Archs oral Pergamon
Bid Vol. 24, pp. 955 lo 963 Press Ltd 19X0. Punted m Great Britam
COMPONENTS OF THE ORGANIC MATRICES RABBIT INCISOR AND MOLAR DENTINE ISOLATED AFTER DIGESTION OF THE DEMINERALIZED TISSUES WITH COLLAGENASE
OF
A. J. SMITH, R. PRICE and A. G. LEAVER Department of Dental Sciences, School of Dental Surgery, Liverpool.
University
of Liverpool.
England
Summary-The insoluble residues remaining after EDTA demineralization of rabbit molar and incisor dentine were digested with collagenase. The soluble non-dialysable material from incisor dentine was fractionated by column chromatography and gel filtration into six fractions; the major one was closely similar to the soluble collagenase-released proteins of human dentine and bovine bone. The insoluble materials, remaining after collagenase digestion of incisor and molar dentine, were extracted with 8 M urea and with urea-mercaptoethanol, giving rise to soluble fractions which, together with three fractions isolated by column chromatography of the 8 M urea extract of the molar tooth preparation, consisted of proteins rich in aspartic acid and serine but with little or no phosphate. One fraction, resembling those isolated from the corresponding insoluble fractions of human dentine, was also found.
INTRODUCTION There has been considerable progress in recent years in the isolation and analysis of the non-collagenous components of the organic matrices of the bone and dentine of various species. The total non-collagenous matrix (NCM) is obtained by EDTA demineralization followed by collagenase digestion, both procedures being carried out within a dialysis sac. Approximately 70 per cent of the NCM goes into solution during demineralization and, if the residual insoluble material is then recovered and digested with collagenase, further NCM components may be isolated for specific study as described here. Leaver et al. (1975) subjected the EDTA-insoluble fractions of bovine bone and human dentine to collagenase digestion in dialysis sacs. Fractionation of the soluble material thus obtained revealed the presence of small amounts of what were presumed to be anionic glycoproteins, sialoproteins and glycosaminoglycans similar to those found in EDTA extracts, but the major fractions comprised collagenase-released proteins (CRPs) of a type previously unrecognized. These soluble CRPs were glycoproteins of relatively low carbohydrate content and of characteristic amino acid composition with approximately 200 residues of glycine and 17-20 residues of hydroxylysine per 1000. Hydroxyproline was totally absent. Subsequently Butler, Mikulski and Urist (1977) obtained similar soluble CRPs from rat dentine. They discarded a small amount of material remaining insoluble after collagenase digestion. The corresponding insoluble collagenase-released fraction (ICR) of human dentine was investigated by Leaver, Price and Smith (1978). Much of the material was soluble in 8 M urea and fractions, some of which closely resembled several of the ‘acid structural proteins’ isolated from various connective tissues (Timpl, Wolff 955
and Weiser, 1969; Wolff et al., 1971). were obtained by gel filtration. We have reported details of the composition of those fractions of rabbit incisor dentine present in an EDTA extract of the tissue (Smith and Leaver, 1979). Some analyses of fractions obtained from molar dentine were carried out but not reported. Our present study was concerned, in particular, with the soluble CRPs of rabbit incisor dentine. Only very small amounts of insoluble material were obtained but these appeared to be of unusual composition. Accordingly, the corresponding insoluble fraction of molar dentine (available in much greater amount) was investigated in more detail. The insoluble CRPs of rabbit molar and incisor dentine were the subject of a brief preliminary report (Price and Leaver, 1979). MATERIALS AND
METHODS
Incisor and molar teeth were extracted from the jaws of 16 albino rabbits, shortly after death. Powdered dentine of particle size ~60 mesh was obtained as described previously (Smith and Leaver, 1979). Demineralization of dentine Samples of powdered incisor and molar dentine were demineralized in 10 per cent (w/v) EDTA at pH 7.5 at 4”C, several changes of EDTA being made until the U.V. absorption of the extracts was minimal and the P content of the insoluble residue, as determined by the method of Chen, Toribara and Warner (1956), reached a constant low level of 0.05 per cent. The insoluble residues were filtered off, washed with distilled water and retained for collagenase digestion. Collagenase digestion of the EDTA-insoluble residue The EDTA-insoluble residues were placed in dialysis sacs, together with 25 ml of tris-calcium acetate
A. J. Smith, R. Price and A. G. Leaver
956
buffer, pH 7.2. Collagenase (8 mg) (Boehringer Grade 1 from Clostridium histolyticum, specific activity 4.0units per mg) was introduced into the sacs which were then suspended in 500 ml of the buffer and incubated in a Dubnoff shaking water-bath at 37°C. After 5 days, a further 4mg of collagenase was added to each sac and incubation continued for a further 5 days after which digestion was complete. The contents of the sacs were then centrifuged at 10,OOOg for 20min in a Beckmann Ultracentrifuge to separate the insoluble collagenase-released material (ICR). The supernatants (CRF) were decanted off, dialysed against distilled water and lyophilized. The insoluble material was washed three times with distilled water, recovered by centrifugation and lyophilized. As these studies were primarily concerned with incisor dentine, no further work was carried out on the soluble collagenase-released material from molar dentine. Because only small amounts of ICR were obtained from incisor dentine, the molar ICR material. obtained in larger amount, was retained for further investigation. Iso-electric focusing of incisor CRF Samples of lyophilized powder were taken up in distilled water and subjected to iso-electric focusing (Karlsson et al., 1973). The complete range of ampholines over the pH range 3.5-10.0 was used in flat-bed gels on an LKB Multiphor 2117 apparatus. The pH gradient was measured at 0.5 cm intervals using an antimony electrode. Focusing was continued for 2 h at 14°C at a constant 2 watts per gel. Gels were then stained with Coomassie blue G.250 (Diezel, Kapperschlager and Hofmann, 1972, as modified by Holbrook and Leaver, 1976). Cellulose acetate electrophoresis phoresis of incisor CRF
and gel
electro-
Electrophoresis was carried out on cellulose acetate strips (Celagram, Shandon Southern Ltd.) in a Shandon Southern apparatus using sodium barbiturate
0:
-.*”
E280nm
-
E
- - -.
Uromc oad
. . ..--
Smhc oad
buffer (0.04 M, pH 8.6) at 200 V for 60 min. A standard sample of human serum proteins (Behring) was run concurrently as a reference. The strips were stained with 0.0015 per cent nigrosine in 2 per cent (v/v) acetic acid and with Alcian blue (0.5 per cent in 3 per cent (v/v) acetic acid), destaining being achieved by the use of 1 per cent (v/v) acetic acid. Disc gel electrophoresis was carried out in a Shandon Southern apparatus in 7 per cent polyacrylamide gels (Davis, 1964). Gels were stained with Coomassie blue G-250 and with Alcian blue. Fractionation of incisor CRF on DEAE-cellulose The total CRF (60mg) was applied to a column (30 x 1.5 cm) of DEAE-cellulose (Whatman DE52, Whatman Biochemicals Ltd., Maidstone) using 400ml of an increasing linear gradient of (M7 M NaCl in 0.05 M tris-HCl buffer, pH 7.2). The effluents were collected in 5.2 ml fractions on an LKB Ultrarac fraction collector and the U.V. absorption of each fraction monitored at 260 and 280nm. Samples of each fraction were taken for the analysis of sialic acid (Skoza and Mohos, 1976) and uranic acid (Brown, 1946). The chloride content of every fifth fraction was determined (Hamilton, 1966). The contents of the tubes were pooled into fractions as designated in Fig. I. They were then dialysed against distilled water and lyophilized. Fractionation by gelfiltration The fractions designated 1 and 2 obtained by DEAE-cellulose chromatography of incisor CRF were further fractionated on columns (35 x 1.6cm) of Ultrogel AcA34, eluting with 0.05 M tris-HCl buffer, pH 7.2. Fractions (1.5 ml) were collected and the U.V. absorption of each fraction measured as before. The contents of the tubes were pooled into fractions, those obtained from fraction 1 being designated as shown in Fig. 2. Only one fraction (2’) resulted from gel filtration of fraction 2. The four fractions were dialysed against distilled water and lyophilized before analysis.
