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The molecular packing of collagen in mineralized and non-mineralized tissues
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE MOLECULARPACKING OF COLLAGEN IN MINERALIZED AND NON-MINERALIZED TISSUES Elton P. Katz and Shu-tung Li Department of Oral Biology University of Connecticut Health Center and I n s t i t u t e of Materials Science Storrs, Connecticut 06268
Received J a n u a r y 4, 1972
SUMMARY The collagen in adult bovine dentine, adult rat bone and t a i l tendon, and purified, reconstituted steerskin collagen were studied by x-ray diffraction in conjunction with a technique for assaying the intermolecular volume of collagen f i b r i l s . These four collagens all have a geometric lateral arrangement compatible with hexagonal or near-hexagonal molecularoarrays. The average intermolecular gap in bone and dentine, however, is 6A whereas in rat t a i l tendon i t is only 3A. Phosphate ions, which have a diameter of approximately 4A, can thus penetrate the collagen f i b r i l s of bone and dentine but not those of rat t a i l tendon. We conclude mineral cannot accumulate within the collagen f i b r i l s of rat t a i l tendon purely for steric reasons. In the process of formation of bone and dentine a mineral is deposited extracellularly in an organic matrix about 90% of which is collagen.
Although
i t has been postulated that collagen induces mineralization in these tissues,
I, 2
the factors determining the induction s p e c i f i c i t y of the collagens of bone and dentine, and their absence from the collagens from other tissues, have not been elucidated. 3' 4 We have been investigating the p o s s i b i l i t y that variations in the molecular packing of collagen from different tissues may be a factor in this phenomonon, and present here our i n i t i a l
findings.
I t is now generally accepted that the principal low-angle meridional x-ray reflection, D, from mammalian collagens is the result of the longitudinal staggering of rod-like molecules with respect to each other
5
by D or some
integral multiple of D, and since the collagen molecule is 4.4 D long, the staggering produces systematic defects or "holes" in the packing, approximately 0.6 D in length 5. lateral packing.
There is less agreement, however, on the nature of the The f i r s t models 6, 7 for the lateral packing proposed that 1368
Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the collagen molecules were organized into an extended hexagonal or pseudohexagonal array.
I t was suggested l a t e r that:
collagen was packaged into
"hexagonal units" containing 7 molecules 8 (in cross section); or into limited lateral arrays containing approximately 150 molecules, but organized either in a cylindrical 9 or a hexagonal I0 l a t t i c e .
The most recent study II has
suggested that an extended array' of pentamer units 12 is compatible with d i f fraction data, but not an array of "tetrads" 13 Since all of the models for the lateral packing of collagen have been based on similar d i f f r a c t i o n data, we have attempted to obviate the ambiguity in the models by evaluating by other means the i n t r a f i b r i l l a r
(intermolecular)
volume per gram of collagen, Vi - a parameter defined by the molecular packing and related to d i f f r a c t i o n measurements as follows: -I Vi = 5 D R2 No/MWc Sin y - Pc
equation 1
where NO is Avogadro's number; Mwc, the molecular weight of collagen; R is the Bragg distance for the strongest equatorial density; and ¥, the packing angle.
reflection; Pc' the (dry)
I t is evident that a determination of Vi ,
in conjunction with diffraction, molecular weight, and density data allows a characterization of the molecular packing in terms of the packing angle y, the intermolecular distance (R/siny), and the intermolecular gap (R/siny-dc), where dc, the diameter of the collagen molecule, is approximated by: dc = 2 (MWc/4.4 D ~PcNo) I / 2
equation 2
EXPERIMENTAL The Bragg distance of the equatorial r e f l e c t i o n , R, and the i n t r a f i b r i l l a r volume, Vi , were determined on: in 0.5 M EDTA, pH 8; "fresh" t a i l
sections of rat t i b i a that had been demineralized tendons 8, II studied immediately after
s a c r i f i c e of adult Sprague Dawley rats; sections of adult bovine dentine, cut perpendicularly to the odontoblastic processes and demineralized as above; and films of p u r i f i e d , reconstituted steerskin collagen (Ethicon Corp., Emoryville, N.J.).
