Splenic Protein Synthesis in Magnesium Mechanism oÃ-the Inhibition
Deficiency:
ABSTRACT To investigate the basis for the depressed protein synthesis in vivo in magnesium deficient spleens, the activities of splenic subcellular fractions in polypeptide synthesis were studied in vitro. Splenic ribosomes from Mg deficient animals were normal structurally and functionally. In contrast, supernatant fractions from the deficient spleens had a reduced ability to incorporate labeled amino acids into protein, both in the presence of endogenous mRNA and in the presence of added polyuridylic acid. The specific defects observed in the Mg deficient supernatants were twofold: There was a modest reduction in the rate of acylation of tRNA and a more marked reduction in the activity of the elongation factors, EF-I and EF-II. The reduction in elongation factor activity was quantitatively sufficient to account for the inhibition of protein synthesis in vivo. J. Nutr. 108: 1635-1641, 1978. INDEXING KEY WORDS magnesium •deficiency • protein • synthesis •ribosome •spleen In a recent study of the effect of mag nesium deficiency on protein and nucleic acid synthesis in vivo, we found the in corporation of [3H]leucine into tissue pro teins reduced by 40% to 50% in spleen and thymus, but not altered in liver (1). The spleen was also distinctive in showing a marked increase ( 350% ) in DN A synthesis. Because of this unique susceptibility of the spleen to the effects of Mg deficiency in vivo, we decided to study the mechanism of the decreased splenic protein synthesis. In an effort to determine which step or steps in protein synthesis were inhibited in Mg deficiency spleens, we studied the activ ities of splenic subcellular fractions in poly peptide synthesis in vitro.
normal and magnesium deficient diets have been described previously (1) except that only 20 mg magnesium was added per kg diet to give the magnesium deficient diet. Measurements of the magnesium content of the diet after mixing varied from 25 to 35 ng/g diet. This diet produced severe Mg deficiency in 120 g rats within 1 to 2 months. The studies of this report were per formed in rats that had consumed this diet for 6 to 8 weeks. At this time spleens were enlarged and bone magnesium, an index of body magnesium depletion, was less than 15% of normal ( 1 ). The controls were pairfed as previously described ( 1 ). ATP,5 GTP,5 polyuridylic acid,5 E. coli tRNA,6 sucrose (density gradient, ribo-
METHODS Materials. Male Sprague-Dawley ratsa were obtained at an average weight of 90 to 100 grams and were fed a stock diet* until they reached approximately 120 g, at which point they were fed the experimental diet. The components of the experimental
Recelved for publication August 17, 1977. 1 Present address : Dept. of Medicine, Richmond Veterans Hospital, Medical College of Virginia. Rich mond, Virginia. 2 Present addressj Hennepln County Medical Center. "Holtzman Rat Co., Madison, Wisconsin. 4 Purinn Rat Chow. 5 P-L Biochemlcals, Milwaukee. Wisconsin. 8 Schwarz/Mann, Orangeburg, New York.
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KENNETH A. FREUDE,1 FRANKLIN J. ZIEVE 1 ANDLESLIE ZIEVE 2 Department of Medicine, Minneapolis Veterans Hospital, University of Minnesota
1636
FREUDE,
ZIEVE AND ZIEVE
nuclease free ) ,6 radioactive amino acids 7 and L-phenylalanyl-tRN A [phenylalanyl14C] ~ were obtained from the indicated
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tion of tRNA by spleen supernatant frac tions was determined by incubating 1 to 3 ¿ig of supernatant protein in a reaction mix ture (250 /xl) containing 50 HIM Tris-Cl commercial sources. Preparation of extracts. Normal or Mg (pH 7.0), 30 mM MgAc2, 40 HIM KCl, 1 mM EDTA, 5 mM ATP, 50 /¿gtRNA, and deficient rats were killed by decapitation, [14C]amino acid (specific activity > and their spleens were removed, minced 40 /»M and homogenized (glass-teflon homogenizer) 200 mCi/mmole ). After the indicated incu in four volumes of medium A (0.01 M Tris bation time at 37°,the labeled product was pH 8.0, 0.1 M KCl, 0.01 M MgAc2, 0.25 M isolated by the method of Mans and Novelli sucrose, 1.6 mg/ml bentonite). The homog(3). Preparation of washed liver ribosomes. enate was centrifuged 15 minutes at 10,000 Ribosomes from rat liver which had little X g, and the supernatant fraction was col lected and recentrifuged at 20,000 X g for or no transfer factor activity were prepared 15 minutes. In some experiments, the 20,000 as follows. Rat lived was minced and ho X g supernatant was diluted with 1.5 vol mogenized in 4 volumes (w/v) of medium of medium B (identical to medium A ex A (see supernatant preparation) and the cept that it contained 0.9 M sucrose) and homogenate centrifuged at 10,000 X g for 15 minutes. The lipid layer was aspirated centrifuged at 105,000 X g for 120 minutes. and the supernatant fraction recentrifuged Amino acid incorporation assays. Incor at 20,000 X g for 15 minutes. The lipid poration of labeled amino acids into protein was measured by two different assays, one layer was again carefully removed by aspi ration and the supernatant collected and utilizing endogenous mRNA, the other uti lizing the exogenous messenger, polyuri- diluted with 1.5 volumes of medium B ( see supernatant preparation). The diluted sudylic acid. The endogenous messenger as say was that of Wust (2). Each reaction pernate was centrifuged at 35,000 X g for mixture (100 p.1) contained 100 HIM Tris- 20 minutes. The resulting supernatant frac acetate (pH 8.0), 20 mM MgAc2, 50 mM tion was made 0.65% in deoxycholate by KCl, 0.8 mM GTP, 0.6 HIM ATP, 20 mM the addition of 5% deoxycholate in water, and then centrifuged for 90 minutes at creatine phosphate, 50 /tg/ml creatine kinase, 5 juM [14C]leucine, 30 to 50 jug/ml 105,000 x g. The resulting pellets were rinsed with medium A lacking bentonite, rat liver ribosomes, and spleen supernatant ( 0.2 to 0.5 mg protein ). Incubation was for suspended in medium A (l/25th the vol 10 minutes at 37°,and the labeled product of the deoxycholate-treated supernatant) was isolated by the method of Mans and by gentle homogenization, and then clari fied with a 10-minute centrifugation at Novelli (3). 35,000 X g. The clarified suspension was The synthesis of [14C]polyphenylalanine centrifuged at 140,000 X g for 120 min from [14C]phenylalanyl-tRNA in the pres utes. The pellets were stored frozen ( —20°) ence of the exogenous messenger, polyovernight, thawed and resuspended in uridylic acid, was carried out by the washing buffer containing 50 HIM Tris-Cl, method of Selawry and Starr (4). pH 7.4; 500 mM KCl; 20 HIMmercaptoethaAssay for elongation factor activities. Activities of the elongation factors EF-I nol. This suspension was gently mixed on and EF-II were measured by the method ice for 20 minutes, after which the ribo of Willis and Starr ( 5 ). The assay mixtures somes were collected by centrifugation at 140,000 X g for 90 minutes. The ribosomes (100 ¿ti)contained 10 mM Tris-HCl (pH at this stage were essentially free of trans 7.2), 9 mM MgAc2, 60 HIM KCl, 2 mM fer factors and were stored as a frozen sus dithiothreitol, 0.2 HIM GTP, 90-100 ¿xg pension in 50 mM Tris-Cl, pH 7.4; 4 mM washed liver ribosomes, and purified elon gation factors as indicated. Purified EF-I MgCL; 350 mM sucrose. Preparation of ribosomes for sucrose and EF-II were prepared as described by density analysis. The procedure is the same Honjo et al. (6). Assay of supernatant fractions for aminoacyl-tRNA ligase activity. The aminoacyla7 New England Nuclear, Boston, Massachusetts.
