Preparative Biochemistry
ISSN: 0032-7484 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb19
Purification of the Neurite Outgrowth Promoting Fragment of Mouse Laminin Sergio R. P. Line To cite this article: Sergio R. P. Line (1992) Purification of the Neurite Outgrowth Promoting Fragment of Mouse Laminin, Preparative Biochemistry, 22:3-4, 229-237, DOI: 10.1080/10826069208021373 To link to this article: http://dx.doi.org/10.1080/10826069208021373
Published online: 24 Sep 2006.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=lpbb20 Download by: [University of California, San Diego]
Date: 28 June 2016, At: 03:03
PREPARATIVE BIOCHEMISTRY,22(3&4), 229-237 (1992)
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
PURIFICATION OF THE NEURITE OUTGROWTH PROMOTING FRAGMENT OF MOUSE LAMININ Sergio. R. P. Line Ludwig Institute for Cancer Research 01509 Sbo Paulo, SP, Brazil Present address: Faculdade de Odontologia de Piracicaba Av. Limeira s/n. Caixa Postal 52 13400 Piracicaba, SP, Brazil
ABSTRACT A method for isolation of the neurite outgrowth promoting fragment of mouse laminin (fragment 8) is described in this paper. Besides producing excellent yields, this method was shown to be fast and practical, since it is based on a single step which consists in an ion exchange chromatography of elastase digested laminin. INTRODUCTION Laminin (880kDa), the major non-collagenous glycoprotein of basement membranes is composed of three polypeptide chains, the A chain (440kDa) and two B chains (220kDa). The three polypeptide chains are spatially arranged forming a cross shaped structure 1. Laminin binds to other basement membrane molecules as type IV collagen, nidogen/entactin, heparan sulfate proteoglycan, and also interacts with cells by means of specific receptors2 . Laminin-cellular receptors interactions are thought to mediate several biological I
229 Copyright 0 1992 by Marcel Dekker, Inc.
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
230
LINE
functions and also to be involved in the process of 4 metastasis . The generation of proteolytic derived fragments has allowed a better understanding of laminin structure and the identification of domains responsible for defined biological functions. A large proteolytic fragment (fragment 1) was identified as consisting of three rod like segments from the center of the short arms that contains a cell binding domain capable of promoting the adhesion of tumor cells5 6 . Another cell binding domain was identified on the elastase derived fragment 8 (280kDa). This fragment located at the end of the long arm was shown to stimulate neurite survival and outgrowth and trigger tyrosine hydroxylase activity in chromaffin cells7I8. Despite its biological importance, the use of fragment 8 has suffered from the lack of a reproducible preparative method for its purification. There exists only one protocol which describes the isolation of biologically active fragment 8 in a reproducible 9 fashion However, the elastase digestion of laminin-nidogen/entactin complex produces also large amounts of fragment 1 (300kDa). Due to their close molecular weights an effective separation of fragments 1 and 8 can only be achieved by two or three passages over a large gel chromatography column, with considerable loss of material. We describe in this paper an improved protocol for obtaining highly purified and biologicaly active fragment 8 from mouse laminin that permits the recovery of almost all the material generated by the elastase digestion.
.
MATERIAL, METHODS AND RESULTS Isolation and analysis of fragment 8 Extraction and purification of the laminin-nidogen complex and further elastase digestion
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
FRAGMENT OF MOUSE LAMININ
23 1
was performed as described by Paulsson et a1 9 . A 50ml aliquot of the digest (lmg/ml) was dialyzed against buffer containing 0.05 M Tris (pH 8.4), 0.025 M NaCl plus 0.5 mM of phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM of N-ethylmaleimide (NEN) as protease inhibitors. The material was then applied to a DEAE-Sephacel column (2.5 x 20 cm, Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in the same buffer. Fragment 8 was eluted by buffer containing 0.05 M Tris (pH 8.4) containing 0.16 M of NaCl plus protease inhibitors (Figure 1). The remaining bound protein was eluted with the same buffer containing 0.5 M NaC1. On a sodium dodecylsulfate-polyacry1ami.de gel ( S DS-PAGE) under non-reducing electrophoresis conditions, fragment 8 migrates as two major bands (Figure 2, lane c). The upper band (subunit E8A, 140kDa) is formed by part of the laminin A chain, while the lower band (subunit E8B, 75kDA) contains fragments corresponding to the B chains. Under reducing conditions, little change was noticed on the migration pattern of the E8A subunit, while E8B split into several low molecular weight bands (Figure 2, lane d) . The electrophoretic configuration of fragment 8 obtained is similar to that of previously described preparations l o . When submitted to molecular sieve cromatography under non-denaturing conditions fragment 8 eluted as a single peak (not shown). Fragment 1 can be eluted in an almost purified form by elution with 0.5 M NaCl (Figure 2, lane b). The first peak observed in figure 1 which represents the non-bound fraction, consisted of several low molecular weight fragments also resulting from the elastase digestion. Purification of fragment 8 was performed 5 times and the method was found to be highly reproducible.