X-ray d i f f r a c t i o n studies were done on samples bathed in solutions con-
taining 0.15 M KCl, pH 7.4, and sealed in c a p i l l a r y tubes, using a camera having
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Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
a specimen-to-film distance of I0 cm.
Vi was determined by using C14 -labelled
polyethylene glycol (New England Nuclear Inc., Boston, Mass.), molecular weight 4000 (PEG*4000) with a Stokes radius of approximately 14A as a (probe) molecule which penetrates into the e x t r a f i b r i l l a r nor penetrates the f i b r i l s .
space, but which neither adsorbs to
The following equation expresses Vi as a function of
the experimental variables of a probe experiment: Vi = (Wt - m*/C*)/WcP
equation 3
where Wt and Wc are the total grams of water and collagen in the sample, respectively; m* is the a c t i v i t y of the sample, C*, the a c t i v i t y per gram water of the solution in equilibrium with the sample, and p, the density of the i n t r a f i b r i l l a r
water, which we assume to be unity.
The experimental procedure was as follows:
the collagen samples were f i r s t
washed at 4° in solutions of 0.15 M KCI, pH 7.4 ("fresh" rat t a i l tendon being the exception), then equilibrated at 25° with a s t e r i l e solution of 0.15 M KCl, pH 7.4, containing PEG* 4000 and either PEG 4000, PEG 6000, or PEG 20,000 (as " c a r r i e r s " , Polyscience Inc., N. J . ) .
The samples were weighed, the PEG* 4000
quantitatively extracted, and the samples dried to constant weight over P205 in vacuo to determine Wt , the water content of the sample.
C* and m* were
determined by radiochemical assay of weighed aliquots of the supernates of the equilibration and extraction solutions, respectively.
The weight of collagen in
the samples, Wc, was in the case of the purified steerskin, the dry weight of the sample.
Wc for the other materials was determined by means of a
hydroxyproline analysis 14 employing a value of 13 residue weight percent for the hydroxyproline content 15 of collagen.
The collagen content determined by
the hydroxyproline method was within 5% of the dried sample weights.
Purified
collagen was employed as a control for the demineralizing procedure.
No changes
in either Vi or R could be detected upon i t s exposure to 0.5 M EDTA, pH 8. RESULTS AND DISCUSSION The molecular packing parameters R and Vi varied for the d i f f e r e n t collagens studied.
They are as follows in order of increasing value: 1370
rat
Vol. 46, No. 3, 1972
tail
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tendon - R, 12.8A (in agreement with l i t e r a t u r e values 8, I I ) ,
and Vi ,
0.68 cc/g; reconstituted steerskin - R, 14.7A (similar to values reported for hydrated bovine achilles tendon 6 and hydrated kangaroo t a i l
tendon 6, 7),
Vi , 1.13 cc/g (comparable to a hydration value of I . I 0 cc/g obtained for Kangaroo t a i l
tendon by a d i f f r a c t i o n method 7); rat t i b i a - R, 15.3A (similar
to a value reported for hydrated chicken bone collagen 16), Vi ' 1.26 cc/g; adult bovine dentine-R, 15.4A, Vi , 1.33 cc/g. In Fig. I , theoretical
curves of Vi against ¥ are shown. They were
computed from equation 1 by employing the above values of R and using values
2.0
1.8
1.6
1.4
1,2
VL 1.0
0,8
0,6
0,4
0.2
0
HEXAGONAL LA TTICE
5'0
6'0
7'o
8'o
9'o
7" Figure 1 - Plots of the i n t r a f i b r i l l a r volume of collagen as a function of packing angle y, employing the principal equatorial reflections of: 15.3A for rat bone; 15.4A for bovine dentine; 14.7A for reconstituted steerskin collagen; and 12.8A for rat t a i l tendon. The arrows are the volumes of the water occupying the i n t r a f i b r i l l a r space of the respective collagens at 25° . The width of the arrows show the uncertainty in ¥ due to the standard error of the determinations.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of 690A for D5, 1.41 f o r Pc
17
, and 283,000 f o r Mwc 18
of Vi are also indicated in Fig. I.
