Enamel Matrix: Structural Proteins J. D. TERMINE, D. A. TORCHIA and K. M. CONN National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. J Dent Res 58(B):773-778, March 1979 Cell-free, fetal bovine enamel tissue was examined or augmented in proteins by exogenous intact by high resolution. 13C Fourier transform, factors. For example, proteins having a nuclear magnetic resonance spectroscopy. Two small amount of internal beta structure types of protein chains were observed under these often exhibit higher proportions of this conditions, one exhibiting rapid mobility and conformation drying when exposed accounting for approximately two-thirds of the 8 to msld heatleg and/or low pH condithonse enamel matrix, while the other exhibited restricted In some smaller polypeptides such as those or anisotropic segmental motion and accounted for the remaining third of the matrix.
comprising the pathogenic deposit known
Sequential extraction of this fetal enamel under non-degradative conditions with dissociative solvents yielded two biochemically distinct populations of natrix protein. As expected, the bulk of the matrix consisted of proline-rich amelogenins, although the SDS-gel electrophoresis molecular weights for these proteins were somewhat higher than those reported using other extraction methods. Approximately fifteen percent of the total matrix consisted of much higher molecular weight phosphoproteins (46,000 72,000 daltons) whose amino acid composition closely resembled that reported for mature enamel protein. These high molecular weight proteins were tightly bound to the fetal enamel apatite crystallites.
amyloid, the enhancement of internal beta structure often leads to fiber formation9. This progression from internal fl-
-
The structural properties of the developing enamel matrix have been discussed extensively in the previous two International Symposia on Tooth Enamel. It is not our intent to recapitulate those discussions, but merely to summarize the two contrasting views on enamel matrix structure that prevail to the present day. These views have been amply aired at this Third Symposium both in formal presentationsl4 and in several discussion sessions. In one view, developing enamel matrix is seen as a thixotropic gel5, generally devoid of extensive structural features as known in other protein systems. Another view looks upon fetal enamel proteins as having secondary and perhaps even quaternary structural properties arising from beta-pleated sheet conformations6. This dichotomy of views was associated with differing experimental observations on the physical structure of enamel matrix when different and even similar instrumental techniques were used in this regard6'7. These apparent differences can be explained, in part, by the ability of the ,Bpleated sheet conformation to be enhanced
as
pleated sheet enhancement to subsequent
fibrilization has been shown to be critically
important in the etiology of amyloidosis imporan
thed
and can
duplicated
amyoisis
etilogye
expersmentally
using
a number of polypeptides such as glucagon,
insulin or the variable segment of some Bence-Jones proteinsl. One such experimental progression is demonstrated in Figure 1, infrared spectroscopy to monitor increases in structure9.
z
j-pleated
sheet
secondary
U~~~
L
~~~~~~~~~b
LU 0
It
LU
1s00
1500
1200
900
600
300
WAVENUMBER (cm-')
Infrared spectra of a Bence-Jones Fig. 1 protein: (a) untreated; (b) after heating to 550 at pH 4.5; and (c) after enzymic digestion to its VL segment at 370 and pH 6.2. This latter treatment resulted in fiber formation. The bands at 1630 and 700 cm-1 are diagnostic for the beta pleated sheet conformation.
In evaluating data then, on the structural properties of many preparations of develop773
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JDent Res Special Issue B, 1979
TERMINE ETA L.
ing enamel proteins, it may well be impossible to ascertain exactly how much ,Bpleated sheet structure is actually present in the native tissue and how much is exogenously induced. This realization led us to examine the structural characteristics of fetal enamel proteins using nuclear magnetic resonance (NMR) spectroscopy on relatively intact, cell-free tissue. Preliminary results of these studies were presented earlier11. Developing enamel tissue was obtained from bovine fetuses, 5-6 months in utero. Preliminary experiments confirmed the presence of a neutral protease in fetal enamel as amply described elsewhere in this volume 12-14. Consequently, protease inhibitors (benzamidine, 5mM; c-amino caproic acid, 1 OOmM; phenylmethyl sulfonyl fluorides, PMSF, 1 mM) were routinely added to all solutions used in our studies and care was taken to remove adhering cells and cell fragments from all tissue examined. The tissue preparation procedure utilized for these experiments is outlined in Figure 2. The bovine tooth matrices were assessed to be cell-free by morphological examination at the light and electron microscopic levels. Scanning electron microscopic examination of tissues similarly treated also revealed loss of adhering cells and cell contact points as well as the loss of surface enamel to a depth of about one micron (A. Boyde, personal communication). Light and transmission electron microscopy were also used to characterize all tissue products isolated, which were then kept frozen at -700 until
Abbatoir Sacrifioe, Fietus Pemved, Head Transferred to Laboratory, (0O)
Preliminiary Dissecticn, Soft Tissue RHwalo, (2'-5°), P.B.sl-S irn-Inhib. Cocktail, pH 7.4
Scnication (4°),
PhIsoatBuffered Saline pH 7.4, Inh. Codctail
cEL-SEE TOMH TRICES (frozen in Lij. N2)
Final Dissectc
ELB
(2°-5°')
e1.tin-Rid, cot nants
ESiADEL IfRAPSiESS Iiq. N2 WMp.
I Fig. 2 - Outline of tissue preparation procedures utilized for these biophysical and biochemical studies.
protein, collagen, do not exhibit NMR spectra unless special high-power, dipolar decoupling techniques are utilized16,17. On the other hand, protein chains exhibiting a high degree of rapid segmental reorientation such as elastin or freely tumbling globular proteins in solution do exhibit NMR signals under normal (scalar-decoupled) utilized. Fourier transform 13C NMR spectro- operating conditions15. When we examined our fetal bovine enamscopy of solid state protein samples is an experimental procedure previously used in el scrapings, we always observed an indigenstudies of connective tissue such as aorta, ous 13C NMR spectrum under normal tendon, cartilage, etc.15. In most instances, decoupling conditions, as shown in Figure natural abundance 13C signals are those. 4. An enhanced NMR signal was also obobserved, but specific 13C-enriched amino served under high-power dipolar decoupling acid residues have been introduced into indicating the presence of segmentally reprotein chains via biosynthesis and studied stricted protein chains in these samples individually as well16. Figure 3 recapitulates along with the considerably more mobile a typical pulse Fourier transform 13C NMR chains giving rise to the scalar decoupled experiment for chick aorta and shows NMR spectra. These findings were verified assignments of the NMR signals to various through additional experiments studying types of carbons in the polypeptide chains. temperature effects, exogenous proteolysis, This scalar decoupled NMR signal arises etc., and using other NMR techniques such as from only those carbons in the sample for cross-polarization methodology18. After takwhich reorientation is isotropic and rapid ing factors such as nuclear Overhauser en(r$ 10-6s). Proteins containing substantial hancement into account, we calculated that structural anisotropy, such as the fibrous approximately two-thirds of the protein Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on June 17, 2015 For personal use only. No other uses without permission.
