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Biosynthesis of thyrotropin releasing factor: Effect of RNase treatment
Brain Researe!~ Bufietin, A11 rights of reproduction
Vol. I, pp. 171-175, 1976. Copyright 0 ANKHO in any form reserved. Printed in the U.S.A.
International
Inc
Biosynthesis of Thyrotropin Releasing Factor: Effect of RNase Treatment Y. GEMS-JORGENSEN
AND J. F. MC KELVY
(Received 28 November
f975)
GRIMM-JGRGENSEN, Y. AND J. P. MC KELVY. Biosynthesis of thyrotropin releasing factor: effect of RNase treatment. BRAIN RES. BULL. l(2) 171.--175, 1976. -. In the present study the role of RNA in the in vitro biosynthesis of thyrotropin releasing factor (TRF) was investigated. The biosynthesis of TRF was assessed by measuring the ability of a 1000 g supernatant fraction of newt brain origin to incorporate (3H)proline into (3H)TRF. It was found that measurable amounts of (3H)TRF are synthesized by newt brain homogenates, that preincubation of these homogenates with pro&se-free RNase cotnpletely abolisftes (3H)TRF synthesis, while preincubation of homogenates with inactivated RNase allows for (3H)TRF synthesis to proceed. These studies suggest that the presence of intact RNA is mandatory for TRF biosynthesis by newt brain homogenates. Biosynthesis
TRF
RNase
Chemicals
THE identification of mammalian thyrotropin releasing factor (TRF) as the tripeptide pGlu-His-ProNH~ [2,3/ and the subsequent availability of the synthetic tripeptide have led to numerous investigations on the mechanism of action of TRF, its distribution in various brain regions of many vertebrate species [ 8, 1 I, 20, 261 and its biosynthesis (for a review, see 1221). Reports from several laboratories have demonstrated that hypothalamic fragments from various mammals are capable of incorporating labeled precursor amino acids into TRF [5, 7, 14, 16, 191. It has also been shown that TRF biosynthesis by hypothalamic fragments in vitro is unaffected by puromycin f 19) or cycloheximid~ (71. Furthermore, it has been claimed that TRF biosynthesis can be demonstrated in a solubIe fraction from hypothalamic extracts of rat and porcine origin [ 191. The synthesis of TRF by this fraction required the presence of the amino acids Glu/Gln, His, and Pro, as well as Mg++and ATP, but was unaffected by RNase treatment. These findings suggested that TRF may be synthesized by a soluble enzyme system not requiring the presence of RNA, and the invoivement of a TRF-synthetase was hypothesized Il9l. We have recently reported the in vitro synthesis of tritium-labeied TRF by newt (Tritums viridescens) hypothalamic and forebrain fragments 161. In order to further characterize TRF synthesis by brain tissue from this amphibian species, we set out to test whether a cell-free homogenate of newt brain origin is capable of 3H-TRF synthesis
and whether
this synthesis
is dependent
(G-3 H)Proline (5 Cijmmol) was obtained from ICN Pharmaceuticals, Inc., Cleveland, Ohio and purified as described earlier [ 61. pGlu-His-(3 H)ProNH, (40 Ci/mmol) and (32P) orthophosphoric acid (carrier-free) were obtained from New England Nuclear, Boston, Mass., RNA (Yeast, type XI) RNase “a” (type Xl-A), DNA (salmon), DNase (type I), and ATP (sodium salt?, were obtained from Sigma Chemical Corp., St. Louis, MO. Procedure Assay for (” H)TRF synthesis by newt brain homogenates. Whole brains of IO newts were homogenized by hand (35 strokes, 3 revolutions each) in 0.25 ml of ice-cold HEPES(N-~-hydroxyethylpiperazine-~-2-ethanesuIfonic acid) buffered amphibian Ringer’s solution [61 at pH 7.6 with a Instrument Company, microhomogenizer (Micrometric Cleveland, Ohio) of 0.5 ml capacity. The homogenizer was rinsed with 0.25 ml of the same incubation medium. The homogenate, the rinse and a further 0.5 ml of incubation medium were then combined, and the mixture was centrifuged for 10 min at 1,000 g at 4°C. The peilet was discarded and the volume of the supernatant adjusted to 1.O ml. The supernatant was then allowed to warm to room temperature (22” C), and 10 ~1 of ATP ( 1(X3M) and 25 r Ci of purified L-f3 H)proline were added to the mixture. Immediately after the addition of the L-(3 H)proline, 200 rri of the incubation mixture were withdrawn and delivered into 2 ml of absolute methanol containing 25 gg of synthetic TRF. This aliquot served as the zero time background control sample and was subtracted from the (’ H)TRF values in all subsequent samples. This background control is of utmost importance in studies where the incorporation of labeled amino acids into labeled TRF is measured because, even after extensive purification of the
on the
presence of intact RNA. METHOD
Animals Adult newts of either sex were obtained from the Mogul Ed Corporation, Oshkosh, Wise. They were fed chopped liver, fortified with calcium, twice per week. 171
172 sample, some labeled contaminants migrate with the TRF peak in various chromatography and electrophoresis systems. At various times after the addtion of the (” H)proline, successive 200 ~1 aliquots were similarly withdrawn. At the end of the incubation period the methanolic extracts were allowed to cool for 30 min at 4°C. The amount of (” H)TRF present in each sample was then determined. The synthesis of TRF was expressed as pmoles of (” H)TRF formed/mg tissue protein. isolation of tritium labeled TRF. (‘H)TRF was extracted from the methanolic extracts of the homogenates by successive column c~omatography and eleetrophoresis at acid and alkaline pH as described in a previous publication [61. Determination of alkali-soluble tritium-labeled proteins. The amount of alkali-soluble radioactivity of the pellet remaining after methanol extraction was determined after extraction with cold and hot trichloroacetic acid and solubilization