- Email: [email protected]
S35 methionine metabolism in rat dentine
Arch. oral Bid. VoLlO. pp.291-297. 1965. Pergamon Press Ltd. Rited in Gt. Britain
Ss5 METHIONINE
METABOLISM
IN RAT DENTINE
G. NIKIFORUKand A. M. HUNT Division of Dental Research, Faculty of Dentistry, University of Toronto Summary-Purified dentine, from young rats injected with Sa5methionine, was analysed. Radioactivity was detected in increasing intensity in the inorganic diffusate and the soluble (presumed to be chondroitin sulphate and protein) and insoluble (primarily collagen) organic components. Total organic and ester Sa6 were measured in barium sulphate precipitates from combusted samples. The precipitates from hydrolysates indicated the levels of S’ in ester sulphate (presumed to be chondroitin sulphate) to be one-seventh and over one-half of the total activity in the insoluble and soluble fractions, respectively. Chromatograms of protein hydrolysates indicated that activity was localized to a single area having an r.f. similar to methionine sulphone (hydrolytic product of methionine). INTRODUCTION THE PRESENCE of
sulphur containing compounds in the organic moiety of teeth was first suggested by histochemical studies which demonstrated metachromasia in dentine and enamel (HOLMGREN;1940; WISLOCKI, SINGER and WALDO, 1948; WISLOCKI and SOGNNAES, 1950; BEVELAN& and JOHNSON,1955). Metachromasia was interpreted as indicating the presence of sulphated mucopolysaccharides (LISON, 1936; SYLVEN, 1941). Such specificity in interpretation is no longer warranted since the finding that metachromasia depends on the availability of consecutive, anionic groups along a carbohydrate chain (CURRAN,1961). The most common functional anionic groups are sulphate, carboxyl and probably phosphoric acid. Biochemical findings supporting the above were reported by P~NCUS(1948) who prepared a fraction from dentine that qualitatively resembled chondroitin sulphuric acid. ROGERS(1949) and STACK(1951) estimated that dentine contains about 0.27; chondroitin sulphuric acid on the basis of its hexosamine content. Both glucosamine and galactosamine have been reported in dentine (NIKIFORUK, BURG= and M~CLAREN, 1959). HESS and LEE (1952) isolated chondroitin sulphuric acid from dentine with a yield of 064%. Studies utilizing sulphur (S%) labelled compounds have extended the above findings. Sulphur, particularly in the form of sulphate esters, has been found to be widely distributed in animal tissues, including bones and teeth (DZIEWIATKOWSKI, 1962). Radioactive sulphate ion is known to be incorporated in the synthesis of sulphated mucopolysaccharides (DZIEWIATKOWSKI, 1951; BOSTR~~M, 1952, DZIEWIATKOWSKI, et al., 1957; SUZUKI and STROMINGER, 1960a, b, and c) and taurine, but not in the synthesis of cystine or methionine (TARVERand SCHMIDT, 1939;BOSTR~~M and AQWT, 1952). BBLANGER(1955) reported that when Ss6 sulphuric acid was 291
292
G. NIKIFORUKAND
A.M.Hum
administered to rats, the radioactivity could be detected in autoradiographs of preenamel, and young enamel, only to disappear gradually as the organic matrix calcified. Also, he observed a rapid incorporation of S36into a hyaluronidase labile matrix in dentine, new bone, and cartilage. Using S35 labelled methionine, BBLANGER (1956) reported that radioactivity could be demonstrated in autoradiographs of the matrices of enamel and dentine. Since hydrolysates of collagen show little methionine (O-7%), the author considered activity in dentine to be due primarily to glycoproteins. GREULICH (1956) and GREULICHand FRIBERG(1957) showed that after ethylenediamine tetracetate demineralization, hyaluronidase removed the S3” label from matrices of cartilage, enamel and dentine. A study was made of the into poration of S3j into bones, cartilage and teeth followmg injection of inorganic sulphur, or organic albumin, and evidence of labelling was found in protein, in the sulphated polysaccharides, and in sulphate. (CREMERand DITTMANN,1956; DITTMANNand CREMER, 1956). The objective of the present study was to determine if repeated loading with radioactive methionine would label rat dentine sufficiently to permit chemical studies of S3j labelled components in this tissue. Repeated injections were considered necessary in view of the low level of sulphur-containing amino acids in collagen, and the low concentration (0.6%) of chondroitin sulphuric acid in dentine (HESS and LEE, 1952). Also, an attempt was made to isolate the main radioactive component of the insoluble and soluble organic dentine fractions. The chemical studies would thus serve to extend previously published S36autoradiographic studies. METHODS AND MATERIALS Thirty 4-day-old rats from three litters were injected intraperitoneally with 0.5 PC of S35methionine per g of body wt on each of 7 consecutive days. The animals were sacrificed when 17 days old at which age the teeth could be easily removed and subsequently purified. The first and second molars and incisors of each jaw were removed, dried at 60°C for 24 hr, pulverized, and the enamel and dentine separated and purified by a flotation procedure using bromoform-acetone mixtures (MANLY and HODGE, 1939). The powdered enamel and dentine were first purified using two successive flotations of bromoform-acetone of specific gravity 2.69. Dentine, recovered as the floating material from the above, was further subjected to two successive purifications using bromoform-acetone mixtures of specific gravity 2.46 and 2.00. This latter procedure separates dentine from cementum and soft tissue. The purified dentine was demineralized, in a dialysis membrane, against 0.1 N hydrochloric acid for 2 days, and subsequently dialysed against distilled water for 24 hr. The diffusate was dried in an oven at 85”C, and the radioactivity determined. The non-dialysable fraction was separated into soluble and insoluble components by centrifugation or by use of a millipore filter. Hydrolysis of each organic fraction was carried out in vials to which was added 6 N hydrochloric acid. The vials were then evacuated, sealed and hydrolysrd at 11O’C for 24 hr. Hydrolysates were dried at 40°C and redissolved in 10 ‘1” isopropyl alcohol. Hydrolysates to be chromatographed were transferred to filter paper (Whatman No. 3 MM, 18 x 22 in.) in 0.01 ml
!?
