5 mM EDTA to the homogenization medium, along with I5O mM KC1, 200 mM NH4C1 and IO mM MgCI~. Conditions of homogenization and centrifugation to provide optimum yields of free and bound polysomes were also established. About 20 % of the polysomes recovered from human placentas were membrane-bound. They were more active than free polysomes in an amino acid incorporating system.
INTRODUCTION
The human placenta secretes protein hormones in considerable amounts into the maternal circulation. For example, chorionic growth hormone-prolactin is secreted at a mean rate of I g per day during the ninth month of pregnancy 1. In the case of the liver, it has been demonstrated that secreted proteins are manufactured by polysomes attached to membranes of the endoplasmic reticulum 2,z, whereas ferritin, a typical retained protein, is formed on free ribosomes not associated with these membranes 4. In order to study the mechanism of synthesis and secretion of protein by the human placenta, it has been necessary to establish conditions for the preparation of both membrane-bound and free polysomes from placenta that will actively incorporate free amino acids into peptides. Cell-free placental systems capable of in vitro amino acid incorporation have been described previously5, but optimal conditions for preparation of free and membrane-bound polysomes from this organ have not been established. Procedures applicable to liver have had to be modified because of the more fibrous nature of the placenta and the presence of an active alkaline ribonuclease. The present investigation establishes optimal conditions for this purpose. METHODS
Materials and Media
Dipotassium EDTA was purchased from Eastman Organic Chemicals, 2mercaptoethanol from Calbiochem and sodium deoxycholate from Mann Research Biochim. Biophys. Acta, 213 (197 o) 391-4oo
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Laboratories, and pancreatic ribonuclease from Nutritional Biochemicals. Guanosine 5-triphosphate trilithium.6 H~O, adenosine 5-triphosphate disodium. 4 H~O, glutathione (reduced, creatine phosphate and creatine phosphokinase were obtained from Calbiochem. The uniformly 14C-labeled L-amino acid mixture (specific activity 5 ° mC/33.6 rag) and uniformly 14C-labeled L-leucine (specific activity 250 mC/mmole) were obtained from New England Nuclear Corporation. Other chemicals were reagent grade. Most of the reagents were made up in 50 mM Tris (pH 7.6)-2o0 mM NH4C115o mM K C I - I o mM MgC12 buffer. The 50 mM Tris (pH 7.6)-15o mM K C I - I o mM MgC12 buffer was modified b y addition of NH4C1 because EARL AND MORGAN6 have found that recovery of polysomes from cardiac muscle is improved by its presence.
Preparation of C-ribosomes, /ree and bound polysomes Fresh placentas (300-600 g) from normal deliveries were rapidly immersed in ice cold modified Tris-KC1-MgCI 2 buffer, transported in an ice-filled polystyrene box and processed within o.5 h after delivery. The placenta was perfused with 1-2 1 of cold Tris-KC1-MgC12 buffer to remove the red blood cells. In the standard procedure finally developed, about 5 g amounts of perfused placental tissue were minced and homogenized with 5 ml of a medium containing 0.25 M sucrose and 0. 5 mM EDTA in Tris-NH4C1-KC1-MgC12 buffer (pH 7.6), using the Sorvall Omni Mixer at 4 ° for I rain at speed setting 7- Cell debris, nuclei and mitochondria were removed by centrifuging the homogenate in the Sorvall RC 2-B centrifuge for 20 min at 12 ooo rev./ rain in the SS-34 rotor. The post-mitochondrial supernatant was filtered through glass wool. The C-ribosome fraction of WETTSTEIN et al. 7 was prepared by layering 5 ml of post-mitochondrial supernatant adjusted to i ~o with deoxycholate on top of 5 ml of 2.0 M sucrose in Tris-NH4C1-KC1-MgC1~buffer containing 0.5 mM EDTA. The free polysomes were prepared by the same procedure but omitting deoxycholate treatment. These two sets of polysomes were harvested by centrifuging in the Spinco L-2 ultracentrifuge at 4 ° for 6 h at 40 ooo rev./min (lO5 ooo ×g) in the Ti-5 o rotor. The pellets were washed twice with cold Tris-NH~C1-KC1-MgC12 buffer, suspended in 0.25 ml of the same buffer, and stored at --4o °. The membrane-bound polysomes were prepared b y collecting the intermediate turbid layer above the interface between the post-mitochondrial supernatant and the sucrose in the tubes used for preparation of the free polysome fraction. This sample was adjusted to i °/o with deoxycholate and layered onto 2.0 M sucrose in Tris-NH4C1-KC1-MgC1 ~ buffer containing o.5 mM E D T A and the solubilized polysomes were harvested as described above.
Sucrose density gradient o[ polysomes The standard polysome patterns were obtained from pelleted total ribosomes, free ribosome and membrane-bound ribosome preparations b y layering a 2o-/,1 sample of dissolved ribosome pellet, diluted with 0.2 ml Tris-NH4C1-KC1-MgClz buffer containing 0.5 mM EDTA on top of a lO-5O ~o linear sucrose gradient made up in the Tris-KC1-MgC12 buffer. The gradients were centrifuged in the Spinco L-2 centrifuge equipped with a SW-5 o rotor at 4 ° for 80 rain at 4 ° ooo rev./min (lO5 ooo ×g). The distribution of polysomal aggregates in the gradient was determined b y puncturing the tube at the bottom and b y passing the effluent of the tube through a o.o25-ml Gilford flow-cell with a 2 m m light path using a Gilford 2000 recording spectrophotometer. The flow rate was kept constant by a Buchler flow p u m p and Biochim. Biophys. -4cta. 213 (197 o) 391-3oo
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the absorption of the effluent at 260 nm was recorded on a moving chart. The proportion of the total profile represented by monosomes and by polysomes (disomes and higher aggregates) was obtained by cutting out each area of the chart paper and weighing it.