in 0.5 N NaOH [ 251. The total amount of protein in the alkali-soluble fraction was then determined as described in [ 131. Assay for RNase activity. The activity of the commercially obtained RNA was measured as described in [9] with yeast RNA as the substrate. In order to determine the amount of RNase needed to achieve adequate hydrolysis of RNA in the homogenates used in the TRF synthesis experiments, the activity of RNase was also measured in HEPES-buffered amphibian Ringer’s solution at pH 7.6 and 22°C. It was found that, under these conditions, the enzyme was much less active, and that 100 pg/ml of RNase were needed to achieve the same degree of hydrolysis of yeast RNA as was achieved by 2 rg of RNase under the assay conditions specified in [ 91. Preliminary studies also showed that, under the experimental conditions used to show (’ H)TRF synthesis, 3 2 P-labeled newt brain RNA was extensively hydrolyzed by 100 pg/ml of RNase. The 3 *P-labeled RNA had been obtained by incubation of newt brain slices in HEPES-buffered amphibian Ringer’s solution containing 100 &X/ml of (‘*~)orthophosphate and subsequent extraction of the labeled RNA as described in [24]. Because of these findings, 100 ug/ml of RNase were added to the incubation mixture when the (3H)TRF synthesis was tested. Assa_v for protease activity, The possible contamination of the RNase preparation with proteases was assayed in two ways. (1) The protease activity of the RNase was compared to that of pronase with azocoll as the substrate, (2) The ability of the RNase preparation to hydrolyze (3H)labeled newt brain proteins was compared to the ability of trypsin to hydrolyze the same proteins. Labeled newt brain proteins had been obtained by incubating brain slices in amphibian Ringer’s solution containing 100 &i/ml of a 3 H-labeled amino acid mixture, homogenizing the tissue, and c~omatographing the homogenate on a column of Sephadex G-75. The void volume peak served as the substrate. Inactivation of RNase. The RNase was inactivated by carboxymethylation of a histidine residue in the active site which leads to inactivation of the enzyme without grossly disturbing its molecular conformation (141 and personal communication). After carboxymethylation, the enzyme was chromatographed on a diethylaminoethylcellulose ion exchange resin (DE-52, hydrogen form) and the combined RNase peak lyophilized and tested for RNase activity as
GRIMM-JORGENSEN
AND MC KELVY
described in 191. It was found that the RNase inactivated by this method had lost 80%~ of its initial ability to hydrolyze RNA. To further ascertain that the carboxymethylation had been successful, an aliquot of the lyophilized material was subjected to acid hydrolysis and amino acid analysis with the Beckman Model 120 C automatic amino acid analyzer [2 1 ] The resuit from this procedure indicated that one His and two Met residues had been alkylated as expected. Determination of degradation of (‘HI-TRF by newt brain homogenates. The ability of the homogenates from the whole newt brains to degrade synthetic (” H)TRF was tested as follows: 1,000 g homogenates were obtained as described above. At zero time 10 @I of ATP ( 10-3M) were added to the homogenate. This was followed by the addition of 25 ~1 (300,000 cpm) of synthetic (“H)TRF which had been purified as described in [ 151. Aliyuots (100 ~1) were withdrawn from the incubation mixture after 10 and 20 min of incubation and delivered into 2 ml of absolute methanol. After refrige~tion overnight, the aliquots were centrifuged at 1,000 g for 20 min, and the supernatants were dried under nitrogen. The residues were suspended in 100 ~1 of absolute methanol.. and 10 ~1 aliquots of each sample were subjected to electrophoresis on cellulose acetate strips in 0.1 M sodium acetate buffer, pII 4.2, at 280 V for 60 min. The electrophoresis strips were then cut into I cm sections and dried, and the radioactivity in each section was determined after the addition of 10 ml of a toluene based scintillation mixture. The amount of radioactivity found in the area of migration of synthetic standard TRF was determined on each electrophoretogram and the amount of ( 3 H)TRF expressed as per cent of the total amount of radioactivity on the eIectrophoresis strip. RESULTS
Figure 1 shows a typical incorporation graph obtained when the 1,000 g supernatant from 10 whole brains was incubated in the presence of 25 PCi of (3H)proline. In this experiment, the rate of incorporation of (‘H)proIine into (3 H)TRF was 0.004 pmoles/min/brain. When the 1,000 g supernatant was preincubated for 15 min with RNase (100 &ml) and then its ability to incorporate (’ H)proline into (” H)TRF was studied, it was found that no (‘H)TRF was produced during the 10 min test period (Fig. 2). On the other hand, when the RNase was inactivated prior to its addition to the homogenate, the synthesis of (” H)TRF was only slightly depressed when compared to control homogenates (Fig. 3). Although the RNase preparation had been shown to be free of protease activity, the possibility was considered that this enzyme might have increased the rate of degradation of TRF, and thus have interfered with the detection of newly formed TRF. Therefore, newt brain homogenates were incubated in the presence of synthetic pClu-His(3H)ProNH, and RNase or inactivated RNase, and the rate of degradation of TRF measured. Table 1 shows that newt brain homogenates, when incubated under the conditions used for (3H)TRF synthesis only minimally degraded exogenous (“H)TRF. After 20 min of incubation, the maximum time used in the synthesis studies, only 10% of the added (‘H)TRF was degraded. The table also shows that neither active RNase nor inactivated RNase increased the rate of TRF degradation.