Vol. 58(B)
775 20 DAY AORTA
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Fig. 3 - Free-induction decay NMR signals from both a single (top) and time-averaged multiple scans (middle) and the resultant Fourier transform spectrum (bottom) for a specimen of chick aorta elastin.
Fig. 4 - Fourier transform 13C NMR spectra from cell-free, fetal bovine enamel tissue. This specimen was examined intact at 370 and analyzed as 48% mineral, 31% water and 21% protein. Both normal scalar [lower-power] (A) and highpower dipolar (b) decoupling spectra are shown.
chains in fetal bovine enamel are highly having a stated 10,000 molecular weight mobile, exhibiting rapid segmental reorien- cut-off. These low molecular weight, low tation with correlation times of 10-100 yield filtrate fractions were not examined nanoseconds. The remaining third exhibits further in this study. restricted motion indicating structural anisoApproximately 85% of the total protein tropy such as found in fibrous or highly in the developing fetal bovine enamel was cross-linked proteins. extracted in the initial 4M guanidine extract. We next attempted to determine if bio- As shown in Table 1, this extract had an chemically distinct protein populations also amino acid composition typical of the ameloexisted in fetal bovine enamel tissue. The genins, being rich in proline, glutamic cell-free enamel scrapings described above acid, leucine and histidine. This fraction were sequentially extracted under non- also contained 0. 14% organic phosphorus. degradative conditions using 4M guanidine In contrast, the sequential "crystal bound" as a dissociative solvent. Some preliminary protein extract had an amino acid composibiochemical results with dissociative sol- tion substantially different from the amelovents were presented earlier19. The sequen- genins. These acidic fetal enamel proteins tial extraction schema used for this study is more closely resembled the proteins of outlined in Figure 5. Transmission electron adult enamel in composition1 and yet commicroscopic examination of the sediment prised 15% of the total protein in fetal from the initial guanidine extract showed bovine enamel tissue. These "crystal bound" only relatively intact, developing enamel proteins contained 0.32% organic phosprisms containing co-oriented apatite crystal- phorus, approximately twice as much per lites. The protein associated with this unit protein as in the initial guanidine, sediment could only be extracted upon amelogenin fraction. Further, while our dissolution of these apatite crystals. Thus, amelogenins did not adhere to DEAEwe labeled this fetal enamel matrix fraction cellulose in 7M urea, the "crystal bound" as "crystal bound" protein. In both the protein fraction was retained entirely, initial guanidine and this latter "crystal only eluting from the column upon addibound" protein extract, only 2-3% of the tion of 0.45 - 0.55 M NaCl. total protein passed through ultrafilters Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on June 17, 2015 For personal use only. No other uses without permission.
776
TERMINE E T AL.
Fig. 5
-
J Den t Res Special Issue B, 1 9 79
Outline of dissociative extraction methods employed in this study.
These two protein fractions were subjected to SDS-urea P.A.G.E. using both the conventional Coomassie Blue and the organic phosphate-sensitive "stains-all"20 staining techniques. Coomassie Blue results for both extracts are shown in Figure 6. Under
TABLE 1 AMINO ACID ANALYSIS OF FETAL BOVINE ENAMEL PROTEINS SEQUENTIALLY EXTRACTED WITH DISSOCIATIVE SOLVENTS*
electrophoresis conditions, our amelogenins ranged in molecular weight from 40,00010,000 daltons, but when urea was excluded from the SDS gels, they only ranged from 28,000-7,000 daltons. As mentioned elsewhere in this volume1 3,1 amelogenins do not behave ideally on SDS-P.A.G.E., an effect most probably related to anomalous internal conformational effects as the above results in the presence and absence of the denaturing solvent, urea, would indicate. Until more details are known about conformational effects and SDS-binding to the proline-rich amelogenins, it is not possible to ascertain true molecular weights for these materials by this procedure. Nevertheless, the apparent SDS-gel molecular weights for fetal bovine amelogenins isolated under the conditions used in this study are somewhat higher than those reported for these proteins when isolated by other extraction methods13,2 1-23.
Guanidine Extract Gu-EDTA Extract
Asp Thr
Ser Glu Pro
Gly Ala
Cys/2 Val Met Ile Leu Tyr Phe His
Lys Arg Hyp
Hyl *:
38 29 46 176 230 56 28 1 46 51 34 102 33 26 68 14 19 2 1
residues/1000; M. wt >
98 55
81 146 139 114 50 2 39 23 28 53 21 49 27 32 40 tr 3 10,000; tr = trace
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Vol. 58(B)
U
777
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:
S MN
SDS-urea reducing polyacrylamide Fig. 6 electrophoresis gels of our initial guanidine extract (left), our sequential Gu-EDTA extract (middle) and a set of molecular weight standards (right), al stained with Coomassie Brilliant Blue R-250. These 7.5% gels contained 8M urea and were run at 3-4 m amps/tube in 0.4 M tris-glycine buffer, pH 8.8 and 0.1% SDS. Samples were dissolved at 650 in electrophoresis buffer containing 8M urea, 1% SDS and 0.1% mercaptoethanol. The molecular weight markers used were phosphorylase b (94,000 daltons), bovine serum albumin (67,000 daltons), ovalbumin (43,000 daltons), carbonic anhydrase (30,000 daltons), soybean trypsin inhibitor (20,000 daltons) and a-lactalbumin (14,000 daltons).