LINE
232
0.6 h
E
C 0.5 0
a
N v
w 0.4 Downloaded by [University of California, San Diego] at 03:03 28 June 2016
0
z a
m 0.3
LT
0
2 0.2 a
0
0.1
I\
0
I\
0 100
0
200
400 500 VOLUME ( m l )
600
300
ELUTION
FIGURE 1 Fractionation of the elastase digested laminin. The digest was applied to a DEAE-Shephacel column equilibrated with 0.05 M Tris (pH 8 . 4 ) , 0.025 M NaCl plus 0.5 mM PMSF and 0.5 mM NEN. The column was eluted with the same buffer containing 0.16 M and 0.5 M NaCl to yield fractions 1 and 8 respectively.
Adhesion assays* The adhesion assays were performed according to Chammas et a1 in RPMI-1640
3
.
Murine melanoma B16F10 cells, cultured
medium
(Gibco, USA) containing
10% fetal
calf serum, were detached by exposure to 0 , 0 2 % EDTA in phosphate-buffered cells were
saline;
resuspended
in
after
centrifugation,
RPMI-1640
medium.
the
Aliquots
(100 ul) containing 1.0 X lo4 cells were incubated in 96
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
FRAGMENT OF MOUSE LAMININ
FIGURE 2 Electrophoretic analysis of the eluted material. 3-15% SDS-PAGE of the total elastase digest (lane a), 0.5 M material (lane b), and material from the 0.16 M NaCl analyzed under non-reducing (lane c) and reducing (lane d) The gel was stained using Coomassie brilliant blue R-250.
233
gradient of NaCl eluted eluted peak conditions.
well polystyrene Falcon plates (Becton Dickinson Co. Oxnard, CA) coated with a 50 ug/ml solution of fragment 8. After 2h incubation period the wells were washed with medium and the adhered cells were fixed for 10 min in ethanol and stained with hematoxylin and eosin. The adhesion experiments showed that fragment 8 was able to promote the adhesion and formation of processes on B16F10 cells. No process formation was observed on cells adhered to plastic alone (Figure 3 ) .
LINE
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
234
FIGURE 3 Adhesion of B16F10 cells to plastic (a) and to purified fragment 8 (b)
-
DISCUSSION
Among the several proteolytic fragments of laminin, fragments 1 and 8 were shown to be of particular importance, since they were identified as the two major cell binding domains of laminin, promoting cell adhesion and spreading of cells in culturel’.Due to its early characterization and easy purification, the study of the biological properties of fragment 1, including the characterization of specific receptors and the intrinsic mechanisms of interaction with cells in normal and neoplastic processes, are now well understood’ l 3 ’ l 4 . Direct evidences supporting the presence of a binding site on the fragment 8 region of laminin, and the identification of putative cell receptors for neurite formation have only recently been
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
FRAGMENT OF MOUSE LAMININ
235
pub1 ished2 f 1 0 r ? 1 The belated investigation of the properties of fragment 8 may be imputed in part to the difficulties involved in its purification. The current protocols for the purification of fragment 8 from the laminin-nidogen/entactin complex are based in a two-step process, which consists first on a molecular sieve chromatography onto a large column. This step does not produce highly purified fragments since peaks containing fragments 1 and 8 are overlapped. The purification of fragment 8 is only achieved by a second step separation which can be either a repetitive 9 molecular sieve chromatography on the same column , or affinity chromatography on heparin-Sepharose column1 5 In both processes there is a substantial loss of material, since the overlapped portion of the two peaks containing fragments 1 and 8, should be discarded to permit an effective separation during the second procedure. If the second step consists of a repeated molecular sieve chromatography, additional loss occurs for the same reason already mentioned. On the other hand, small contaminations of fragment 1 were detected in the eluted material when using heparin-Sepharose affinity chromatography. This can be explained by the fact that even though the major heparin binding site of laminin is located on fragment 8, two other distinct heparin binding sites were found to exist on fragment
.