173
TRH AND RNase TREATMENT
MINUTES FIG, 1. Incorporation of (3Hjproline into t3H)TRF by a cell free extract from whole newt brains: Whole brains from 10 newts were homogenized by hand in a total volume of 1.0 ml of HEPESbuffered amphibian Ringer’s solution (pH 7.6). After centrifugation for 10 min at 1,000 g the supernatant was reconstituted with amphibian Ringer’s solution to a to t~$ volume of 1 .O ml and 25 r&i of (3 H)proline and 10 (~1of ATP (10 j were added. 200 ~1 aliquots were withdrawn at the times indicated and the (3HfTRF isolated. The values represent the amount of (aH)TRF (pmoles) produced by the whole extract. 0.6 0.5
i.k “0 h
0.4.
k R
0.3.
Control
RNorr lOOpa
FIG. 3. The effect of RNase and inactivated RNase on the ~corporat~on of (3H)p~~ne into (3HjTRF by a cell-free extract from whole newt brains: whole brains from 10 newts were homogenized by hand in a total volume of 3.0 ml of HEPESbuffered amphibian Ringer’s solution (pH 7.6). After centrifugation at 1,000 g, the supematant was reconstituted to a total volume of 1.0 ml with amphibian Ringer’s solution. After the addition of 100 gg of RNase, 100 pg of inactivated RNase, or an equal volume of incubation medium, the extract was preincuba ed for 15 min. Then 25 &Ji of (3H]proIine and IO PI of ATP (lo- 5Mj were added and 200 ~1 aliquots withdrawn from each tube. The incubation was continued for 10 min and a second 200 ~1 aliquot was withdrawn. (3H)TRF was then isolated as described in methods. The values represent the amount of f3H)TRF (pmoles) produced by the extracts during a 10 min period. TABLE 1 EFFECT OF RNase ON (3H)TRF DEGRADATION
;;; B 0.2. E P 0.1
Treatment
RNase
-----_c----+ 5
100 pg
10 MINUTES
FIG. 2. The effect of RNase on the incorporation of (3H)Pro into (3H)TRF by a cell-free extract from whole newt brains. Whole brains from 10 newts were homogenized by hand in a total volume of 1.0 ml of HEPES buffered amphibian Ringer’s solution (pH 7.6). After centrifugation for IO min at 1000 g, the supernatant was reconstituted to a volume of 1.0 ml. After the addition of 100 r.zgof RNase (or an equal volume of incubation medium for the control), the extract was preincub-fed for 15 min. Then 25 &i of (aH)Pro and 10 &f of ATP (10 M) were added. 200 ~1 aliquots were withdrawn from the homogenates at the times indicated. (‘HjTRF was isolated as described in methods. The values represent the amount of (‘HITRF (pmates) produced by the whok extract.
Control RNase ( 100 ~g/mI j &activated RNase (100 rgimlf
Percent of Applied Radioactivity in the TRF-area 0 min 10 min 20 min 88.8 85.3 88.0
84.5 85.6 86.9
80.6 85.4 85.1
The 1,000 g supernatants of homogenates from 10 newt brains were incubated with synthetic (3HjTRF for the time indicated. At the end of each incubation period the proteins were precipitated with methanol and the methano~ic supernatants subjected to electrop~oresis as described under methods.