Either in the presence or the absence of urea, the "crystal bound" protein fraction described above consisted of three major species on SDS-P.A.G.E. having molecular weights of 72,000, 56,000 and 48,000 daltons. Ferguson plots [log Rf vs % acrylamide (24)] for these three proteins in gels with differing acrylamide concentrations showed ideality coincident with molecular weight standards in the 40,000 - 90,000 dalton range. This indicates that the SDSP.A.G.E. molecular weights obtained for these "crystal bound" proteins are reliable25. Figure 7 shows SDS-urea gels for these proteins stained with "stains-all", a dye which specifically stains phosphoproteins deep blue20. All three of our higher molecular weight, "crystal bound" fetal enamel proteins stained blue with this dye, suggesting that they are phosphoproteins,
SDS-urea P.A.G.E. (as in Figure 6) Fig. 7 of bovine serum albumin (left), our "crystal bound" proteins (center) and egg yolk phosvitin (right) stained with "stains-all" dye. The albumin, not being phosphorylated, stained pink on a pale pink background (pink bands are also photosensitive in this procedure) and thus is not sharply delineated on this black and white reproduction. Phosvitin, being extensively phosphorylated, stained intensely blue as did the 72,000 and 56,000 dalton bands in the "crystal bound" protein extract. The 48,000 dalton "crystalbound" protein band stained pale blue by this technique. The thin line at ca. 96,000 daltons in this middle gel is the blue staining dentin phosphoprotein, a minor contaminant in this particular tissue preparation.
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7787JDent TERMINE ETA L. Res Special Issue B, 19 79
as also indicated by the organic phosphate chemical data described above. None of the amelogenins stained for phosphate with this dye, suggesting that their organic phosphate, perhaps being evenly distributed over a larger number of individual species, is below the limits of detection for this technique. It is not possible to correlate these biochemically different protein populations with the differently mobile peptide chain components detected by 13C NMR procedures. In fact, each set of different protein populations may encompass or overlap the other. Each set also poses interesting questions and perplexing problems. For example, what is the morphological counterpart to the segmentally restricted, anisotropic protein chains found in our NMR experiments? What is the role, if any, of the predominant highly mobile protein chains in enamel apatite crystal development? Further, are the high molecular weight fetal enamel phosphoproteins found in this study the precursors of the mature enamel proteins? What is their relationship, if any, to the amelogenins and how do they affect enamel mineralization in view of their high affinity for enamel crystals? All of these questions arise from the spirit of this conference and could well be with us for several conferences to come. REFERENCES 1. EASTOE, J. E.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 2. GLIMCHER, M. J.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 3. FEARNHEAD, R. -W.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. trom this book, J. Dent. Res. Suppl., 1979. 4. NYLEN, M. U.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 5. EASTOE, J. E.: In: Comprehensive Biochemistry 26C (FLORKIN, M., STOTZ, E. H., eds) pp. 785-834. Amsterdam: Elsevier, 1971. 6. BONAR, L. C., GLIMCHER, M. J., MECHANIC, G. L.: J. Lltrastruc. Res. 13:308-318 (1965).
7. FEARNHEAD, R. W.: In: Tooth Enamel (STACK, M. V., FEARNHEAD, R. W., eds) pp. 127-131. Bristol: John Wright and Sons, Ltd., 1965. 8. AMBROSE, E. J., ELLIOTT, A.: Proc. Roy. Soc. London, A208, 75-84 (1951). 9. TERMINE, J. D., EANES, E. D., EIN, D., GLENNER, G. G.: Biopolymers 11:1103-
1113 (1972). 10. GLENNER, G. G., EANES, E. D., BLADEN, H. A., LINKE, R. P., TERMINE, J. D.: J. Histochem. Cytochem. 22:1141-1158 (1974). 11. TERMINE, J. D., TORCHIA, D. A.: J.Dent. Res., 56A, A55 (1977). 12. SHIMIZU, M., TANABE, T., FUKAE, M.: In: Proceedings of Third International Symposium on Tooth Eanmel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 13. SASAKI, S., SHIMOKAWA, H.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 14. MOE, D., BIRKEDAL-HANSEN, H.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 15. TORCHIA, D. A., VANDERHART, D. L.: In: Topics in Carbon 13 NMR Spectroscopy (G. C. LEVY, ed), In press. Wiley, New York,
1978. 16. TORCHIA, D. A., VANDERHART, D. L.: J. Mol. Biol. 104:315-321 (1967). 17. TORCHIA, D. A., PIEZ, K. A.: J. Mol. Biol. 76:419-424 (1973). 18. PINES, A., GIBBY, M. G., WAUGH, J. S.: J. Chem. Phys. 59:569-589 (1973). 19. SHARKEY, M. A., CONN, K. M., TERMINE, J. D.: J. Dent. Res. 56A, A82 (1977). 20. GREEN, M. R., PASTEWKA, J. V., PEACOCK, A. C.: Analyt. Biochem. 56:43-51 (1973). 21. ROBINSON, C., BRIGGS, H. D., ATKINSON, P. J., WEATHERELL, J. A.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 22. EGGERT, F. M., ALLEN, G. A., BURGESS, R. C.: Biochem. J. 131:471484 (1973). 23. FUKAE, M., SHIMIZU, M.: Arch. Oral Biol. 19:381-386 (1974). 24. FERGUSON, K. A.: Metabolism 13:9851002 (1964). 25. RODBARD, D.: In: Methods of Protein Separation 2 (CATSIMPOOLAS, N. ed) pp. 145-218. New York: Plenum Press, 1976.