PROTEIN
SYNTHESIS
IN MAGNESIUM
1637
DEFICIENCY
as for washed liver ribosomes except the washing buffer was replaced with 10 HIM Tris-acétate,pH 8.0; 50 IHM KCl; and 10 niM magnesium acetate. Sucrose gradient anahjsis of ribosomes. Sucrose density profiles were run in 50 HIM Tris-Cl, pH 7.8; 25 HIMKCl; 5 HIM MgCl2; containing a 10% to 34% linear gradient of sucrose. Approximately 100 to 200 /xg of ribosomal RNA were analyzed per gradient. The profiles were developed using a SW-40 rotor8 at 27,500 rpm for 120 minutes. Statistical analysis. Standard procedures were used for calculating means, standard deviations, standard error of means ( SEM), and tests of significance of differences be tween means (7).
TABLE 1 Protein synthesis by ribosomes from normal and Mg deficient spleens in the presence of normal liver supernatant
RESULTS Ribosomes. We first attempted to deter mine whether the defect in spleen protein synthesis resided in the ribosomes or in the supernatant fraction. To ascertain whether
ribosomal structure was grossly altered, we examined the sedimentation profiles of polysomes in extracts from normal and Mg deficient spleens. As shown in figure 1, the polysomal profiles were identical in the con trol and deficient rats. This finding indi cated that no gross disruption of ribosomal or polysomal structure had occurred. Very little low molecular weight material was present, as shown by the position of the 80S ribosomal marker; this showed that ex tensive polysomal breakdown during the preparation had not taken place. The functional activity of spleen ribo somes was tested by measuring their ability to support protein synthesis in the presence of a ribosome-free supernatant fraction from normal liver. Two different assays were used, one of which measured the in corporation of [14C]leucine into protein utilizing endogenous mRNA, the other of which measured the incorporation of [14C]phenylalanine in the presence of added poly U. As shown in table 1, neither assay revealed any difference between control ribosomes and Mg deficient ribosomes. We conclude that, within the limits of sensitiv ity of these measurements, the functional capacity of spleen ribosomes was unaffected by Mg deficiency. Supernatants. Since it appeared from the above studies that the decreased protein synthesis observed in vivo in Mg deficient rat spleen was not due to a defect in ribo somes, we examined the activity of splenic
Fig. 1 Sedimentation profiles of spleen polysomes from control and magnesium deficient (MgD) rats. Extracts were prepared and centrifuged as described under Methods. Fractions (300 /¿])were collected by displacement from the top of the tube using a Hoefer density gradient fractionator. The direction of sedimentation is from left to right, and the arrow represents the position of an 80S ribosomal marker. Each extract was from the pooled spleens of four rats. Similar profiles were obtained from at least three other preparations of normal and Mg deficient spleen.
.Source of ribosomesNormal
messenger U assay169±19assay3,499
spleens ±382 Mg deficient spleensEndogenous 155±22Poly3,399 ±347 1Results are expressed as cpm/10 /ig ribosomal RNA. 2 Each value listed represents the mean ±SD for three separate preparations of spleen ribosomes.
8Becknian Instruments, Fullerton, California.
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Bottom
["CjAmino acid incorporation1'2
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FREUDE, ZIEVE AND ZIEVE TABLE 2
Protein synthesis by supernatant fractions from norma and Mg deficient spleens in the presence of normal liver ribosomes
Xg
ratsControl of
incorporation1'2832±
150
Normal
21 61,957±147 330±
deficientControl Mg 105,000 X gTreatment Mg deficient["C]Leucine 974±65 1Results are expressed as cpm/mg supernatant protein. *Each value represents the mean±SD for two separate preparations of spleen supernatant.
100
q
q 3^ c
MgD I
supernatant fractions. The experimental design was analogous to the ribosome ex periments described above. Spleen super natant fractions were tested for their ability to incorporate [UC] leucine into protein in the presence of normal rat liver ribosomes. Table 2 shows the activities of the 20,000 X g and 105,000 x g supernatant fractions from normal and Mg deficient spleens. In both preparations the activity of the Mg deficient supernatants was approximately one-half that of the controls. This decrease was comparable to that seen in the in vivo leucine incorporation studies reported pre viously (1). Thus it appeared that the factors responsible for diminished splenic protein synthesis in Mg deficiency were to be sought in the postmicrosomal superna tant fraction. The 105,000 X g supernatant fraction contains several components that are neces sary for protein synthesis including amino acyl-tRNA ligases, tRNA, mRNA, and elongation factors. Activities of aminoacyltRNA ligases were assessed by measuring the acylation of rat liver tRNA with [14C]phenylalanine or [14C]leucine in the pres ence of spleen supernatant fractions. The ligase activities were measured under con ditions of saturating substrate. A typical experiment is shown in figure 2, in which the rate of conversion of [14C]phenylalanine into phenylalanyl-tRNA was reduced by about one-third in the Mg deficient supernatant. A similar modest reduction was observed when [14C]leucine was used as the substrate. We conclude that the ac tivity of at least two aminoacyl-tRNA li gases was reduced in the Mg deficient spleens.