METHIONINE
METABOLISM
IN RAT DENTINE
293
aliquots. Each aliquot was allowed to dry before the next was applied. Since filter paper absorbs the weak j? emissions of Ss6, it was necessary to transfer several aliquots to the filter paper until the activity of the spot on the paper was about 200 c.p.m. The chromatogram was then double developed in butanol-acetic acid-water (250: 60:25Ov/v/v), cut into appropriate strips, and scanned to locate the radioactive areas. The relative activity of SW in the organic moiety of dentine, and in the sulphate ester of chondroitin sulphuric acid, was determined by preparing barium sulphate precipitates from hydrolysed and non-hydrolysed fractions. Total sulphur was determined by converting the organic sulphur to the inorganic form by the Schijniger combustion technique as modified by LYSYJand ZAREMBO(1958). The procedure for combustion of samples was as follows. Organic samples to be combusted were weighed on filter paper, placed in a platinum spiral, ignited and then inserted into a 500 ml iodine flask previously flushed with oxygen and containing 10 ml of 0.1 N NaOH and four drops of 30 % hydrogen peroxide. After the sample was burned completely, the flask was shaken vigorously for a few minutes and then let stand for 10 min, in order to absorb the products of combustion. Two or three drops of phenolphthalein were added, the sample boiled. and 0.2 N nitric acid added drop-wise until the solution remained acid. The sample was concentrated to about 5 ml and cooled. One ml of carrier ammonium sulphate (5 mg), 2 ml of acetate buffer (pH 4.0,O.l M), 10 ml of 50 % ethyl alcohol, and 30 mg of barium chloranilate were added to the flask. The contents were shaken, and filtered through a millipore filter. The active Ss6, precipitated on a millipore filter disc as barium sulphate, was mounted on a planchet, dried at 60°C and counted. Barium sulphate precipitates, containing the ester Ss5sulphate only, were prepared from hydrolysates as above, except that the combustion of the sample was omitted. Radioactivity of all powdered or dried samples was measured in a stainless steel planchet (7 cma), using a gas flow system with a micromil window. The location of radioactivity on chromatograms was determined by using a radiochromatogram scanner. RESULTS
Removal and pulverization of all the first and second molars and incisors from thirty rats yielded approximately 1.5 g of powdered tooth substance. After purification, approximately 1.0 g of pure dentine and 50 mg of enamel were obtained. The activity of an infinitely thick sample of undemineralized dentine (135 mg/cm%) was 3910 c.p.m. Activities of the insoluble (21 mg/cm2) and soluble (1.4 mg/cm*) organic fractions from, demineralized dentine are indicated in Table 1. The insoluble organic portion of dentine and a Ss5 methionine standard were hydrolysed separately, and the hydrolysates chromatographed. A scan of these chromatographs is reproduced in Fig. 1. Only one peak occurred at the r.f. of methionine sulphone. The hydrolysis conditions used in this study resulted in conversion of methionine to methionine sulphone. A scan of a chromatogram from a hydrolysate of the dentine insoluble organic fraction, to which was added some G
294
G. NIKIFORUK
AND
A. M. HUNT
methionine P, again revealed only one principal area of activity. were obtained with the soluble organic portion. TABLE 1. Ss5 ACTMTYOFORGANICFRACTIONSOP
Insoluble organic Soluble organic
Weight (mg)
Measured activity (c.p.m.)
148.7 10
14,960 4,620
Similar results
DENTINE
Activity (c.p.m.) corrected for self absorption 59,840 5.430
Purified dentine powder was demineralized in a dialysis membrane against 0.1 N HCl. The nondialysable fraction was separated into a soluble and insoluble organic fraction by centrifugation. The radioactivity was measured by a Geiger-Mueller gas flow counter.
Activity of barium sulphate precipitates prepared from hydrolysates, and therefore containing the hydrolysable ester sulphate only, and from combusted samples containing the total sulphur, was determined in the soluble and insoluble organic fractions (Table 2). TABLE 2. Ac~rvrru(c.p.m.)
OF BaSa601 PRECIPITATESFROMSOLUBLEANDINSOLUBLEORGANIC FRACTIONS
Insoluble organic Schiiniger Hydrolysate Combusted Fraction (Total Sulphur) (Ester Linked Sulphur) (equal wt.) 616 442
Soluble organic Schiiniger Combusted Fraction Hydrolysate (Total Sulphur) (Ester Linked Sulphur) (equal vol.)
76 64
34 -
18 -
One-half portion of the insoluble and soluble organic fraction was combusted by the Schoniger (1955) technique to convet t organic sulphur to the inorganic fotm. The other half portion of each fraction was hydrolysed, thus releasing the weakly bound ester sulphates. Barium eulphate precipitates were ptepared from each fraction, as described in the text, and the relative activity of the total and ester linked sulphate determined.