Preparation o/cell sap/actors/rom placenta The pH 5 enzyme fraction was obtained by omitting deoxycholate treatment of the post-mitochondrial supernatant but adding 5 mM 2-mercaptoethanol. This post-mitochondrial supernatant was immediately centrifuged in the Spinco L-2 at 4 ° for 4 h in a Ti-5o rotor at 4 ° ooo rev./min (lO5 ooo ×g) to remove microsomes and free ribosomes 8. To remove factors inhibiting amino acid incorporation, the cell sap obtained as a clear supernatant after centrifugation was passed through a column of Sephadex G-25 that had been equilibrated with Tris-KC1-MgCI~ buffer containing 5 mM 2-mercaptoethanol at pH 7.6 (ref. 9). The pH 5 precipitate was obtained by slow addition of I M acetic acid at o ° and removed by centrifugation in a Sorvall RC-2B centrifuge at 4 ° for 15 min in the SS-34 rotor at 13 ooo rev./min (20 ooo ×g). The pH 5 enzyme protein was dissolved in 2 ml 50 mM Tris (pH 7.6)-1 mM E D T A I mM reduced glutathione buffer, and stored at --4 o°.
Cell-/ree amino acid incorporation system The reaction mixture contained 5o mM "Iris, 5 mM MgCI~, 8o mM NH4C1, 2 mM ATP, I mM GTP, 8 mM GSH, 0.02 mM [14C]leucine (0.5 #C), 1. 5 mg placental pH 5 enzyme protein and 200-500 #g placental polysome RNA in a volume of 0. 5 ml incubation mixture. In one series of experiments (see Fig. 8), an ATP regenerating system (IO #moles creatine phosphate and IOO/~g creatine phosphokinase per tube) was added to the incubation mixture. Following incubation at 37 ° for 30 min, the reaction was stopped by addition of an equal amount of Io% (w/v) trichloroacetic acid solution. The solution was then heated to 9 °0 for 15 min and the insoluble protein was collected on a glass fiber filter, washed with 5 % trichloroacetic acid and dried before counting the radioactivity. Blank incubations were run without polysomes and the incorporation by these was subtracted from the standard incubation.
Protein and RNA determinations Protein was measured by the method of LOWRYet al. 1°. rRNA was determined by measuring the absorbance at 260 nm and correcting for protein present using the formula of FLECK AND MUNRO 11. RESULTS
Recovery o[ polysomes ]tom placenta Attempts to prepare polysomes (C-ribosomes) by techniques used to give liver polysomes failed to yield more than monosomes and small oligosomes. MATTHAEI AND SCHOECH5 used EDTA in the homogenizing medium to obtain active ribosome populations for protein synthesis. We therefore explored the recovery of polysomes using different concentrations of EDTA in the homogenizing medium. Fig. I shows that increasing concentrations of EDTA up to 0. 5 mM greatly increased polysome recovery, but that higher levels of this chelator were less efficient. These findings were Biochim. Biophys. Acta, 213 (197 o) 391-4oo
E.M. LAGA et al
394 I OC Top~ OmM 10 0 ImM EDTA A
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050
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Fig. i. Effect of v a r y i n g E D T A concentration in the h o m o g e n i z i n g medium on the sucrose gradient profiles of total (A), free (]3) and m e m b r a n e - b o u n d (C) p o l y s o m e preparations obtained from equivalent a m o u n t s of h u m a n placenta. The E D T A concentrations used were o, o.I, 0.25, 0.5, I, and 5 raM.
similar for mixed polysome populations (Fig. IA), free polysomes (Fig. IB) and membrane-bound polysomes (Fig. IC). As shown in the diagram, there was better recovery of polysomes from the bound than from the free population at low levels of EDTA, presumably due to stabilization of the polysomes through attachment to the membranes. The action of o. 5 mM EDTA in preserving polysomes was confirmed by electron microscopy of the C-ribosome pellet. Fig. 2B shows that in the presence of o.5 mM EDTA the preparations exhibit much more of polysome aggregates than in the absence of this chelating agent (Fig. 2A). Consequently, o.5 mM EDTA was used in the subsequent
studies.
Recovery o] /ree and membrane-bound polysomes BLOBEL AND POTTER12,13,z° have described techniques for preparing free and membrane-bound polysome populations separately from the liver. These techniques were applied in slightly modified form to human placenta. The post-mitochondrial supernatant was applied directly over 2.o M buffered sucrose containing 0. 5 mM EDTA and ribosomes free of membrane were sedimented through this layer. The lower half of the supernatant fluid was then made I °/o with deoxycholate and this was then spun down through 2.0 M buffered sucrose containing EDTA to yield a pellet of membrane-bound ribosomes. Another portion of the post-mitochondrial supernatant l?,iochim. Biophys. Acta, 213 (197 o) 3 9 1 - 4 o o
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Fig. 2. Electron micrographs of polysomes prepared from human placenta without EDTA (A), or with o. 5 mM EDTA (B) in the homogenizing medium and negatively stained with uranyl acetate ( × 50 ooo).