50
a 012345 Incubation
Time (minutas)
Fig. 2 Phenylalanyl-tRNA ligase activity of spleen supernatant fractions from normal and Mg deficient (MgD) rats. Assays were carried out as described under Methods. The curves shown are representative of the results of four separate ex periments using different splenic extracts.
While the reduction in aminoacyl-tRNA ligase activity could cause a reduction in [14C]leucine incorporation into spleen pro tein in vivo, the lowering of ligase activity was quantitatively rather small. We there fore examined further the protein synthetic activity of the spleen supernatant fraction. In the experiment shown in table 3, splenic supernatants were tested for their ability to catalyze the synthesis of polyphenylalanine, using [14C]phenylalanyl-tRNA as the sub strate. Since this assay system utilizes aminoacylated tRNA as the substrate, it is TABLE 3 Synthesis of polyphenylalanine by 106,000 X g supernatant fractions from normal and Mg deficient spleens
Source of supernatantControl
incorporation1-135.9±9.3
Mg deficient[14C]Phenylalanine 14.7±6.7 1Results are expressed as cpm/^g protein. 2Each value represents the mean±SD for three separate preparations of spleen supernatant.
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Supernatant fraction20,000
200
sK
PROTEIN
SYNTHESIS
IN MAGNESIUM
zooo
*—(EF-eNormal) —
1600
1200
800 •
400 -
IZ Fraction
1fo 20
Z4
28
Number
Fig. 3 Separation of splenic elongation factors by Sephadex G-200 gel filtration. High-speed supernatants from normal and magnesium deficient (MgD) spleens were prepared as described under Methods. Aliquots (1.5 ml) of the supernatants were applied to a column (1.5x90 cm) of Sephadex G-200. The column was eluted with 20 HIM potassium phosphate (pH 7.0) containing 1 HIM dithiothreitol at a flow rate of 20 ml/hr. The first 40 ml of effluent were discarded, after which 2-ml fractions were collected. Assays for EF-I and EF-II were as described under Methods. Similar elution profiles were obtained using six different extracts of normal and Mg deficient spleens.
deficient supernatants and by a mixture of equal amounts of the two. The mixture gave an intermediate value, and there was no evidence of inhibition of the normal supernatant by the deficient supernatant. TABLE 4 Synthesis of polyphenylalanine by splenic 105,000 X g superno/ani« Supernatant
added
Control (10 Ml) Mg deficient (10 ¿d) Control (5 ¿il)+ Mg deficient (5 ^1)44.8
["C]Phenylalaiiine incorporation1 21.630.9
1Results are expressed as cpm/Vg protein.
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independent of aminoacyl-tRNA ligase ac tivity, as well as being independent of the content of tRNA and mRNA in the supernatants. As demonstrated in table 3, the rate of polyphenylalanine synthesis was re duced in supernatants from Mg deficient spleens. A number of similar experiments were carried out, and in each case the per cent inhibition of polyphenylalanine syn thesis in the deficient supernatants was similar whether the labeled substrate was [MC]phenylalanine or [14C]phenylalanyltRNA. It therefore appeared that the re duced protein synthesis in Mg deficient spleens was due primarily to a reduction in the activity of one or both of the elonga tion factors, EF-I and EF-II. The activity of the two elongation factors in splenic extracts were assayed by com plementation in polyphenylalanine synthe sis, using ribosomes washed free of elonga tion factors and purified EF-I and EF-II from normal liver. The 105,000 X g super natant fractions from normal and Mg de ficient spleens showed considerable indi vidual variation in absolute levels of elon gation factors, as previously noted by Willis and Starr ( 5 ) in supernatant fractions from immunized rat spleens. Despite this vari ability, the Mg deficient spleen supernatant fractions consistently showed a reduced level of one or both elongation factors when compared with the normal. A typical ex periment is illustrated in figure 3, in which EF-I and EF-II have been separated by Sephadex G-200 chromatography. Both EF-I and EF-II activity were reduced in the Mg deficient supernatant. Other Mg deficient supernatants showed a preponder ant reduction in the activity of one or the other elongation factor. We conclude that Mg deficiency causes a decrease in the splenic content of one or both elongation factors, and that the reduced elongation factor activity is largely responsible for the reduced splenic protein synthesis observed in vivo. It was also possible that the Mg deficient extracts contained an inhibitor of elonga tion factor activity. To rule out this possi bility, mixing experiments were carried out using normal and Mg deficient spleen su pernatants. Table 4 gives the rates of poly phenylalanine synthesis by control and Mg
1639
DEFICIENCY
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FREUDE, ZIEVE AND ZIEVE DISCUSSION
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Inhibition of protein synthesis in Mg de ficiency. We previously reported (1) that the rate of incorporation of [3H]leucine into splenic protein in vivo was depressed in the Mg deficient rat. Inhibition of pro tein synthesis in Mg deficiency was not un expected, since Mg ion is required for virtually every step in protein synthesis. The present experiments have localized the site of inhibition to the postmicrosomal supernatant fraction, with the most signifi cant change being reduced activity of the elongation factors EF-I and EF-II. Aminoacyl-tRNA ligase activity was also reduced in the Mg deficient spleen extracts. Because the difference between control and Mg de ficient extracts persisted in a system inde pendent of ligase activity (table 3), it ap peared that ligase activity was not ratelimiting, and that the inhibition of protein synthesis was primarily due to reduced elongation factor activity. A similar reduction in the protein syn thetic activity of the postmicrosomal super natant fraction was found by Schwartz et al. ( 8 ) in Mg deficient liver. These workers found, in agreement with our results (1), that in vivo incorporation of a labeled amino acid into total liver protein was un affected by Mg deficiency; in contrast, [14C]valine incorporation into serum albu min was reduced. In vitro, they were able to demonstrate reduced [14C]valine incor porating activity of the supernatant frac tion, while ribosomal activity appeared normal. While the component(s) of the supernatant showing reduced activity were not investigated by Schwartz et al., it is quite conceivable that reduced elongation factor activity is responsible for the de creased albumin synthesis by Mg deficient liver. Protein synthesis is also inhibited in Mg deficient bacteria (9, 10) but the mecha nism is completely different from that we have observed in spleen. When bacteria (11, 12) or yeast (13) are grown in Mgfree media, they lose the majority of their ribosomes. The degradation of ribosomes appears to be a stepwise process, in which polysomes are first broken down to 70S ribosomes, which are then converted to 30S and 50S subunits, then to smaller ribonu-