, 300
260nm
04
200 03 s m 2
w 02
s 100
01
0
0 1
0
10
20
30
40 Froctm
Fig. I. Fractionation
of incisor
CRF
on
50
60
70
80
90
No
DEAE-cellulose.
Volume
of each fraction
5.2 ml.
Collagenase-released
0 6w
components of rabbit dentine matrix
-E
260nm
.--
E 280 nm
957
0 4-
Fraction No. Fig. 2. Fractionation
of incisor CRF-fraction
Analysis of fractions from column chromatography gel filtration
on Ultrogel
and
Samples of the fractions isolated by gel filtration and column chromatography were hydrolysed with 6 M HCI for 20 h at 105°C. The amino acid compositions of the hydrolysates were then determined using a Bio-Cal BCIOOL autoanalyser. The sugar compositions of fractions IB and 1C were analysed by gas-liquid chromatography on a Pye-Unicam Series 204 chromatograph. Monosaccharides were determined as o-trimethylsilyl ethers of the methyl glycosides following methanolysis of the fractions in 1 M methanolic HCl (Chambers and Clamp, 1971). Approximate estimations of the molecular weights of fractions 1B and IC were carried out using the SDS gel electrophoretic method of Weber and Osborn (1969). Fraction 4 was eluted from DEAE-cellulose at a higher salt concentration (0.5 M NaCI) and was rich in uranic acid. A sample was hydrolysed with 6 M HCl for determination of amino acid composition on the autoanalyser. Further samples were digested with Pronase (Sigma P-150 Type VI from Streptococcus griseus) for 48 h at 55°C prior to electrophoresis on cellulose acetate (Stanbury and Embery. 1977) and determination of hexosamine and uranic acid (Blumenkrantz and Asboe-Hansen, 1973, 1976). Investigations of the insoluble fraction of rabbit incisor dentine
collagenase-released
Only small amounts of incisor ICR (IICR) were available. Samples were analysed for hydroxyproline (Woessner, 1961) hexose (Trevelyan and Harrison, 1952) and P (Chen et a/., 1956). A further sample was hydrolysed with 6 M HCl for determination of amino acid composition on the Autoanalyser. The remaining material was then extracted with 8 M urea until the absorption of the extracts at 280 nm became minimal. The extracts were dialysed against distilled water and lyophilized. A sample of the lyophilized powder (IICR-U) was hydrolysed for the determination of amino acid composition and further samples taken for determination of hexose and phosphorus. The material remaining insoluble after urea extraction was
AcA 34. Volume
of each fraction
1.5 ml.
then extracted with 8 M urea contacning 0.1 M mercaptoethanol, which appeared to dissolve this small residue. After dialysis against distilled water and lyophilization, a sample of lyophilized powder (IICR-UM) was hydrolysed for determination of amino acid composition. Samples of IICR and IICR-U were dissolved in SDS-mercaptoethanol for determination of their approximate molecular weights by gel electrophoresis (Weber and Osborn, 1969). Investigation of the insoluble collagenose-releused tion of rahbit molar dentine (MICR)
fruc-
Samples of MICR were taken for the determination of amino acid composition, hexose, hydroxyproline, phosphorus and approximate molecular weight by the methods referred to above. The remaining material was then extracted with 8 M urea until the absorption of the extracts at 280 nm became minimal. After dialysis and lyophilization, samples of this fraction (MICR-U) were taken for determination of amino acid composition, phosphorus, hexose and approximate molecular weight. DEAE-cellulose
chromatography
of MICR-C’
The lyophilized powder was now readily soluble in distilled water. It was taken up in tris-HCI buffer (0.05 M, pH 7.2) and applied to a column (30 x 1.5 cm) of DEAE-cellulose. Elution was continued using an increasing linear gradient of O-O.7 M NaCl in the same buffer. The effluents were collected in the usual way and the U.V. absorption of each 5.2 ml fraction measured at 280 nm. Chloride determinations were carried out on every fifth fraction. The contents of the tubes were pooled into fractions as designated in Fig. 3, dialysed against distilled water and lyophilized. Analysis of MICR-U fractions from tography
column chroma-
Samples of the three fractions obtained from DEAE-cellulose chromatography of MICR-U were taken for the determination of hexose and P and for the approximate determination of molecular weight by the usual methods. Further samples were hydro-
958
A. J. Smith, R. Price and A. G. Leaver 0’~
r
M-U,
r M-&t
-j
M-U,1
0 16.
: 8 N w
1.0 0.9 i 0.6 0.7
010. 08 ,/'
06
/'
04
Froctton
Fig. 3. Fractionation
of molar
fraction
ICR-U
lysed with 6 M HCI before determination of their amino acid compositions on the Autoanalyser. Urea-insoluble
residue of rabbit molar ICR
The residual material after extraction with 8 M urea was subsequently extracted with 8 M urea containing 0.1 M mercaptoethanol which appeared to solubilize the residue totally. After dialysis and lyophilization. the resulting powder (MICR-UM) was water-soluble. Samples were taken for the determination of hexose. P, and approximate molecular weight. A further sample was hydrolysed for amino acid determination. Figure 4 summarizes the various treatments and separation procedures.
RESULTS
The total collagenase-released fraction 0.36 per cent by weight of whole dentine
comprised and repre-
0.6 05 0.4 03
6 2 &
No
on DEAE-cellulose.
Volume of each fraction 5.2 ml.
sented approximately 5 per cent of the total NCM which itself comprised 25-30 per cent of the total matrix, a considerably higher proportion than that found in human dentine. Iso-electric focusing of the soluble CRF material resulted in one main band of pI 4.17, together with three minor bands with pI values in the range 3.38-4.25. Cellulose acetate electrophoresis revealed three bands staining with nigrosine, one of which coincided with a band staining with Alcian blue. The bands exhibited mobilities approaching that of serum albumin; two bands of higher mobility, staining only with Alcian blue were also observed. Polyacrylamide gel electrophoresis showed a fastmoving component of mobility corresponding to that of the electrophoretic front and staining with both Alcian blue and Coomassie blue. Two slower-moving bands staining only with Coomassie blue were also noted.
/-iyq Deminerallse
1
DEAE Ceilulose
Fig. 4. Fractionation
EM-Urea + 0 IM-mercaptoethanol
of soluble
EDTA
I
Dfs:ard
6M-ikea
Gel Flltratlon Uitrogel
I
and
8M-brea
DEAE-Cellulose chromatography
insoluble collagenase-released molar dentine.
8M -Urea + O.IM-mercoptaethonol
material
from
rabbit
incisor
and
Collagenase-released
components of rabbit dentine matrix
Table 1. Amino acid compositions
Hydroxyproline Aspartic acid Threonine Serine (and P-Serine) Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysine Arginine
959
of soluble CRF fractions
1A
1B
1C
2’
3
4
91 53 149 143 43 141 91
90 60 143 119 59 64 78
122 32 104 137 85 180 82
59 42 214 154 28 186 84
133 31 145 135 53 166 69
142 45 93 141 79 140 84
33
59
32
23
25
31 56 22 32
31 88 33 37
42 39 35
28 73 38
34 33 38 23 6 20 37 35
22 28 14 18 93 23 12
21 26 34 76 26 6 22 28 29
26 39 56 22 28 37 43
Values expressed as residues per 1000 total residues.