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Session III Discussion Dr. Reith: I take it you began your project with the expectation that you might find some structural protein positioned in some way which would account for the organization and orientation of the crystallites and the enamel prisms. I am not certain that an oriented structural protein is necessary. Some years ago Paul Weiss showed that, in tissue culture preparations, by putting a slight amount of tension on the gel substrate on which the cells were grown, he could cause these cells to assume an orientation similar to the direction of the tension which was applied. It seems to me that from what you have described, you have molecular components of such a size that they would display properties of viscous solutions. These molecules could be aligned if subject to flow or stress. As I recall at the Tooth Enamel I Conference, Dr. Boyde pointed out that the ameloblasts were continuously moving during the production of enamel matrix and I wondered if their continuous movement could set up some sort of flow tension which would account for an orientation of molecules such as you have described and thereby account for crystallites. Theoretically, this would cease once the cell movement ended. It would also cease if you disrupt the system as in test tube experiments. This may be one of the reasons why it is so very hard to find the basis for orientation in in vitro preparations. Would you care to comment on that? Dr. Termine: One of the models which fit the NMR data very aptly is elastin, and if you remember, elastin is composed of interconnected and cross-linked polypeptide chains. At any point of close .attachment, this protein is very rigid, that is, it doesn't show much segmental motion. On the other hand, further away from the cross-links, there is a considerable degree of segmental motion. Perhaps this could be a model for enamel where inter-chain hydrogen bonding or even protein-apatite bonds might serve as analagous points of attachment. Thus, both flow and chemical forces could possibly act in concert under cellular control in such a model system. Dr. Reith: The question I want to ask is, are we overlooking the movement of cells
as a possible orienting factor for enamel crystallites. Dr. Termine: That is possible, but I have no comment on this point. Dr. Glimcher: I would like to point out that there have already been published a number of studies concerning the molecular sturcture of embryonic enamel proteins, both in the solid state and in solution (J. Mol. Biol. 3:541-546, 1961; J. Ultrastruct. Res. 13:281-295, 296-307 and 308-317, 1965; Calc. Tissue Res. 2, Suppl, 1, 1968). In these publications we have pointed out the importance of distinguishing between the primary structure, secondary structure and other higher ordered structures and what is meant by and the significance of the term "gel" (Ferry, Adv. Protein Chem. 4:1-78, 1948). Let me emphasize that whether a protein system exists in solution or as a hydrated precipitate or a gel is an altogether different issue from the nature of the primary, secondary, or tertiary structure of the protein (Calc. Tiss. Res. 2, Suppl. 1, 1968). To say that the enamel proteins are or cannot be in a particular configuration like the 3-pleated sheet or a a helix because they are aggregated as a gel is equivalent to saying "this pencil is not made of wood, it is red." Further, we demonstrated that embryonic enamel matrix has components in it whose secondary structure is in the 13-pleated sheet configuration. Moreover, the fact that the two major reflections of the 13-pleated configuration are at right angles to one another indicates that the "crystallites" of the 13pleated protein are packed in what has been described as a "cross-4" arrangement, as opposed to a parallel-b packing. We were not able to induce this 13-pleated sheet secondary structure or the cross-,B packing arrangement in a variety of other proteins treated with EDTA and other solvents used in preparing the enamel. Further, the cross-b patterns were generated in both the dry and hydrated- states, so that they did not result artifactually from drying. Also, it is important to note that the 13-pleated sheet secondary structure and the tertiary cross-4 configuration were obtained from whole, intact enamel matrix as well as from the neutral soluble proteins which had been extracted, 779
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JDent Res Special Issue B, 1979
put into solution, and then re-formed into the solid state either as films or oriented
threads. Preliminary results with the pure phosphopeptides E3 and E4 (FEBS Lett., 79: 276-280, 1977; Biochim. Biophys. Acta 493:441451, 1977) have shown similar results: A sufficient amount of the peptide is in the pleated sheet configuration to generate the characteristic fiber diffraction pattern representative of this configuration. An examination of the amino acid composition of either the entire embryonic enamel matrix, the neutral soluble embryonic enamel proteins, or the pure E3 and E4 peptides also makes it abundantly clear that the majority of the proteins cannot be in an a-helical configuration because of their high contents of proline (Lindley, Biochim. Biophys. Acta 18:194, 1955). However, Ramachandran (Collagen, Ramachandran, ed. New York: Wiley Interscience, 1962, p. 27) has proposed a modification of the pleated sheet structures which can accommodate large amounts of proline. Thus the data that have been presented previously are strongly in favor of a (3-pleated sheet configuration for components in the developing enamel matrix, especially in the solid state. These are, in addition, packed in a cross-j configuration. At a minimum, one can conclude that this is the most favorable solid state configuration for these components, since they re-form into this configuration when aggregated from solution to the solid state. I want to emphasize very strongly that nothing has been published in the literature or presented today by Dr. Termine which would alter these conclusions. His data simply confirm our earlier studies that some of the enamel components are in a specific configuration and are not randomly ordered. How much of the protein, or how many of the components are organized in the (-pleated sheet configuration in the solid state cannot be determined from the x-ray data alone. However, using optical rotary dispersion we have estimated that, in solution, 6-1 0% of the components are in a (-pleated sheet configuration (J. Ultrastruct. Res. 13:296-307, 1965). Dr. Termine: What I meant to imply and what our own and the other data indicate is that very little of the protein in enamel is in a (-pleated sheet conformation. There is no question that some structural protein does exist in fetal enamel. The question as
to the proportion of :-pleated sheet protein relative to unordered protein in this fetal tissue remains to be determined. Dr. Veis: I'd like to ask Dr. Termine what the significance of the structures he measures in enamel protein is when you consider that the protein may have a different structure in the presence of all the mineral in the bound state. Dr. Termine: Dr. Veis, the NMR data that I showed was taken on scrapings from developing bovine enamel which contained about 50% mineral. These tissues were examined as dissected with no further treatment. Dr. Roufosse: Dr. Termine, I would like to ask if, in your 13C NMR study of the enamel proteins, you have assigned specific resonances to certain molecular groups. Dr. Termine: What we see are averaged resonance lines from the peptide backbone carbons. We have not done anything on individual sidechains as yet. Dr. Roufosse: Your conclusion that some segments of the backbone do not rotate are based on just the large values of the linewidths of unassigned resonances. Could these large values result from insufficient proton decoupling? Dr. Termine: No, that is not the case based on many different NMR data in this regard, including cross-polarization studies. Dr. Fearnhead: I would like to address a short question to Dr. Termine. It is related to his crystal-bound protein which he extracts with 0.5 molar EDTA. Dr. Termine: These proteins were extracted in four molar guanidine mixed with 0.5 molar EDTA. Dr. Fearnhead: I think you said that it all went into solution. I have tried t;6 do a similar thing, but I have only used .5 molar and .01 molar EDTA and I nearly always get a very small residue. I wonder what temperature you are making your extraction at, or can you explain the difference? Dr. Termine: Our tissues were extracted at 4 degrees, but I think the key to total dissolution is the guanidine. In guanidine, you denature the protein as you extract it and it comes out in random conformation. If you just use EDTA, it doesn't all come out.
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Session I Commentary Dr. Mechanic: Ten years ago at the Tooth Enamel II Conference, I succeeded in confusing everybody with the data I had. I am still confused. Now, for instance, Fukae and Shimizu in their poster at this meeting show very nicely a separation from porcine embryonic enamel of at least eight components of similar amino acid composition. We showed a minimum of about eleven different proteins based on amino acid analysis considerations, although we did not isolate any one of them in a pure form. What is confusing me now is the enzymes that are present in the enamel matrix. Drs. Sasaki and Shimokawa have an enzyme that acts at pH 6, Drs. Moe and Birkedal-Hansen, in their poster, show a protease that acts at a pH optimum of 8-9,
and Drs. Fukae and Shimizu have isolated an enzyme that acts at neutral pH. So now something else has to be straightened out with the enamel matrix. The last question concerns molecular weight. The highest molecular weight component previously reported from the University of Southern California is a proenamelin of about 58,000 daltons. Dr. Sasaki reports a proenamelin of 25,000 M.W., but has now revised his to 15,000 because of the high amount of proline it contains. And now Dr. Termine has proteins present in enamel upwards of 70,000 molecular weight down to 10,000. So, needless to say, the protein chemistry of the enamel matrix is still in a slightly confusing state, although many advances have been made since the last tooth enamel symposium.