cleoprotein particles, and finally to acidsoluble materials (11, 12). In contrast, in Mg deficient spleen, we found both polysome sedimentation profiles (fig. 1) and the protein synthetic capacity of ribosomes ( table 1 ) to be normal, despite the signifi cant inhibition of splenic protein synthesis. This contrast between bacteria and spleen probably reflects different mecha nisms of response to Mg deficiency. In bac teria, the loss of ribosomes is likely due simply to a reduced cellular concentration of Mg ion, which is required for the preser vation of ribosomal structure (13). In spleen, the decreased elongation factor ac tivity cannot be explained by a simple re duction of cellular Mg concentration, since optimal amounts of Mg were added to the assay mixtures. In any case, the Mg con tent of the spleen was normal in our Mg deficient animals (1). Thus the reduced elongation factor activity must reflect a more complex mechanism than the simple unavailability of an ion required for catalysis. One logical question for future consider ation is whether the reduction in assayable elongation factor activity is due to a re duced amount of elongation factor protein or to the presence of elongation factors of subnormal catalytic activity. A reduction in the quantity of elongation factors present might be the ultimate response of the cell to a sustained inhibition of protein syn thesis. Relationship to the immune response. Mg deficient mice have a significant impair ment of both humoral and cell-mediated immune response, as reflected by decreased immunoglobulin concentrations (15, 16) and decreased antibody plaque-forming cells in the spleen ( 16). Willis and Starr (5) have provided evidence that splenic elongation factor EF-I can serve as a translational regulatory factor during the im mune response. It is therefore possible that the decreased elongation factor activity of Mg deficient spleens could partially account for the impaired immune responses of these animals. Relationship to lymphoproliferative re sponse. In our previous work ( 1 ), we found that Mg deficiency produced a reduction of splenic protein synthesis and an increase in
PROTEIN SYNTHESIS IN MAGNESIUM DEFICIENCY
ACKNOWLEDGMENT
We thank Connie Faltynek and Sandra Gould for technical assistance. LITERATURE CITED 1. Zieve F. J., Freude, K. A. & Zieve, L. ( 1977) Effects of magnesium deficiency on protein and nucleic acid synthesis in vivo. J. Nutr. 107, 2178-2188. 2. Wust, C. J. (1967) Studies on ribosomes of rat spleen during the immune response, Arch. Biochem. Biophys. 118, 568-576. 3. Mans, R. J. & Novelli, G. D. (1961) Mea surement of the incorporation of radioactive amino acids into protein by a filler-paper disk method. Arch. Biochem. Biophys. 94, 48-53. 4. Selawry, H. S. & Starr, J. L. (1971) Protein biosynthesis in the spleen. I. Effect of primary immunization on microsomal and ribosomal function in vitro. J. Immunol. 106, 349-357. 5. Willis, D. B. & Starr, J. L. (1971) Protein biosynthesis in the spleen. III. Aminoacyltransferase I as a translational regulatory factor during the immune response. J. Biol. Chem. 246, 2828-2834. 6. Honjo, T., Nishizuka, Y., Kato, I. & Hayaishi, O. ( 1971 ) Adenosine diphosphate ribosyla-
tion of aminoacyl transferase II and inhibition of protein synthesis by diphtheria toxin. J. Biol. Chem. 246, 4251-4260. 7. Fisher, R. A. (1938) Statistical methods for research workers. Oliver and Boyd, Lon don. 8. Schwartz, R., Woodcock, N. A., Blakely, J. D., Wang, F. L. & Khairallah, E. A. (1970) Effect of magnesium deficiency in growing rats on synthesis of liver proteins and serum albumin. J. Nutr. 100, 123-128. 9. Kennell, D. & Kotoulas, A. (1967) Mag nesium starvation of Aerobacter aerogenes. II. Rates of nucleic acid synthesis and methods of their measurement. J. Bacteriol. 93. 345356. 10. Marchesi, S. L. & Kennell, D. (1967) Mag nesium starvation of Aerobacter aerogenes. III. Protein metabolism. J. Bacteriol. 93, 357366. 11. McCarthy, B. J. ( 1962) The effects of mag nesium starvation on the ribosome content of Escherichia coli. Biochim. Biophys. Acta 55, 880-888. 12. Kennell, D. & Kotoulas, A. (1962) Mag nesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition. J. Bac teriol. 93, 334-344. 13. Beck, G., Aubel-Sadron, G. & Ebel, J. (1967) Etude de l'acide ribonucleique ribosomal de levures carencees en magnesium. Bull. Soc. Chim. Biol. 49, 349-360. 14. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. R. (1959) Ribonucleoprotein particles from Escherichia coli. J. Molec. Biol. 1, 221-233. 15. Alcock, N. W. & Shils, M. E. ( 1974) Serum immunoglobulin G in the magnesium-depleted •rat.Proc. Soc. Exp. Biol. Med. 145, 855-858. 16. Elin, R. J. (1975) The effect of magnesium deficiency in mice on serum immunoglobulin concentrations and antibody plaque-Torming cells. Proc. Soc. Exp. Biol. Med. 148, 620624. 17. Clifford, J. I., Rees, K. R. & Stevens, M. E. M. (1967) The effect of the aflatoxins B,, G> and G2 on protein and nucleic acid synthesis in rat liver. Biochem. J. 103, 258-261.
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splenic DNA synthesis. Considering the high incidence of lymphoid neoplasms in Mg deficient rats, we suggested that the increased DNA synthesis might represent an early stage in a lymphoproliferative process. The role of the inhibition of pro tein synthesis in such a process is purely speculative. A number of chemical carcino gens have among their early effects the in hibition of protein synthesis in target tis sues ( 17), and it is conceivable that the chronic inhibition of protein synthesis could produce a regenerative response which might eventually lead to cellular prolifera tion and neoplasia.
