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Subunits of the collagen molecule
PRELIMINARY NOTES
of the collagen molecule from some tissues by a variety of lvents. solution and purified, are very sim erhaps have shown that cold aq. NaC1 e most solutions such as dilute acetic ack buffers n, and insoluble collagen represen t fracred in solution (by heating at a pH , or by rmed 3. ro different components of unequa faster se have been designated the ~- a~d ~J-components~, the tt latter : t clear he ultracentrifuge. Although several suggestions have haw been m~ )llagen ,ther or to tl t relationship these components have to each othez larifies )resent evi& ~cule3, 4. It is the purpose of this communication to pre~ situation. fro: )f 5o-g The present experiments were all performed with collagen col ~mples extracted at 5 ° with either i M NaC1 or o.5 M acetic acid. Bot acetic purified by repeated precipitation with 5 % NaC1, dissoluti( )matoLgen sampl , and centrifugation at 3oooo x g for z h. The collag ~t presi )hed on a 2.2 /, 18 cm column of CM-cellulose in a manner i olumn )onents isolatec tsly describedq Samples of collagen or the componenl :l were tomatic inst ~ents were taken for amino acid analysis on an auton nined in the ultracentrifuge. cted collagen sedimented with In the ultracentrifuge, denatured (4o°) salt-extracte rial one large boundary and a small amount of heavier material while acid-extracted collagen &components. These results are zed the two boundaries typical of the ~- and fi-con showed ;trated in Fig. I. The relative sizes of the peaks are, distorted by the Johnstonillustrated are actually present ton effect ; the two components in the acid-extracted collagen c Ogston tifferent sedimentation rates of early the same amounts (see below). The slightly diff( in nearl
| i ,!
0. 7 ~o Fig. i. S e d i m e n t a t i o n p a t t e r n s of d e n a t u r e d rat-skin collagen. U p p e r p a t t e r n (wedge cell), 0.7% salt-extracted collagen; lower p a t t e r n (standard cell), 0 . 7 % acid-extracted collagen. Sodium ar 5), i28 m i n at 5978o rev./min, 4 o°, i2 m m K e l - F ceils, 7o ° bbat f o r m a t e buffer (pH 3.75, I o.I5), a r ttgle, s e d i m e n t a t i o n from left to right.
Biochim. Biophys. Actc~, 53 (I96I) 596-598 596-59~
PRELIMINARY NOTES
samples can be explained by the ]
tration
ured acid-extracted collagen by c phy on :e of four major components. On iso omatoltracentrifuge with the original unfn filagen, ¢¢hich we will arbitrarily call aI and dimena-component while the other two, flI and onent (Fig. 2). This correspondent er subthat the salt-extracted collagen c gely of and a2 (see Fig. 2) as would be predicted from the ultracentri u on the fractionated sample. In the sample of which the chromatograli chrol Fig. 2, is ratio ~ ratio of (total =):(total fl) was 85:15 while for acid-extracted acid-~ s close to i. The ratio of aI :a2, calculated from the areas un ks, was :ays close to 2 for both salt- and acid-extracted coll~ collagen, ave in five :omatograms. Three of the four major chromatographic peaks were isolated t tracted lagen essentially free of each other as judged by rechromatc re L ultrattrifugation. The sample of f12 contained appreciably amounts, s found amino acid analysis (Table I) that =I and a2 differed lfrom each mmber amino acids, particularly hydroxyproline, proline, alanine, eucine, droxylysine, lysine and histidine, flz had a composition equivale nixture xZ and =2, while f12 did not differ from aI to any signific~ nllacant d~ nificant degree, this mtormatlon, nation [ether with the knowledge that the fl-components have hay about twice the weight of ot the; a-components3, 4, provides good evidence that the two types of fl-components ents n rSlS cross-links. The analysis can1 be considered chain pairs probably joined by one or more of pz flI suggests that it is composed of one aI chain and one ~2 chain. Similarly, f12 g2 appears to contain two aI chains. On the basis of our evidence we propose that the ne newly formed molecule, which Ly is the :he major species in salt-extracted collagen, consists of 0 subunits of approximately equal ml size (a-components) but which differ in composit )osition. The apparent 2:1 stoitoichiometr~ ometry of =I : a2 is most easily explained by assuming t h a t each molecule contains tins three subunits of which one is a2 and two are ~I. This is also consistent with the amino tO acid composition of the or~ riginal collagen. Though direct evidence is lacking, it is lS 1
i
J
i
i
J
¢11
[
Ioo
zoo
=z
300 40o sOO ML EFFLUENT
600
700
Fig. 2. E l u t i o n p a t t e r n s of d e n[atured r a t - s k i n collagen c h r o m a t o g r a p h e d on CM-cellulose a t 4 o°. Solid line, 20 m g s a l t - e x t r a c t e d collagen; d a s h line, 2o m g a c i d - e x t r a c t e d collagen. A linear g r a d i e n t b e t w e e n o.o 7 and o.I 7 I a c e t a t ee buffer (pH 4.83) w a s e m p l o y e d . T h e labels refer to b o t h patterns.