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was made 1 % with deoxycholate and spun immediately through 2 M sucrose with E D T A to provide the total ribosome population. BLOBEL AND POTTER12,13,2° have emphasized that the recovery of polysomes in all fractions from rat liver can be improved b y adding extra rat liver cell sap to the homogenizing medium. However, we have not found that rat liver cell sap has this protective effect when used in place of E D T A in the homogenizing medium to harvest placental polysomes. Various factors in obtaining m a x i m u m yields of these different populations of ribosomes were investigated by measurement of RNA recovered in the pellets. Fig. 3 shows the effect of varying the ratio of tissue to homogenizing medium; a ratio of I g tissue to I ml fluid gave maximal recoveries of both free and bound ribosomes, most notably in the case of the free ribosomes. Next, the loss of bound and free polysomes into the mitochondrial and cell debris fraction was investigated. Fig. 4 shows that recovery of bound ribosomes is very much dependent on this step,
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Fig. 3. E f f e c t of ratio of a m o u n t of p l a c e n t a l tissue (g) to a m o u n t of h o m o g e n i z a t i o n m e d i u m (ml) on yield of r R N A in free a n d m e m b r a n e - b o u n d p o l y s o m e pellets. H o m o g e n i z a t i o n w a s p e r f o r m e d in t h e Sorvall O m n i Mixer r a t s p e e d s e t t i n g 7 for I min, u s i n g a Tris-NH4C1-KC1-MgC12 buffer w i t h a d d i t i o n of 0.25 M sucose a n d 0. 5 m M E D T A . T h e pellets of free a n d m e m b r a n e b o u n d r i b o s o m e s were o b t a i n e d b y c e n t r i f u g a t i o n of t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t . T h e pellets were dissolved in Tris-KC1-MgC12 b u f f e r a n d t h e i r a b s o r b a n c e w a s m e a s u r e d a t 26o n m . Fig. 4- E f f e c t of v a r y i n g speed of c e n t r i f u g a t i o n of p l a c e n t a l h o m o g e n a t e on yield of r R b l A in free a n d m e m b r a n e - b o u n d p o l y s o m e pellets. C e n t r i f u g a t i o n w a s p e r f o r m e d in t h e Sorvall R C 2 - B c e n t r i f u g e a t 4 ° for 20 m i n w i t h t h e SS-34 rotor a t speeds f r o m 12 to 17 ooo r e v . / m i n . Free a n d m e m b r a n e - b o u n d p o l y s o m e pellets were p r e p a r e d f r o m t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t b y c e n t r i f u g a t i o n , r R N A in t h e dissolved pellet w a s m e a s u r e d a t 260 n m .
presumably because polysomes attached to large membraneous masses of endoplasmic reticulum are precipitated even at low speeds. Consequently, the optimal speed chosen was 12 ooo rev./min on the Sorvall RC 2-B for 20 rain using SS-34 rotor. Next, the time course of sedimenting ribosomes through 2.o M sucrose from the postmitochondrial supernatant fraction was examined. Fig. 5 demonstrates that at least 5 h of spinning is needed to sediment the free polysomes without leaving some behind at the interface in among the membrane-bound ribosomes. The effect of ions in the homogenizing medium was next investigated as a factor in polysome recovery, using both total rRNA recoveries and gradient profiles to evaluate the effects produced. Fig. 6 shows that increasing the concentration of KC1 in the medium affects the recovery of free and bound ribosomes. Recovery of Biochim. Biophys. Acta, 213 (197 o) 391-4oo
PLACENTAL POLYSOMES
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Fig. 5. Effect of v a r y i n g the d u r a t i o n of centrifugation of p o s t - m i t o c h o n d r i a l s u p e r n a t a n t t h r o u g h 2.o M buffered sucrose on the yield of r R N A . Free ribosome pellets were h a r v e s t e d after I, 2, 3, 4, 5, 6, 7 and 8 h of centrifugation in the Spinco L-2 at 4 ° in the Ti-5o r o t o r at lO5 ooo ×g. At every h, t u b e s were removed; the pellet of free ribosomes at the b o t t o m of the t u b e was harvested and the interface material was used for p r e p a r a t i o n of m e m b r a n e - b o u n d polysomes. r R N A in each fraction was m e a s u r e d at 260 nm. Fig. 6. Effect of v a r y i n g concentration of KCI in the homogenization m e d i u m on the yield ot r R N A and on the p o l y s o m e / m o n o s o m e ratios of the free and m e m b r a n e - b o u n d ribosomes. The KCI concentration ranged from o to 60o raM. r R N A in the dissolved pellets was m e a s u r e d at 26o nm. The p o l y s o m e / m o n o s o m e ratios were c o m p u t e d from s t a n d a r d polysome profiles obtained after lO-5O % continuous sucrose gradient centrifugation.
free ribosomes increases up to 5O-lOO mM KC1. Above 3oo mM, the recovery of bound ribosomes falls off and the free population rises sharply. Presumably this last effect represents detachment of ribosomes from the membrane at the high salt concentra-
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Fig. 7. Effect of varying MgC12 and E D T A concentration in the homogenization medium on the yield of rIRNA from the free and m e m b r a n e - b o u n d ribosomes, r R N A in the pellets was m e a s u r e d at 260 nm. The concentration of Mg ~+ in the homogenization buffer was 2, io, and 15 mM in the presence of respectively o, 0. 5, I.O, 1.5 and 2.0 mlV[ E D T A .
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tion. E x a m i n a t i o n of the polysome profiles at these various c o n c e n t r a t i o n s shows t h a t high salt levels lead to disaggregation, as shown b y the fall in polysome/monosome ratio. I n the light of these experiments, 15o mM KC1 provides m a x i m a l recovery of ribosomes a n d m a x i m a l polysome/monosome ratio in b o t h populations of ribosomes. Fig. 7 shows the effects of v a r y i n g both Mg 2+ a n d E D T A c o n t e n t of the homogenizing i n e d i u m on recovery of free a n d b o u n d ribosomes. The recovery of free ribosomes is m a x i m a l in Io mM Mg ~+ at 0.5 n a n E D T A , whereas better recoveries of b o u n d polysomes are o b t a i n e d with 15 mM Mg z+ a n d o.5 mM E D T A . As a compromise the Io mM Mg 2+ was chosen for isolation of b o t h populations. Fig. IA, I B a n d IC show the profiles of total, free a n d m e m b r a n e - b o u n d polysomes prepared from h u m a n placenta. W h e n prepared u n d e r o p t i m a l conditions (o.5 mM E D T A ) from e q u i v a l e n t weights of placenta, the yield of m e m b r a n e - b o u n d ribosomes is seen to be low, some 2 o ± 5 °/0 of the t o t a l ribosome p o p u l a t i o n being in this form. I t will also be noted t h a t most of the monosomes a n d disomes in the t o t a l polysome p o p u l a t i o n are derived from the free polysome fraction a n d are poorly represented in the b o u n d ribosome profile.