1641
Deficiency:
ABSTRACT To investigate the basis for the depressed protein synthesis in vivo in magnesium deficient spleens, the activities of splenic subcellular fractions in polypeptide synthesis were studied in vitro. Splenic ribosomes from Mg deficient animals were normal structurally and functionally. In contrast, supernatant fractions from the deficient spleens had a reduced ability to incorporate labeled amino acids into protein, both in the presence of endogenous mRNA and in the presence of added polyuridylic acid. The specific defects observed in the Mg deficient supernatants were twofold: There was a modest reduction in the rate of acylation of tRNA and a more marked reduction in the activity of the elongation factors, EF-I and EF-II. The reduction in elongation factor activity was quantitatively sufficient to account for the inhibition of protein synthesis in vivo. J. Nutr. 108: 1635-1641, 1978. INDEXING KEY WORDS magnesium •deficiency • protein • synthesis •ribosome •spleen In a recent study of the effect of mag nesium deficiency on protein and nucleic acid synthesis in vivo, we found the in corporation of [3H]leucine into tissue pro teins reduced by 40% to 50% in spleen and thymus, but not altered in liver (1). The spleen was also distinctive in showing a marked increase ( 350% ) in DN A synthesis. Because of this unique susceptibility of the spleen to the effects of Mg deficiency in vivo, we decided to study the mechanism of the decreased splenic protein synthesis. In an effort to determine which step or steps in protein synthesis were inhibited in Mg deficiency spleens, we studied the activ ities of splenic subcellular fractions in poly peptide synthesis in vitro.
normal and magnesium deficient diets have been described previously (1) except that only 20 mg magnesium was added per kg diet to give the magnesium deficient diet. Measurements of the magnesium content of the diet after mixing varied from 25 to 35 ng/g diet. This diet produced severe Mg deficiency in 120 g rats within 1 to 2 months. The studies of this report were per formed in rats that had consumed this diet for 6 to 8 weeks. At this time spleens were enlarged and bone magnesium, an index of body magnesium depletion, was less than 15% of normal ( 1 ). The controls were pairfed as previously described ( 1 ). ATP,5 GTP,5 polyuridylic acid,5 E. coli tRNA,6 sucrose (density gradient, ribo-
METHODS Materials. Male Sprague-Dawley ratsa were obtained at an average weight of 90 to 100 grams and were fed a stock diet* until they reached approximately 120 g, at which point they were fed the experimental diet. The components of the experimental
Recelved for publication August 17, 1977. 1 Present address : Dept. of Medicine, Richmond Veterans Hospital, Medical College of Virginia. Rich mond, Virginia. 2 Present addressj Hennepln County Medical Center. "Holtzman Rat Co., Madison, Wisconsin. 4 Purinn Rat Chow. 5 P-L Biochemlcals, Milwaukee. Wisconsin. 8 Schwarz/Mann, Orangeburg, New York.
1635
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KENNETH A. FREUDE,1 FRANKLIN J. ZIEVE 1 ANDLESLIE ZIEVE 2 Department of Medicine, Minneapolis Veterans Hospital, University of Minnesota
1636
FREUDE,
ZIEVE AND ZIEVE
nuclease free ) ,6 radioactive amino acids 7 and L-phenylalanyl-tRN A [phenylalanyl14C] ~ were obtained from the indicated
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tion of tRNA by spleen supernatant frac tions was determined by incubating 1 to 3 ¿ig of supernatant protein in a reaction mix ture (250 /xl) containing 50 HIM Tris-Cl commercial sources. Preparation of extracts. Normal or Mg (pH 7.0), 30 mM MgAc2, 40 HIM KCl, 1 mM EDTA, 5 mM ATP, 50 /¿gtRNA, and deficient rats were killed by decapitation, [14C]amino acid (specific activity > and their spleens were removed, minced 40 /»M and homogenized (glass-teflon homogenizer) 200 mCi/mmole ). After the indicated incu in four volumes of medium A (0.01 M Tris bation time at 37°,the labeled product was pH 8.0, 0.1 M KCl, 0.01 M MgAc2, 0.25 M isolated by the method of Mans and Novelli sucrose, 1.6 mg/ml bentonite). The homog(3). Preparation of washed liver ribosomes. enate was centrifuged 15 minutes at 10,000 Ribosomes from rat liver which had little X g, and the supernatant fraction was col lected and recentrifuged at 20,000 X g for or no transfer factor activity were prepared 15 minutes. In some experiments, the 20,000 as follows. Rat lived was minced and ho X g supernatant was diluted with 1.5 vol mogenized in 4 volumes (w/v) of medium of medium B (identical to medium A ex A (see supernatant preparation) and the cept that it contained 0.9 M sucrose) and homogenate centrifuged at 10,000 X g for 15 minutes. The lipid layer was aspirated centrifuged at 105,000 X g for 120 minutes. and the supernatant fraction recentrifuged Amino acid incorporation assays. Incor at 20,000 X g for 15 minutes. The lipid poration of labeled amino acids into protein was measured by two different assays, one layer was again carefully removed by aspi ration and the supernatant collected and utilizing endogenous mRNA, the other uti lizing the exogenous messenger, polyuri- diluted with 1.5 volumes of medium B ( see supernatant preparation). The diluted sudylic acid. The endogenous messenger as say was that of Wust (2). Each reaction pernate was centrifuged at 35,000 X g for mixture (100 p.1) contained 100 HIM Tris- 20 minutes. The resulting supernatant frac acetate (pH 8.0), 20 mM MgAc2, 50 mM tion was made 0.65% in deoxycholate by KCl, 0.8 mM GTP, 0.6 HIM ATP, 20 mM the addition of 5% deoxycholate in water, and then centrifuged for 90 minutes at creatine phosphate, 50 /tg/ml creatine kinase, 5 juM [14C]leucine, 30 to 50 jug/ml 105,000 x g. The resulting pellets were rinsed with medium A lacking bentonite, rat liver ribosomes, and spleen supernatant ( 0.2 to 0.5 mg protein ). Incubation was for suspended in medium A (l/25th the vol 10 minutes at 37°,and the labeled product of the deoxycholate-treated supernatant) was isolated by the method of Mans and by gentle homogenization, and then clari fied with a 10-minute centrifugation at Novelli (3). 35,000 X g. The clarified suspension was The synthesis of [14C]polyphenylalanine centrifuged at 140,000 X g for 120 min from [14C]phenylalanyl-tRNA in the pres utes. The pellets were stored frozen ( —20°) ence of the exogenous messenger, polyovernight, thawed and resuspended in uridylic acid, was carried out by the washing buffer containing 50 HIM Tris-Cl, method of Selawry and Starr (4). pH 7.4; 500 mM KCl; 20 HIMmercaptoethaAssay for elongation factor activities. Activities of the elongation factors EF-I nol. This suspension was gently mixed on and EF-II were measured by the method ice for 20 minutes, after which the ribo of Willis and Starr ( 5 ). The assay mixtures somes were collected by centrifugation at 140,000 X g for 90 minutes. The ribosomes (100 ¿ti)contained 10 mM Tris-HCl (pH at this stage were essentially free of trans 7.2), 9 mM MgAc2, 60 HIM KCl, 2 mM fer factors and were stored as a frozen sus dithiothreitol, 0.2 HIM GTP, 90-100 ¿xg pension in 50 mM Tris-Cl, pH 7.4; 4 mM washed liver ribosomes, and purified elon gation factors as indicated. Purified EF-I MgCL; 350 mM sucrose. Preparation of ribosomes for sucrose and EF-II were prepared as described by density analysis. The procedure is the same Honjo et al. (6). Assay of supernatant fractions for aminoacyl-tRNA ligase activity. The aminoacyla7 New England Nuclear, Boston, Massachusetts.