The elution profile of the total CRF material eluted from DEAE-cellulose is shown in Fig. 1. Subsequent amino acid analysis showed that the pre-gradient fraction consisted mainly of degraded collagenous material. Fraction 1 was eluted within the concentration range associated with less-acidic glycoproteins, (Fig. 2) whereas the minor fractions 2 and 3, appearing within the range 0.25-0.3 M NaCI, might be expected to represent anionic glycoproteins. A further fraction (4). rich in uranic acid was eluted at a salt concentration of 0.5 M. Gel filtration of fraction 1 gave rise to three peaks eluting at the void volume (lA), within the gel (1B) and at the column volume (1C). Fractions 1A and 1C differed from 1B in that their U.V. absorption was higher at 260nm than at 280nm. Only one peak (2’) was obtained by gel filtration of the more acidic fraction 2. This was eluted at the column volume and it, too, exhibited higher U.V.absorption at 260nm than at 280nm. The bulk of the material in fraction 1 appeared in 1C after gel filtration whereas, despite its relatively low U.V.absorption, fraction 1B contained more material than either 1A or the single fraction (2’) recovered from fraction 2 after gel filtration. Details of the amino acid compositions of these four fractions, together with those of the minor fraction (3) and the uranic-acid-rich fraction (4) obtained directly from DEAE-cellulose chromatography, are given in Table 1. Three of the fractions (lA, 2’ and 3) were present in only small amounts and were not investigated further. The amino acid composition of 1A is of the less-acidic glycoprotein type, its acidic amino acid content of 234 residues/l000 being similar to several of the less acidic glycoproteins isolated from human dentine by Thomas and Leaver (1977). It did not, however, resemble any of the EDTA-soluble fractions isolated from rabbit incisor dentine (Smith and Leaver, 1979). Fraction 3, on the other hand, was distinctly similar
to the EDTA-soluble fraction 4B we then reported, except in its higher glycine content. The composition of fraction 2’ is unusual, exhibiting a high level of serine coinciding with a low aspartic acid content. The amino acid composition of fraction 4 was similar to that of the proteoglycan fraction isolated previously from EDTA extracts of rabbit incisor dentine. The total hexosamine and uranic acid contents of this fraction determined after prior digestion with pronase were 18.4 per cent and 14.8 per cent respectively. Cellulose acetate electrophoresis of the pronasedigested fraction 4 gave rise to a single band, staining with Alcian blue and of corresponding mobility to an authentic sample of chondroitin-4-sulphate. Fractions 1B and 1C were analysed for total carbohydrate content and individual sugar composition by GLC. (Table 2). Both fractions ran as single entities, both in polyacrylamide gel electrophoresis and when run in SDS-gels for molecular weight determination. Their approximate molecular weights were 66,000
Table 2. Sugar contents of fractions 1B and 1C
Total carbohydrate (%) Individual sugars* Xylose Fucose Mannose Galactose Glucose N-Acetyl galactosamine N-Acetyl glucosamine Sialic acid
1B
1c
6.32
3.30
65.6 135.9 126.9 13.3 5.4 -
* Expressed as n moles per mg sample. - Not detected.
55.5 57.0 56.0 8.5 3.6
A. J. Smith, R. Price and A. G. Leaver
960
Table 3. Analytical data of insoluble collagenase-released fractions of rabbit incisor dentine (amino acid compositions as residues/l000 total residues) IICR Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine lso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysinc Arginine Hydroxyproline (:,) Hexose (92) Phosphorus (:I:,) Approx. mol. wt. Approx. mol. wt.
Trace 194 42 261 I9 30 103 57 Trace 63 23 46 14 17
8 50 57 18 0.05
I.25 0.20 24,000 and 10.000
IICR-U
IICR-UM
300 24 343 53 33 70 23 14 22 Trace I3 I6 Trace I3 Trace I9 40 I6
209 41 211 72 I03 80 46 Trace 55
N.D. 0.22 Trace 32,000, 25.000 and 10,000
N.D. N.D. N.D. N.D.
24 46 30 6 I’ 4; 22
N.D. = Not determined.
(IB) and 10,000 (IC). The amino acid composition of IB did not resemble any one of the less acidic glycoproteins found in rabbit incisor EDTA extracts. Its low carbohydrate content and lack of fucose contrasted with the less-acidic glycoproteins of human dentine (Thomas and Leaver, 1977). The carbohydrate moiety appears to comprise glucose and galactose in approximately equimolecular proportions together with about half as much mannose and smaller amounts of acetylated hexosamines. The major fraction IC contained only 3 per cent total carbohydrate with apparently equimolecular proportions of glucose, galactose and mannose and smaller amounts of acelyated hexosamines. Its amino acid composition was characterized by a high level of glycine, a total acidic amino acid content (259 residues/lOOO) midway between those of less-acidic and anionic glycoproteins, and by the presence of hydroxylysine. Cornpositio~ of’ insoluble
collugenuse-released
fractions
Analytical details of the insoluble collagenasereleased fractions of rabbit incisor dentine are presented in Table 3. The amino acid composition of the total insoluble fraction (IICR) was typified by a high combined level of aspartic acid and serine (455 residues per 1000). The urea extract (IICR-U) exhibited similar features, with 643 residues per 1000 of those two amino acids. Glycine was again the most abundant of the remaining amino acids, while a definite level of cystine was noted although only trace
amounts of hydroxylysine were present. The origmal fraction (IICR) contained only 0.20 per cent phosphorus which was reduced to trace quantities in the urea extract. An apparent anomaly was the appearance in SDS-gel electrophoresis of IICR-U of a faint band of higher molecular weight than those recognized in IICR. Although only minimal amounts of fraction IICR-UM were obtained, its amino composition was broadly similar to that of the original insoluble material (IICR). Because of the unexpected nature of the IICR fractions further studies were made of the corresponding fraction of molar dentine, available in considerably greater amount. Table 4 shows analytical details of the correspondmolar fractions (MICR, MICR-U and ing MICR-UM) together with those of the three fractions (MU-A, MU-B and MU-C) isolated by column urea-soluble fraction chromatography of the MICR-U. The amino acid compositions of the molar ICR and urea extracts (MICR-U) were similar to those of the corresponding incisor fractions. with very high combined levels of aspartic acid and serine. though with higher levels of glutamic acid and less glycine. They appear to consist of fractions of similar molecular size and to contain little organic P. The final fraction (MICR-UM) extracted only with ureamercaptoethanol had a very high level of aspartic acid and serine but lacked organic phosphate, again confirming that such proteins can be highly insoluble. Once solubilized, however, these fractions all became
Collagenase-released Table 4. Analytical data of insoluble (amino acid composition MICR
components of rabbit dentine matrix
961
collagenase-released fractions of rabbit molar expressed as residues/1000 total residues) MICR-U
MICR-UM
MU-A
MU-B
MU-C
97 57 77 118 57 170 70 Trace 49
330 22 362 55 31 48 21
300 21 333 53 49 65 30
14
Trace 12 22 15 19 8 9 44 20
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysine Arginine
Trace 235 42 221 81 44 61 44 -
262 36 250 75 44 68 40
173 48 222 58 42 121 71
35
33
47
21 55 15 22 I 23 61 33
20 46 Trace 22 7 17 52 28
19 36 14 19 4 60 43 24
36 79 26 45 31 46 41
8 17 7 11 10 11 39 14
Hydroxyproline (%) Hexose (%) Phosphorus (%) Mol. wt.
0.05 4.40 0.05 27,000 22,500 10,000
N.D.
N.D.
N.D.
Trace
~
Trace
1.25 Trace
dentine
N.D. 0.08 -10,000
24,000 10,000
N.D. = Not determined.
water-soluble after dialysis and lyophilization. Two of the fractions obtained by column chromatography (MU-A and MU-C) were of the high aspartic acid-serine type, whereas the third, minor, fraction (MU-B) had a quite different amino acid composition.