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comprising the pathogenic deposit known
Sequential extraction of this fetal enamel under non-degradative conditions with dissociative solvents yielded two biochemically distinct populations of natrix protein. As expected, the bulk of the matrix consisted of proline-rich amelogenins, although the SDS-gel electrophoresis molecular weights for these proteins were somewhat higher than those reported using other extraction methods. Approximately fifteen percent of the total matrix consisted of much higher molecular weight phosphoproteins (46,000 72,000 daltons) whose amino acid composition closely resembled that reported for mature enamel protein. These high molecular weight proteins were tightly bound to the fetal enamel apatite crystallites.
amyloid, the enhancement of internal beta structure often leads to fiber formation9. This progression from internal fl-
-
The structural properties of the developing enamel matrix have been discussed extensively in the previous two International Symposia on Tooth Enamel. It is not our intent to recapitulate those discussions, but merely to summarize the two contrasting views on enamel matrix structure that prevail to the present day. These views have been amply aired at this Third Symposium both in formal presentationsl4 and in several discussion sessions. In one view, developing enamel matrix is seen as a thixotropic gel5, generally devoid of extensive structural features as known in other protein systems. Another view looks upon fetal enamel proteins as having secondary and perhaps even quaternary structural properties arising from beta-pleated sheet conformations6. This dichotomy of views was associated with differing experimental observations on the physical structure of enamel matrix when different and even similar instrumental techniques were used in this regard6'7. These apparent differences can be explained, in part, by the ability of the ,Bpleated sheet conformation to be enhanced
as
pleated sheet enhancement to subsequent
fibrilization has been shown to be critically
important in the etiology of amyloidosis imporan
thed
and can
duplicated
amyoisis
etilogye
expersmentally
using
a number of polypeptides such as glucagon,
insulin or the variable segment of some Bence-Jones proteinsl. One such experimental progression is demonstrated in Figure 1, infrared spectroscopy to monitor increases in structure9.
z
j-pleated
sheet
secondary
U~~~
L
~~~~~~~~~b
LU 0
It
LU
1s00
1500
1200
900
600
300
WAVENUMBER (cm-')
Infrared spectra of a Bence-Jones Fig. 1 protein: (a) untreated; (b) after heating to 550 at pH 4.5; and (c) after enzymic digestion to its VL segment at 370 and pH 6.2. This latter treatment resulted in fiber formation. The bands at 1630 and 700 cm-1 are diagnostic for the beta pleated sheet conformation.
In evaluating data then, on the structural properties of many preparations of develop773
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TERMINE ETA L.
ing enamel proteins, it may well be impossible to ascertain exactly how much ,Bpleated sheet structure is actually present in the native tissue and how much is exogenously induced. This realization led us to examine the structural characteristics of fetal enamel proteins using nuclear magnetic resonance (NMR) spectroscopy on relatively intact, cell-free tissue. Preliminary results of these studies were presented earlier11. Developing enamel tissue was obtained from bovine fetuses, 5-6 months in utero. Preliminary experiments confirmed the presence of a neutral protease in fetal enamel as amply described elsewhere in this volume 12-14. Consequently, protease inhibitors (benzamidine, 5mM; c-amino caproic acid, 1 OOmM; phenylmethyl sulfonyl fluorides, PMSF, 1 mM) were routinely added to all solutions used in our studies and care was taken to remove adhering cells and cell fragments from all tissue examined. The tissue preparation procedure utilized for these experiments is outlined in Figure 2. The bovine tooth matrices were assessed to be cell-free by morphological examination at the light and electron microscopic levels. Scanning electron microscopic examination of tissues similarly treated also revealed loss of adhering cells and cell contact points as well as the loss of surface enamel to a depth of about one micron (A. Boyde, personal communication). Light and transmission electron microscopy were also used to characterize all tissue products isolated, which were then kept frozen at -700 until
Abbatoir Sacrifioe, Fietus Pemved, Head Transferred to Laboratory, (0O)
Preliminiary Dissecticn, Soft Tissue RHwalo, (2'-5°), P.B.sl-S irn-Inhib. Cocktail, pH 7.4
Scnication (4°),
PhIsoatBuffered Saline pH 7.4, Inh. Codctail
cEL-SEE TOMH TRICES (frozen in Lij. N2)
Final Dissectc
ELB
(2°-5°')
e1.tin-Rid, cot nants
ESiADEL IfRAPSiESS Iiq. N2 WMp.
I Fig. 2 - Outline of tissue preparation procedures utilized for these biophysical and biochemical studies.
protein, collagen, do not exhibit NMR spectra unless special high-power, dipolar decoupling techniques are utilized16,17. On the other hand, protein chains exhibiting a high degree of rapid segmental reorientation such as elastin or freely tumbling globular proteins in solution do exhibit NMR signals under normal (scalar-decoupled) utilized. Fourier transform 13C NMR spectro- operating conditions15. When we examined our fetal bovine enamscopy of solid state protein samples is an experimental procedure previously used in el scrapings, we always observed an indigenstudies of connective tissue such as aorta, ous 13C NMR spectrum under normal tendon, cartilage, etc.15. In most instances, decoupling conditions, as shown in Figure natural abundance 13C signals are those. 4. An enhanced NMR signal was also obobserved, but specific 13C-enriched amino served under high-power dipolar decoupling acid residues have been introduced into indicating the presence of segmentally reprotein chains via biosynthesis and studied stricted protein chains in these samples individually as well16. Figure 3 recapitulates along with the considerably more mobile a typical pulse Fourier transform 13C NMR chains giving rise to the scalar decoupled experiment for chick aorta and shows NMR spectra. These findings were verified assignments of the NMR signals to various through additional experiments studying types of carbons in the polypeptide chains. temperature effects, exogenous proteolysis, This scalar decoupled NMR signal arises etc., and using other NMR techniques such as from only those carbons in the sample for cross-polarization methodology18. After takwhich reorientation is isotropic and rapid ing factors such as nuclear Overhauser en(r$ 10-6s). Proteins containing substantial hancement into account, we calculated that structural anisotropy, such as the fibrous approximately two-thirds of the protein Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on June 17, 2015 For personal use only. No other uses without permission.