PROTEIN
SYNTHESIS
IN MAGNESIUM
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DEFICIENCY
as for washed liver ribosomes except the washing buffer was replaced with 10 HIM Tris-acétate,pH 8.0; 50 IHM KCl; and 10 niM magnesium acetate. Sucrose gradient anahjsis of ribosomes. Sucrose density profiles were run in 50 HIM Tris-Cl, pH 7.8; 25 HIMKCl; 5 HIM MgCl2; containing a 10% to 34% linear gradient of sucrose. Approximately 100 to 200 /xg of ribosomal RNA were analyzed per gradient. The profiles were developed using a SW-40 rotor8 at 27,500 rpm for 120 minutes. Statistical analysis. Standard procedures were used for calculating means, standard deviations, standard error of means ( SEM), and tests of significance of differences be tween means (7).
TABLE 1 Protein synthesis by ribosomes from normal and Mg deficient spleens in the presence of normal liver supernatant
RESULTS Ribosomes. We first attempted to deter mine whether the defect in spleen protein synthesis resided in the ribosomes or in the supernatant fraction. To ascertain whether
ribosomal structure was grossly altered, we examined the sedimentation profiles of polysomes in extracts from normal and Mg deficient spleens. As shown in figure 1, the polysomal profiles were identical in the con trol and deficient rats. This finding indi cated that no gross disruption of ribosomal or polysomal structure had occurred. Very little low molecular weight material was present, as shown by the position of the 80S ribosomal marker; this showed that ex tensive polysomal breakdown during the preparation had not taken place. The functional activity of spleen ribo somes was tested by measuring their ability to support protein synthesis in the presence of a ribosome-free supernatant fraction from normal liver. Two different assays were used, one of which measured the in corporation of [14C]leucine into protein utilizing endogenous mRNA, the other of which measured the incorporation of [14C]phenylalanine in the presence of added poly U. As shown in table 1, neither assay revealed any difference between control ribosomes and Mg deficient ribosomes. We conclude that, within the limits of sensitiv ity of these measurements, the functional capacity of spleen ribosomes was unaffected by Mg deficiency. Supernatants. Since it appeared from the above studies that the decreased protein synthesis observed in vivo in Mg deficient rat spleen was not due to a defect in ribo somes, we examined the activity of splenic
Fig. 1 Sedimentation profiles of spleen polysomes from control and magnesium deficient (MgD) rats. Extracts were prepared and centrifuged as described under Methods. Fractions (300 /¿])were collected by displacement from the top of the tube using a Hoefer density gradient fractionator. The direction of sedimentation is from left to right, and the arrow represents the position of an 80S ribosomal marker. Each extract was from the pooled spleens of four rats. Similar profiles were obtained from at least three other preparations of normal and Mg deficient spleen.
.Source of ribosomesNormal
messenger U assay169±19assay3,499
spleens ±382 Mg deficient spleensEndogenous 155±22Poly3,399 ±347 1Results are expressed as cpm/10 /ig ribosomal RNA. 2 Each value listed represents the mean ±SD for three separate preparations of spleen ribosomes.
8Becknian Instruments, Fullerton, California.
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Bottom
["CjAmino acid incorporation1'2
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FREUDE, ZIEVE AND ZIEVE TABLE 2
Protein synthesis by supernatant fractions from norma and Mg deficient spleens in the presence of normal liver ribosomes
Xg
ratsControl of
incorporation1'2832±
150
Normal
21 61,957±147 330±
deficientControl Mg 105,000 X gTreatment Mg deficient["C]Leucine 974±65 1Results are expressed as cpm/mg supernatant protein. *Each value represents the mean±SD for two separate preparations of spleen supernatant.
100
q
q 3^ c
MgD I
supernatant fractions. The experimental design was analogous to the ribosome ex periments described above. Spleen super natant fractions were tested for their ability to incorporate [UC] leucine into protein in the presence of normal rat liver ribosomes. Table 2 shows the activities of the 20,000 X g and 105,000 x g supernatant fractions from normal and Mg deficient spleens. In both preparations the activity of the Mg deficient supernatants was approximately one-half that of the controls. This decrease was comparable to that seen in the in vivo leucine incorporation studies reported pre viously (1). Thus it appeared that the factors responsible for diminished splenic protein synthesis in Mg deficiency were to be sought in the postmicrosomal superna tant fraction. The 105,000 X g supernatant fraction contains several components that are neces sary for protein synthesis including amino acyl-tRNA ligases, tRNA, mRNA, and elongation factors. Activities of aminoacyltRNA ligases were assessed by measuring the acylation of rat liver tRNA with [14C]phenylalanine or [14C]leucine in the pres ence of spleen supernatant fractions. The ligase activities were measured under con ditions of saturating substrate. A typical experiment is shown in figure 2, in which the rate of conversion of [14C]phenylalanine into phenylalanyl-tRNA was reduced by about one-third in the Mg deficient supernatant. A similar modest reduction was observed when [14C]leucine was used as the substrate. We conclude that the ac tivity of at least two aminoacyl-tRNA li gases was reduced in the Mg deficient spleens.