Incorporation o/ amino acids by placental polysomes The capacity of the p l a c e n t a l polysomes to incorporate labeled a m i n o acids in a n in vitro system with p l a c e n t a l p H 5 enzymes was examined. Fig. 8 shows t h a t incorporation after 60 rain i n c u b a t i o n was m a x i m a l when mixed polysomes prepared in the presence of 0.5 mM E D T A were used, a n d was m u c h reduced at either lower or higher c o n c e n t r a t i o n s of E D T A . This coincides with the o p t i m a l conditions for potysome recovery established earlier. The u p t a k e of a m i n o acids into i n d i v i d u a l polysome fractions was also examined. The total, free a n d b o u n d ribosome fractions were each i n c u b a t e d with co-factors a n d !14C]leucine a n d showed active u p t a k e into peptide E
0
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ol
~
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I000
0 0 EDTA (ram)
] 15
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Fig. 8. Effect of varying the EDTA concentration in the homogenizing medium on amino acid incorporation by the placental cell-free system. The standard incorporation mixture included 2oo pg rRNA from mixed placental polysomes, 1.5 mg pH 5 enzyme protein, 5°/~g tRNA, and an ATP regenerating system in a final volume of o. 5 ml medium. Incubation at 37° was stopped at 6o rain by addition of cold trichloroacetic acid. The concentrations of EDTA used in the homogenizing medium were o, 0.5, i.o, 1.5, 2, 5, and io raM. Fig. 9. Time course of [14C]leucineincorporation in a standard placental cell-free incorporation system using total, free and membrane-bound polysome preparations, rRNA concentration was o.25 nag and pH 5 enzyme protein concentration 1.5 mg/o. 5 ml final volume. Samples of the incubation mixture were obtained at 5, I5, 3°, 45 and 6o nun and the reactions stopped by addition of cold trichloroaeetic acid.
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bonds (Fig. 9). I t will be noted that the free polysomes are only active for 15 rain whereas the bound polysomes continue to incorporate for at least 6o rain. The optimum concentration of p H 5 fraction protein to obtain this incorporation was found to be 1-2 mg per 0.5 ml of the incubation mixture. Incorporation was proportional to addition of polysomes over the range O.l-O.3 mg per 0.5 ml incubation medium.
DISCUSSION
Techniques for the recovery of aggregated polysomes capable of in vitro incorporation of amino acids have been well established for the liver and some other major mammalian tissues. The human placenta presents difficulties because of its considerable content of fibrous tissue and of ribonuclease. It was found that the Sorvall Omni Mixer was most efficient in homogenizing this tissue. In order to inactivate alkaline ribonuclease in rat liver, addition of rat liver cell sap has been found to be effective 13,14, since the sap contains a ribonuclease inhibitor 15. However, we have found that the addition of rat liver cell sap to placental homogenates fails to prevent polysome disaggregation. Other methods of ribonuclease control have been proposed. These include addition of large amounts of substrate in the form of yeast RNA 1°, of heparin 17 and of bentonite TM. We have not observed any beneficial action of bentonite or of Triton X - I o o on polysome preservation in placental homogenates. EARL AND MORGAN6 found with cardiac muscle that inclusion of EDTA and NH4C1 in the homogenizing medium reduced nuclease activity and yielded polysomes that incorporated amino acids into protein more effectively in a cell-free system. MATTHAEI AND S C H O E C H 5 have observed that placental ribosomes prepared in the presence of E D T A incorporate amino acids more actively. Our data confirm this observation (Fig. 8) and show that the phenomenon is due to better preservation of polysomes harvested in the presence of 0.5 mM EDTA. The action of this chelating agent on alkaline ribonuclease has long been known. ELLEM AND COLTER19examined its action on the ribonucleases of mouse liver and kidney and found in homogenates prepared from both organs that o.i mM E D T A completely inhibits the activity of alkaline ribonuclease, with little or no action on acid ribonuclease. In our hands, EDTA has proved the most effective agent for inhibiting polysome degradation in placental tissue. Since it also binds some of the Mg 2+ in the isolation medium, the Mg 2+ content of the medium was increased to IO mM, which was demonstrated to provide optimal levels (Fig. 7).
ACKNOWLEDGMENTS
We are grateful to Dr. S. Driscoll and the nursing staff of The Boston Hospital for Women (Lying-In Division), Boston, Mass., for arranging the collection of placentas. Miss K. Sargent kindly prepared the electron micrographs of placental polysomes shown in Fig. 2. The investigation was supported by Grant No. H D o469O-Ol b y the U. S. Public Health Service. Biochim. Biophys. Acta, 213 (197 o) 3 9 1 - 4 o o
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REFERENCES i M. M. GRUMBACH, S. L. I~APLAN, J. J. SCIARNA AND I. M. BURR, Ann. N. Y. Acad. Sci., 148 (1968) 5Ol. 2 C. M. REDMAN, Biochem. Biophys. Res. Commun., 31 (1968) 845. 3 M. TAKAGI AND K. OGATA, Biochem. Biophys. Res. Commun., 33 (1968) 55. 4 S. J. HICKS, J. W. DRYSDALE AND 1~. N. MUNRO, Science, 164 (1969) 584 . 5 J. H. MATTHAEI AND G. SCHOECH, Biochem. Biophys. Res. Commun., 27 (1967) 638. 6 D. C. N. EARL AND H. E. MORGAN, Arch. Biochem. Biophys., 128 (I968) 460. 7 F. O. WETTSTEIN, T. STAEHELIN AND H. NOLL, Nature, 197 (1963) 43 o. 8 W. H. \V[INNER, J. BELL AND H. N. MUNRO, Biochem. J., IOI (1966) 417 . 9 B. S. BALIGA, A. x/V. PRONCZUK AND H. N. MUNRO, dr. Mol. Biol., 34 (1968) 199. IO O. FI. LOWRY, N. J. ROSEBROUGH, A. I,. FARR AND R. J. I~ANDALL,dr. Biol. Chem., 193 (1951) Io 5. I I A. FLECK AND H. N. MUNRO, Biochim. Biophys. Acta, 55 (1962) 571. 12 G. BLOBEL AND V. R. POTTER, J. Mol. Biol., 26 (1967) 279. 13 G. BLOBEL AND V. R. POTTER, ]. Mol. Biol., 28 (1967) 539. 14 G. R. LAWFORD, P. LANGFORD AND H. SCHACHTER, J. Biol. Chem., 241 (1966) 1835. 15 J. S. ROTH, dr . Biol. Chem., 231 (1958 ) lO85 . 16 A. E. V. HASCHEMEYER AND J. GRoss, Biochim. Biophys. Acta, 145 (1967) 76. 17 P. T. RO~A~LEYAND J, MORRIS, Exptl. Cell Res., 45 (1966) 494. 18 R. L. V~rATTS AND A. P. MATHIAS, Bioehim. Biophys. Acta, 145 (1967) 828. 19 K. A. O. ELLE~,t AND J. S. COLTER, J. Cellular Comp. Physiol., 58 (1963) 267. 20 G. BLOBEL AND V. R. POTTER, J. Mol. Biol., 26 (1967) 293.