The experimental values
I t can be seen that within the uncertainty
of the measurements ¥ is equal to 60° f o r all the collagens studied, a value consistant with hexagonal packing
8, I0
We have considered two " m i c r o f i b r i l " on the basis of d i f f r a c t i o n data.
models proposed f o r rat t a i l
The minimal i n t r a f i b r i l l a r
tendon
volumes possible
for the pentamer and the heptamer models were calculated to be 1.41cc/g and 0.72 cc/g, respectively. tail
The i n t r a f i b r i l l a r
volume actually observed f o r rat
tendon (0.68cc) is closest to that predicted f o r a heptamer model.
hexagonally-packed heptamer is not, however, the only m i c r o f i b r i l compatible with our results.
A
model that is
An hexagonal array of nonomers (9 molecules
approximately hexagonally-packed) is an example of another p o s s i b i l i t y . Since collagen is f i r s t
aggregated into i t s f i b r i l l a r
state before becoming
impregnated with mineral 3, the mineral constituents have to be transported into the f i b r i l s
in a form smaller than the average gap between molecules.
Phosphate
ions, which have a diameter of approximately 4 A, are able to penetrate into the collagen f i b r i l s
of bone and dentine, which have an intermolecular gap of
approximately 6A but they would have to do so e s s e n t i a l l y by ionic d i f f u s i o n 4 I t is p a r t i c u l a r l y i n t e r e s t i n g , however, that in r a t t a i l
tendon, the i n t e r -
molecular gap (3 A) is in a l l p r o b a b i l i t y too small to allow phosphate ion to penetrate and thus for s t e r i c reasons alone mineral could not accumulate intrafibrillarly
for this collagenous tissue.
between the molecular packing of rat t a i l
We suggest that the difference
tendon and rat bone is a s u f f i c i e n t
reason why the former does not mineralize where the l a t t e r does. ACKNOWLEDGMENTS The authors g r a t e f u l l y acknowledge helpful discussions with Drs. J. Knox, R. Schor and G. Rodan. This work was supported by grants from the National I n s t i t u t e s of Health (DE-O2953) and University of Connecticut Research Foundation.
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REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
Glimcher, M. J., Hodge, A. J. and Schmitt, F. 0., Proc. Natl. Acad. Sci. U. S., 43, 860, (1957). Strates, B. G., Neuman, W. F. and Levinskas, G. J., J. Phys. Chem., 61, 279, (1957). Glimcher, M. J. and Krane, S. M., in "Treatise on Collagen", ed. B. S. Gould, (Academic Press, New York) Vol. 2, p. 68, (1968). Katz, E. P., Biochim. Biophys. Acta, 194, 121, (1969). Hodge, A. J. and Petruska, J. A., In G. N. Ramachandran "Aspects of Protein Structure" (Academic Press, London), p. 239, (1963). Bear, R. S., Adv. Protein Chem. ~, 69, (1952). Bear, R. S., J. Biophys. Biochem. Cytol., 2, 363, (1956). North, A. C. T., Cowan, P. M. and Randall, J. T., Nature, 17A, 1142, (1954). Ramachandran, C. N. and Sasisekharan, V., Arch. Biochem. Biophys., 63, 255, (1956). Burge, R. E., J. Mol. B i o l . , 7, 219, (1963). Miller, A. and Wray, J. S., Nature, 230, 437, (1971). Smith, J. W., Nature, 219, 157, (1968T. Veis, A., Anesey, J. and Mussell, S., Nature, 215, 931, (1967). Neuman, R. and Logan, M., J. Biol. Chem., 1 8 4 , ~ 9 , (1955). Eastoe, J. E. in "Treatise on Collagen", ed, B. S. Gould (Academic Press, New York), Vol. I , p. I , (1965). Eanes, E. D. and M i l l e r , E. S., Arch. Biophys. Biochem., 129, 769 (1969). Richelle, L. J. and Onkelinx, C. in "Mineral Metabolism",~. C. Comar and F. Bronner, (Academic Press, New York), Vol. I I I , 123, (1969). Katz, E. P., Francois, C. and Glimcher, M. J., Biochemistry, 8, 2009, (1969).