DISCUSSION
Soluble colluymase-released
fractions
When soluble CRPs were first isolated from ox bone and human dentine (Leaver et al., 1975) minor peaks observed during DEAE-cellulose chromatography and gel filtration were not analysed, although it was assumed, from the distribution of sialit and uranic acids, that they represented small amounts of those fractions previously isolated from EDTA extracts of the tissues. In our present study all fractions were subjected to analysis although three were present in only very small amounts. One, fraction 3. appeared to be similar to a fraction previously found in EDTA extracts. A proteoglycan fraction (4) similar to that found in the EDTA extract, was also found in the soluble fraction resulting from collagenase digestion of demineralized rabbit dentine. One of the two remaining fractions (1B) appeared to be of the less-acidic glycoprotein type in amino acid composition but with a low carbohydrate content which did not include fucose. a sugar typical of glycoproteins of this type. The major fraction (IQ. representing about 50 per OR 24
I?-
6
cent of the total collagenase-released material, resembled the soluble CRP of human dentine (Leaver et al., 1975) in several respects. Glycine (180 residues per 1000) was the most abundant amino acid; the combined levels of acidic amino acids were also comparable; the presence of hydroxylysine, although at a lower level, was a characteristic feature. Low levels of carbohydrate are typical of CRPs. Thus the rabbit CRP-IC contained only 3 per cent of carbohydrate, cornprizing mannose, glucose and galactose m equimolar proportions. Human dentine CRP contained less than 2 per cent of carbohydrate, with a hexosamine: hexose ratio of 2:l. This close similarity between the major soluble CRPs of human and rabbit dentine and bovine bone is of interest as the ICR fractions show considerable differences. None of the soluble fractions resembled collagenase, which is characterized by alanine being the most abundant amino acid and a low level of serine. Butler et al. (1977) subjected demineralized rat dentine to collagenase digestion and obtained three soluble fractions after gel filtration and column chromatography. Two were of the same genera1 type as the soluble CRPs of human and rabbit dentine, though not identical in amino acid composition. No carbohydrate analyses were reported. A third CRP was of an entirely different type; it contained over 800 residues per 1000 of aspartic acid, serine and phosphoserine and was regarded as identical to the phosphoprotein previously isolated from the soluble fraction of rat incisors (Butler, Finch and Desteno. 1972). This
962
A. J. Smith, R. Price and A. G. Leaver
observation led Butler et al. (1977) to concede that a collagen-bound phosphoprotein is present in rat dentine, albeit in small amount. Such collagen-bound phosphoproteins had been found in bovine dentine (Veis and Perry, 1967; Carmichael, Veis and Wang, 1971). It appears, therefore, that both soluble and collagenbound phosphoproteins of the high aspartic acid and serine type are present in rat and bovine dentine. Although we did not find phosphoproteins of this type in EDTA extracts of rabbit dentine (Smith and Leaver. 1979), such components were isolated from this tissue by Richardson rt ul. (1977) using 0.05 M tris/HCl/l M NaCl extraction after initial demineralization with 0.5 M acetic acid. Insol~hle collagenase-released
fractions
In our present study. none of the soluble CRF fractions were of this type, but the insoluble collagenasereleased (IICR) fractions of rabbit incisor dentine appeared to consist entirely of proteins of the high aspartic acid and serine type which, however, contained little or no phosphate. Although we have reservations about the use of rabbit molar dentine and have not reported on our investigations of the EDTA extracts and soluble CRF fractions obtained from this tissue. the insoluble fractions (MICR) obtained from collagenase digestion of demineralized molar dentine were studied. All fractions except one were of the high aspartic acid and serine type, with only small or trace amounts of phosphate. One fraction (MU-B), isolated by column chromatography, was similar in amino acid composition to human dentine ICR (Leaver. Price and Smith. 1978) and therefore bore a general resemblance to the acid structural proteins. Both incisor and molar dentine contained ICR material, which could only be solubilized by 8 M urea containing mercaptoethanol and which was also of the high aspartic acid and serine type. Whereas it seems reasonable to assume that soluble CRPs are firmly bound to collagen in human and rabbit dentine and in bovine bone, we have reservations in respect of the human dentine ICR fractions. which. being similar to structural glycoproteins, might exist alongside, rather than attached to collagen in the intact tissue. The same considerations must apply to the rabbit fraction MICR-UB which is of this type. As phosphoproteins of the high aspartic acid and serine type appear to exist in association with collagen in both bovine and rat dentine, it seems likely that rabbit dentine ICR fractions, rich in these amino acids, represent material originally bound to collagen. Their insolubility might be ascribed to the absence of phosphate but, once solubilized by urea or urea/mercaptoethanol, they become water-soluble. This might suggest that their insolubility is due to a high degree of association, but this would suggest that they exist as separate entities, rather than bound to collagen in the intact tissues. If, on the other hand, dentine proteins rich in aspartic acid and serine but containing little or no P are inherently insoluble, it is necessary to ascertain whether their existence specifically in rabbit dentine is of significance. The insoluble collagenasereleased fraction of rat dentine, described by Butler ef al. (1977), would repay investigation. The possibility
that the phosphoproteins might lose phosphate fortuitously, as an artifact of isolation, resulting in this insolubility, must be borne in mind. Acknowledgements-We thank Dr. S. D. Hogg and Mr. P. D. Wilkes for carrying out the GLC analyses and isoelectric focusing respectively and Mr. J. S. Bailie for his assistance in the preparation of the figures.
REFERENCES Blumenkrantz N. and Asboe-Hansen G. 1973. New method for quantitative determination of uranic acids Analyt. Biochem 54, 484-489. Blumenkrantz N. and Asboe-Hansen G. 1976. An assay for total hexosamine and a differential assay for glucosamine and galactosamine. Clin. Biochem. 9, 269-274. Brown A. H. 1946. Determination of pentoses in the presence of a large quantity of glucose. Archs Biochem. 11, 269-278. Butler W. T., Finch J. E. and Desteno W. 1972. Chemical character of proteins in rat incisors. Biochim. biophys. Acta 257, 167-171. Butler W. T., Mikulski A. and Urist M. R. 1977. Noncollagenous proteins of a rat dentine matrix possessing bone morphogenetic activity. J. dent. Res. 56, 228-232. Carmichael D. J.. Veis A. and Wang E. T. 1971. Dentin matrix collagens. Evidence for a covalently linked phosphoprotein attachment. Cal. Tiss. Rex 7, 331-344. Chambers R. E. and Clamp J. R. 1971. An assessment of methanolysis and other factors used in the analysis of carbohydrate containing materials. Biochem. J. 125, 1009-1018. Chen P. S., ToFibara T. Y. and Warner H. 1956. Microdetermination of phosphorus. Analyt. Chem. 28, 1956-1958. Davis B. J. 1964. Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Diezel W., Kapperschlager G. and Hofmann E. 1972. An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue. Analyt. Biochem. 48, 617-620. Hamilton T. H. 1966. A direct photoelectric method for chloride in biological fluids employing mercuric thiocyanate and perchloric acid. Clin. Chem. 12, l--17. Holbrook I. B. and Leaver A. G. 1976. A procedure to increase the sensitivity of staining by Coomassie Brilliant Blue-G250-perchloric acid solution. Analyt. Biochem. 75, 634-636. Karlsson C., Davies H. I., Ohmann J. and Andersson U. B. 1973. LKB 2117 Multiphor-1. Analytical thin-gel electrofocussing in polyacrylamide gel. LKB Application Note 75. Leaver A. G., Holbrook I. B., Jones I. L., Thomas M. and Sheil L. 1975. Components of the organic matrices of bone and dentine isolated only after digestion with collagenase. Archs oral Bio/. 20, 21 l-216. Leaver A. G., Price R. and Smith A. J. 1978. The insoluble fraction isolated after digestion of the demineralized human dentine matrix with collagenase. Archs oral Biof. 23, 511-513. Price R. and Leaver A. G. 1978. Insoluble collagenasereleased proteins of rabbit molar and incisor dentine. J. dent. Res. 57, 1271. Richardson W. S., Beagle W. F., Butler W. T. and Munksgaard E. G. 1977. The phosphoprotein of rabbit incisors. .I. dent. Res. 54, 233-236. Skoza L. and Mohos S. 1976. Stable thiobarbituric acid chromophore with dimethyl sulphoxide-application to sialic acid assay in analytical de-0-acetylation. Biochem. .I. 159, 457-462.