DISCUSSION Preliminary studies had shown that the biosynthesis of (‘H)TRF by newt hypothalamic fragments in vitro is not influenced by the protein synthesis inhibitors cyefoheximide, c~o~rnp~e~i~o~, and diphtheria toxin f 17 1,
173
confirming previous reports from other laboratories [ 7,191. However. these findings cannot definitely rule out a ribosomal participation in the synthesis of this peptide. since the residual amount of protein synthesis which proceeds in the presence of the protein synthesis inhibitors may be sufficient to account for the small amount of TRF synthesized during the incubation period. To further study the possible role of RNA in TRF synthesis, the effect of RNase treatment on the incorporation of (” H)TRF by a cell-free newt brain homogenate was investigated in this study. While (” H)TRF was formed by control homogenates, no (” H)TRF was produced by homogenates which had been ~reincubated with RNase prior to the addition of (” H)proline. The effect of RNase cannot be attributed to a non-specific chemical property of this enzyme, e.g. its polycationic nature, since inactivated RNase, which differs from RNase only in 3 amino acid residues, did not have a similar effect on TRF synthesis. The slight inhibition of (“H)TRF synthesis which was observed when the homogenate was exposed to inactivated RNase prior to the addition of (3H)prohne (Fig. 3) can be attributed to the fact that. after carboxymethylation, the enzyme had retained approximately 20% of its activity. It is also unlikely that the abolition of (3 H)TRF synthesis by RNase is due to contamination of the pancreatic enzyme with proteases, since no such activity could be detected when the enzyme was tested in 2 protease assays using either azocoll or tritium-labeled newt brain-proteins as the substrates. Since it has been reported that in crude preparations of
Bacillus
brevis, DNase treatment is capable of dissociating enzyme complex responsible for tyrocidine synthesis [ 131. the possibility ihat the effect of RNase on TRF biosynthesis may be due to contaminating DNase was considered. However, it was found that the RNasz was devoid of such activity. The possibility that RNase may have Increased the degradation of TRF was ruled out, since no such effect was observed when homogenates were incubated with Rnase and tritium labeled synthetic TRF. While these studies suggest that RNA is involved in the synthesis of TRF, they cannot explain the mechanism of this involvement. It is possible that TRF synthesis proceeds via a ribosomal mechanism and that the failure to observe inhibition of TRF biosynthesis in vitro in the presence of various protein synthesis inhibitors is due to the residual ongoing protein synthesis. On the other hand TRF biosynthesis may be non-ribosomal, but requiring RNA. Several possibifities for such an RNA requirement exist. For example, RNA may be involved in maintaining the structural integrity of the TRF synthesizing enzymes. On the other hand, it is possible that the addition of one or more amino acids to the growing molecule involves the participation of an aminoacyl-t-RNA-transferase. It has been demonstrated that amino acids can be added to dipeptides by such enzymes 1231 and that aminoacyl-t-RNAtransferase activity is present in brain homogenates [ I I. Further studies will be necessary to detertnine which of these, or other, mechanisms are operating in TRF hiosynthesis.
the
REFERENCES 1. Barra, H. S., J. A. Rodriguez, C. A. Arce and K. Caputto. A soluble preparation from rat brain that incorporates into its own proteins ’ 4C-arginine by a ribonuclease-sensitive system system. J. and ’ 4C-tyrosine by a ribonuclease-insensitive Neurochem.
20: 97-109,1973.
2. Bdler. J.. F. Enzmann. K. Folkers. C. Y. Bowers and A. V. Shalli. The identity of chemicai and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl prohneamide. Biochem. bjophys. Rex Comrn~~n. 37: 7055710, 1969. 3. Burgus, R., T. F. Dunn, D. Desiderio and R. Guillemin. Structure moleculaire du facteur hypothalamique hypophysiotrope TRF d’origine ovine: evidence par spectrometrie de masse de la sequence PCA-His-Pro-NH,. C. R. Hebd. S&W Acad. Sci., Paris 269:
1870-1873,
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4. Goren, H. J. and E. A. Barnard. Relations of reactivity to structure in pancreatic ribonuclease II. Positions of residues alkylated in certain conditions by bromoacetate. Biochemistry 9: 974-983,197O. 5. Grimm, Y. and S. Reichlin. Thyrotropin releasing hormone (TRH): Neurotransmitter regulation of secretion by mouse hypothalamic tissue in vitro. Endocrinology 93: 626-631, 1973. Y. and J. F. McKelvy. Biosynthesis of 6. Grimm-Jbgensen, thyrotropin releasing factor by newt (triturus vkidescens) brain in r~itro. Isolation and characterization of thyrotropin releasing factor. J. Neurochem. 23: 471--478, 1974. I. Guillemin. R. Biosynthesis of the hypothalamic peptide-amide TRF. Program of-the 1st annual meeting of the society for neuroscience, p, 70 (abstract), 1971. 8. Jackson, I. M. D. and S. Reichhn. Thyrotropin releasing hormone (TRH): Distribution in hypotha~dmic and extrahypothalamic brain tissues of mammalian and submammalian chordates. Endocrinology 95: 816-824, 1974.
9. Kalnitsky, G., J. P. Hummel and C. Dierks. Some factors which affect the enzymatic digestion of ribonucleic acid. .I biol. Chem. 234:
1512-1516,
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10. Kunitz, M. Crystalline desoxyribonuclease. I. Isolation and general properties. Spectropho tometric method for the measurement of desoxyribonuclease activity. J. gen. Physiol. 33: 3499362, 19.50. 11. Krulich, L., M. Quijada, E. Hefco and D. K. Sundberg. Localization of thyrotropin-rei~ds~ng factor (TRF) in the hypothalamus of the rat. Endocrinology 95: 9917, 1974. 12. Lee, S. G., R. Roskosky, Jr., K. Bauer and t:. Lipmann. Purification of the polyenzymes responsible for tyrocidine synthesis and their dissociation into subunits. Biochemistry 12: 398-405, 1973. 13. Leggett Bailey, J. Techniques in Protein Clzemistry. New York: Clsevier Publishing Company, 1967, p. 340. I. Biosyn14. McKeivy, J. F. Biochemical neuroendocrinology. thesis of thyrotropin releasing hormone (TRH) by organ Bra&z Res 65: cultures of mammalian hypothalamus. 489%502,1974. 15. McKeivy, J. 1:. A rapid and quantitative procedure for the purification of picomote quantities of salt-free synthetic ]3H-Pro] thyrotropjn releasing hormone. ,4tzat_vt. Biochem. 64: 609-614,1975.