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THE MOLECULARPACKING OF COLLAGEN IN MINERALIZED AND NON-MINERALIZED TISSUES Elton P. Katz and Shu-tung Li Department of Oral Biology University of Connecticut Health Center and I n s t i t u t e of Materials Science Storrs, Connecticut 06268
Received J a n u a r y 4, 1972
SUMMARY The collagen in adult bovine dentine, adult rat bone and t a i l tendon, and purified, reconstituted steerskin collagen were studied by x-ray diffraction in conjunction with a technique for assaying the intermolecular volume of collagen f i b r i l s . These four collagens all have a geometric lateral arrangement compatible with hexagonal or near-hexagonal molecularoarrays. The average intermolecular gap in bone and dentine, however, is 6A whereas in rat t a i l tendon i t is only 3A. Phosphate ions, which have a diameter of approximately 4A, can thus penetrate the collagen f i b r i l s of bone and dentine but not those of rat t a i l tendon. We conclude mineral cannot accumulate within the collagen f i b r i l s of rat t a i l tendon purely for steric reasons. In the process of formation of bone and dentine a mineral is deposited extracellularly in an organic matrix about 90% of which is collagen.
Although
i t has been postulated that collagen induces mineralization in these tissues,
I, 2
the factors determining the induction s p e c i f i c i t y of the collagens of bone and dentine, and their absence from the collagens from other tissues, have not been elucidated. 3' 4 We have been investigating the p o s s i b i l i t y that variations in the molecular packing of collagen from different tissues may be a factor in this phenomonon, and present here our i n i t i a l
findings.
I t is now generally accepted that the principal low-angle meridional x-ray reflection, D, from mammalian collagens is the result of the longitudinal staggering of rod-like molecules with respect to each other
5
by D or some
integral multiple of D, and since the collagen molecule is 4.4 D long, the staggering produces systematic defects or "holes" in the packing, approximately 0.6 D in length 5. lateral packing.
There is less agreement, however, on the nature of the The f i r s t models 6, 7 for the lateral packing proposed that 1368
Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the collagen molecules were organized into an extended hexagonal or pseudohexagonal array.
I t was suggested l a t e r that:
collagen was packaged into
"hexagonal units" containing 7 molecules 8 (in cross section); or into limited lateral arrays containing approximately 150 molecules, but organized either in a cylindrical 9 or a hexagonal I0 l a t t i c e .
The most recent study II has
suggested that an extended array' of pentamer units 12 is compatible with d i f fraction data, but not an array of "tetrads" 13 Since all of the models for the lateral packing of collagen have been based on similar d i f f r a c t i o n data, we have attempted to obviate the ambiguity in the models by evaluating by other means the i n t r a f i b r i l l a r
(intermolecular)
volume per gram of collagen, Vi - a parameter defined by the molecular packing and related to d i f f r a c t i o n measurements as follows: -I Vi = 5 D R2 No/MWc Sin y - Pc
equation 1
where NO is Avogadro's number; Mwc, the molecular weight of collagen; R is the Bragg distance for the strongest equatorial density; and ¥, the packing angle.
reflection; Pc' the (dry)
I t is evident that a determination of Vi ,
in conjunction with diffraction, molecular weight, and density data allows a characterization of the molecular packing in terms of the packing angle y, the intermolecular distance (R/siny), and the intermolecular gap (R/siny-dc), where dc, the diameter of the collagen molecule, is approximated by: dc = 2 (MWc/4.4 D ~PcNo) I / 2
equation 2
EXPERIMENTAL The Bragg distance of the equatorial r e f l e c t i o n , R, and the i n t r a f i b r i l l a r volume, Vi , were determined on: in 0.5 M EDTA, pH 8; "fresh" t a i l
sections of rat t i b i a that had been demineralized tendons 8, II studied immediately after
s a c r i f i c e of adult Sprague Dawley rats; sections of adult bovine dentine, cut perpendicularly to the odontoblastic processes and demineralized as above; and films of p u r i f i e d , reconstituted steerskin collagen (Ethicon Corp., Emoryville, N.J.).