16 1 .
The protocol described in this paper is highly advantageous when compared to previously described ones. It permits an effective separation of fragments 1 and 8, leading consequently to the isolation of practically all of the fragment 8 generated by the elastase digestion, i.e., 12-14 mg of fragment 8 from 100 mg of laminin-nidogen/entactin complex. This can be considered a high yield (60%) taking into account losses of 15-50%,
LINE
236
that occur due to further degradation of fragment 8 during the digestion procedure 9 This protocol offers two additional advantages. It permits the isolation of fragment 8 from relatively small amounts of the complex, and it also avoids extensive handling that includes repeated passages over large gel filtration columns which can lead to further degradation and/or denaturation of the digested material. Additionally, the whole mass of fragment 1 generated by the elastase digestion can be easily purified by a further passage over a gel filtration column (not shown) , providing also an increased output of this fragment.
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
.
1.
REFERENCES G.R. Martin and R. Timpl, Ann. Rev. Cell Biol. 57-85
2.
3.
4. 5.
2,
(1987).
H.X. Kleinman, R.C. Ogle, F.B. Cannon, C.D. Little, T.M. Sweeney and L. Luckenbill-Edds, Proc. Natl. Acad. Sci. USA. 82, 1282- 1286 (1988). R. Chammas, S.S. Veiga, S . Line, P. Potocnjak and R.R. Brentani, J. Biol. Chem. 2 2 , 3349-3355 (1991). V. P. Terranova, J.E. Williams, L.A. Liotta and G.R. Martin, Science 226, 982-985 (1984). J . Engel, E. Odermatt, A. Engel, J.A. Madri, H. Furthmayr, H. Rohde and R. Timpl, J. Mol. Biol. 150,
.
97-120 (1981) 6.
S.R.P. Line and S.M.F. Ribeiro, Arq. Biol. Tecnol. 31, 21 (1988). -
7.
D.Edgar, R. 1363-1368
8.
9.
Timpl
and
H.
Thoenen, EMBO
J.
3, -
(1984).
A. Acheson, D. Edgar, R. Timpl and H.J. Thoenen, J. Cell Biol. 102, 151-159 (1986). M. Paulsson, M. Aumailley, R. Deutzmann, R. Timpl, K. Beck and J . Engel, Eur J. Biochem 166, - 11-19 (1987).
FRAGMENT OF MOUSE LAMININ
237
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
10. S.L. Goodman, R. Deutzmann and X von der Mark, J .
Cell Biol. 105, 589-598 (1987). 11. M. Aumailley, V. Nurcombe , D. Edgard, M. Paulsson and R. Timpl, J. Biol. Chem. 262, 11532-11536 (1987). 12. S.H. Barsky, C.N. Rao, J . E . Williams and L.A. Liotta, J Clin. Invest. 74, - 843-848 (1984) 13. Y. Iwamoto, F. Robey, J. Graf, M. Sasaki, H.K. Kleinman, Y. Yamada and G. Martin, Science 238,
.
1132-1134 (1987)
J.B. McCarthy, A.P.N. Skubitz, S . L . Palm and L.T. Furcht, J . Natl. Cancer Inst. 80, - 108-115 (1988). 15. M. Paulsson, R. Deutzmann, R. Timpl, D. Dalzoppo, E. Odermatt and J. Engel, EMBO J. 4, 309-316 (1985). 16. A.P.N. Skubitz, J . B . MacCarthy, A.S. Charonis and L. T. Furcht, J. Biol. Chem. 263, - 4861-4868 (1988). 14.