Vol. 58(B)
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Fig. 3 - Free-induction decay NMR signals from both a single (top) and time-averaged multiple scans (middle) and the resultant Fourier transform spectrum (bottom) for a specimen of chick aorta elastin.
Fig. 4 - Fourier transform 13C NMR spectra from cell-free, fetal bovine enamel tissue. This specimen was examined intact at 370 and analyzed as 48% mineral, 31% water and 21% protein. Both normal scalar [lower-power] (A) and highpower dipolar (b) decoupling spectra are shown.
chains in fetal bovine enamel are highly having a stated 10,000 molecular weight mobile, exhibiting rapid segmental reorien- cut-off. These low molecular weight, low tation with correlation times of 10-100 yield filtrate fractions were not examined nanoseconds. The remaining third exhibits further in this study. restricted motion indicating structural anisoApproximately 85% of the total protein tropy such as found in fibrous or highly in the developing fetal bovine enamel was cross-linked proteins. extracted in the initial 4M guanidine extract. We next attempted to determine if bio- As shown in Table 1, this extract had an chemically distinct protein populations also amino acid composition typical of the ameloexisted in fetal bovine enamel tissue. The genins, being rich in proline, glutamic cell-free enamel scrapings described above acid, leucine and histidine. This fraction were sequentially extracted under non- also contained 0. 14% organic phosphorus. degradative conditions using 4M guanidine In contrast, the sequential "crystal bound" as a dissociative solvent. Some preliminary protein extract had an amino acid composibiochemical results with dissociative sol- tion substantially different from the amelovents were presented earlier19. The sequen- genins. These acidic fetal enamel proteins tial extraction schema used for this study is more closely resembled the proteins of outlined in Figure 5. Transmission electron adult enamel in composition1 and yet commicroscopic examination of the sediment prised 15% of the total protein in fetal from the initial guanidine extract showed bovine enamel tissue. These "crystal bound" only relatively intact, developing enamel proteins contained 0.32% organic phosprisms containing co-oriented apatite crystal- phorus, approximately twice as much per lites. The protein associated with this unit protein as in the initial guanidine, sediment could only be extracted upon amelogenin fraction. Further, while our dissolution of these apatite crystals. Thus, amelogenins did not adhere to DEAEwe labeled this fetal enamel matrix fraction cellulose in 7M urea, the "crystal bound" as "crystal bound" protein. In both the protein fraction was retained entirely, initial guanidine and this latter "crystal only eluting from the column upon addibound" protein extract, only 2-3% of the tion of 0.45 - 0.55 M NaCl. total protein passed through ultrafilters Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on June 17, 2015 For personal use only. No other uses without permission.
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Fig. 5
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J Den t Res Special Issue B, 1 9 79
Outline of dissociative extraction methods employed in this study.
These two protein fractions were subjected to SDS-urea P.A.G.E. using both the conventional Coomassie Blue and the organic phosphate-sensitive "stains-all"20 staining techniques. Coomassie Blue results for both extracts are shown in Figure 6. Under
TABLE 1 AMINO ACID ANALYSIS OF FETAL BOVINE ENAMEL PROTEINS SEQUENTIALLY EXTRACTED WITH DISSOCIATIVE SOLVENTS*
electrophoresis conditions, our amelogenins ranged in molecular weight from 40,00010,000 daltons, but when urea was excluded from the SDS gels, they only ranged from 28,000-7,000 daltons. As mentioned elsewhere in this volume1 3,1 amelogenins do not behave ideally on SDS-P.A.G.E., an effect most probably related to anomalous internal conformational effects as the above results in the presence and absence of the denaturing solvent, urea, would indicate. Until more details are known about conformational effects and SDS-binding to the proline-rich amelogenins, it is not possible to ascertain true molecular weights for these materials by this procedure. Nevertheless, the apparent SDS-gel molecular weights for fetal bovine amelogenins isolated under the conditions used in this study are somewhat higher than those reported for these proteins when isolated by other extraction methods13,2 1-23.
Guanidine Extract Gu-EDTA Extract
Asp Thr
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38 29 46 176 230 56 28 1 46 51 34 102 33 26 68 14 19 2 1
residues/1000; M. wt >
98 55
81 146 139 114 50 2 39 23 28 53 21 49 27 32 40 tr 3 10,000; tr = trace
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Vol. 58(B)
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SDS-urea reducing polyacrylamide Fig. 6 electrophoresis gels of our initial guanidine extract (left), our sequential Gu-EDTA extract (middle) and a set of molecular weight standards (right), al stained with Coomassie Brilliant Blue R-250. These 7.5% gels contained 8M urea and were run at 3-4 m amps/tube in 0.4 M tris-glycine buffer, pH 8.8 and 0.1% SDS. Samples were dissolved at 650 in electrophoresis buffer containing 8M urea, 1% SDS and 0.1% mercaptoethanol. The molecular weight markers used were phosphorylase b (94,000 daltons), bovine serum albumin (67,000 daltons), ovalbumin (43,000 daltons), carbonic anhydrase (30,000 daltons), soybean trypsin inhibitor (20,000 daltons) and a-lactalbumin (14,000 daltons).
Either in the presence or the absence of urea, the "crystal bound" protein fraction described above consisted of three major species on SDS-P.A.G.E. having molecular weights of 72,000, 56,000 and 48,000 daltons. Ferguson plots [log Rf vs % acrylamide (24)] for these three proteins in gels with differing acrylamide concentrations showed ideality coincident with molecular weight standards in the 40,000 - 90,000 dalton range. This indicates that the SDSP.A.G.E. molecular weights obtained for these "crystal bound" proteins are reliable25. Figure 7 shows SDS-urea gels for these proteins stained with "stains-all", a dye which specifically stains phosphoproteins deep blue20. All three of our higher molecular weight, "crystal bound" fetal enamel proteins stained blue with this dye, suggesting that they are phosphoproteins,
SDS-urea P.A.G.E. (as in Figure 6) Fig. 7 of bovine serum albumin (left), our "crystal bound" proteins (center) and egg yolk phosvitin (right) stained with "stains-all" dye. The albumin, not being phosphorylated, stained pink on a pale pink background (pink bands are also photosensitive in this procedure) and thus is not sharply delineated on this black and white reproduction. Phosvitin, being extensively phosphorylated, stained intensely blue as did the 72,000 and 56,000 dalton bands in the "crystal bound" protein extract. The 48,000 dalton "crystalbound" protein band stained pale blue by this technique. The thin line at ca. 96,000 daltons in this middle gel is the blue staining dentin phosphoprotein, a minor contaminant in this particular tissue preparation.