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BBA 96561
ISOLATION AND P R O P E R T I E S OF POLYSOMES FROM HUMAN PLACENTA E. M. L A G A , B. S. B A L I G A AND H. N. M U N R O
Physiological Chemistry Laboratories, Department o/ Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass., o2139 (U.S.,4.) (Received M a r c h 6th, 197 o)
SUMMARY
Conditions have been investigated for obtaining free and membrane-bound polysomes from human placenta that can incorporate free amino acids into peptide bonds in a cell-free protein-synthesizing system. Optimum recoveries of polysomes were obtained when ribonuclease activity was inhibited by addition of 0. 5 mM EDTA to the homogenization medium, along with I5O mM KC1, 200 mM NH4C1 and IO mM MgCI~. Conditions of homogenization and centrifugation to provide optimum yields of free and bound polysomes were also established. About 20 % of the polysomes recovered from human placentas were membrane-bound. They were more active than free polysomes in an amino acid incorporating system.
INTRODUCTION
The human placenta secretes protein hormones in considerable amounts into the maternal circulation. For example, chorionic growth hormone-prolactin is secreted at a mean rate of I g per day during the ninth month of pregnancy 1. In the case of the liver, it has been demonstrated that secreted proteins are manufactured by polysomes attached to membranes of the endoplasmic reticulum 2,z, whereas ferritin, a typical retained protein, is formed on free ribosomes not associated with these membranes 4. In order to study the mechanism of synthesis and secretion of protein by the human placenta, it has been necessary to establish conditions for the preparation of both membrane-bound and free polysomes from placenta that will actively incorporate free amino acids into peptides. Cell-free placental systems capable of in vitro amino acid incorporation have been described previously5, but optimal conditions for preparation of free and membrane-bound polysomes from this organ have not been established. Procedures applicable to liver have had to be modified because of the more fibrous nature of the placenta and the presence of an active alkaline ribonuclease. The present investigation establishes optimal conditions for this purpose. METHODS
Materials and Media
Dipotassium EDTA was purchased from Eastman Organic Chemicals, 2mercaptoethanol from Calbiochem and sodium deoxycholate from Mann Research Biochim. Biophys. Acta, 213 (197 o) 391-4oo
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Laboratories, and pancreatic ribonuclease from Nutritional Biochemicals. Guanosine 5-triphosphate trilithium.6 H~O, adenosine 5-triphosphate disodium. 4 H~O, glutathione (reduced, creatine phosphate and creatine phosphokinase were obtained from Calbiochem. The uniformly 14C-labeled L-amino acid mixture (specific activity 5 ° mC/33.6 rag) and uniformly 14C-labeled L-leucine (specific activity 250 mC/mmole) were obtained from New England Nuclear Corporation. Other chemicals were reagent grade. Most of the reagents were made up in 50 mM Tris (pH 7.6)-2o0 mM NH4C115o mM K C I - I o mM MgC12 buffer. The 50 mM Tris (pH 7.6)-15o mM K C I - I o mM MgC12 buffer was modified b y addition of NH4C1 because EARL AND MORGAN6 have found that recovery of polysomes from cardiac muscle is improved by its presence.
Preparation of C-ribosomes, /ree and bound polysomes Fresh placentas (300-600 g) from normal deliveries were rapidly immersed in ice cold modified Tris-KC1-MgCI 2 buffer, transported in an ice-filled polystyrene box and processed within o.5 h after delivery. The placenta was perfused with 1-2 1 of cold Tris-KC1-MgC12 buffer to remove the red blood cells. In the standard procedure finally developed, about 5 g amounts of perfused placental tissue were minced and homogenized with 5 ml of a medium containing 0.25 M sucrose and 0. 5 mM EDTA in Tris-NH4C1-KC1-MgC12 buffer (pH 7.6), using the Sorvall Omni Mixer at 4 ° for I rain at speed setting 7- Cell debris, nuclei and mitochondria were removed by centrifuging the homogenate in the Sorvall RC 2-B centrifuge for 20 min at 12 ooo rev./ rain in the SS-34 rotor. The post-mitochondrial supernatant was filtered through glass wool. The C-ribosome fraction of WETTSTEIN et al. 7 was prepared by layering 5 ml of post-mitochondrial supernatant adjusted to i ~o with deoxycholate on top of 5 ml of 2.0 M sucrose in Tris-NH4C1-KC1-MgC1~buffer containing 0.5 mM EDTA. The free polysomes were prepared by the same procedure but omitting deoxycholate treatment. These two sets of polysomes were harvested by centrifuging in the Spinco L-2 ultracentrifuge at 4 ° for 6 h at 40 ooo rev./min (lO5 ooo ×g) in the Ti-5 o rotor. The pellets were washed twice with cold Tris-NH~C1-KC1-MgC12 buffer, suspended in 0.25 ml of the same buffer, and stored at --4o °. The membrane-bound polysomes were prepared b y collecting the intermediate turbid layer above the interface between the post-mitochondrial supernatant and the sucrose in the tubes used for preparation of the free polysome fraction. This sample was adjusted to i °/o with deoxycholate and layered onto 2.0 M sucrose in Tris-NH4C1-KC1-MgC1 ~ buffer containing o.5 mM E D T A and the solubilized polysomes were harvested as described above.