The diffusate, obtained after demineralizing the dentine powder, consisted primarily of inorganic salts and a small amount of diffusible organic components. This fraction was evaporated to dryness and the activity of the infinitely thick sample was determined to be 159 c.p.m. When redissolved and subjected to a barium chloride precipitation, an active precipitate (243 c.p.m.j, assumed to be P in inorganic form, was obtained. The organic components of the diffusate were not studied; DISCUSSION
In dcntine sulphur is primarily a component of the protein and the chondroitin sulphuric acid fractions. The amount of methionine in dentine collagen hydrolysates is about O*7Oo/o(PIE& 1963), and the quantity of mucopolysaccharide in dentine powder is approximately 0.6% (HESS and LEE, 1952) of which about 5.27% is
s”
METHIONINE
METAROUSM
IN RAT
DENTINE
295
sulphur. Although the. sulphur content of these tissues is low, intraperitoneal injections of SU methionine into Cday-old rats at a level of 03 PC/g of body weight, on each of 7 consecutive days, resulted in a sufficient accumulation of !3= compounds to permit studies of sulphur metabolism in rat dentine. Since the mineral phase absorbed a significant number of the weak S3s p emissions, a relatively higher activity was detected in the demineralized as compared to the mineralized dentine powder. Radioactivity was detected in the demineralized insoluble and soluble organic moieties and in the diffusate. Activity in the insoluble organic matrix was about 10 times greater than in the soluble organic component. Hydrolysis of the insoluble fraction, and subsequent separation of the amino acid components of the hydrolysate by paper chromatography, yielded a radioactive spot having an r.f. corresponding to methionine sulphone. No other peaks were observed. Highly active barium sulphate precipitates were prepared from hydrolysates of the soluble and insoluble organic fractions. The weak ester linkages of chondroitin sulphuric acid would be readily broken under the conditions of hydrolysis used in this study, and it was assumed that the chondroitin sulphuric acid was the major source of the Sas in these precipitates. Precipitates from cornbusted samples indicated the total sulphur activity. In the insoluble organic component, activity residing in the ester linkages contributed only one-seventh of the total activity; however, over half of the ,F activity was present in ester sulphate in the soluble component. These results show that methionine Sss is incorporated into the collagen of dentine and into the chondroitin sulphate and its protein complex, and that activity is not primarily due to glycoprotein as was suggested by the autoradiographic interpretations (BBLANGER,1956). This work. will be extended to a study of the sulphur metabolism of immature enamel. R--De
la dentine purifi&e provenant de jeunes rats, inject& avec de la methionine radioactive S 35, est a&y&e. Une intensit6 croissante de radioactivite est no& dans la fraction diffusante inorganique et les composes organiques solubles (probablement de nature chrondroitine sulfate et proteinique) et insoluble (essentiellement collagenique). Le S 35 total organique et esterifie est mesure dam des precipitb de sulfate de baryum, obtenus a partir d’echantillons calcines. Les precipitb d’hydrolysats donnent des quantit&s de S 35 dans les sulfates esterifib (constitues vraisemblablement de chondroitine sulfate) de l/7 et plus de la moitit de l’activite totale dans les fractions insoluble et soluble respectivement. Des chromatogrammes d’hydrolysats proteiniques indiquent que l’activite est localis& a une seule region ayant un r.f. semblable au sulfone de mtthionine (produit hydrolytique de la mbthionine). Zusamtn&m--Es wurde gereinigtes Dentin von jungen Ratten analysiert, die mit “S-Methionin injiziert worden waren. Radioaktivitat ansteigender Intensitiit wurde im anorganischen Diffusat und in den liislichen (vermutlich Chondroitinsulfat und Protein) und unl&lichen (in enter Linie Kollagen) organ&hen Komponenten festgestellt. Das gesamte organische und Ei.ster-85S wurde in Rariumsulfatniederschl&gen aus veraschten Proben bestimmt. Die Niederschlage aus Hydrolysaten zeigten, daj3 die Mengen von =S im Estersulfat (vermutlich Chondroitinsulfat) in den unloslichen und l&lichen Fraktionen vorhanden sind. Chromatogramme von ProteinHydrolysaten wiesen darauf hin, da8 die Aktivitat in einem einzigen Fleck lokalisiert war, der einen Rt-Wet-t iihnlich wie Methionin-sulfon (hydrolytisches Produkt aus Methionin) besitzt.