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7787JDent TERMINE ETA L. Res Special Issue B, 19 79
as also indicated by the organic phosphate chemical data described above. None of the amelogenins stained for phosphate with this dye, suggesting that their organic phosphate, perhaps being evenly distributed over a larger number of individual species, is below the limits of detection for this technique. It is not possible to correlate these biochemically different protein populations with the differently mobile peptide chain components detected by 13C NMR procedures. In fact, each set of different protein populations may encompass or overlap the other. Each set also poses interesting questions and perplexing problems. For example, what is the morphological counterpart to the segmentally restricted, anisotropic protein chains found in our NMR experiments? What is the role, if any, of the predominant highly mobile protein chains in enamel apatite crystal development? Further, are the high molecular weight fetal enamel phosphoproteins found in this study the precursors of the mature enamel proteins? What is their relationship, if any, to the amelogenins and how do they affect enamel mineralization in view of their high affinity for enamel crystals? All of these questions arise from the spirit of this conference and could well be with us for several conferences to come. REFERENCES 1. EASTOE, J. E.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 2. GLIMCHER, M. J.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 3. FEARNHEAD, R. -W.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. trom this book, J. Dent. Res. Suppl., 1979. 4. NYLEN, M. U.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 5. EASTOE, J. E.: In: Comprehensive Biochemistry 26C (FLORKIN, M., STOTZ, E. H., eds) pp. 785-834. Amsterdam: Elsevier, 1971. 6. BONAR, L. C., GLIMCHER, M. J., MECHANIC, G. L.: J. Lltrastruc. Res. 13:308-318 (1965).
7. FEARNHEAD, R. W.: In: Tooth Enamel (STACK, M. V., FEARNHEAD, R. W., eds) pp. 127-131. Bristol: John Wright and Sons, Ltd., 1965. 8. AMBROSE, E. J., ELLIOTT, A.: Proc. Roy. Soc. London, A208, 75-84 (1951). 9. TERMINE, J. D., EANES, E. D., EIN, D., GLENNER, G. G.: Biopolymers 11:1103-
1113 (1972). 10. GLENNER, G. G., EANES, E. D., BLADEN, H. A., LINKE, R. P., TERMINE, J. D.: J. Histochem. Cytochem. 22:1141-1158 (1974). 11. TERMINE, J. D., TORCHIA, D. A.: J.Dent. Res., 56A, A55 (1977). 12. SHIMIZU, M., TANABE, T., FUKAE, M.: In: Proceedings of Third International Symposium on Tooth Eanmel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 13. SASAKI, S., SHIMOKAWA, H.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 14. MOE, D., BIRKEDAL-HANSEN, H.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 15. TORCHIA, D. A., VANDERHART, D. L.: In: Topics in Carbon 13 NMR Spectroscopy (G. C. LEVY, ed), In press. Wiley, New York,
1978. 16. TORCHIA, D. A., VANDERHART, D. L.: J. Mol. Biol. 104:315-321 (1967). 17. TORCHIA, D. A., PIEZ, K. A.: J. Mol. Biol. 76:419-424 (1973). 18. PINES, A., GIBBY, M. G., WAUGH, J. S.: J. Chem. Phys. 59:569-589 (1973). 19. SHARKEY, M. A., CONN, K. M., TERMINE, J. D.: J. Dent. Res. 56A, A82 (1977). 20. GREEN, M. R., PASTEWKA, J. V., PEACOCK, A. C.: Analyt. Biochem. 56:43-51 (1973). 21. ROBINSON, C., BRIGGS, H. D., ATKINSON, P. J., WEATHERELL, J. A.: In: Proceedings of Third International Symposium on Tooth Enamel (NYLEN, M. U., TERMINE, J. D., eds) pp. from this book, J. Dent. Res. Suppl., 1979. 22. EGGERT, F. M., ALLEN, G. A., BURGESS, R. C.: Biochem. J. 131:471484 (1973). 23. FUKAE, M., SHIMIZU, M.: Arch. Oral Biol. 19:381-386 (1974). 24. FERGUSON, K. A.: Metabolism 13:9851002 (1964). 25. RODBARD, D.: In: Methods of Protein Separation 2 (CATSIMPOOLAS, N. ed) pp. 145-218. New York: Plenum Press, 1976.
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Session III Discussion Dr. Reith: I take it you began your project with the expectation that you might find some structural protein positioned in some way which would account for the organization and orientation of the crystallites and the enamel prisms. I am not certain that an oriented structural protein is necessary. Some years ago Paul Weiss showed that, in tissue culture preparations, by putting a slight amount of tension on the gel substrate on which the cells were grown, he could cause these cells to assume an orientation similar to the direction of the tension which was applied. It seems to me that from what you have described, you have molecular components of such a size that they would display properties of viscous solutions. These molecules could be aligned if subject to flow or stress. As I recall at the Tooth Enamel I Conference, Dr. Boyde pointed out that the ameloblasts were continuously moving during the production of enamel matrix and I wondered if their continuous movement could set up some sort of flow tension which would account for an orientation of molecules such as you have described and thereby account for crystallites. Theoretically, this would cease once the cell movement ended. It would also cease if you disrupt the system as in test tube experiments. This may be one of the reasons why it is so very hard to find the basis for orientation in in vitro preparations. Would you care to comment on that? Dr. Termine: One of the models which fit the NMR data very aptly is elastin, and if you remember, elastin is composed of interconnected and cross-linked polypeptide chains. At any point of close .attachment, this protein is very rigid, that is, it doesn't show much segmental motion. On the other hand, further away from the cross-links, there is a considerable degree of segmental motion. Perhaps this could be a model for enamel where inter-chain hydrogen bonding or even protein-apatite bonds might serve as analagous points of attachment. Thus, both flow and chemical forces could possibly act in concert under cellular control in such a model system. Dr. Reith: The question I want to ask is, are we overlooking the movement of cells
as a possible orienting factor for enamel crystallites. Dr. Termine: That is possible, but I have no comment on this point. Dr. Glimcher: I would like to point out that there have already been published a number of studies concerning the molecular sturcture of embryonic enamel proteins, both in the solid state and in solution (J. Mol. Biol. 3:541-546, 1961; J. Ultrastruct. Res. 13:281-295, 296-307 and 308-317, 1965; Calc. Tissue Res. 2, Suppl, 1, 1968). In these publications we have pointed out the importance of distinguishing between the primary structure, secondary structure and other higher ordered structures and what is meant by and the significance of the term "gel" (Ferry, Adv. Protein Chem. 4:1-78, 1948). Let me emphasize that whether a protein system exists in solution or as a hydrated precipitate or a gel is an altogether different issue from the nature of the primary, secondary, or tertiary structure of the protein (Calc. Tiss. Res. 2, Suppl. 