Sucrose density gradient o[ polysomes The standard polysome patterns were obtained from pelleted total ribosomes, free ribosome and membrane-bound ribosome preparations b y layering a 2o-/,1 sample of dissolved ribosome pellet, diluted with 0.2 ml Tris-NH4C1-KC1-MgClz buffer containing 0.5 mM EDTA on top of a lO-5O ~o linear sucrose gradient made up in the Tris-KC1-MgC12 buffer. The gradients were centrifuged in the Spinco L-2 centrifuge equipped with a SW-5 o rotor at 4 ° for 80 rain at 4 ° ooo rev./min (lO5 ooo ×g). The distribution of polysomal aggregates in the gradient was determined b y puncturing the tube at the bottom and b y passing the effluent of the tube through a o.o25-ml Gilford flow-cell with a 2 m m light path using a Gilford 2000 recording spectrophotometer. The flow rate was kept constant by a Buchler flow p u m p and Biochim. Biophys. -4cta. 213 (197 o) 391-3oo
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the absorption of the effluent at 260 nm was recorded on a moving chart. The proportion of the total profile represented by monosomes and by polysomes (disomes and higher aggregates) was obtained by cutting out each area of the chart paper and weighing it.
Preparation o/cell sap/actors/rom placenta The pH 5 enzyme fraction was obtained by omitting deoxycholate treatment of the post-mitochondrial supernatant but adding 5 mM 2-mercaptoethanol. This post-mitochondrial supernatant was immediately centrifuged in the Spinco L-2 at 4 ° for 4 h in a Ti-5o rotor at 4 ° ooo rev./min (lO5 ooo ×g) to remove microsomes and free ribosomes 8. To remove factors inhibiting amino acid incorporation, the cell sap obtained as a clear supernatant after centrifugation was passed through a column of Sephadex G-25 that had been equilibrated with Tris-KC1-MgCI~ buffer containing 5 mM 2-mercaptoethanol at pH 7.6 (ref. 9). The pH 5 precipitate was obtained by slow addition of I M acetic acid at o ° and removed by centrifugation in a Sorvall RC-2B centrifuge at 4 ° for 15 min in the SS-34 rotor at 13 ooo rev./min (20 ooo ×g). The pH 5 enzyme protein was dissolved in 2 ml 50 mM Tris (pH 7.6)-1 mM E D T A I mM reduced glutathione buffer, and stored at --4 o°.
Cell-/ree amino acid incorporation system The reaction mixture contained 5o mM "Iris, 5 mM MgCI~, 8o mM NH4C1, 2 mM ATP, I mM GTP, 8 mM GSH, 0.02 mM [14C]leucine (0.5 #C), 1. 5 mg placental pH 5 enzyme protein and 200-500 #g placental polysome RNA in a volume of 0. 5 ml incubation mixture. In one series of experiments (see Fig. 8), an ATP regenerating system (IO #moles creatine phosphate and IOO/~g creatine phosphokinase per tube) was added to the incubation mixture. Following incubation at 37 ° for 30 min, the reaction was stopped by addition of an equal amount of Io% (w/v) trichloroacetic acid solution. The solution was then heated to 9 °0 for 15 min and the insoluble protein was collected on a glass fiber filter, washed with 5 % trichloroacetic acid and dried before counting the radioactivity. Blank incubations were run without polysomes and the incorporation by these was subtracted from the standard incubation.
Protein and RNA determinations Protein was measured by the method of LOWRYet al. 1°. rRNA was determined by measuring the absorbance at 260 nm and correcting for protein present using the formula of FLECK AND MUNRO 11. RESULTS
Recovery o[ polysomes ]tom placenta Attempts to prepare polysomes (C-ribosomes) by techniques used to give liver polysomes failed to yield more than monosomes and small oligosomes. MATTHAEI AND SCHOECH5 used EDTA in the homogenizing medium to obtain active ribosome populations for protein synthesis. We therefore explored the recovery of polysomes using different concentrations of EDTA in the homogenizing medium. Fig. I shows that increasing concentrations of EDTA up to 0. 5 mM greatly increased polysome recovery, but that higher levels of this chelator were less efficient. These findings were Biochim. Biophys. Acta, 213 (197 o) 391-4oo
E.M. LAGA et al
394 I OC Top~ OmM 10 0 ImM EDTA A
O50
O25
LO0 Top~ 075
fi O
I-
OmM to OI mM EOTA |
/3
t
~
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EDTA
f L
050
J
025
I00
075
Top ~ OmM to OImM EOTA
~lmM
to 5ram
EDTA
C
J
050
025
Fig. i. Effect of v a r y i n g E D T A concentration in the h o m o g e n i z i n g medium on the sucrose gradient profiles of total (A), free (]3) and m e m b r a n e - b o u n d (C) p o l y s o m e preparations obtained from equivalent a m o u n t s of h u m a n placenta. The E D T A concentrations used were o, o.I, 0.25, 0.5, I, and 5 raM.
similar for mixed polysome populations (Fig. IA), free polysomes (Fig. IB) and membrane-bound polysomes (Fig. IC). As shown in the diagram, there was better recovery of polysomes from the bound than from the free population at low levels of EDTA, presumably due to stabilization of the polysomes through attachment to the membranes. The action of o. 5 mM EDTA in preserving polysomes was confirmed by electron microscopy of the C-ribosome pellet. Fig. 2B shows that in the presence of o.5 mM EDTA the preparations exhibit much more of polysome aggregates than in the absence of this chelating agent (Fig. 2A). Consequently, o.5 mM EDTA was used in the subsequent
studies.
Recovery o] /ree and membrane-bound polysomes BLOBEL AND POTTER12,13,z° have described techniques for preparing free and membrane-bound polysome populations separately from the liver. These techniques were applied in slightly modified form to human placenta. The post-mitochondrial supernatant was applied directly over 2.o M buffered sucrose containing 0. 5 mM EDTA and ribosomes free of membrane were sedimented through this layer. The lower half of the supernatant fluid was then made I °/o with deoxycholate and this was then spun down through 2.0 M buffered sucrose containing EDTA to yield a pellet of membrane-bound ribosomes. Another portion of the post-mitochondrial supernatant l?,iochim. Biophys. Acta, 213 (197 o) 3 9 1 - 4 o o
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Fig. 2. Electron micrographs of polysomes prepared from human placenta without EDTA (A), or with o. 5 mM EDTA (B) in the homogenizing medium and negatively stained with uranyl acetate ( × 50 ooo).