50
a 012345 Incubation
Time (minutas)
Fig. 2 Phenylalanyl-tRNA ligase activity of spleen supernatant fractions from normal and Mg deficient (MgD) rats. Assays were carried out as described under Methods. The curves shown are representative of the results of four separate ex periments using different splenic extracts.
While the reduction in aminoacyl-tRNA ligase activity could cause a reduction in [14C]leucine incorporation into spleen pro tein in vivo, the lowering of ligase activity was quantitatively rather small. We there fore examined further the protein synthetic activity of the spleen supernatant fraction. In the experiment shown in table 3, splenic supernatants were tested for their ability to catalyze the synthesis of polyphenylalanine, using [14C]phenylalanyl-tRNA as the sub strate. Since this assay system utilizes aminoacylated tRNA as the substrate, it is TABLE 3 Synthesis of polyphenylalanine by 106,000 X g supernatant fractions from normal and Mg deficient spleens
Source of supernatantControl
incorporation1-135.9±9.3
Mg deficient[14C]Phenylalanine 14.7±6.7 1Results are expressed as cpm/^g protein. 2Each value represents the mean±SD for three separate preparations of spleen supernatant.
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Supernatant fraction20,000
200
sK
PROTEIN
SYNTHESIS
IN MAGNESIUM
zooo
*—(EF-eNormal) —
1600
1200
800 •
400 -
IZ Fraction
1fo 20
Z4
28
Number
Fig. 3 Separation of splenic elongation factors by Sephadex G-200 gel filtration. High-speed supernatants from normal and magnesium deficient (MgD) spleens were prepared as described under Methods. Aliquots (1.5 ml) of the supernatants were applied to a column (1.5x90 cm) of Sephadex G-200. The column was eluted with 20 HIM potassium phosphate (pH 7.0) containing 1 HIM dithiothreitol at a flow rate of 20 ml/hr. The first 40 ml of effluent were discarded, after which 2-ml fractions were collected. Assays for EF-I and EF-II were as described under Methods. Similar elution profiles were obtained using six different extracts of normal and Mg deficient spleens.
deficient supernatants and by a mixture of equal amounts of the two. The mixture gave an intermediate value, and there was no evidence of inhibition of the normal supernatant by the deficient supernatant. TABLE 4 Synthesis of polyphenylalanine by splenic 105,000 X g superno/ani« Supernatant
added
Control (10 Ml) Mg deficient (10 ¿d) Control (5 ¿il)+ Mg deficient (5 ^1)44.8
["C]Phenylalaiiine incorporation1 21.630.9
1Results are expressed as cpm/Vg protein.
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independent of aminoacyl-tRNA ligase ac tivity, as well as being independent of the content of tRNA and mRNA in the supernatants. As demonstrated in table 3, the rate of polyphenylalanine synthesis was re duced in supernatants from Mg deficient spleens. A number of similar experiments were carried out, and in each case the per cent inhibition of polyphenylalanine syn thesis in the deficient supernatants was similar whether the labeled substrate was [MC]phenylalanine or [14C]phenylalanyltRNA. It therefore appeared that the re duced protein synthesis in Mg deficient spleens was due primarily to a reduction in the activity of one or both of the elonga tion factors, EF-I and EF-II. The activity of the two elongation factors in splenic extracts were assayed by com plementation in polyphenylalanine synthe sis, using ribosomes washed free of elonga tion factors and purified EF-I and EF-II from normal liver. The 105,000 X g super natant fractions from normal and Mg de ficient spleens showed considerable indi vidual variation in absolute levels of elon gation factors, as previously noted by Willis and Starr ( 5 ) in supernatant fractions from immunized rat spleens. Despite this vari ability, the Mg deficient spleen supernatant fractions consistently showed a reduced level of one or both elongation factors when compared with the normal. A typical ex periment is illustrated in figure 3, in which EF-I and EF-II have been separated by Sephadex G-200 chromatography. Both EF-I and EF-II activity were reduced in the Mg deficient supernatant. Other Mg deficient supernatants showed a preponder ant reduction in the activity of one or the other elongation factor. We conclude that Mg deficiency causes a decrease in the splenic content of one or both elongation factors, and that the reduced elongation factor activity is largely responsible for the reduced splenic protein synthesis observed in vivo. It was also possible that the Mg deficient extracts contained an inhibitor of elonga tion factor activity. To rule out this possi bility, mixing experiments were carried out using normal and Mg deficient spleen su pernatants. Table 4 gives the rates of poly phenylalanine synthesis by control and Mg
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FREUDE, ZIEVE AND ZIEVE DISCUSSION
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Inhibition of protein synthesis in Mg de ficiency. We previously reported (1) that the rate of incorporation of [3H]leucine into splenic protein in vivo was depressed in the Mg deficient rat. Inhibition of pro tein synthesis in Mg deficiency was not un expected, since Mg ion is required for virtually every step in protein synthesis. The present experiments have localized the site of inhibition to the postmicrosomal supernatant fraction, with the most signifi cant change being reduced activity of the elongation factors EF-I and EF-II. Aminoacyl-tRNA ligase activity was also reduced in the Mg deficient spleen extracts. Because the difference between control and Mg de ficient extracts persisted in a system inde pendent of ligase activity (table 3), it ap peared that ligase activity was not ratelimiting, and that the inhibition of protein synthesis was primarily due to reduced elongation factor activity. A similar reduction in the protein syn thetic activity of the postmicrosomal super natant fraction was found by Schwartz et al. ( 8 ) in Mg deficient liver. These workers found, in agreement with our results (1), that in vivo incorporation of a labeled amino acid into total liver protein was un affected by Mg deficiency; in contrast, [14C]valine incorporation into serum albu min was reduced. In vitro, they were able to demonstrate reduced [14C]valine incor porating activity of the supernatant frac tion, while ribosomal activity appeared normal. While the component(s) of the supernatant showing reduced activity were not investigated by Schwartz et al., it is quite conceivable that reduced elongation factor activity is responsible for the de creased albumin synthesis by Mg deficient liver. Protein synthesis is also inhibited in Mg deficient bacteria (9, 10) but the mecha nism is completely different from that we have observed in spleen. When bacteria (11, 12) or yeast (13) are grown in Mgfree media, they lose the majority of their ribosomes. The degradation of ribosomes appears to be a stepwise process, in which polysomes are first broken down to 70S ribosomes, which are then converted to 30S and 50S subunits, then to smaller ribonu-