X-ray d i f f r a c t i o n studies were done on samples bathed in solutions con-
taining 0.15 M KCl, pH 7.4, and sealed in c a p i l l a r y tubes, using a camera having
1369
Vol. 46, No. 3, 1972
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
a specimen-to-film distance of I0 cm.
Vi was determined by using C14 -labelled
polyethylene glycol (New England Nuclear Inc., Boston, Mass.), molecular weight 4000 (PEG*4000) with a Stokes radius of approximately 14A as a (probe) molecule which penetrates into the e x t r a f i b r i l l a r nor penetrates the f i b r i l s .
space, but which neither adsorbs to
The following equation expresses Vi as a function of
the experimental variables of a probe experiment: Vi = (Wt - m*/C*)/WcP
equation 3
where Wt and Wc are the total grams of water and collagen in the sample, respectively; m* is the a c t i v i t y of the sample, C*, the a c t i v i t y per gram water of the solution in equilibrium with the sample, and p, the density of the i n t r a f i b r i l l a r
water, which we assume to be unity.
The experimental procedure was as follows:
the collagen samples were f i r s t
washed at 4° in solutions of 0.15 M KCI, pH 7.4 ("fresh" rat t a i l tendon being the exception), then equilibrated at 25° with a s t e r i l e solution of 0.15 M KCl, pH 7.4, containing PEG* 4000 and either PEG 4000, PEG 6000, or PEG 20,000 (as " c a r r i e r s " , Polyscience Inc., N. J . ) .
The samples were weighed, the PEG* 4000
quantitatively extracted, and the samples dried to constant weight over P205 in vacuo to determine Wt , the water content of the sample.
C* and m* were
determined by radiochemical assay of weighed aliquots of the supernates of the equilibration and extraction solutions, respectively.
The weight of collagen in
the samples, Wc, was in the case of the purified steerskin, the dry weight of the sample.
Wc for the other materials was determined by means of a
hydroxyproline analysis 14 employing a value of 13 residue weight percent for the hydroxyproline content 15 of collagen.
The collagen content determined by
the hydroxyproline method was within 5% of the dried sample weights.
Purified
collagen was employed as a control for the demineralizing procedure.
No changes
in either Vi or R could be detected upon i t s exposure to 0.5 M EDTA, pH 8. RESULTS AND DISCUSSION The molecular packing parameters R and Vi varied for the d i f f e r e n t collagens studied.
They are as follows in order of increasing value: 1370
rat
Vol. 46, No. 3, 1972
tail
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tendon - R, 12.8A (in agreement with l i t e r a t u r e values 8, I I ) ,
and Vi ,
0.68 cc/g; reconstituted steerskin - R, 14.7A (similar to values reported for hydrated bovine achilles tendon 6 and hydrated kangaroo t a i l
tendon 6, 7),
Vi , 1.13 cc/g (comparable to a hydration value of I . I 0 cc/g obtained for Kangaroo t a i l
tendon by a d i f f r a c t i o n method 7); rat t i b i a - R, 15.3A (similar
to a value reported for hydrated chicken bone collagen 16), Vi ' 1.26 cc/g; adult bovine dentine-R, 15.4A, Vi , 1.33 cc/g. In Fig. I , theoretical
curves of Vi against ¥ are shown. They were
computed from equation 1 by employing the above values of R and using values
2.0
1.8
1.6
1.4
1,2
VL 1.0
0,8
0,6
0,4
0.2
0
HEXAGONAL LA TTICE
5'0
6'0
7'o
8'o
9'o
7" Figure 1 - Plots of the i n t r a f i b r i l l a r volume of collagen as a function of packing angle y, employing the principal equatorial reflections of: 15.3A for rat bone; 15.4A for bovine dentine; 14.7A for reconstituted steerskin collagen; and 12.8A for rat t a i l tendon. The arrows are the volumes of the water occupying the i n t r a f i b r i l l a r space of the respective collagens at 25° . The width of the arrows show the uncertainty in ¥ due to the standard error of the determinations.
1371
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of 690A for D5, 1.41 f o r Pc
17
, and 283,000 f o r Mwc 18
of Vi are also indicated in Fig. I.