296
G. NIKIFORUKAND A. M. HUNT REFERENCES
B~LANGER,L. F. 1955. Autoradiographic detection of radiosulfate incorporation by the grobing enamel of rats and hamsters. J. dent. Res. 34 20-27. BELANCER,L. F. 1956. Autoradiographic studies of the formation of the organic matrix of cartilage, bone and the tissues of teeth. Ciba Found. Symposium on Bone Structure and Metabolism. p. 75-87. BEVELANDER, G. and JOHNSON,P. L. 1955. The localization of polysaccharides in the developing teeth. J. dent. Res. 34, 123-131. B~STR~M, H. 1952. On the metabolism of the sulfate group of chondroitin sulfuric acid. J. biol. Chem. 196, 47748 I. BOSTR~M,H. and AQVLW, S. 1952. Utilization of Ss5 labelied sodium sulfate in the synthesis of chondroitin sulfuric acid, taurine, methionine and cystine. Acta them. stand. 6, 1557-1559. CREMER,H. D. and D~I-~MANN,G. 1956. Einbau von organischem (Eiweiss-) und anorganischem (Sulfat-) Schwefel in Knorpel, Knochen und Zahnen. [Incorporation of organic (protein) and inorganic (sulfate) sulfur in bones, cartilage and teeth.] Biochem. Z. 327, 377-382. CURRAN, R. C. 1961. The histological demonstration of connective tissue mucopolysaccharides. Biochem. sot. Symposia 20, 24-38. DITTMANN,G. and CREMER,H. D. 1956. Chondroitinschwefelsaiire in Knorpel, Knochen und Ziihnen. [Chondroitin sulfuric acid in cartilage, bones and teeth.] Biochem. Z. 327, 368-376. DZIEWIATKOWSKI,D. D. 1951. Isolation of chondroitin sulfate Sa5 from articular cartilage of rats. J. biof. Chem. 189, 187-190. DZIEWIATKOWSKI,D. D. 1962. Autoradiographic studies of bone growth with P sulfate. Radioisotopes and Bone Symposium. p. 277. Blackwell Scientific, Oxford. DZIEWIATKOWSKI,D. D., DI FERRAN~E,N., BRONNER,F. and OKINAKA, G. 1957. Turnover of YS sulfate in epiphyses and diaphyses of suckling rats; nature of the Y3 labelled compounds. J. exp. Med. 106.509-524. GREULICH,R. C. 1956. Contribution of sulfate ion to the formation of organic bone matrix. (abs.) Anat. Rec. 124,404405. GREULICH,R. C. and FRIBERG,U. 1957. Histochemical studies of sulfomucopotysaccharides in-the organic matrices of mineralized tissues. Exp. Cell Res. 12, 685-689. HESS,W. C. and LEE, C. 1952. The isolation of chondroitin sulfuric acid from dentin. J. dent. Res. 31,793-797. HOLMGREN,H. 1940. Studien tiber Verbreuung und Bedeutung der chromotropen substanz. Z. mikr.-anat. Forsch. 47.489-521. LLWN, L. 1936. Histochemie AnimaIe. Guathier-Villars, Paris. LYSYJ, I. and ZAREMBO,J. E. 1958. Rapid quantitative determination of sulfur in organic compounds. Analyt. Chem. 30, 428430. MANLY, R. S. and HODGE, H. C. 1939. Density and refractive index studies of dental hard tissues. J. dent. Res. 18. 133-141. NIKIFORUK, G., BURGESS,R. C. and MACLAREN,C. 1959. Amino sugars in dentin and enamel. J. dent. Res. 38, 675. PIEZ. K. A. 1963. The amino acid chemistry of some calcified tissues. Ann. N. Y. Acad. Sci. 109,256. PINCUS, P. 1948. Relation of enamel protein to dental caries.
Nature, Land. 161, 1014. and distribution of polysaccharides in human cortical bone and dentin of teeth. Nature. Land. 164, 625-626. STACK, M. V. 1951. Organic constituents of dentine. Brit. dent. J. 90, 173-181. SUZUKI, S. and STROMINGER. J. L. 1960. Enzymatic sulfation of mucopolysaccharides in hen oviduct. 1. Transfer of sulfate from 3,-phosphoadenosine 5’-phosphosulfate to mucopolysaccharides. J. biol. Chem. US, 257-266. SUZUKI, S. and STROMINGER, J. L. 1960. Enzymatic sulfation of mucopolysaccharides in hen oviduct. II. Mechanism of the reaction studied with oligosacchatides and monosaccharides as acceptors. J. biol. Chem. 235, 267-273.
ROGERS,H. J. 1949. Concentration
SM METHlONINE METABOLISMIN RAT DENTINE Suzu~r.
S.
297
and STROMDJOER, J. L. 1960. Enzymatic sulfation of mucopolysaccharides in hen oviduct. III. Mechanism of sulfation of chondroitin and chondroitin sulfate A. /. biol. Gem. 235,274276. SYLVEN, B. 1941. f_ber das Vorkommen von hochmolekularen Esterschwefelsiiuren im Granulationsgewebe und bei der Epithelregeneration: Experimentelle und pathiologisch-anatomische Untersuchungen tiber das Granulationsgewebe und die Regeneration von Plattenepithel mit besonderer Beriicksichtigung des Vorkommens und der Bedeutung auf&tender hochmolekularer Eaterschwefelsiiuren und Mastzellen. Acta chir. scund. 86, l-l 51. TARVER, H. and Scrn.rm~, C. L. A. 1939. The conversion of methionine to cystine: Experiments with radioactive sulfur. J. biol. Gem. 130,67-80. WISLOCKI,.G. B.. SINGER, M. and WALDO, C. M. 1948. Some histochemical reactions of mucopolysaccharides, glycogen. lipids and other substances in teeth. Anat. Rec. 101.487-5 14. WLSLOCKI,G. B. and SOGNNAES, R. F. 1950. Histochemical reactions of normal teeth. Amer. J. Anor. 87, 239-275.
PLATE 1 OVERLEAF
s3' METHIONINE METABOLISM IN RAT DENTINE
i___r__r_..*- .