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was made 1 % with deoxycholate and spun immediately through 2 M sucrose with E D T A to provide the total ribosome population. BLOBEL AND POTTER12,13,2° have emphasized that the recovery of polysomes in all fractions from rat liver can be improved b y adding extra rat liver cell sap to the homogenizing medium. However, we have not found that rat liver cell sap has this protective effect when used in place of E D T A in the homogenizing medium to harvest placental polysomes. Various factors in obtaining m a x i m u m yields of these different populations of ribosomes were investigated by measurement of RNA recovered in the pellets. Fig. 3 shows the effect of varying the ratio of tissue to homogenizing medium; a ratio of I g tissue to I ml fluid gave maximal recoveries of both free and bound ribosomes, most notably in the case of the free ribosomes. Next, the loss of bound and free polysomes into the mitochondrial and cell debris fraction was investigated. Fig. 4 shows that recovery of bound ribosomes is very much dependent on this step,
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Fig. 3. E f f e c t of ratio of a m o u n t of p l a c e n t a l tissue (g) to a m o u n t of h o m o g e n i z a t i o n m e d i u m (ml) on yield of r R N A in free a n d m e m b r a n e - b o u n d p o l y s o m e pellets. H o m o g e n i z a t i o n w a s p e r f o r m e d in t h e Sorvall O m n i Mixer r a t s p e e d s e t t i n g 7 for I min, u s i n g a Tris-NH4C1-KC1-MgC12 buffer w i t h a d d i t i o n of 0.25 M sucose a n d 0. 5 m M E D T A . T h e pellets of free a n d m e m b r a n e b o u n d r i b o s o m e s were o b t a i n e d b y c e n t r i f u g a t i o n of t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t . T h e pellets were dissolved in Tris-KC1-MgC12 b u f f e r a n d t h e i r a b s o r b a n c e w a s m e a s u r e d a t 26o n m . Fig. 4- E f f e c t of v a r y i n g speed of c e n t r i f u g a t i o n of p l a c e n t a l h o m o g e n a t e on yield of r R b l A in free a n d m e m b r a n e - b o u n d p o l y s o m e pellets. C e n t r i f u g a t i o n w a s p e r f o r m e d in t h e Sorvall R C 2 - B c e n t r i f u g e a t 4 ° for 20 m i n w i t h t h e SS-34 rotor a t speeds f r o m 12 to 17 ooo r e v . / m i n . Free a n d m e m b r a n e - b o u n d p o l y s o m e pellets were p r e p a r e d f r o m t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t b y c e n t r i f u g a t i o n , r R N A in t h e dissolved pellet w a s m e a s u r e d a t 260 n m .
presumably because polysomes attached to large membraneous masses of endoplasmic reticulum are precipitated even at low speeds. Consequently, the optimal speed chosen was 12 ooo rev./min on the Sorvall RC 2-B for 20 rain using SS-34 rotor. Next, the time course of sedimenting ribosomes through 2.o M sucrose from the postmitochondrial supernatant fraction was examined. Fig. 5 demonstrates that at least 5 h of spinning is needed to sediment the free polysomes without leaving some behind at the interface in among the membrane-bound ribosomes. The effect of ions in the homogenizing medium was next investigated as a factor in polysome recovery, using both total rRNA recoveries and gradient profiles to evaluate the effects produced. Fig. 6 shows that increasing the concentration of KC1 in the medium affects the recovery of free and bound ribosomes. Recovery of Biochim. Biophys. Acta, 213 (197 o) 391-4oo
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397
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Fig. 5. Effect of v a r y i n g the d u r a t i o n of centrifugation of p o s t - m i t o c h o n d r i a l s u p e r n a t a n t t h r o u g h 2.o M buffered sucrose on the yield of r R N A . Free ribosome pellets were h a r v e s t e d after I, 2, 3, 4, 5, 6, 7 and 8 h of centrifugation in the Spinco L-2 at 4 ° in the Ti-5o r o t o r at lO5 ooo ×g. At every h, t u b e s were removed; the pellet of free ribosomes at the b o t t o m of the t u b e was harvested and the interface material was used for p r e p a r a t i o n of m e m b r a n e - b o u n d polysomes. r R N A in each fraction was m e a s u r e d at 260 nm. Fig. 6. Effect of v a r y i n g concentration of KCI in the homogenization m e d i u m on the yield ot r R N A and on the p o l y s o m e / m o n o s o m e ratios of the free and m e m b r a n e - b o u n d ribosomes. The KCI concentration ranged from o to 60o raM. r R N A in the dissolved pellets was m e a s u r e d at 26o nm. The p o l y s o m e / m o n o s o m e ratios were c o m p u t e d from s t a n d a r d polysome profiles obtained after lO-5O % continuous sucrose gradient centrifugation.
free ribosomes increases up to 5O-lOO mM KC1. Above 3oo mM, the recovery of bound ribosomes falls off and the free population rises sharply. Presumably this last effect represents detachment of ribosomes from the membrane at the high salt concentra-
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l
0 1.5
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o
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Fig. 7. Effect of varying MgC12 and E D T A concentration in the homogenization medium on the yield of rIRNA from the free and m e m b r a n e - b o u n d ribosomes, r R N A in the pellets was m e a s u r e d at 260 nm. The concentration of Mg ~+ in the homogenization buffer was 2, io, and 15 mM in the presence of respectively o, 0. 5, I.O, 1.5 and 2.0 mlV[ E D T A .