cleoprotein particles, and finally to acidsoluble materials (11, 12). In contrast, in Mg deficient spleen, we found both polysome sedimentation profiles (fig. 1) and the protein synthetic capacity of ribosomes ( table 1 ) to be normal, despite the signifi cant inhibition of splenic protein synthesis. This contrast between bacteria and spleen probably reflects different mecha nisms of response to Mg deficiency. In bac teria, the loss of ribosomes is likely due simply to a reduced cellular concentration of Mg ion, which is required for the preser vation of ribosomal structure (13). In spleen, the decreased elongation factor ac tivity cannot be explained by a simple re duction of cellular Mg concentration, since optimal amounts of Mg were added to the assay mixtures. In any case, the Mg con tent of the spleen was normal in our Mg deficient animals (1). Thus the reduced elongation factor activity must reflect a more complex mechanism than the simple unavailability of an ion required for catalysis. One logical question for future consider ation is whether the reduction in assayable elongation factor activity is due to a re duced amount of elongation factor protein or to the presence of elongation factors of subnormal catalytic activity. A reduction in the quantity of elongation factors present might be the ultimate response of the cell to a sustained inhibition of protein syn thesis. Relationship to the immune response. Mg deficient mice have a significant impair ment of both humoral and cell-mediated immune response, as reflected by decreased immunoglobulin concentrations (15, 16) and decreased antibody plaque-forming cells in the spleen ( 16). Willis and Starr (5) have provided evidence that splenic elongation factor EF-I can serve as a translational regulatory factor during the im mune response. It is therefore possible that the decreased elongation factor activity of Mg deficient spleens could partially account for the impaired immune responses of these animals. Relationship to lymphoproliferative re sponse. In our previous work ( 1 ), we found that Mg deficiency produced a reduction of splenic protein synthesis and an increase in
PROTEIN SYNTHESIS IN MAGNESIUM DEFICIENCY
ACKNOWLEDGMENT
We thank Connie Faltynek and Sandra Gould for technical assistance. LITERATURE CITED 1. Zieve F. J., Freude, K. A. & Zieve, L. ( 1977) Effects of magnesium deficiency on protein and nucleic acid synthesis in vivo. J. Nutr. 107, 2178-2188. 2. Wust, C. J. (1967) Studies on ribosomes of rat spleen during the immune response, Arch. Biochem. Biophys. 118, 568-576. 3. Mans, R. J. & Novelli, G. D. (1961) Mea surement of the incorporation of radioactive amino acids into protein by a filler-paper disk method. Arch. Biochem. Biophys. 94, 48-53. 4. Selawry, H. S. & Starr, J. L. (1971) Protein biosynthesis in the spleen. I. Effect of primary immunization on microsomal and ribosomal function in vitro. J. Immunol. 106, 349-357. 5. Willis, D. B. & Starr, J. L. (1971) Protein biosynthesis in the spleen. III. Aminoacyltransferase I as a translational regulatory factor during the immune response. J. Biol. Chem. 246, 2828-2834. 6. Honjo, T., Nishizuka, Y., Kato, I. & Hayaishi, O. ( 1971 ) Adenosine diphosphate ribosyla-
tion of aminoacyl transferase II and inhibition of protein synthesis by diphtheria toxin. J. Biol. Chem. 246, 4251-4260. 7. Fisher, R. A. (1938) Statistical methods for research workers. Oliver and Boyd, Lon don. 8. Schwartz, R., Woodcock, N. A., Blakely, J. D., Wang, F. L. & Khairallah, E. A. (1970) Effect of magnesium deficiency in growing rats on synthesis of liver proteins and serum albumin. J. Nutr. 100, 123-128. 9. Kennell, D. & Kotoulas, A. (1967) Mag nesium starvation of Aerobacter aerogenes. II. Rates of nucleic acid synthesis and methods of their measurement. J. Bacteriol. 93. 345356. 10. Marchesi, S. L. & Kennell, D. (1967) Mag nesium starvation of Aerobacter aerogenes. III. Protein metabolism. J. Bacteriol. 93, 357366. 11. McCarthy, B. J. ( 1962) The effects of mag nesium starvation on the ribosome content of Escherichia coli. Biochim. Biophys. Acta 55, 880-888. 12. Kennell, D. & Kotoulas, A. (1962) Mag nesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition. J. Bac teriol. 93, 334-344. 13. Beck, G., Aubel-Sadron, G. & Ebel, J. (1967) Etude de l'acide ribonucleique ribosomal de levures carencees en magnesium. Bull. Soc. Chim. Biol. 49, 349-360. 14. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. R. (1959) Ribonucleoprotein particles from Escherichia coli. J. Molec. Biol. 1, 221-233. 15. Alcock, N. W. & Shils, M. E. ( 1974) Serum immunoglobulin G in the magnesium-depleted •rat.Proc. Soc. Exp. Biol. Med. 145, 855-858. 16. Elin, R. J. (1975) The effect of magnesium deficiency in mice on serum immunoglobulin concentrations and antibody plaque-Torming cells. Proc. Soc. Exp. Biol. Med. 148, 620624. 17. Clifford, J. I., Rees, K. R. & Stevens, M. E. M. (1967) The effect of the aflatoxins B,, G> and G2 on protein and nucleic acid synthesis in rat liver. Biochem. J. 103, 258-261.
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splenic DNA synthesis. Considering the high incidence of lymphoid neoplasms in Mg deficient rats, we suggested that the increased DNA synthesis might represent an early stage in a lymphoproliferative process. The role of the inhibition of pro tein synthesis in such a process is purely speculative. A number of chemical carcino gens have among their early effects the in hibition of protein synthesis in target tis sues ( 17), and it is conceivable that the chronic inhibition of protein synthesis could produce a regenerative response which might eventually lead to cellular prolifera tion and neoplasia.
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