The experimental values
I t can be seen that within the uncertainty
of the measurements ¥ is equal to 60° f o r all the collagens studied, a value consistant with hexagonal packing
8, I0
We have considered two " m i c r o f i b r i l " on the basis of d i f f r a c t i o n data.
models proposed f o r rat t a i l
The minimal i n t r a f i b r i l l a r
tendon
volumes possible
for the pentamer and the heptamer models were calculated to be 1.41cc/g and 0.72 cc/g, respectively. tail
The i n t r a f i b r i l l a r
volume actually observed f o r rat
tendon (0.68cc) is closest to that predicted f o r a heptamer model.
hexagonally-packed heptamer is not, however, the only m i c r o f i b r i l compatible with our results.
A
model that is
An hexagonal array of nonomers (9 molecules
approximately hexagonally-packed) is an example of another p o s s i b i l i t y . Since collagen is f i r s t
aggregated into i t s f i b r i l l a r
state before becoming
impregnated with mineral 3, the mineral constituents have to be transported into the f i b r i l s
in a form smaller than the average gap between molecules.
Phosphate
ions, which have a diameter of approximately 4 A, are able to penetrate into the collagen f i b r i l s
of bone and dentine, which have an intermolecular gap of
approximately 6A but they would have to do so e s s e n t i a l l y by ionic d i f f u s i o n 4 I t is p a r t i c u l a r l y i n t e r e s t i n g , however, that in r a t t a i l
tendon, the i n t e r -
molecular gap (3 A) is in a l l p r o b a b i l i t y too small to allow phosphate ion to penetrate and thus for s t e r i c reasons alone mineral could not accumulate intrafibrillarly
for this collagenous tissue.
between the molecular packing of rat t a i l
We suggest that the difference
tendon and rat bone is a s u f f i c i e n t
reason why the former does not mineralize where the l a t t e r does. ACKNOWLEDGMENTS The authors g r a t e f u l l y acknowledge helpful discussions with Drs. J. Knox, R. Schor and G. Rodan. This work was supported by grants from the National I n s t i t u t e s of Health (DE-O2953) and University of Connecticut Research Foundation.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
Glimcher, M. J., Hodge, A. J. and Schmitt, F. 0., Proc. Natl. Acad. Sci. U. S., 43, 860, (1957). Strates, B. G., Neuman, W. F. and Levinskas, G. J., J. Phys. Chem., 61, 279, (1957). Glimcher, M. J. and Krane, S. M., in "Treatise on Collagen", ed. B. S. Gould, (Academic Press, New York) Vol. 2, p. 68, (1968). Katz, E. P., Biochim. Biophys. Acta, 194, 121, (1969). Hodge, A. J. and Petruska, J. A., In G. N. Ramachandran "Aspects of Protein Structure" (Academic Press, London), p. 239, (1963). Bear, R. S., Adv. Protein Chem. ~, 69, (1952). Bear, R. S., J. Biophys. Biochem. Cytol., 2, 363, (1956). North, A. C. T., Cowan, P. M. and Randall, J. T., Nature, 17A, 1142, (1954). Ramachandran, C. N. and Sasisekharan, V., Arch. Biochem. Biophys., 63, 255, (1956). Burge, R. E., J. Mol. B i o l . , 7, 219, (1963). Miller, A. and Wray, J. S., Nature, 230, 437, (1971). Smith, J. W., Nature, 219, 157, (1968T. Veis, A., Anesey, J. and Mussell, S., Nature, 215, 931, (1967). Neuman, R. and Logan, M., J. Biol. Chem., 1 8 4 , ~ 9 , (1955). Eastoe, J. E. in "Treatise on Collagen", ed, B. S. Gould (Academic Press, New York), Vol. I , p. I , (1965). Eanes, E. D. and M i l l e r , E. S., Arch. Biophys. Biochem., 129, 769 (1969). Richelle, L. J. and Onkelinx, C. in "Mineral Metabolism",~. C. Comar and F. Bronner, (Academic Press, New York), Vol. I I I , 123, (1969). Katz, E. P., Francois, C. and Glimcher, M. J., Biochemistry, 8, 2009, (1969).
1373