1, 1968). To say that the enamel proteins are or cannot be in a particular configuration like the 3-pleated sheet or a a helix because they are aggregated as a gel is equivalent to saying "this pencil is not made of wood, it is red." Further, we demonstrated that embryonic enamel matrix has components in it whose secondary structure is in the 13-pleated sheet configuration. Moreover, the fact that the two major reflections of the 13-pleated configuration are at right angles to one another indicates that the "crystallites" of the 13pleated protein are packed in what has been described as a "cross-4" arrangement, as opposed to a parallel-b packing. We were not able to induce this 13-pleated sheet secondary structure or the cross-,B packing arrangement in a variety of other proteins treated with EDTA and other solvents used in preparing the enamel. Further, the cross-b patterns were generated in both the dry and hydrated- states, so that they did not result artifactually from drying. Also, it is important to note that the 13-pleated sheet secondary structure and the tertiary cross-4 configuration were obtained from whole, intact enamel matrix as well as from the neutral soluble proteins which had been extracted, 779
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put into solution, and then re-formed into the solid state either as films or oriented
threads. Preliminary results with the pure phosphopeptides E3 and E4 (FEBS Lett., 79: 276-280, 1977; Biochim. Biophys. Acta 493:441451, 1977) have shown similar results: A sufficient amount of the peptide is in the pleated sheet configuration to generate the characteristic fiber diffraction pattern representative of this configuration. An examination of the amino acid composition of either the entire embryonic enamel matrix, the neutral soluble embryonic enamel proteins, or the pure E3 and E4 peptides also makes it abundantly clear that the majority of the proteins cannot be in an a-helical configuration because of their high contents of proline (Lindley, Biochim. Biophys. Acta 18:194, 1955). However, Ramachandran (Collagen, Ramachandran, ed. New York: Wiley Interscience, 1962, p. 27) has proposed a modification of the pleated sheet structures which can accommodate large amounts of proline. Thus the data that have been presented previously are strongly in favor of a (3-pleated sheet configuration for components in the developing enamel matrix, especially in the solid state. These are, in addition, packed in a cross-j configuration. At a minimum, one can conclude that this is the most favorable solid state configuration for these components, since they re-form into this configuration when aggregated from solution to the solid state. I want to emphasize very strongly that nothing has been published in the literature or presented today by Dr. Termine which would alter these conclusions. His data simply confirm our earlier studies that some of the enamel components are in a specific configuration and are not randomly ordered. How much of the protein, or how many of the components are organized in the (-pleated sheet configuration in the solid state cannot be determined from the x-ray data alone. However, using optical rotary dispersion we have estimated that, in solution, 6-1 0% of the components are in a (-pleated sheet configuration (J. Ultrastruct. Res. 13:296-307, 1965). Dr. Termine: What I meant to imply and what our own and the other data indicate is that very little of the protein in enamel is in a (-pleated sheet conformation. There is no question that some structural protein does exist in fetal enamel. The question as
to the proportion of :-pleated sheet protein relative to unordered protein in this fetal tissue remains to be determined. Dr. Veis: I'd like to ask Dr. Termine what the significance of the structures he measures in enamel protein is when you consider that the protein may have a different structure in the presence of all the mineral in the bound state. Dr. Termine: Dr. Veis, the NMR data that I showed was taken on scrapings from developing bovine enamel which contained about 50% mineral. These tissues were examined as dissected with no further treatment. Dr. Roufosse: Dr. Termine, I would like to ask if, in your 13C NMR study of the enamel proteins, you have assigned specific resonances to certain molecular groups. Dr. Termine: What we see are averaged resonance lines from the peptide backbone carbons. We have not done anything on individual sidechains as yet. Dr. Roufosse: Your conclusion that some segments of the backbone do not rotate are based on just the large values of the linewidths of unassigned resonances. Could these large values result from insufficient proton decoupling? Dr. Termine: No, that is not the case based on many different NMR data in this regard, including cross-polarization studies. Dr. Fearnhead: I would like to address a short question to Dr. Termine. It is related to his crystal-bound protein which he extracts with 0.5 molar EDTA. Dr. Termine: These proteins were extracted in four molar guanidine mixed with 0.5 molar EDTA. Dr. Fearnhead: I think you said that it all went into solution. I have tried t;6 do a similar thing, but I have only used .5 molar and .01 molar EDTA and I nearly always get a very small residue. I wonder what temperature you are making your extraction at, or can you explain the difference? Dr. Termine: Our tissues were extracted at 4 degrees, but I think the key to total dissolution is the guanidine. In guanidine, you denature the protein as you extract it and it comes out in random conformation. If you just use EDTA, it doesn't all come out.
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Session I Commentary Dr. Mechanic: Ten years ago at the Tooth Enamel II Conference, I succeeded in confusing everybody with the data I had. I am still confused. Now, for instance, Fukae and Shimizu in their poster at this meeting show very nicely a separation from porcine embryonic enamel of at least eight components of similar amino acid composition. We showed a minimum of about eleven different proteins based on amino acid analysis considerations, although we did not isolate any one of them in a pure form. What is confusing me now is the enzymes that are present in the enamel matrix. Drs. Sasaki and Shimokawa have an enzyme that acts at pH 6, Drs. Moe and Birkedal-Hansen, in their poster, show a protease that acts at a pH optimum of 8-9,
and Drs. Fukae and Shimizu have isolated an enzyme that acts at neutral pH. So now something else has to be straightened out with the enamel matrix. The last question concerns molecular weight. The highest molecular weight component previously reported from the University of Southern California is a proenamelin of about 58,000 daltons. Dr. Sasaki reports a proenamelin of 25,000 M.W., but has now revised his to 15,000 because of the high amount of proline it contains. And now Dr. Termine has proteins present in enamel upwards of 70,000 molecular weight down to 10,000. So, needless to say, the protein chemistry of the enamel matrix is still in a slightly confusing state, although many advances have been made since the last tooth enamel symposium.
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