.. - ._ I-
._
:--
,*
d+&,W . _
___i
FIG. 1. Scan of two paper chromatograms containing S3” methionine standard (top scan) and the hydrolysate of a dentine insoluble organic fraction lsbelled with Y5 methionine in viva (bottom scan). The single radioactive peak corresponds to the r.f. of methionine sulphone (hydrolytic product of methionine). The slight discrepancy in the peaks is due to the s!cewing of the solvent front.
f.p. 298
PLATE
1
Ss5 METHIONINE
METABOLISM
IN RAT DENTINE
G. NIKIFORUKand A. M. HUNT Division of Dental Research, Faculty of Dentistry, University of Toronto Summary-Purified dentine, from young rats injected with Sa5methionine, was analysed. Radioactivity was detected in increasing intensity in the inorganic diffusate and the soluble (presumed to be chondroitin sulphate and protein) and insoluble (primarily collagen) organic components. Total organic and ester Sa6 were measured in barium sulphate precipitates from combusted samples. The precipitates from hydrolysates indicated the levels of S’ in ester sulphate (presumed to be chondroitin sulphate) to be one-seventh and over one-half of the total activity in the insoluble and soluble fractions, respectively. Chromatograms of protein hydrolysates indicated that activity was localized to a single area having an r.f. similar to methionine sulphone (hydrolytic product of methionine). INTRODUCTION THE PRESENCE of
sulphur containing compounds in the organic moiety of teeth was first suggested by histochemical studies which demonstrated metachromasia in dentine and enamel (HOLMGREN;1940; WISLOCKI, SINGER and WALDO, 1948; WISLOCKI and SOGNNAES, 1950; BEVELAN& and JOHNSON,1955). Metachromasia was interpreted as indicating the presence of sulphated mucopolysaccharides (LISON, 1936; SYLVEN, 1941). Such specificity in interpretation is no longer warranted since the finding that metachromasia depends on the availability of consecutive, anionic groups along a carbohydrate chain (CURRAN,1961). The most common functional anionic groups are sulphate, carboxyl and probably phosphoric acid. Biochemical findings supporting the above were reported by P~NCUS(1948) who prepared a fraction from dentine that qualitatively resembled chondroitin sulphuric acid. ROGERS(1949) and STACK(1951) estimated that dentine contains about 0.27; chondroitin sulphuric acid on the basis of its hexosamine content. Both glucosamine and galactosamine have been reported in dentine (NIKIFORUK, BURG= and M~CLAREN, 1959). HESS and LEE (1952) isolated chondroitin sulphuric acid from dentine with a yield of 064%. Studies utilizing sulphur (S%) labelled compounds have extended the above findings. Sulphur, particularly in the form of sulphate esters, has been found to be widely distributed in animal tissues, including bones and teeth (DZIEWIATKOWSKI, 1962). Radioactive sulphate ion is known to be incorporated in the synthesis of sulphated mucopolysaccharides (DZIEWIATKOWSKI, 1951; BOSTR~~M, 1952, DZIEWIATKOWSKI, et al., 1957; SUZUKI and STROMINGER, 1960a, b, and c) and taurine, but not in the synthesis of cystine or methionine (TARVERand SCHMIDT, 1939;BOSTR~~M and AQWT, 1952). BBLANGER(1955) reported that when Ss6 sulphuric acid was 291
292
G. NIKIFORUKAND
A.M.Hum
administered to rats, the radioactivity could be detected in autoradiographs of preenamel, and young enamel, only to disappear gradually as the organic matrix calcified. Also, he observed a rapid incorporation of S36into a hyaluronidase labile matrix in dentine, new bone, and cartilage. Using S35 labelled methionine, BBLANGER (1956) reported that radioactivity could be demonstrated in autoradiographs of the matrices of enamel and dentine. Since hydrolysates of collagen show little methionine (O-7%), the author considered activity in dentine to be due primarily to glycoproteins. GREULICH (1956) and GREULICHand FRIBERG(1957) showed that after ethylenediamine tetracetate demineralization, hyaluronidase removed the S3” label from matrices of cartilage, enamel and dentine. A study was made of the into poration of S3j into bones, cartilage and teeth followmg injection of inorganic sulphur, or organic albumin, and evidence of labelling was found in protein, in the sulphated polysaccharides, and in sulphate. (CREMERand DITTMANN,1956; DITTMANNand CREMER, 1956). The objective of the present study was to determine if repeated loading with radioactive methionine would label rat dentine sufficiently to permit chemical studies of S3j labelled components in this tissue. Repeated injections were considered necessary in view of the low level of sulphur-containing amino acids in collagen, and the low concentration (0.6%) of chondroitin sulphuric acid in dentine (HESS and LEE, 1952). Also, an attempt was made to isolate the main radioactive component of the insoluble and soluble organic dentine fractions. The chemical studies would thus serve to extend previously published S36autoradiographic studies. METHODS AND MATERIALS Thirty 4-day-old rats from three litters were injected intraperitoneally with 0.5 PC of S35methionine per g of body wt on each of 7 consecutive days. The animals were sacrificed when 17 days old at which age the teeth could be easily removed and subsequently purified. The first and second molars and incisors of each jaw were removed, dried at 60°C for 24 hr, pulverized, and the enamel and dentine separated and purified by a flotation procedure using bromoform-acetone mixtures (MANLY and HODGE, 1939). The powdered enamel and dentine were first purified using two successive flotations of bromoform-acetone of specific gravity 2.69. Dentine, recovered as the floating material from the above, was further subjected to two successive purifications using bromoform-acetone mixtures of specific gravity 2.46 and 2.00. This latter procedure separates dentine from cementum and soft tissue. The purified dentine was demineralized, in a dialysis membrane, against 0.1 N hydrochloric acid for 2 days, and subsequently dialysed against distilled water for 24 hr. The diffusate was dried in an oven at 85”C, and the radioactivity determined. The non-dialysable fraction was separated into soluble and insoluble components by centrifugation or by use of a millipore filter. Hydrolysis of each organic fraction was carried out in vials to which was added 6 N hydrochloric acid. The vials were then evacuated, sealed and hydrolysrd at 11O’C for 24 hr. Hydrolysates were dried at 40°C and redissolved in 10 ‘1” isopropyl alcohol. Hydrolysates to be chromatographed were transferred to filter paper (Whatman No. 3 MM, 18 x 22 in.) in 0.01 ml
!?