Biochim. Biophys. Acta, 213 (197 o) 391-4oo
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398
tion. E x a m i n a t i o n of the polysome profiles at these various c o n c e n t r a t i o n s shows t h a t high salt levels lead to disaggregation, as shown b y the fall in polysome/monosome ratio. I n the light of these experiments, 15o mM KC1 provides m a x i m a l recovery of ribosomes a n d m a x i m a l polysome/monosome ratio in b o t h populations of ribosomes. Fig. 7 shows the effects of v a r y i n g both Mg 2+ a n d E D T A c o n t e n t of the homogenizing i n e d i u m on recovery of free a n d b o u n d ribosomes. The recovery of free ribosomes is m a x i m a l in Io mM Mg ~+ at 0.5 n a n E D T A , whereas better recoveries of b o u n d polysomes are o b t a i n e d with 15 mM Mg z+ a n d o.5 mM E D T A . As a compromise the Io mM Mg 2+ was chosen for isolation of b o t h populations. Fig. IA, I B a n d IC show the profiles of total, free a n d m e m b r a n e - b o u n d polysomes prepared from h u m a n placenta. W h e n prepared u n d e r o p t i m a l conditions (o.5 mM E D T A ) from e q u i v a l e n t weights of placenta, the yield of m e m b r a n e - b o u n d ribosomes is seen to be low, some 2 o ± 5 °/0 of the t o t a l ribosome p o p u l a t i o n being in this form. I t will also be noted t h a t most of the monosomes a n d disomes in the t o t a l polysome p o p u l a t i o n are derived from the free polysome fraction a n d are poorly represented in the b o u n d ribosome profile.
Incorporation o/ amino acids by placental polysomes The capacity of the p l a c e n t a l polysomes to incorporate labeled a m i n o acids in a n in vitro system with p l a c e n t a l p H 5 enzymes was examined. Fig. 8 shows t h a t incorporation after 60 rain i n c u b a t i o n was m a x i m a l when mixed polysomes prepared in the presence of 0.5 mM E D T A were used, a n d was m u c h reduced at either lower or higher c o n c e n t r a t i o n s of E D T A . This coincides with the o p t i m a l conditions for potysome recovery established earlier. The u p t a k e of a m i n o acids into i n d i v i d u a l polysome fractions was also examined. The total, free a n d b o u n d ribosome fractions were each i n c u b a t e d with co-factors a n d !14C]leucine a n d showed active u p t a k e into peptide E
0
~u
ol
~
o0o o: I ~oOYA'
2000
I000
0 0 EDTA (ram)
] 15
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__
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Fig. 8. Effect of varying the EDTA concentration in the homogenizing medium on amino acid incorporation by the placental cell-free system. The standard incorporation mixture included 2oo pg rRNA from mixed placental polysomes, 1.5 mg pH 5 enzyme protein, 5°/~g tRNA, and an ATP regenerating system in a final volume of o. 5 ml medium. Incubation at 37° was stopped at 6o rain by addition of cold trichloroacetic acid. The concentrations of EDTA used in the homogenizing medium were o, 0.5, i.o, 1.5, 2, 5, and io raM. Fig. 9. Time course of [14C]leucineincorporation in a standard placental cell-free incorporation system using total, free and membrane-bound polysome preparations, rRNA concentration was o.25 nag and pH 5 enzyme protein concentration 1.5 mg/o. 5 ml final volume. Samples of the incubation mixture were obtained at 5, I5, 3°, 45 and 6o nun and the reactions stopped by addition of cold trichloroaeetic acid.
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bonds (Fig. 9). I t will be noted that the free polysomes are only active for 15 rain whereas the bound polysomes continue to incorporate for at least 6o rain. The optimum concentration of p H 5 fraction protein to obtain this incorporation was found to be 1-2 mg per 0.5 ml of the incubation mixture. Incorporation was proportional to addition of polysomes over the range O.l-O.3 mg per 0.5 ml incubation medium.
DISCUSSION
Techniques for the recovery of aggregated polysomes capable of in vitro incorporation of amino acids have been well established for the liver and some other major mammalian tissues. The human placenta presents difficulties because of its considerable content of fibrous tissue and of ribonuclease. It was found that the Sorvall Omni Mixer was most efficient in homogenizing this tissue. In order to inactivate alkaline ribonuclease in rat liver, addition of rat liver cell sap has been found to be effective 13,14, since the sap contains a ribonuclease inhibitor 15. However, we have found that the addition of rat liver cell sap to placental homogenates fails to prevent polysome disaggregation. Other methods of ribonuclease control have been proposed. These include addition of large amounts of substrate in the form of yeast RNA 1°, of heparin 17 and of bentonite TM. We have not observed any beneficial action of bentonite or of Triton X - I o o on polysome preservation in placental homogenates. EARL AND MORGAN6 found with cardiac muscle that inclusion of EDTA and NH4C1 in the homogenizing medium reduced nuclease activity and yielded polysomes that incorporated amino acids into protein more effectively in a cell-free system. MATTHAEI AND S C H O E C H 5 have observed that placental ribosomes prepared in the presence of E D T A incorporate amino acids more actively. Our data confirm this observation (Fig. 8) and show that the phenomenon is due to better preservation of polysomes harvested in the presence of 0.5 mM EDTA. The action of this chelating agent on alkaline ribonuclease has long been known. ELLEM AND COLTER19examined its action on the ribonucleases of mouse liver and kidney and found in homogenates prepared from both organs that o.i mM E D T A completely inhibits the activity of alkaline ribonuclease, with little or no action on acid ribonuclease. In our hands, EDTA has proved the most effective agent for inhibiting polysome degradation in placental tissue. Since it also binds some of the Mg 2+ in the isolation medium, the Mg 2+ content of the medium was increased to IO mM, which was demonstrated to provide optimal levels (Fig. 7).
ACKNOWLEDGMENTS
We are grateful to Dr. S. Driscoll and the nursing staff of The Boston Hospital for Women (Lying-In Division), Boston, Mass., for arranging the collection of placentas. Miss K. Sargent kindly prepared the electron micrographs of placental polysomes shown in Fig. 2. The investigation was supported by Grant No. H D o469O-Ol b y the U. S. Public Health Service. Biochim. Biophys. Acta, 213 (197 o) 3 9 1 - 4 o o
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