Biockira. Biophys. Acta, 53 (z96I) 596-598
PRELIMINARY NOTES TABLE I OSITION
OF
IDUES/IO00
RAT-SKIN TOTAL
COLLAGEN
AND
]
RESIDUES)
presented. Glycine, included as a criteri ted differed n o t at all or only v e r y slig]
and the
Subunit
Hypro Pro Ala Val lieu Leu Hylys Lys His
97 I2O Io2 23 i o. 7 24 6. i 28 4.7
339 94 127 108 21 6. 7 18.2 4.9 31 1.7
341 85 II 4 99 32 16.1 31 8-4 24 7 .8
339 89 I2o 103 27 12.I 26 6.9 26 4 .8
' The s a m p l e of f12 analyzed contained a b o u t 3o % of ~I.
;onable to presume that these subunits represent singlle chains :h may md the full length of the molecule. An important feature featu of the :ueture rot, as the molecule matures, and can be readily extracted extr~ wit] Lot salt .tions, the chains cross-link intramolecularly in pairs (fl-components). It is likely )onents are observed in the ultrathat: the cross-links are covalent bonds since fl-componenl :rifuge in various denatming solvents including 5 M guanidine. centrifu
National Institutes of Health, Bethesda, Md. and Massachusetts General Hospital, Boston, Mass., (U.S.A.)
KARL A. PIEZ MARC S. L E w i s GEORGE R .
MARTIN
JEROME GROSS
ective Tissue, Thrombosis and A therosc[erosis, Academic Press, Inc., 1 j. GROSS, in I, H. PAGE, Connective 1959, p. 77. D. S. JACKSON AND J. P. BENTLEY 4TLEY, J. Biop~ys. Biochem. Cytol., 7 (196o) 373 V. N. OREKHOVICH, V. O. SHPIKITER 'IRITER, V. I. MAZUROV AND O. V. KOUNINA, Bull. soc. chim. biol 4 2 {I900) 5 0 5 • 4 I4. A. PIEZ, E. WEISS AND M.• S. LEwis, J. Biol. Chem., 235 {196o ) 1987. K. A. PIEZ AND L. MORRIS, Anal. [nal. Biochem., 1 (196o) I87.
Received July 3Ist, 1961 Biochim. Biophys. Acta, 53 (1961) 596-598
of the collagen molecule from some tissues by a variety of lvents. solution and purified, are very sim erhaps have shown that cold aq. NaC1 e most solutions such as dilute acetic ack buffers n, and insoluble collagen represen t fracred in solution (by heating at a pH , or by rmed 3. ro different components of unequa faster se have been designated the ~- a~d ~J-components~, the tt latter : t clear he ultracentrifuge. Although several suggestions have haw been m~ )llagen ,ther or to tl t relationship these components have to each othez larifies )resent evi& ~cule3, 4. It is the purpose of this communication to pre~ situation. fro: )f 5o-g The present experiments were all performed with collagen col ~mples extracted at 5 ° with either i M NaC1 or o.5 M acetic acid. Bot acetic purified by repeated precipitation with 5 % NaC1, dissoluti( )matoLgen sampl , and centrifugation at 3oooo x g for z h. The collag ~t presi )hed on a 2.2 /, 18 cm column of CM-cellulose in a manner i olumn )onents isolatec tsly describedq Samples of collagen or the componenl :l were tomatic inst ~ents were taken for amino acid analysis on an auton nined in the ultracentrifuge. cted collagen sedimented with In the ultracentrifuge, denatured (4o°) salt-extracte rial one large boundary and a small amount of heavier material while acid-extracted collagen &components. These results are zed the two boundaries typical of the ~- and fi-con showed ;trated in Fig. I. The relative sizes of the peaks are, distorted by the Johnstonillustrated are actually present ton effect ; the two components in the acid-extracted collagen c Ogston tifferent sedimentation rates of early the same amounts (see below). The slightly diff( in nearl
| i ,!
0. 7 ~o Fig. i. S e d i m e n t a t i o n p a t t e r n s of d e n a t u r e d rat-skin collagen. U p p e r p a t t e r n (wedge cell), 0.7% salt-extracted collagen; lower p a t t e r n (standard cell), 0 . 7 % acid-extracted collagen. Sodium ar 5), i28 m i n at 5978o rev./min, 4 o°, i2 m m K e l - F ceils, 7o ° bbat f o r m a t e buffer (pH 3.75, I o.I5), a r ttgle, s e d i m e n t a t i o n from left to right.