METHIONINE
METABOLISM
IN RAT DENTINE
293
aliquots. Each aliquot was allowed to dry before the next was applied. Since filter paper absorbs the weak j? emissions of Ss6, it was necessary to transfer several aliquots to the filter paper until the activity of the spot on the paper was about 200 c.p.m. The chromatogram was then double developed in butanol-acetic acid-water (250: 60:25Ov/v/v), cut into appropriate strips, and scanned to locate the radioactive areas. The relative activity of SW in the organic moiety of dentine, and in the sulphate ester of chondroitin sulphuric acid, was determined by preparing barium sulphate precipitates from hydrolysed and non-hydrolysed fractions. Total sulphur was determined by converting the organic sulphur to the inorganic form by the Schijniger combustion technique as modified by LYSYJand ZAREMBO(1958). The procedure for combustion of samples was as follows. Organic samples to be combusted were weighed on filter paper, placed in a platinum spiral, ignited and then inserted into a 500 ml iodine flask previously flushed with oxygen and containing 10 ml of 0.1 N NaOH and four drops of 30 % hydrogen peroxide. After the sample was burned completely, the flask was shaken vigorously for a few minutes and then let stand for 10 min, in order to absorb the products of combustion. Two or three drops of phenolphthalein were added, the sample boiled. and 0.2 N nitric acid added drop-wise until the solution remained acid. The sample was concentrated to about 5 ml and cooled. One ml of carrier ammonium sulphate (5 mg), 2 ml of acetate buffer (pH 4.0,O.l M), 10 ml of 50 % ethyl alcohol, and 30 mg of barium chloranilate were added to the flask. The contents were shaken, and filtered through a millipore filter. The active Ss6, precipitated on a millipore filter disc as barium sulphate, was mounted on a planchet, dried at 60°C and counted. Barium sulphate precipitates, containing the ester Ss5sulphate only, were prepared from hydrolysates as above, except that the combustion of the sample was omitted. Radioactivity of all powdered or dried samples was measured in a stainless steel planchet (7 cma), using a gas flow system with a micromil window. The location of radioactivity on chromatograms was determined by using a radiochromatogram scanner. RESULTS
Removal and pulverization of all the first and second molars and incisors from thirty rats yielded approximately 1.5 g of powdered tooth substance. After purification, approximately 1.0 g of pure dentine and 50 mg of enamel were obtained. The activity of an infinitely thick sample of undemineralized dentine (135 mg/cm%) was 3910 c.p.m. Activities of the insoluble (21 mg/cm2) and soluble (1.4 mg/cm*) organic fractions from, demineralized dentine are indicated in Table 1. The insoluble organic portion of dentine and a Ss5 methionine standard were hydrolysed separately, and the hydrolysates chromatographed. A scan of these chromatographs is reproduced in Fig. 1. Only one peak occurred at the r.f. of methionine sulphone. The hydrolysis conditions used in this study resulted in conversion of methionine to methionine sulphone. A scan of a chromatogram from a hydrolysate of the dentine insoluble organic fraction, to which was added some G
294
G. NIKIFORUK
AND
A. M. HUNT
methionine P, again revealed only one principal area of activity. were obtained with the soluble organic portion. TABLE 1. Ss5 ACTMTYOFORGANICFRACTIONSOP
Insoluble organic Soluble organic
Weight (mg)
Measured activity (c.p.m.)
148.7 10
14,960 4,620
Similar results
DENTINE
Activity (c.p.m.) corrected for self absorption 59,840 5.430
Purified dentine powder was demineralized in a dialysis membrane against 0.1 N HCl. The nondialysable fraction was separated into a soluble and insoluble organic fraction by centrifugation. The radioactivity was measured by a Geiger-Mueller gas flow counter.
Activity of barium sulphate precipitates prepared from hydrolysates, and therefore containing the hydrolysable ester sulphate only, and from combusted samples containing the total sulphur, was determined in the soluble and insoluble organic fractions (Table 2). TABLE 2. Ac~rvrru(c.p.m.)
OF BaSa601 PRECIPITATESFROMSOLUBLEANDINSOLUBLEORGANIC FRACTIONS
Insoluble organic Schiiniger Hydrolysate Combusted Fraction (Total Sulphur) (Ester Linked Sulphur) (equal wt.) 616 442
Soluble organic Schiiniger Combusted Fraction Hydrolysate (Total Sulphur) (Ester Linked Sulphur) (equal vol.)
76 64
34 -
18 -
One-half portion of the insoluble and soluble organic fraction was combusted by the Schoniger (1955) technique to convet t organic sulphur to the inorganic fotm. The other half portion of each fraction was hydrolysed, thus releasing the weakly bound ester sulphates. Barium eulphate precipitates were ptepared from each fraction, as described in the text, and the relative activity of the total and ester linked sulphate determined.