16. McKelvy, J. F., M. Sheridan, S. Joseph, C. H. Phelps, and S. Perrie. Biosynthesis of thyrotropin-releasing hormone in organ cultures of the guinea pig median eminence. Endocrinology 97: 908-918,197s. 17. McKelvy, J. I;., and Y. Grimm-Sjlrgensen. Hypothalamic Hormones:
Chemistry Physiology,
Pharmacology
and Clinical
Uses, edited by L. Martini. New York: Academic Press, 13-25, 1975. hor18. Mitnick, M. A. and S. Reichlin. Thyrotropin-releasing mone: Biosynthesis by rat hypothalamic fragments in vitro. Science 172: 1241-1243, 1971.
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TRH AND RNase TREATMENT
19.
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Mitnick, M. A. and S. Reichlin. Enzymatic synthesis of thyrotropin releasing hormone (TRH) by hypothalamic “TRH-synthetase.“Endocrinology 91: 1145 -1153, 1972. Oliver, C., R. L. Eskay, N. Ben-Johathan and J. C. Porter. Distribution and concentration of TRH in the rat brain.
EndocrinokJgy 95: 540-546,1974. Ozols, J. Cytochrome b, from
microsomal membranes of equine, bovine, and procine livers. Isolation and properties of preparations containing the membrane segment. Biochemistry
13: 426-434, 22.
23. 24.
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Reichtin, S.. I. M. D. Jackson, L. E, Seyler and Y. GrimmJorgensen. Frontiers in Neurology and Neuroscience Research 1974, edited by P. Seeman and G. M. Brown. Toronto: Toronto ~iniversity press. 1974, pp. 43-59.
Soffer, R. L. Peptide acceptors in the arginine transfer reaction, J. biol. Chem. 248: 2918-2921, 1973. Spear, B. D. and J. C. Gall. Independent control of ribonsomal gene replication in polytene -chronosomes of Drosophila melanoeaster. Froc. natn. Acad. Sci. U.S.A. 70: 1359-1363. 1973. ” Sunde, D., J. F. McKelvy and H. Sachs. Studies on RNA methylation and dissociation between RNA and protein biosynthesis in neural lobes of dehydrated rats. Brain Res. 47:
237-253.1972. 26.
Winters, A. J., R. L. Eskay and J. C. Porter. Concentration and distribution of TRH and LRH in the human fetal brain.
Et~docrjnol. Metub. 39: 960-963,
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Vol. I, pp. 171-175, 1976. Copyright 0 ANKHO in any form reserved. Printed in the U.S.A.
International
Inc
Biosynthesis of Thyrotropin Releasing Factor: Effect of RNase Treatment Y. GEMS-JORGENSEN
AND J. F. MC KELVY
(Received 28 November
f975)
GRIMM-JGRGENSEN, Y. AND J. P. MC KELVY. Biosynthesis of thyrotropin releasing factor: effect of RNase treatment. BRAIN RES. BULL. l(2) 171.--175, 1976. -. In the present study the role of RNA in the in vitro biosynthesis of thyrotropin releasing factor (TRF) was investigated. The biosynthesis of TRF was assessed by measuring the ability of a 1000 g supernatant fraction of newt brain origin to incorporate (3H)proline into (3H)TRF. It was found that measurable amounts of (3H)TRF are synthesized by newt brain homogenates, that preincubation of these homogenates with pro&se-free RNase cotnpletely abolisftes (3H)TRF synthesis, while preincubation of homogenates with inactivated RNase allows for (3H)TRF synthesis to proceed. These studies suggest that the presence of intact RNA is mandatory for TRF biosynthesis by newt brain homogenates. Biosynthesis
TRF
RNase
Chemicals
THE identification of mammalian thyrotropin releasing factor (TRF) as the tripeptide pGlu-His-ProNH~ [2,3/ and the subsequent availability of the synthetic tripeptide have led to numerous investigations on the mechanism of action of TRF, its distribution in various brain regions of many vertebrate species [ 8, 1 I, 20, 261 and its biosynthesis (for a review, see 1221). Reports from several laboratories have demonstrated that hypothalamic fragments from various mammals are capable of incorporating labeled precursor amino acids into TRF [5, 7, 14, 16, 191. It has also been shown that TRF biosynthesis by hypothalamic fragments in vitro is unaffected by puromycin f 19) or cycloheximid~ (71. Furthermore, it has been claimed that TRF biosynthesis can be demonstrated in a solubIe fraction from hypothalamic extracts of rat and porcine origin [ 191. The synthesis of TRF by this fraction required the presence of the amino acids Glu/Gln, His, and Pro, as well as Mg++and ATP, but was unaffected by RNase treatment. These findings suggested that TRF may be synthesized by a soluble enzyme system not requiring the presence of RNA, and the invoivement of a TRF-synthetase was hypothesized Il9l. We have recently reported the in vitro synthesis of tritium-labeied TRF by newt (Tritums viridescens) hypothalamic and forebrain fragments 161. In order to further characterize TRF synthesis by brain tissue from this amphibian species, we set out to test whether a cell-free homogenate of newt brain origin is capable of 3H-TRF synthesis