Biochim. Biophys. Actc~, 53 (I96I) 596-598 596-59~
PRELIMINARY NOTES
samples can be explained by the ]
tration
ured acid-extracted collagen by c phy on :e of four major components. On iso omatoltracentrifuge with the original unfn filagen, ¢¢hich we will arbitrarily call aI and dimena-component while the other two, flI and onent (Fig. 2). This correspondent er subthat the salt-extracted collagen c gely of and a2 (see Fig. 2) as would be predicted from the ultracentri u on the fractionated sample. In the sample of which the chromatograli chrol Fig. 2, is ratio ~ ratio of (total =):(total fl) was 85:15 while for acid-extracted acid-~ s close to i. The ratio of aI :a2, calculated from the areas un ks, was :ays close to 2 for both salt- and acid-extracted coll~ collagen, ave in five :omatograms. Three of the four major chromatographic peaks were isolated t tracted lagen essentially free of each other as judged by rechromatc re L ultrattrifugation. The sample of f12 contained appreciably amounts, s found amino acid analysis (Table I) that =I and a2 differed lfrom each mmber amino acids, particularly hydroxyproline, proline, alanine, eucine, droxylysine, lysine and histidine, flz had a composition equivale nixture xZ and =2, while f12 did not differ from aI to any signific~ nllacant d~ nificant degree, this mtormatlon, nation [ether with the knowledge that the fl-components have hay about twice the weight of ot the; a-components3, 4, provides good evidence that the two types of fl-components ents n rSlS cross-links. The analysis can1 be considered chain pairs probably joined by one or more of pz flI suggests that it is composed of one aI chain and one ~2 chain. Similarly, f12 g2 appears to contain two aI chains. On the basis of our evidence we propose that the ne newly formed molecule, which Ly is the :he major species in salt-extracted collagen, consists of 0 subunits of approximately equal ml size (a-components) but which differ in composit )osition. The apparent 2:1 stoitoichiometr~ ometry of =I : a2 is most easily explained by assuming t h a t each molecule contains tins three subunits of which one is a2 and two are ~I. This is also consistent with the amino tO acid composition of the or~ riginal collagen. Though direct evidence is lacking, it is lS 1
i
J
i
i
J
¢11
[
Ioo
zoo
=z
300 40o sOO ML EFFLUENT
600
700
Fig. 2. E l u t i o n p a t t e r n s of d e n[atured r a t - s k i n collagen c h r o m a t o g r a p h e d on CM-cellulose a t 4 o°. Solid line, 20 m g s a l t - e x t r a c t e d collagen; d a s h line, 2o m g a c i d - e x t r a c t e d collagen. A linear g r a d i e n t b e t w e e n o.o 7 and o.I 7 I a c e t a t ee buffer (pH 4.83) w a s e m p l o y e d . T h e labels refer to b o t h patterns.
Biockira. Biophys. Acta, 53 (z96I) 596-598
PRELIMINARY NOTES TABLE I OSITION
OF
IDUES/IO00
RAT-SKIN TOTAL
COLLAGEN
AND
]
RESIDUES)
presented. Glycine, included as a criteri ted differed n o t at all or only v e r y slig]
and the
Subunit
Hypro Pro Ala Val lieu Leu Hylys Lys His
97 I2O Io2 23 i o. 7 24 6. i 28 4.7
339 94 127 108 21 6. 7 18.2 4.9 31 1.7
341 85 II 4 99 32 16.1 31 8-4 24 7 .8
339 89 I2o 103 27 12.I 26 6.9 26 4 .8
' The s a m p l e of f12 analyzed contained a b o u t 3o % of ~I.
;onable to presume that these subunits represent singlle chains :h may md the full length of the molecule. An important feature featu of the :ueture rot, as the molecule matures, and can be readily extracted extr~ wit] Lot salt .tions, the chains cross-link intramolecularly in pairs (fl-components). It is likely )onents are observed in the ultrathat: the cross-links are covalent bonds since fl-componenl :rifuge in various denatming solvents including 5 M guanidine. centrifu
National Institutes of Health, Bethesda, Md. and Massachusetts General Hospital, Boston, Mass., (U.S.A.)
KARL A. PIEZ MARC S. L E w i s GEORGE R .
MARTIN
JEROME GROSS
ective Tissue, Thrombosis and A therosc[erosis, Academic Press, Inc., 1 j. GROSS, in I, H. PAGE, Connective 1959, p. 77. D. S. JACKSON AND J. P. BENTLEY 4TLEY, J. Biop~ys. Biochem. Cytol., 7 (196o) 373 V. N. OREKHOVICH, V. O. SHPIKITER 'IRITER, V. I. MAZUROV AND O. V. KOUNINA, Bull. soc. chim. biol 4 2 {I900) 5 0 5 • 4 I4. A. PIEZ, E. WEISS AND M.• S. LEwis, J. Biol. Chem., 235 {196o ) 1987. K. A. PIEZ AND L. MORRIS, Anal. [nal. Biochem., 1 (196o) I87.
Received July 3Ist, 1961 Biochim. Biophys. Acta, 53 (1961) 596-598