ISSN: 0032-7484 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb19
Purification of the Neurite Outgrowth Promoting Fragment of Mouse Laminin Sergio R. P. Line To cite this article: Sergio R. P. Line (1992) Purification of the Neurite Outgrowth Promoting Fragment of Mouse Laminin, Preparative Biochemistry, 22:3-4, 229-237, DOI: 10.1080/10826069208021373 To link to this article: http://dx.doi.org/10.1080/10826069208021373
Published online: 24 Sep 2006.
Submit your article to this journal
Article views: 3
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=lpbb20 Download by: [University of California, San Diego]
Date: 28 June 2016, At: 03:03
PREPARATIVE BIOCHEMISTRY,22(3&4), 229-237 (1992)
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
PURIFICATION OF THE NEURITE OUTGROWTH PROMOTING FRAGMENT OF MOUSE LAMININ Sergio. R. P. Line Ludwig Institute for Cancer Research 01509 Sbo Paulo, SP, Brazil Present address: Faculdade de Odontologia de Piracicaba Av. Limeira s/n. Caixa Postal 52 13400 Piracicaba, SP, Brazil
ABSTRACT A method for isolation of the neurite outgrowth promoting fragment of mouse laminin (fragment 8) is described in this paper. Besides producing excellent yields, this method was shown to be fast and practical, since it is based on a single step which consists in an ion exchange chromatography of elastase digested laminin. INTRODUCTION Laminin (880kDa), the major non-collagenous glycoprotein of basement membranes is composed of three polypeptide chains, the A chain (440kDa) and two B chains (220kDa). The three polypeptide chains are spatially arranged forming a cross shaped structure 1. Laminin binds to other basement membrane molecules as type IV collagen, nidogen/entactin, heparan sulfate proteoglycan, and also interacts with cells by means of specific receptors2 . Laminin-cellular receptors interactions are thought to mediate several biological I
229 Copyright 0 1992 by Marcel Dekker, Inc.
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
230
LINE
functions and also to be involved in the process of 4 metastasis . The generation of proteolytic derived fragments has allowed a better understanding of laminin structure and the identification of domains responsible for defined biological functions. A large proteolytic fragment (fragment 1) was identified as consisting of three rod like segments from the center of the short arms that contains a cell binding domain capable of promoting the adhesion of tumor cells5 6 . Another cell binding domain was identified on the elastase derived fragment 8 (280kDa). This fragment located at the end of the long arm was shown to stimulate neurite survival and outgrowth and trigger tyrosine hydroxylase activity in chromaffin cells7I8. Despite its biological importance, the use of fragment 8 has suffered from the lack of a reproducible preparative method for its purification. There exists only one protocol which describes the isolation of biologically active fragment 8 in a reproducible 9 fashion However, the elastase digestion of laminin-nidogen/entactin complex produces also large amounts of fragment 1 (300kDa). Due to their close molecular weights an effective separation of fragments 1 and 8 can only be achieved by two or three passages over a large gel chromatography column, with considerable loss of material. We describe in this paper an improved protocol for obtaining highly purified and biologicaly active fragment 8 from mouse laminin that permits the recovery of almost all the material generated by the elastase digestion.
.
MATERIAL, METHODS AND RESULTS Isolation and analysis of fragment 8 Extraction and purification of the laminin-nidogen complex and further elastase digestion
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
FRAGMENT OF MOUSE LAMININ
23 1
was performed as described by Paulsson et a1 9 . A 50ml aliquot of the digest (lmg/ml) was dialyzed against buffer containing 0.05 M Tris (pH 8.4), 0.025 M NaCl plus 0.5 mM of phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM of N-ethylmaleimide (NEN) as protease inhibitors. The material was then applied to a DEAE-Sephacel column (2.5 x 20 cm, Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in the same buffer. Fragment 8 was eluted by buffer containing 0.05 M Tris (pH 8.4) containing 0.16 M of NaCl plus protease inhibitors (Figure 1). The remaining bound protein was eluted with the same buffer containing 0.5 M NaC1. On a sodium dodecylsulfate-polyacry1ami.de gel ( S DS-PAGE) under non-reducing electrophoresis conditions, fragment 8 migrates as two major bands (Figure 2, lane c). The upper band (subunit E8A, 140kDa) is formed by part of the laminin A chain, while the lower band (subunit E8B, 75kDA) contains fragments corresponding to the B chains. Under reducing conditions, little change was noticed on the migration pattern of the E8A subunit, while E8B split into several low molecular weight bands (Figure 2, lane d) . The electrophoretic configuration of fragment 8 obtained is similar to that of previously described preparations l o . When submitted to molecular sieve cromatography under non-denaturing conditions fragment 8 eluted as a single peak (not shown). Fragment 1 can be eluted in an almost purified form by elution with 0.5 M NaCl (Figure 2, lane b). The first peak observed in figure 1 which represents the non-bound fraction, consisted of several low molecular weight fragments also resulting from the elastase digestion. Purification of fragment 8 was performed 5 times and the method was found to be highly reproducible.