and whether
this synthesis
is dependent
(G-3 H)Proline (5 Cijmmol) was obtained from ICN Pharmaceuticals, Inc., Cleveland, Ohio and purified as described earlier [ 61. pGlu-His-(3 H)ProNH, (40 Ci/mmol) and (32P) orthophosphoric acid (carrier-free) were obtained from New England Nuclear, Boston, Mass., RNA (Yeast, type XI) RNase “a” (type Xl-A), DNA (salmon), DNase (type I), and ATP (sodium salt?, were obtained from Sigma Chemical Corp., St. Louis, MO. Procedure Assay for (” H)TRF synthesis by newt brain homogenates. Whole brains of IO newts were homogenized by hand (35 strokes, 3 revolutions each) in 0.25 ml of ice-cold HEPES(N-~-hydroxyethylpiperazine-~-2-ethanesuIfonic acid) buffered amphibian Ringer’s solution [61 at pH 7.6 with a Instrument Company, microhomogenizer (Micrometric Cleveland, Ohio) of 0.5 ml capacity. The homogenizer was rinsed with 0.25 ml of the same incubation medium. The homogenate, the rinse and a further 0.5 ml of incubation medium were then combined, and the mixture was centrifuged for 10 min at 1,000 g at 4°C. The peilet was discarded and the volume of the supernatant adjusted to 1.O ml. The supernatant was then allowed to warm to room temperature (22” C), and 10 ~1 of ATP ( 1(X3M) and 25 r Ci of purified L-f3 H)proline were added to the mixture. Immediately after the addition of the L-(3 H)proline, 200 rri of the incubation mixture were withdrawn and delivered into 2 ml of absolute methanol containing 25 gg of synthetic TRF. This aliquot served as the zero time background control sample and was subtracted from the (’ H)TRF values in all subsequent samples. This background control is of utmost importance in studies where the incorporation of labeled amino acids into labeled TRF is measured because, even after extensive purification of the
on the
presence of intact RNA. METHOD
Animals Adult newts of either sex were obtained from the Mogul Ed Corporation, Oshkosh, Wise. They were fed chopped liver, fortified with calcium, twice per week. 171
172 sample, some labeled contaminants migrate with the TRF peak in various chromatography and electrophoresis systems. At various times after the addtion of the (” H)proline, successive 200 ~1 aliquots were similarly withdrawn. At the end of the incubation period the methanolic extracts were allowed to cool for 30 min at 4°C. The amount of (” H)TRF present in each sample was then determined. The synthesis of TRF was expressed as pmoles of (” H)TRF formed/mg tissue protein. isolation of tritium labeled TRF. (‘H)TRF was extracted from the methanolic extracts of the homogenates by successive column c~omatography and eleetrophoresis at acid and alkaline pH as described in a previous publication [61. Determination of alkali-soluble tritium-labeled proteins. The amount of alkali-soluble radioactivity of the pellet remaining after methanol extraction was determined after extraction with cold and hot trichloroacetic acid and solubilization in 0.5 N NaOH [ 251. The total amount of protein in the alkali-soluble fraction was then determined as described in [ 131. Assay for RNase activity. The activity of the commercially obtained RNA was measured as described in [9] with yeast RNA as the substrate. In order to determine the amount of RNase needed to achieve adequate hydrolysis of RNA in the homogenates used in the TRF synthesis experiments, the activity of RNase was also measured in HEPES-buffered amphibian Ringer’s solution at pH 7.6 and 22°C. It was found that, under these conditions, the enzyme was much less active, and that 100 pg/ml of RNase were needed to achieve the same degree of hydrolysis of yeast RNA as was achieved by 2 rg of RNase under the assay conditions specified in [ 91. Preliminary studies also showed that, under the experimental conditions used to show (’ H)TRF synthesis, 3 2 P-labeled newt brain RNA was extensively hydrolyzed by 100 pg/ml of RNase. The 3 *P-labeled RNA had been obtained by incubation of newt brain slices in HEPES-buffered amphibian Ringer’s solution containing 100 &X/ml of (‘*~)orthophosphate and subsequent extraction of the labeled RNA as described in [24]. Because of these findings, 100 ug/ml of RNase were added to the incubation mixture when the (3H)TRF synthesis was tested. Assa_v for protease activity, The possible contamination of the RNase preparation with proteases was assayed in two ways. (1) The protease activity of the RNase was compared to that of pronase with azocoll as the substrate, (2) The ability of the RNase preparation to hydrolyze (3H)labeled newt brain proteins was compared to the ability of trypsin to hydrolyze the same proteins. Labeled newt brain proteins had