Collagenase-released
components of rabbit dentine matrix
Smith A. J. and Leaver A. G. 1979. Non-collagenous components of the organic matrix of rabbit incisor dentine. Archs oral Biol. 2$ 449-454.
Stanbury J. B. and Embery Cl. 1977. An improved electrophoretic procedure for the detection of acidic glycosaminoglycans (mucopolysaccharides). Med. Lab. Sci. 34. 267-269.
Thomas M. and Leaver A. G. 1977. The less-acidic glycoproteins of the organic matrix of human dentine Archs &al Biol. 22, 54S549.
Timol R.. Wolff 1. and Weiser M. 1969. Acid structural pr’oteins of connective tissue--I. Solubilization and preliminary characterization. Biochim. hiophys. Acta 194. 112-120. Trevelyan W. E. and Harrison J. S. 1952. Studies on yeast
963
metabolism--I. Fractionation and microdetermination of cell carbohydrates. Biochem. J. 50, 298-309. Veis A. and Perry 1967. The phosphoprotein of the dentin matrix. Biockkstry 6, 2469-2416. . Weber K. and Osborn M. 1969. The reliabilitv of molecular weight determinations by dodecylsulphate-polyacrylamide gel electrophoresis. J: biol. C&m. i24, 44064412. Woessner J. F. 1961. The determination of hvdroxvnroline in tissue and protein samples containing small proportions of this amino acid. Archs Biochem. Biophys. 93. 44&447.
Wolff I., Fuchswans W., Weiser M., Furthmayr H. and Timpl R. 1971. Acid structural proteins of connective tissue. Characterization of their heterogeneous nature. Eur. J. Biochem. 20, 426-431.
Bid Vol. 24, pp. 955 lo 963 Press Ltd 19X0. Punted m Great Britam
COMPONENTS OF THE ORGANIC MATRICES RABBIT INCISOR AND MOLAR DENTINE ISOLATED AFTER DIGESTION OF THE DEMINERALIZED TISSUES WITH COLLAGENASE
OF
A. J. SMITH, R. PRICE and A. G. LEAVER Department of Dental Sciences, School of Dental Surgery, Liverpool.
University
of Liverpool.
England
Summary-The insoluble residues remaining after EDTA demineralization of rabbit molar and incisor dentine were digested with collagenase. The soluble non-dialysable material from incisor dentine was fractionated by column chromatography and gel filtration into six fractions; the major one was closely similar to the soluble collagenase-released proteins of human dentine and bovine bone. The insoluble materials, remaining after collagenase digestion of incisor and molar dentine, were extracted with 8 M urea and with urea-mercaptoethanol, giving rise to soluble fractions which, together with three fractions isolated by column chromatography of the 8 M urea extract of the molar tooth preparation, consisted of proteins rich in aspartic acid and serine but with little or no phosphate. One fraction, resembling those isolated from the corresponding insoluble fractions of human dentine, was also found.
INTRODUCTION There has been considerable progress in recent years in the isolation and analysis of the non-collagenous components of the organic matrices of the bone and dentine of various species. The total non-collagenous matrix (NCM) is obtained by EDTA demineralization followed by collagenase digestion, both procedures being carried out within a dialysis sac. Approximately 70 per cent of the NCM goes into solution during demineralization and, if the residual insoluble material is then recovered and digested with collagenase, further NCM components may be isolated for specific study as described here. Leaver et al. (1975) subjected the EDTA-insoluble fractions of bovine bone and human dentine to collagenase digestion in dialysis sacs. Fractionation of the soluble material thus obtained revealed the presence of small amounts of what were presumed to be anionic glycoproteins, sialoproteins and glycosaminoglycans similar to those found in EDTA extracts, but the major fractions comprised collagenase-released proteins (CRPs) of a type previously unrecognized. These soluble CRPs were glycoproteins of relatively low carbohydrate content and of characteristic amino acid composition with approximately 200 residues of glycine and 17-20 residues of hydroxylysine per 1000. Hydroxyproline was totally absent. Subsequently Butler, Mikulski and Urist (1977) obtained similar soluble CRPs from rat dentine. They discarded a small amount of material remaining insoluble after collagenase digestion. The corresponding insoluble collagenase-released fraction (ICR) of human dentine was investigated by Leaver, Price and Smith (1978). Much of the material was soluble in 8 M urea and fractions, some of which closely resembled several of the ‘acid structural proteins’ isolated from various connective tissues (Timpl, Wolff 955
and Weiser, 1969; Wolff et al., 1971). were obtained by gel filtration. We have reported details of the composition of those fractions of rabbit incisor dentine present in an EDTA extract of the tissue (Smith and Leaver, 1979). Some analyses of fractions obtained from molar dentine were carried out but not reported. Our present study was concerned, in particular, with the soluble CRPs of rabbit incisor dentine. Only very small amounts of insoluble material were obtained but these appeared to be of unusual composition. Accordingly, the corresponding insoluble fraction of molar dentine (available in much greater amount) was investigated in more detail. The insoluble CRPs of rabbit molar and incisor dentine were the subject of a brief preliminary report (Price and Leaver, 1979). MATERIALS AND
METHODS
Incisor and molar teeth were extracted from the jaws of 16 albino rabbits, shortly after death. Powdered dentine of particle size ~60 mesh was obtained as described previously (Smith and Leaver, 1979). Demineralization of dentine Samples of powdered incisor and molar dentine were demineralized in 10 per cent (w/v) EDTA at pH 7.5 at 4”C, several changes of EDTA being made until the U.V. absorption of the extracts was minimal and the P content of the insoluble residue, as determined by the method of Chen, Toribara and Warner (1956), reached a constant low level of 0.05 per cent. The insoluble residues were filtered off, washed with distilled water and retained for collagenase digestion. Collagenase digestion of the EDTA-insoluble residue The EDTA-insoluble residues were placed in dialysis sacs, together with 25 ml of tris-calcium acetate
A. J. Smith, R. Price and A. G. Leaver
956
buffer, pH 7.2. Collagenase (8 mg) (Boehringer Grade 1 from Clostridium histolyticum, specific activity 4.0units per mg) was introduced into the sacs which were then suspended in 500 ml of the buffer and incubated in a Dubnoff shaking water-bath at 37°C. After 5 days, a further 4mg of collagenase was added to each sac and incubation continued for a further 5 days after which digestion was complete. The contents of the sacs were then centrifuged at 10,OOOg for 20min in a Beckmann Ultracentrifuge to separate the insoluble collagenase-released material (ICR). The supernatants (CRF) were decanted off, dialysed against distilled water and lyophilized. The insoluble material was washed three times with distilled water, recovered by centrifugation and lyophilized. As these studies were primarily concerned with incisor dentine, no further work was carried out on the soluble collagenase-released material from molar dentine. Because only small amounts of ICR were obtained from incisor dentine, the molar ICR material. obtained in larger amount, was retained for further investigation. Iso-electric focusing of incisor CRF Samples of lyophilized powder were taken up in distilled water and subjected to iso-electric focusing (Karlsson et al., 1973). The complete range of ampholines over the pH range 3.5-10.0 was used in flat-bed gels on an LKB Multiphor 2117 apparatus. The pH gradient was measured at 0.5 cm intervals using an antimony electrode. Focusing was continued for 2 h at 14°C at a constant 2 watts per gel. Gels were then stained with Coomassie blue G.250 (Diezel, Kapperschlager and Hofmann, 1972, as modified by Holbrook and Leaver, 1976). Cellulose acetate electrophoresis phoresis of incisor CRF
and gel
electro-
Electrophoresis was carried out on cellulose acetate strips (Celagram, Shandon Southern Ltd.) in a Shandon Southern apparatus using sodium barbiturate
0:
-.*”
E280nm
-
E
- - -.