LINE
232
0.6 h
E
C 0.5 0
a
N v
w 0.4 Downloaded by [University of California, San Diego] at 03:03 28 June 2016
0
z a
m 0.3
LT
0
2 0.2 a
0
0.1
I\
0
I\
0 100
0
200
400 500 VOLUME ( m l )
600
300
ELUTION
FIGURE 1 Fractionation of the elastase digested laminin. The digest was applied to a DEAE-Shephacel column equilibrated with 0.05 M Tris (pH 8 . 4 ) , 0.025 M NaCl plus 0.5 mM PMSF and 0.5 mM NEN. The column was eluted with the same buffer containing 0.16 M and 0.5 M NaCl to yield fractions 1 and 8 respectively.
Adhesion assays* The adhesion assays were performed according to Chammas et a1 in RPMI-1640
3
.
Murine melanoma B16F10 cells, cultured
medium
(Gibco, USA) containing
10% fetal
calf serum, were detached by exposure to 0 , 0 2 % EDTA in phosphate-buffered cells were
saline;
resuspended
in
after
centrifugation,
RPMI-1640
medium.
the
Aliquots
(100 ul) containing 1.0 X lo4 cells were incubated in 96
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
FRAGMENT OF MOUSE LAMININ
FIGURE 2 Electrophoretic analysis of the eluted material. 3-15% SDS-PAGE of the total elastase digest (lane a), 0.5 M material (lane b), and material from the 0.16 M NaCl analyzed under non-reducing (lane c) and reducing (lane d) The gel was stained using Coomassie brilliant blue R-250.
233
gradient of NaCl eluted eluted peak conditions.
well polystyrene Falcon plates (Becton Dickinson Co. Oxnard, CA) coated with a 50 ug/ml solution of fragment 8. After 2h incubation period the wells were washed with medium and the adhered cells were fixed for 10 min in ethanol and stained with hematoxylin and eosin. The adhesion experiments showed that fragment 8 was able to promote the adhesion and formation of processes on B16F10 cells. No process formation was observed on cells adhered to plastic alone (Figure 3 ) .
LINE
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
234
FIGURE 3 Adhesion of B16F10 cells to plastic (a) and to purified fragment 8 (b)
-
DISCUSSION
Among the several proteolytic fragments of laminin, fragments 1 and 8 were shown to be of particular importance, since they were identified as the two major cell binding domains of laminin, promoting cell adhesion and spreading of cells in culturel’.Due to its early characterization and easy purification, the study of the biological properties of fragment 1, including the characterization of specific receptors and the intrinsic mechanisms of interaction with cells in normal and neoplastic processes, are now well understood’ l 3 ’ l 4 . Direct evidences supporting the presence of a binding site on the fragment 8 region of laminin, and the identification of putative cell receptors for neurite formation have only recently been
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
FRAGMENT OF MOUSE LAMININ
235
pub1 ished2 f 1 0 r ? 1 The belated investigation of the properties of fragment 8 may be imputed in part to the difficulties involved in its purification. The current protocols for the purification of fragment 8 from the laminin-nidogen/entactin complex are based in a two-step process, which consists first on a molecular sieve chromatography onto a large column. This step does not produce highly purified fragments since peaks containing fragments 1 and 8 are overlapped. The purification of fragment 8 is only achieved by a second step separation which can be either a repetitive 9 molecular sieve chromatography on the same column , or affinity chromatography on heparin-Sepharose column1 5 In both processes there is a substantial loss of material, since the overlapped portion of the two peaks containing fragments 1 and 8, should be discarded to permit an effective separation during the second procedure. If the second step consists of a repeated molecular sieve chromatography, additional loss occurs for the same reason already mentioned. On the other hand, small contaminations of fragment 1 were detected in the eluted material when using heparin-Sepharose affinity chromatography. This can be explained by the fact that even though the major heparin binding site of laminin is located on fragment 8, two other distinct heparin binding sites were found to exist on fragment
.