been obtained by incubating brain slices in amphibian Ringer’s solution containing 100 &i/ml of a 3 H-labeled amino acid mixture, homogenizing the tissue, and c~omatographing the homogenate on a column of Sephadex G-75. The void volume peak served as the substrate. Inactivation of RNase. The RNase was inactivated by carboxymethylation of a histidine residue in the active site which leads to inactivation of the enzyme without grossly disturbing its molecular conformation (141 and personal communication). After carboxymethylation, the enzyme was chromatographed on a diethylaminoethylcellulose ion exchange resin (DE-52, hydrogen form) and the combined RNase peak lyophilized and tested for RNase activity as
GRIMM-JORGENSEN
AND MC KELVY
described in 191. It was found that the RNase inactivated by this method had lost 80%~ of its initial ability to hydrolyze RNA. To further ascertain that the carboxymethylation had been successful, an aliquot of the lyophilized material was subjected to acid hydrolysis and amino acid analysis with the Beckman Model 120 C automatic amino acid analyzer [2 1 ] The resuit from this procedure indicated that one His and two Met residues had been alkylated as expected. Determination of degradation of (‘HI-TRF by newt brain homogenates. The ability of the homogenates from the whole newt brains to degrade synthetic (” H)TRF was tested as follows: 1,000 g homogenates were obtained as described above. At zero time 10 @I of ATP ( 10-3M) were added to the homogenate. This was followed by the addition of 25 ~1 (300,000 cpm) of synthetic (“H)TRF which had been purified as described in [ 151. Aliyuots (100 ~1) were withdrawn from the incubation mixture after 10 and 20 min of incubation and delivered into 2 ml of absolute methanol. After refrige~tion overnight, the aliquots were centrifuged at 1,000 g for 20 min, and the supernatants were dried under nitrogen. The residues were suspended in 100 ~1 of absolute methanol.. and 10 ~1 aliquots of each sample were subjected to electrophoresis on cellulose acetate strips in 0.1 M sodium acetate buffer, pII 4.2, at 280 V for 60 min. The electrophoresis strips were then cut into I cm sections and dried, and the radioactivity in each section was determined after the addition of 10 ml of a toluene based scintillation mixture. The amount of radioactivity found in the area of migration of synthetic standard TRF was determined on each electrophoretogram and the amount of ( 3 H)TRF expressed as per cent of the total amount of radioactivity on the eIectrophoresis strip. RESULTS
Figure 1 shows a typical incorporation graph obtained when the 1,000 g supernatant from 10 whole brains was incubated in the presence of 25 PCi of (3H)proline. In this experiment, the rate of incorporation of (‘H)proIine into (3 H)TRF was 0.004 pmoles/min/brain. When the 1,000 g supernatant was preincubated for 15 min with RNase (100 &ml) and then its ability to incorporate (’ H)proline into (” H)TRF was studied, it was found that no (‘H)TRF was produced during the 10 min test period (Fig. 2). On the other hand, when the RNase was inactivated prior to its addition to the homogenate, the synthesis of (” H)TRF was only slightly depressed when compared to control homogenates (Fig. 3). Although the RNase preparation had been shown to be free of protease activity, the possibility was considered that this enzyme might have increased the rate of degradation of TRF, and thus have interfered with the detection of newly formed TRF. Therefore, newt brain homogenates were incubated in the presence of synthetic pClu-His(3H)ProNH, and RNase or inactivated RNase, and the rate of degradation of TRF measured. Table 1 shows that newt brain homogenates, when incubated under the conditions used for (3H)TRF synthesis only minimally degraded exogenous (“H)TRF. After 20 min of incubation, the maximum time used in the synthesis studies, only 10% of the added (‘H)TRF was degraded. The table also shows that neither active RNase nor inactivated RNase increased the rate of TRF degradation.
173
TRH AND RNase TREATMENT
MINUTES FIG, 1. Incorporation of (3Hjproline into t3H)TRF by a cell free extract from whole newt brains: Whole brains from 10 newts were homogenized by hand in a total volume of 1.0 ml of HEPESbuffered amphibian Ringer’s solution (pH 7.6). After centrifugation for 10 min at 1,000 g the supernatant was reconstituted with amphibian Ringer’s solution to a to t~$ volume of 1 .O ml and 25 r&i of (3 H)proline and 10 (~1of ATP (10 j were added. 200 ~1 aliquots were withdrawn at the times indicated and the (3HfTRF isolated. The values represent the amount of (aH)TRF (pmoles) produced by the whole extract. 0.6 0.5
i.k “0 h
0.4.
k R
0.3.