Uromc oad
. . ..--
Smhc oad
buffer (0.04 M, pH 8.6) at 200 V for 60 min. A standard sample of human serum proteins (Behring) was run concurrently as a reference. The strips were stained with 0.0015 per cent nigrosine in 2 per cent (v/v) acetic acid and with Alcian blue (0.5 per cent in 3 per cent (v/v) acetic acid), destaining being achieved by the use of 1 per cent (v/v) acetic acid. Disc gel electrophoresis was carried out in a Shandon Southern apparatus in 7 per cent polyacrylamide gels (Davis, 1964). Gels were stained with Coomassie blue G-250 and with Alcian blue. Fractionation of incisor CRF on DEAE-cellulose The total CRF (60mg) was applied to a column (30 x 1.5 cm) of DEAE-cellulose (Whatman DE52, Whatman Biochemicals Ltd., Maidstone) using 400ml of an increasing linear gradient of (M7 M NaCl in 0.05 M tris-HCl buffer, pH 7.2). The effluents were collected in 5.2 ml fractions on an LKB Ultrarac fraction collector and the U.V. absorption of each fraction monitored at 260 and 280nm. Samples of each fraction were taken for the analysis of sialic acid (Skoza and Mohos, 1976) and uranic acid (Brown, 1946). The chloride content of every fifth fraction was determined (Hamilton, 1966). The contents of the tubes were pooled into fractions as designated in Fig. I. They were then dialysed against distilled water and lyophilized. Fractionation by gelfiltration The fractions designated 1 and 2 obtained by DEAE-cellulose chromatography of incisor CRF were further fractionated on columns (35 x 1.6cm) of Ultrogel AcA34, eluting with 0.05 M tris-HCl buffer, pH 7.2. Fractions (1.5 ml) were collected and the U.V. absorption of each fraction measured as before. The contents of the tubes were pooled into fractions, those obtained from fraction 1 being designated as shown in Fig. 2. Only one fraction (2’) resulted from gel filtration of fraction 2. The four fractions were dialysed against distilled water and lyophilized before analysis.
, 300
260nm
04
200 03 s m 2
w 02
s 100
01
0
0 1
0
10
20
30
40 Froctm
Fig. I. Fractionation
of incisor
CRF
on
50
60
70
80
90
No
DEAE-cellulose.
Volume
of each fraction
5.2 ml.
Collagenase-released
0 6w
components of rabbit dentine matrix
-E
260nm
.--
E 280 nm
957
0 4-
Fraction No. Fig. 2. Fractionation
of incisor CRF-fraction
Analysis of fractions from column chromatography gel filtration
on Ultrogel
and
Samples of the fractions isolated by gel filtration and column chromatography were hydrolysed with 6 M HCI for 20 h at 105°C. The amino acid compositions of the hydrolysates were then determined using a Bio-Cal BCIOOL autoanalyser. The sugar compositions of fractions IB and 1C were analysed by gas-liquid chromatography on a Pye-Unicam Series 204 chromatograph. Monosaccharides were determined as o-trimethylsilyl ethers of the methyl glycosides following methanolysis of the fractions in 1 M methanolic HCl (Chambers and Clamp, 1971). Approximate estimations of the molecular weights of fractions 1B and IC were carried out using the SDS gel electrophoretic method of Weber and Osborn (1969). Fraction 4 was eluted from DEAE-cellulose at a higher salt concentration (0.5 M NaCI) and was rich in uranic acid. A sample was hydrolysed with 6 M HCl for determination of amino acid composition on the autoanalyser. Further samples were digested with Pronase (Sigma P-150 Type VI from Streptococcus griseus) for 48 h at 55°C prior to electrophoresis on cellulose acetate (Stanbury and Embery. 1977) and determination of hexosamine and uranic acid (Blumenkrantz and Asboe-Hansen, 1973, 1976). Investigations of the insoluble fraction of rabbit incisor dentine
collagenase-released
Only small amounts of incisor ICR (IICR) were available. Samples were analysed for hydroxyproline (Woessner, 1961) hexose (Trevelyan and Harrison, 1952) and P (Chen et a/., 1956). A further sample was hydrolysed with 6 M HCl for determination of amino acid composition on the Autoanalyser. The remaining material was then extracted with 8 M urea until the absorption of the extracts at 280 nm became minimal. The extracts were dialysed against distilled water and lyophilized. A sample of the lyophilized powder (IICR-U) was hydrolysed for the determination of amino acid composition and further samples taken for determination of hexose and phosphorus. The material remaining insoluble after urea extraction was
AcA 34. Volume
of each fraction
1.5 ml.
then extracted with 8 M urea contacning 0.1 M mercaptoethanol, which appeared to dissolve this small residue. After dialysis against distilled water and lyophilization, a sample of lyophilized powder (IICR-UM) was hydrolysed for determination of amino acid composition. Samples of IICR and IICR-U were dissolved in SDS-mercaptoethanol for determination of their approximate molecular weights by gel electrophoresis (Weber and Osborn, 1969). Investigation of the insoluble collagenose-releused tion of rahbit molar dentine (MICR)
fruc-
Samples of MICR were taken for the determination of amino acid composition, hexose, hydroxyproline, phosphorus and approximate molecular weight by the methods referred to above. The remaining material was then extracted with 8 M urea until the absorption of the extracts at 280 nm became minimal. After dialysis and lyophilization, samples of this fraction (MICR-U) were taken for determination of amino acid composition, phosphorus, hexose and approximate molecular weight. DEAE-cellulose
chromatography
of MICR-C’
The lyophilized powder was now readily soluble in distilled water. It was taken up in tris-HCI buffer (0.05 M, pH 7.2) and applied to a column (30 x 1.5 cm) of DEAE-cellulose. Elution was continued using an increasing linear gradient of O-O.7 M NaCl in the same buffer. The effluents were collected in the usual way and the U.V. absorption of each 5.2 ml fraction measured at 280 nm. Chloride determinations were carried out on every fifth fraction. The contents of the tubes were pooled into fractions as designated in Fig. 3, dialysed against distilled water and lyophilized. Analysis of MICR-U fractions from tography
column chroma-
Samples of the three fractions obtained from DEAE-cellulose chromatography of MICR-U were taken for the determination of hexose and P and for the approximate determination of molecular weight by the usual methods. Further samples were hydro-
958
A. J. Smith, R. Price and A. G. Leaver 0’~
r
M-U,
r M-&t
-j
M-U,1
0 16.
: 8 N w
1.0 0.9 i 0.6 0.7
010. 08 ,/'
06
/'
04
Froctton
Fig. 3. Fractionation
of molar
fraction
ICR-U
lysed with 6 M HCI before determination of their amino acid compositions on the Autoanalyser. Urea-insoluble
residue of rabbit molar ICR
The residual material after extraction with 8 M urea was subsequently extracted with 8 M urea containing 0.1 M mercaptoethanol which appeared to solubilize the residue totally. After dialysis and lyophilization. the resulting powder (MICR-UM) was water-soluble. Samples were taken for the determination of hexose. P, and approximate molecular weight. A further sample was hydrolysed for amino acid determination. Figure 4 summarizes the various treatments and separation procedures.
RESULTS
The total collagenase-released fraction 0.36 per cent by weight of whole dentine
comprised and repre-
0.6 05 0.4 03
6 2 &
No
on DEAE-cellulose.