The diffusate, obtained after demineralizing the dentine powder, consisted primarily of inorganic salts and a small amount of diffusible organic components. This fraction was evaporated to dryness and the activity of the infinitely thick sample was determined to be 159 c.p.m. When redissolved and subjected to a barium chloride precipitation, an active precipitate (243 c.p.m.j, assumed to be P in inorganic form, was obtained. The organic components of the diffusate were not studied; DISCUSSION
In dcntine sulphur is primarily a component of the protein and the chondroitin sulphuric acid fractions. The amount of methionine in dentine collagen hydrolysates is about O*7Oo/o(PIE& 1963), and the quantity of mucopolysaccharide in dentine powder is approximately 0.6% (HESS and LEE, 1952) of which about 5.27% is
s”
METHIONINE
METAROUSM
IN RAT
DENTINE
295
sulphur. Although the. sulphur content of these tissues is low, intraperitoneal injections of SU methionine into Cday-old rats at a level of 03 PC/g of body weight, on each of 7 consecutive days, resulted in a sufficient accumulation of !3= compounds to permit studies of sulphur metabolism in rat dentine. Since the mineral phase absorbed a significant number of the weak S3s p emissions, a relatively higher activity was detected in the demineralized as compared to the mineralized dentine powder. Radioactivity was detected in the demineralized insoluble and soluble organic moieties and in the diffusate. Activity in the insoluble organic matrix was about 10 times greater than in the soluble organic component. Hydrolysis of the insoluble fraction, and subsequent separation of the amino acid components of the hydrolysate by paper chromatography, yielded a radioactive spot having an r.f. corresponding to methionine sulphone. No other peaks were observed. Highly active barium sulphate precipitates were prepared from hydrolysates of the soluble and insoluble organic fractions. The weak ester linkages of chondroitin sulphuric acid would be readily broken under the conditions of hydrolysis used in this study, and it was assumed that the chondroitin sulphuric acid was the major source of the Sas in these precipitates. Precipitates from cornbusted samples indicated the total sulphur activity. In the insoluble organic component, activity residing in the ester linkages contributed only one-seventh of the total activity; however, over half of the ,F activity was present in ester sulphate in the soluble component. These results show that methionine Sss is incorporated into the collagen of dentine and into the chondroitin sulphate and its protein complex, and that activity is not primarily due to glycoprotein as was suggested by the autoradiographic interpretations (BBLANGER,1956). This work. will be extended to a study of the sulphur metabolism of immature enamel. R--De
la dentine purifi&e provenant de jeunes rats, inject& avec de la methionine radioactive S 35, est a&y&e. Une intensit6 croissante de radioactivite est no& dans la fraction diffusante inorganique et les composes organiques solubles (probablement de nature chrondroitine sulfate et proteinique) et insoluble (essentiellement collagenique). Le S 35 total organique et esterifie est mesure dam des precipitb de sulfate de baryum, obtenus a partir d’echantillons calcines. Les precipitb d’hydrolysats donnent des quantit&s de S 35 dans les sulfates esterifib (constitues vraisemblablement de chondroitine sulfate) de l/7 et plus de la moitit de l’activite totale dans les fractions insoluble et soluble respectivement. Des chromatogrammes d’hydrolysats proteiniques indiquent que l’activite est localis& a une seule region ayant un r.f. semblable au sulfone de mtthionine (produit hydrolytique de la mbthionine). Zusamtn&m--Es wurde gereinigtes Dentin von jungen Ratten analysiert, die mit “S-Methionin injiziert worden waren. Radioaktivitat ansteigender Intensitiit wurde im anorganischen Diffusat und in den liislichen (vermutlich Chondroitinsulfat und Protein) und unl&lichen (in enter Linie Kollagen) organ&hen Komponenten festgestellt. Das gesamte organische und Ei.ster-85S wurde in Rariumsulfatniederschl&gen aus veraschten Proben bestimmt. Die Niederschlage aus Hydrolysaten zeigten, daj3 die Mengen von =S im Estersulfat (vermutlich Chondroitinsulfat) in den unloslichen und l&lichen Fraktionen vorhanden sind. Chromatogramme von ProteinHydrolysaten wiesen darauf hin, da8 die Aktivitat in einem einzigen Fleck lokalisiert war, der einen Rt-Wet-t iihnlich wie Methionin-sulfon (hydrolytisches Produkt aus Methionin) besitzt.