Control
RNorr lOOpa
FIG. 3. The effect of RNase and inactivated RNase on the ~corporat~on of (3H)p~~ne into (3HjTRF by a cell-free extract from whole newt brains: whole brains from 10 newts were homogenized by hand in a total volume of 3.0 ml of HEPESbuffered amphibian Ringer’s solution (pH 7.6). After centrifugation at 1,000 g, the supematant was reconstituted to a total volume of 1.0 ml with amphibian Ringer’s solution. After the addition of 100 gg of RNase, 100 pg of inactivated RNase, or an equal volume of incubation medium, the extract was preincuba ed for 15 min. Then 25 &Ji of (3H]proIine and IO PI of ATP (lo- 5Mj were added and 200 ~1 aliquots withdrawn from each tube. The incubation was continued for 10 min and a second 200 ~1 aliquot was withdrawn. (3H)TRF was then isolated as described in methods. The values represent the amount of f3H)TRF (pmoles) produced by the extracts during a 10 min period. TABLE 1 EFFECT OF RNase ON (3H)TRF DEGRADATION
;;; B 0.2. E P 0.1
Treatment
RNase
-----_c----+ 5
100 pg
10 MINUTES
FIG. 2. The effect of RNase on the incorporation of (3H)Pro into (3H)TRF by a cell-free extract from whole newt brains. Whole brains from 10 newts were homogenized by hand in a total volume of 1.0 ml of HEPES buffered amphibian Ringer’s solution (pH 7.6). After centrifugation for IO min at 1000 g, the supernatant was reconstituted to a volume of 1.0 ml. After the addition of 100 r.zgof RNase (or an equal volume of incubation medium for the control), the extract was preincub-fed for 15 min. Then 25 &i of (aH)Pro and 10 &f of ATP (10 M) were added. 200 ~1 aliquots were withdrawn from the homogenates at the times indicated. (‘HjTRF was isolated as described in methods. The values represent the amount of (‘HITRF (pmates) produced by the whok extract.
Control RNase ( 100 ~g/mI j &activated RNase (100 rgimlf
Percent of Applied Radioactivity in the TRF-area 0 min 10 min 20 min 88.8 85.3 88.0
84.5 85.6 86.9
80.6 85.4 85.1
The 1,000 g supernatants of homogenates from 10 newt brains were incubated with synthetic (3HjTRF for the time indicated. At the end of each incubation period the proteins were precipitated with methanol and the methano~ic supernatants subjected to electrop~oresis as described under methods.
DISCUSSION Preliminary studies had shown that the biosynthesis of (‘H)TRF by newt hypothalamic fragments in vitro is not influenced by the protein synthesis inhibitors cyefoheximide, c~o~rnp~e~i~o~, and diphtheria toxin f 17 1,
173
confirming previous reports from other laboratories [ 7,191. However. these findings cannot definitely rule out a ribosomal participation in the synthesis of this peptide. since the residual amount of protein synthesis which proceeds in the presence of the protein synthesis inhibitors may be sufficient to account for the small amount of TRF synthesized during the incubation period. To further study the possible role of RNA in TRF synthesis, the effect of RNase treatment on the incorporation of (” H)TRF by a cell-free newt brain homogenate was investigated in this study. While (” H)TRF was formed by control homogenates, no (” H)TRF was produced by homogenates which had been ~reincubated with RNase prior to the addition of (” H)proline. The effect of RNase cannot be attributed to a non-specific chemical property of this enzyme, e.g. its polycationic nature, since inactivated RNase, which differs from RNase only in 3 amino acid residues, did not have a similar effect on TRF synthesis. The slight inhibition of (“H)TRF synthesis which was observed when the homogenate was exposed to inactivated RNase prior to the addition of (3H)prohne (Fig. 3) can be attributed to the fact that. after carboxymethylation, the enzyme had retained approximately 20% of its activity. It is also unlikely that the abolition of (3 H)TRF synthesis by RNase is due to contamination of the pancreatic enzyme with proteases, since no such activity could be detected when the enzyme was tested in 2 protease assays using either azocoll or tritium-labeled newt brain-proteins as the substrates. Since it has been reported that in crude preparations of
Bacillus
brevis, DNase treatment is capable of dissociating enzyme complex responsible for tyrocidine synthesis [ 131. the possibility ihat the effect of RNase on TRF biosynthesis may be due to contaminating DNase was considered. However, it was found that the RNasz was devoid of such activity. The possibility that RNase may have Increased the degradation of TRF was ruled out, since no such effect was observed when homogenates were incubated with Rnase and tritium labeled synthetic TRF. While these studies suggest that RNA is involved in the synthesis of TRF, they cannot explain the mechanism of this involvement. It is possible that TRF synthesis proceeds via a ribosomal mechanism and that the failure to observe inhibition of TRF biosynthesis in vitro in the presence of various protein synthesis inhibitors is due to the residual ongoing protein synthesis. On the other hand TRF biosynthesis may be non-ribosomal, but requiring RNA. Several possibifities for such an RNA requirement exist. For example, RNA may be involved in maintaining the structural integrity of the TRF synthesizing enzymes. On the other hand, it is possible that the addition of one or more amino acids to the growing molecule involves the participation of an aminoacyl-t-RNA-transferase. It has been demonstrated that amino acids can be added to dipeptides by such enzymes 1231 and that aminoacyl-t-RNAtransferase activity is present in brain homogenates [ I I. Further studies will be necessary to detertnine which of these, or other, mechanisms are operating in TRF hiosynthesis.
the
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