Volume of each fraction 5.2 ml.
sented approximately 5 per cent of the total NCM which itself comprised 25-30 per cent of the total matrix, a considerably higher proportion than that found in human dentine. Iso-electric focusing of the soluble CRF material resulted in one main band of pI 4.17, together with three minor bands with pI values in the range 3.38-4.25. Cellulose acetate electrophoresis revealed three bands staining with nigrosine, one of which coincided with a band staining with Alcian blue. The bands exhibited mobilities approaching that of serum albumin; two bands of higher mobility, staining only with Alcian blue were also observed. Polyacrylamide gel electrophoresis showed a fastmoving component of mobility corresponding to that of the electrophoretic front and staining with both Alcian blue and Coomassie blue. Two slower-moving bands staining only with Coomassie blue were also noted.
/-iyq Deminerallse
1
DEAE Ceilulose
Fig. 4. Fractionation
EM-Urea + 0 IM-mercaptoethanol
of soluble
EDTA
I
Dfs:ard
6M-ikea
Gel Flltratlon Uitrogel
I
and
8M-brea
DEAE-Cellulose chromatography
insoluble collagenase-released molar dentine.
8M -Urea + O.IM-mercoptaethonol
material
from
rabbit
incisor
and
Collagenase-released
components of rabbit dentine matrix
Table 1. Amino acid compositions
Hydroxyproline Aspartic acid Threonine Serine (and P-Serine) Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysine Arginine
959
of soluble CRF fractions
1A
1B
1C
2’
3
4
91 53 149 143 43 141 91
90 60 143 119 59 64 78
122 32 104 137 85 180 82
59 42 214 154 28 186 84
133 31 145 135 53 166 69
142 45 93 141 79 140 84
33
59
32
23
25
31 56 22 32
31 88 33 37
42 39 35
28 73 38
34 33 38 23 6 20 37 35
22 28 14 18 93 23 12
21 26 34 76 26 6 22 28 29
26 39 56 22 28 37 43
Values expressed as residues per 1000 total residues.
The elution profile of the total CRF material eluted from DEAE-cellulose is shown in Fig. 1. Subsequent amino acid analysis showed that the pre-gradient fraction consisted mainly of degraded collagenous material. Fraction 1 was eluted within the concentration range associated with less-acidic glycoproteins, (Fig. 2) whereas the minor fractions 2 and 3, appearing within the range 0.25-0.3 M NaCI, might be expected to represent anionic glycoproteins. A further fraction (4). rich in uranic acid was eluted at a salt concentration of 0.5 M. Gel filtration of fraction 1 gave rise to three peaks eluting at the void volume (lA), within the gel (1B) and at the column volume (1C). Fractions 1A and 1C differed from 1B in that their U.V. absorption was higher at 260nm than at 280nm. Only one peak (2’) was obtained by gel filtration of the more acidic fraction 2. This was eluted at the column volume and it, too, exhibited higher U.V.absorption at 260nm than at 280nm. The bulk of the material in fraction 1 appeared in 1C after gel filtration whereas, despite its relatively low U.V.absorption, fraction 1B contained more material than either 1A or the single fraction (2’) recovered from fraction 2 after gel filtration. Details of the amino acid compositions of these four fractions, together with those of the minor fraction (3) and the uranic-acid-rich fraction (4) obtained directly from DEAE-cellulose chromatography, are given in Table 1. Three of the fractions (lA, 2’ and 3) were present in only small amounts and were not investigated further. The amino acid composition of 1A is of the less-acidic glycoprotein type, its acidic amino acid content of 234 residues/l000 being similar to several of the less acidic glycoproteins isolated from human dentine by Thomas and Leaver (1977). It did not, however, resemble any of the EDTA-soluble fractions isolated from rabbit incisor dentine (Smith and Leaver, 1979). Fraction 3, on the other hand, was distinctly similar
to the EDTA-soluble fraction 4B we then reported, except in its higher glycine content. The composition of fraction 2’ is unusual, exhibiting a high level of serine coinciding with a low aspartic acid content. The amino acid composition of fraction 4 was similar to that of the proteoglycan fraction isolated previously from EDTA extracts of rabbit incisor dentine. The total hexosamine and uranic acid contents of this fraction determined after prior digestion with pronase were 18.4 per cent and 14.8 per cent respectively. Cellulose acetate electrophoresis of the pronasedigested fraction 4 gave rise to a single band, staining with Alcian blue and of corresponding mobility to an authentic sample of chondroitin-4-sulphate. Fractions 1B and 1C were analysed for total carbohydrate content and individual sugar composition by GLC. (Table 2). Both fractions ran as single entities, both in polyacrylamide gel electrophoresis and when run in SDS-gels for molecular weight determination. Their approximate molecular weights were 66,000
Table 2. Sugar contents of fractions 1B and 1C
Total carbohydrate (%) Individual sugars* Xylose Fucose Mannose Galactose Glucose N-Acetyl galactosamine N-Acetyl glucosamine Sialic acid
1B
1c
6.32
3.30
65.6 135.9 126.9 13.3 5.4 -
* Expressed as n moles per mg sample. - Not detected.
55.5 57.0 56.0 8.5 3.6
A. J. Smith, R. Price and A. G. Leaver
960
Table 3. Analytical data of insoluble collagenase-released fractions of rabbit incisor dentine (amino acid compositions as residues/l000 total residues) IICR Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine lso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysinc Arginine Hydroxyproline (:,) Hexose (92) Phosphorus (:I:,) Approx. mol. wt. Approx. mol. wt.
Trace 194 42 261 I9 30 103 57 Trace 63 23 46 14 17
8 50 57 18 0.05
I.25 0.20 24,000 and 10.000
IICR-U
IICR-UM
300 24 343 53 33 70 23 14 22 Trace I3 I6 Trace I3 Trace I9 40 I6
209 41 211 72 I03 80 46 Trace 55
N.D. 0.22 Trace 32,000, 25.000 and 10,000
N.D. N.D. N.D. N.D.
24 46 30 6 I’ 4; 22
N.D. = Not determined.
(IB) and 10,000 (IC). The amino acid composition of IB did not resemble any one of the less acidic glycoproteins found in rabbit incisor EDTA extracts. Its low carbohydrate content and lack of fucose contrasted with the less-acidic glycoproteins of human dentine (Thomas and Leaver, 1977). The carbohydrate moiety appears to comprise glucose and galactose in approximately equimolecular proportions together with about half as much mannose and smaller amounts of acetylated hexosamines. The major fraction IC contained only 3 per cent total carbohydrate with apparently equimolecular proportions of glucose, galactose and mannose and smaller amounts of acelyated hexosamines. Its amino acid composition was characterized by a high level of glycine, a total acidic amino acid content (259 residues/lOOO) midway between those of less-acidic and anionic glycoproteins, and by the presence of hydroxylysine. Cornpositio~ of’ insoluble
collugenuse-released
fractions
Analytical details of the insoluble collagenasereleased fractions of rabbit incisor dentine are presented in Table 3. The amino acid composition of the total insoluble fraction (IICR) was typified by a high combined level of aspartic acid and serine (455 residues per 1000). The urea extract (IICR-U) exhibited similar features, with 643 residues per 1000 of those two amino acids. Glycine was again the most abundant of the remaining amino acids, while a definite level of cystine was noted although only trace
amounts of hydroxylysine were present. The origmal fraction (IICR) contained only 0.20 per cent phosphorus which was reduced to trace quantities in the urea extract. An apparent anomaly was the appearance in SDS-gel electrophoresis of IICR-U of a faint band of higher molecular weight than those recognized in IICR. Although only minimal amounts of fraction IICR-UM were obtained, its amino composition was broadly similar to that of the original insoluble material (IICR). Because of the unexpected nature of the IICR fractions further studies were made of the corresponding fraction of molar dentine, available in considerably greater amount. Table 4 shows analytical details of the correspondmolar fractions (MICR, MICR-U and ing MICR-UM) together with those of the three fractions (MU-A, MU-B and MU-C) isolated by column urea-soluble fraction chromatography of the MICR-U. The amino acid compositions of the molar ICR and urea extracts (MICR-U) were similar to those of the corresponding incisor fractions. with very high combined levels of aspartic acid and serine. though with higher levels of glutamic acid and less glycine. They appear to consist of fractions of similar molecular size and to contain little organic P. The final fraction (MICR-UM) extracted only with ureamercaptoethanol had a very high level of aspartic acid and serine but lacked organic phosphate, again confirming that such proteins can be highly insoluble. Once solubilized, however, these fractions all became
Collagenase-released Table 4. Analytical data of insoluble (amino acid composition MICR
components of rabbit dentine matrix
961
collagenase-released fractions of rabbit molar expressed as residues/1000 total residues) MICR-U
MICR-UM
MU-A
MU-B
MU-C
97 57 77 118 57 170 70 Trace 49
330 22 362 55 31 48 21
300 21 333 53 49 65 30
14
Trace 12 22 15 19 8 9 44 20
Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Hydroxylysine Histidine Lysine Arginine
Trace 235 42 221 81 44 61 44 -
262 36 250 75 44 68 40
173 48 222 58 42 121 71
35
33
47
21 55 15 22 I 23 61 33
20 46 Trace 22 7 17 52 28
19 36 14 19 4 60 43 24
36 79 26 45 31 46 41
8 17 7 11 10 11 39 14
Hydroxyproline (%) Hexose (%) Phosphorus (%) Mol. wt.