296
G. NIKIFORUKAND A. M. HUNT REFERENCES
B~LANGER,L. F. 1955. Autoradiographic detection of radiosulfate incorporation by the grobing enamel of rats and hamsters. J. dent. Res. 34 20-27. BELANCER,L. F. 1956. Autoradiographic studies of the formation of the organic matrix of cartilage, bone and the tissues of teeth. Ciba Found. Symposium on Bone Structure and Metabolism. p. 75-87. BEVELANDER, G. and JOHNSON,P. L. 1955. The localization of polysaccharides in the developing teeth. J. dent. Res. 34, 123-131. B~STR~M, H. 1952. On the metabolism of the sulfate group of chondroitin sulfuric acid. J. biol. Chem. 196, 47748 I. BOSTR~M,H. and AQVLW, S. 1952. Utilization of Ss5 labelied sodium sulfate in the synthesis of chondroitin sulfuric acid, taurine, methionine and cystine. Acta them. stand. 6, 1557-1559. CREMER,H. D. and D~I-~MANN,G. 1956. Einbau von organischem (Eiweiss-) und anorganischem (Sulfat-) Schwefel in Knorpel, Knochen und Zahnen. [Incorporation of organic (protein) and inorganic (sulfate) sulfur in bones, cartilage and teeth.] Biochem. Z. 327, 377-382. CURRAN, R. C. 1961. The histological demonstration of connective tissue mucopolysaccharides. Biochem. sot. Symposia 20, 24-38. DITTMANN,G. and CREMER,H. D. 1956. Chondroitinschwefelsaiire in Knorpel, Knochen und Ziihnen. [Chondroitin sulfuric acid in cartilage, bones and teeth.] Biochem. Z. 327, 368-376. DZIEWIATKOWSKI,D. D. 1951. Isolation of chondroitin sulfate Sa5 from articular cartilage of rats. J. biof. Chem. 189, 187-190. DZIEWIATKOWSKI,D. D. 1962. Autoradiographic studies of bone growth with P sulfate. Radioisotopes and Bone Symposium. p. 277. Blackwell Scientific, Oxford. DZIEWIATKOWSKI,D. D., DI FERRAN~E,N., BRONNER,F. and OKINAKA, G. 1957. Turnover of YS sulfate in epiphyses and diaphyses of suckling rats; nature of the Y3 labelled compounds. J. exp. Med. 106.509-524. GREULICH,R. C. 1956. Contribution of sulfate ion to the formation of organic bone matrix. (abs.) Anat. Rec. 124,404405. GREULICH,R. C. and FRIBERG,U. 1957. Histochemical studies of sulfomucopotysaccharides in-the organic matrices of mineralized tissues. Exp. Cell Res. 12, 685-689. HESS,W. C. and LEE, C. 1952. The isolation of chondroitin sulfuric acid from dentin. J. dent. Res. 31,793-797. HOLMGREN,H. 1940. Studien tiber Verbreuung und Bedeutung der chromotropen substanz. Z. mikr.-anat. Forsch. 47.489-521. LLWN, L. 1936. Histochemie AnimaIe. Guathier-Villars, Paris. LYSYJ, I. and ZAREMBO,J. E. 1958. Rapid quantitative determination of sulfur in organic compounds. Analyt. Chem. 30, 428430. MANLY, R. S. and HODGE, H. C. 1939. Density and refractive index studies of dental hard tissues. J. dent. Res. 18. 133-141. NIKIFORUK, G., BURGESS,R. C. and MACLAREN,C. 1959. Amino sugars in dentin and enamel. J. dent. Res. 38, 675. PIEZ. K. A. 1963. The amino acid chemistry of some calcified tissues. Ann. N. Y. Acad. Sci. 109,256. PINCUS, P. 1948. Relation of enamel protein to dental caries.
Nature, Land. 161, 1014. and distribution of polysaccharides in human cortical bone and dentin of teeth. Nature. Land. 164, 625-626. STACK, M. V. 1951. Organic constituents of dentine. Brit. dent. J. 90, 173-181. SUZUKI, S. and STROMINGER. J. L. 1960. Enzymatic sulfation of mucopolysaccharides in hen oviduct. 1. Transfer of sulfate from 3,-phosphoadenosine 5’-phosphosulfate to mucopolysaccharides. J. biol. Chem. US, 257-266. SUZUKI, S. and STROMINGER, J. L. 1960. Enzymatic sulfation of mucopolysaccharides in hen oviduct. II. Mechanism of the reaction studied with oligosacchatides and monosaccharides as acceptors. J. biol. Chem. 235, 267-273.
ROGERS,H. J. 1949. Concentration
SM METHlONINE METABOLISMIN RAT DENTINE Suzu~r.
S.
297
and STROMDJOER, J. L. 1960. Enzymatic sulfation of mucopolysaccharides in hen oviduct. III. Mechanism of sulfation of chondroitin and chondroitin sulfate A. /. biol. Gem. 235,274276. SYLVEN, B. 1941. f_ber das Vorkommen von hochmolekularen Esterschwefelsiiuren im Granulationsgewebe und bei der Epithelregeneration: Experimentelle und pathiologisch-anatomische Untersuchungen tiber das Granulationsgewebe und die Regeneration von Plattenepithel mit besonderer Beriicksichtigung des Vorkommens und der Bedeutung auf&tender hochmolekularer Eaterschwefelsiiuren und Mastzellen. Acta chir. scund. 86, l-l 51. TARVER, H. and Scrn.rm~, C. L. A. 1939. The conversion of methionine to cystine: Experiments with radioactive sulfur. J. biol. Gem. 130,67-80. WISLOCKI,.G. B.. SINGER, M. and WALDO, C. M. 1948. Some histochemical reactions of mucopolysaccharides, glycogen. lipids and other substances in teeth. Anat. Rec. 101.487-5 14. WLSLOCKI,G. B. and SOGNNAES, R. F. 1950. Histochemical reactions of normal teeth. Amer. J. Anor. 87, 239-275.
PLATE 1 OVERLEAF
s3' METHIONINE METABOLISM IN RAT DENTINE
i___r__r_..*- .
.. - ._ I-
._
:--
,*
d+&,W . _
___i
FIG. 1. Scan of two paper chromatograms containing S3” methionine standard (top scan) and the hydrolysate of a dentine insoluble organic fraction lsbelled with Y5 methionine in viva (bottom scan). The single radioactive peak corresponds to the r.f. of methionine sulphone (hydrolytic product of methionine). The slight discrepancy in the peaks is due to the s!cewing of the solvent front.
f.p. 298
PLATE
1