Polymer Journal, Vol. 24, No. 5, pp 465---472 (1992)

Liquid-Phase Synthesis and Cell Attachment Activity of Bioactive Pentapeptide YIGSR

Yoshiaki HIRANO, Motoya OKUNO, Toshio HAYASHI,* and Akio NAKAJIMAt

Department of Applied Chemistry, Osaka Institute of Technology, Ohmiya, Asahi-ku, Osaka 535, Japan *Research Center for Engineering, Kyoto University, Kawahara-cho, Sakyo-ku, Kyoto 606, Japan

(Received October 3, 1991)

ABSTRACT: Pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR), an amino acid sequence existing in the cell attachment domain of the Bl chain of laminin, was synthesized by an improved liquid­ phase procedure. Bioactivity of YIGSR as cell recognition determinant was investigated using L-929 fibroblast cell and A431 epidermoid cell. The YIGSR pentapeptide was immobilized to ethylene-acrylic acid copolymer (PEA) film, and the number of cells attached to the immobilized film was counted. In addition, the cell-attachment activity of YIGSR toward the cells was also evaluated by the cell inhibition test. These two tests led to a conclusion that YIGSR acts as a cell determinant toward L-929 and A431 cells. KEY WORDS Tyr-Ile-Gly-Ser-Arg (YIGSR) Pentapeptide / Liquid-Phase Peptide Synthesis I Cell Attachment Activity I YIGSR-Immobilized Ethylene-Acrylic Acid Copolymer/ L-929 Fibroblast Cell/ A431 Epidermoid Cell/

Laminin, the major non-collagenous glyco­ cell-spreading and chemotaxis. In addition, protein distributing only in the basement Iwamoto and co-workers7 have demonstrated membrane, an extracellular matrix between that the synthetic pentapeptide YIGSR in­ epithelial cell and stroma, has various bio­ hibited the formation of metastasis by logical activities, such as adhesion, migration, malignant melanoma cells. In 1989, Murata differentiation, and growth, of cell. 1 •2 Laminin and co-workers10 reported that synthetic is composed of three chains designated as A, sequential polypeptide poly(YIGSR) drasti­ B I, and B2, which are arranged in a cross­ cally inhibited the metastatic formation of shaped structure. 3 These three chains of B16--BLI6 melanoma cells. laminin have been cloned and sequenced, and The aim of this work is to establish a several active sites in the molecule have been method to synthesize YIGSR pentapeptide in identified. 4 - 6 Iwamoto et al. 7 and Graf et al. 8 •9 satisfactory yields by an improved liquid­ suggested that the peptide sequence Cys-Asp­ phase procedure, and to elucidate the cell­ Pro-Gly-Tyr-Ile-Gly-Ser-Arg (CDPGYIGSR, attachment activity of the pentapeptide and in one letter amino acid code) in the B I chain of pentapeptide7immobilized polymer films. concerns with cell-attachment activity, and, The reason why we use the liquid-phase in particular, pentapeptide sequence YIGSR procedure for peptide synthesis is that we can plays important role for cell-attachment, obtain much more amounts of pentapeptide

t To whom correspondence should be addressed.

465 Y. HIRANO eta/. in one cycle compared with the use of (Peptide Institute, Inc.) were used as deblock­ automatic peptide synthesizer. The polymer ing agents. Laminin sample used for coating used for immobilization is an ethylene-acrylic of cell culture dishes was purchased from Iwaki acid copolymer (PEA) containing 8.9mol% Glass, Co. All the solvents and chemicals used (20 wt%) acrylic acid. The cells used are were of reagent grade. fibroblast L-929 cell originating in mouse, and epidermoid A431 cell originating in human. Synthesis of Tyr-Ile-Gly-Ser-Arg ( YIGSR) by Liquid-Phase Procedure EXPERIMENTAL NG-Tosyi-L-arginine methyl ester (NH2- Arg(Tos)-0Me) was synthesized11 from Boc­ Materials Arg(Tos)-OH, and Boc-Tyr-0Su12 was from t-Butoxycarbonyl-L-tyrosine (Boc-Tyr), t• Boc-Tyr-O H. Scheme of synthesis of YIGSR butoxycarbonyl-0-benzyl-L-serine (Boc­ by liquid-phase procedure is shown in Figure Ser(Bzl)), glycine methyl ester hydrochloride 1. Fragment condensation method was used. (Giy-OMe · HCl), L-isoleucine methyl ester First, Boc-Tyr-OSu and NHz-IIe-OMe were (Ile-OMe · HCl), were purchased from Koku­ coupled by N-succinimide procedure, secondly, san Chemical Works, Ltd., and t-butoxy­ Boc-Tyr-IIe-NHNH2 was coupled with NH2- carbonyl-N0-tosyl-L-arginine (Boc-Arg(Tos)) Gly-0Me by azide procedure. On one hand, was from Peptide Institute, Inc. Dicyclohexyl­ Boc-Ser(Bzl) was condensed with NH2-Arg­ carbodiimide (DCC) used as a dehydration (Tos)-0Me by DCC procedure. Finally, Boc­ agent, and N-hydroxysuccinimide (HOSu) Tyr-IIe-Gly-NHNH2 was coupled with NH2- were purchased from Peptide Institute, Inc. Ser(Bzl)-Arg(Tos)-0Me by azide procedure, Triethylamine (TEA) used as a neutralization and the product obtained was deblocked by agent, and hydroxybenzotriazole (HOBt) were saponification and TFMSA to obtain H-Tyr­ purchased from Katayama Chemicals, and IIe-Gly-Ser-Arg-OH. Kokusan Chemical Works, respectively. Tri­ Step 1: To 3.48g (1.9x 10-2mol) NH2-Ile- fiuoromethanesulfonic acid (TFMSA) (Kanto 0Me · HCl dissolved in 35 ml tetrahydrofuran Chemicals), and trifiuoroacetic acid (TF A) (THF), equimolar TEA was added. To the Tyr lle Gly Ser Arg