16 1 .
The protocol described in this paper is highly advantageous when compared to previously described ones. It permits an effective separation of fragments 1 and 8, leading consequently to the isolation of practically all of the fragment 8 generated by the elastase digestion, i.e., 12-14 mg of fragment 8 from 100 mg of laminin-nidogen/entactin complex. This can be considered a high yield (60%) taking into account losses of 15-50%,
LINE
236
that occur due to further degradation of fragment 8 during the digestion procedure 9 This protocol offers two additional advantages. It permits the isolation of fragment 8 from relatively small amounts of the complex, and it also avoids extensive handling that includes repeated passages over large gel filtration columns which can lead to further degradation and/or denaturation of the digested material. Additionally, the whole mass of fragment 1 generated by the elastase digestion can be easily purified by a further passage over a gel filtration column (not shown) , providing also an increased output of this fragment.
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
.
1.
REFERENCES G.R. Martin and R. Timpl, Ann. Rev. Cell Biol. 57-85
2.
3.
4. 5.
2,
(1987).
H.X. Kleinman, R.C. Ogle, F.B. Cannon, C.D. Little, T.M. Sweeney and L. Luckenbill-Edds, Proc. Natl. Acad. Sci. USA. 82, 1282- 1286 (1988). R. Chammas, S.S. Veiga, S . Line, P. Potocnjak and R.R. Brentani, J. Biol. Chem. 2 2 , 3349-3355 (1991). V. P. Terranova, J.E. Williams, L.A. Liotta and G.R. Martin, Science 226, 982-985 (1984). J . Engel, E. Odermatt, A. Engel, J.A. Madri, H. Furthmayr, H. Rohde and R. Timpl, J. Mol. Biol. 150,
.
97-120 (1981) 6.
S.R.P. Line and S.M.F. Ribeiro, Arq. Biol. Tecnol. 31, 21 (1988). -
7.
D.Edgar, R. 1363-1368
8.
9.
Timpl
and
H.
Thoenen, EMBO
J.
3, -
(1984).
A. Acheson, D. Edgar, R. Timpl and H.J. Thoenen, J. Cell Biol. 102, 151-159 (1986). M. Paulsson, M. Aumailley, R. Deutzmann, R. Timpl, K. Beck and J . Engel, Eur J. Biochem 166, - 11-19 (1987).
FRAGMENT OF MOUSE LAMININ
237
Downloaded by [University of California, San Diego] at 03:03 28 June 2016
10. S.L. Goodman, R. Deutzmann and X von der Mark, J .
Cell Biol. 105, 589-598 (1987). 11. M. Aumailley, V. Nurcombe , D. Edgard, M. Paulsson and R. Timpl, J. Biol. Chem. 262, 11532-11536 (1987). 12. S.H. Barsky, C.N. Rao, J . E . Williams and L.A. Liotta, J Clin. Invest. 74, - 843-848 (1984) 13. Y. Iwamoto, F. Robey, J. Graf, M. Sasaki, H.K. Kleinman, Y. Yamada and G. Martin, Science 238,
.
1132-1134 (1987)
J.B. McCarthy, A.P.N. Skubitz, S . L . Palm and L.T. Furcht, J . Natl. Cancer Inst. 80, - 108-115 (1988). 15. M. Paulsson, R. Deutzmann, R. Timpl, D. Dalzoppo, E. Odermatt and J. Engel, EMBO J. 4, 309-316 (1985). 16. A.P.N. Skubitz, J . B . MacCarthy, A.S. Charonis and L. T. Furcht, J. Biol. Chem. 263, - 4861-4868 (1988). 14.