0.05 4.40 0.05 27,000 22,500 10,000
N.D.
N.D.
N.D.
Trace
~
Trace
1.25 Trace
dentine
N.D. 0.08 -10,000
24,000 10,000
N.D. = Not determined.
water-soluble after dialysis and lyophilization. Two of the fractions obtained by column chromatography (MU-A and MU-C) were of the high aspartic acid-serine type, whereas the third, minor, fraction (MU-B) had a quite different amino acid composition.
DISCUSSION
Soluble colluymase-released
fractions
When soluble CRPs were first isolated from ox bone and human dentine (Leaver et al., 1975) minor peaks observed during DEAE-cellulose chromatography and gel filtration were not analysed, although it was assumed, from the distribution of sialit and uranic acids, that they represented small amounts of those fractions previously isolated from EDTA extracts of the tissues. In our present study all fractions were subjected to analysis although three were present in only very small amounts. One, fraction 3. appeared to be similar to a fraction previously found in EDTA extracts. A proteoglycan fraction (4) similar to that found in the EDTA extract, was also found in the soluble fraction resulting from collagenase digestion of demineralized rabbit dentine. One of the two remaining fractions (1B) appeared to be of the less-acidic glycoprotein type in amino acid composition but with a low carbohydrate content which did not include fucose. a sugar typical of glycoproteins of this type. The major fraction (IQ. representing about 50 per OR 24
I?-
6
cent of the total collagenase-released material, resembled the soluble CRP of human dentine (Leaver et al., 1975) in several respects. Glycine (180 residues per 1000) was the most abundant amino acid; the combined levels of acidic amino acids were also comparable; the presence of hydroxylysine, although at a lower level, was a characteristic feature. Low levels of carbohydrate are typical of CRPs. Thus the rabbit CRP-IC contained only 3 per cent of carbohydrate, cornprizing mannose, glucose and galactose m equimolar proportions. Human dentine CRP contained less than 2 per cent of carbohydrate, with a hexosamine: hexose ratio of 2:l. This close similarity between the major soluble CRPs of human and rabbit dentine and bovine bone is of interest as the ICR fractions show considerable differences. None of the soluble fractions resembled collagenase, which is characterized by alanine being the most abundant amino acid and a low level of serine. Butler et al. (1977) subjected demineralized rat dentine to collagenase digestion and obtained three soluble fractions after gel filtration and column chromatography. Two were of the same genera1 type as the soluble CRPs of human and rabbit dentine, though not identical in amino acid composition. No carbohydrate analyses were reported. A third CRP was of an entirely different type; it contained over 800 residues per 1000 of aspartic acid, serine and phosphoserine and was regarded as identical to the phosphoprotein previously isolated from the soluble fraction of rat incisors (Butler, Finch and Desteno. 1972). This
962
A. J. Smith, R. Price and A. G. Leaver
observation led Butler et al. (1977) to concede that a collagen-bound phosphoprotein is present in rat dentine, albeit in small amount. Such collagen-bound phosphoproteins had been found in bovine dentine (Veis and Perry, 1967; Carmichael, Veis and Wang, 1971). It appears, therefore, that both soluble and collagenbound phosphoproteins of the high aspartic acid and serine type are present in rat and bovine dentine. Although we did not find phosphoproteins of this type in EDTA extracts of rabbit dentine (Smith and Leaver. 1979), such components were isolated from this tissue by Richardson rt ul. (1977) using 0.05 M tris/HCl/l M NaCl extraction after initial demineralization with 0.5 M acetic acid. Insol~hle collagenase-released
fractions
In our present study. none of the soluble CRF fractions were of this type, but the insoluble collagenasereleased (IICR) fractions of rabbit incisor dentine appeared to consist entirely of proteins of the high aspartic acid and serine type which, however, contained little or no phosphate. Although we have reservations about the use of rabbit molar dentine and have not reported on our investigations of the EDTA extracts and soluble CRF fractions obtained from this tissue. the insoluble fractions (MICR) obtained from collagenase digestion of demineralized molar dentine were studied. All fractions except one were of the high aspartic acid and serine type, with only small or trace amounts of phosphate. One fraction (MU-B), isolated by column chromatography, was similar in amino acid composition to human dentine ICR (Leaver. Price and Smith. 1978) and therefore bore a general resemblance to the acid structural proteins. Both incisor and molar dentine contained ICR material, which could only be solubilized by 8 M urea containing mercaptoethanol and which was also of the high aspartic acid and serine type. Whereas it seems reasonable to assume that soluble CRPs are firmly bound to collagen in human and rabbit dentine and in bovine bone, we have reservations in respect of the human dentine ICR fractions. which. being similar to structural glycoproteins, might exist alongside, rather than attached to collagen in the intact tissue. The same considerations must apply to the rabbit fraction MICR-UB which is of this type. As phosphoproteins of the high aspartic acid and serine type appear to exist in association with collagen in both bovine and rat dentine, it seems likely that rabbit dentine ICR fractions, rich in these amino acids, represent material originally bound to collagen. Their insolubility might be ascribed to the absence of phosphate but, once solubilized by urea or urea/mercaptoethanol, they become water-soluble. This might suggest that their insolubility is due to a high degree of association, but this would suggest that they exist as separate entities, rather than bound to collagen in the intact tissues. If, on the other hand, dentine proteins rich in aspartic acid and serine but containing little or no P are inherently insoluble, it is necessary to ascertain whether their existence specifically in rabbit dentine is of significance. The insoluble collagenasereleased fraction of rat dentine, described by Butler ef al. (1977), would repay investigation. The possibility
that the phosphoproteins might lose phosphate fortuitously, as an artifact of isolation, resulting in this insolubility, must be borne in mind. Acknowledgements-We thank Dr. S. D. Hogg and Mr. P. D. Wilkes for carrying out the GLC analyses and isoelectric focusing respectively and Mr. J. S. Bailie for his assistance in the preparation of the figures.
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Collagenase-released
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Thomas M. and Leaver A. G. 1977. The less-acidic glycoproteins of the organic matrix of human dentine Archs &al Biol. 22, 54S549.
Timol R.. Wolff 1. and Weiser M. 1969. Acid structural pr’oteins of connective tissue--I. Solubilization and preliminary characterization. Biochim. hiophys. Acta 194. 112-120. Trevelyan W. E. and Harrison J. S. 1952. Studies on yeast
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Wolff I., Fuchswans W., Weiser M., Furthmayr H. and Timpl R. 1971. Acid structural proteins of connective tissue. Characterization of their heterogeneous nature. Eur. J. Biochem. 20, 426-431.