Soc --osu NH2- f-OMe Mixed Soc OMe NH2NH2 Tos Soc NHNH2 NH 2- -OMe Soc- OH NH2-L OMe Azide 1/Szl DCC Tos Soc OMe Soc / OMe NH2NH2 Szl TFA Tos 1/ / Soc NHNH2 NH2 OMe Azide 1/Szl Tos Soc / OMe NoOH/MeOH 1/Szl Tos Soc OH TFMSA

NH 2 OH Figure 1. Scheme of Tyr-Ile-Gly-Ser-Arg synthesis by liquid phase-procedure.

466 Polym. J., Vol. 24, No. 5, 1992 Synthesis and Cell-Attachment Activity of YIGSR

solution obtained, 6.05 g (1.6 x 10-2 mol) Boc­ 5% citric acid and 5% NaHC03 solution. Tyr-OSu was added, and then the mixture kept Purity of the product was checked by TLC. alkaline by TEA was stirred at 4oC for 24h. Further, the extract was washed with NaCl­ After 24 h, the solution was filtered and the saturated water, dried over Na2S04 , and then filtrate was evaporated in high vacuum. The evaporated. Boc-Tyr-Ile-Gly-OMe (2.45 g) ob­ resulting precipitate was extracted with ethyl tained was dissolved in a small amount of acetate, and the extract was washed with 5% MeOH, to which 5 times the molar quantity citric acid and 5% sodium hydrogencarbonate of hydrazine hydrate was added, and the

(NaHC03 ). Purity of the product was checked solution was kept at room temperature by using thin-layer chromatography (TLC; overnight. The gelatinous mass formed was Merck) (CHC1 3 : MeOH: H20 = 8: 3: 1). The collected by filtration, and the filtrate was extract was further washed with NaCI­ evaporated. The resulting solid was re­ saturated water, dried over Na2S04 and then crystallized from acetonitrile and vacuum evaporated. Boc-Tyr-Ile-OMe obtained was dried. Thus, Boc-Tyr-Ile-Gly-NHNH 2 (2.14 g; dissolved in small amounts of MeOH, to 0.46 x 10-2 mol) was obtained. which, 5 times the molar quantity of hydrazine Step 3: 2.95 g (1.0 x 10-2 mol) Boc-Ser(Bzl)­ hydrate was added, and the solution was OH dissolved in 50 ml THF was mixed with kept at room temperature overnight. The 1.2 times the molar quantity of DCC and gelatinous mass formed was collected by HOBt under stirring in an ice bath for 30 min. filtration and the filtrate was evaporated. The 4.53 g (1.2 x 10-2 mol) NH2-Arg(Tos)-0Me· resulting solid was recrystallized from acetoni­ HCl was dissolved in 40 ml DMF and trile, and vacuum dried. Thus, Boc-Tyr-Ile­ equimolar TEA was added. The Boc-Ser(Bzl)­ NHNH2 (3.92 g; 0.96 x 10-2 mol) was ob­ OH solution was mixed with the NH2- tained. Arg(Tos)-0Me· HCl solution in a manner as Step 2: At a low temperature cooled down mentioned above, and stirred at 4oc for 24 h. with ice and NaCl, 4N HCl-dioxane (5.8 ml) After 24 h, the Boc-Ser(Bzl)-Arg(Tos)-OMe and isoamylnitrile (1.3 ml) were added succes­ obtained was purified by the same method sively to a solution of 3.92 g (0.96 x 10-2 mol) mentioned in step 2. The resulting solid was Boc-Tyr-Ile-NHNH2 in 30ml dimethylforma­ dissolved in 5 ml anisole. TFA (1 0 ml) was mide (DMF), and the mixture was stirred for added to remove Boc group at a low tem­ 30 min, where the hydrazine test was carried perature cooled down in ice bath for 60 min. out. For hydrazine test, 1% FeC1 3 /2M acetic The solvent was evaporated, and the resulting acid and 1% K 3 [Fe(CN)6] aqueous solution solid was recrystallized from diethylether and mixture (1: 1) was used as a color-producing vacuum dried. Thus, solid NH2 -Ser(Bzl)­ reagent. The solution was neutralized with Arg(Tos)-0Me (2.80g; 0.54x 10- 2 mol) was TEA. 1.44 g (1.2 x 10-2 mol) NH2-Gly-0Me · obtained. HCl was dissolved in 20m! DMF, to which Step 4: At a low temperature cooled with equimolar TEA was added. Now, the Boc­ ice and NaCI, 4N HCl-dioxane (2.8 ml) and Tyr-Ile-NHNH2 solution was mixed with the isoamylnitrile (0. 7 ml) were added successively NH2-Gly-0Me · HCl solution, and stirred at to a solution of 2.14 g (0.46 x 10-2 mol) ooc for 24 h, whereby, the pH of the mixture Boc-Tyr-Ile-Gly-NHNH2 in 30ml DMF, and was kept alkaline. The mixture was neutralized the mixture was stirred for 30 min, while the with acetic acid, filtered and then the filtrate hydrazine test was carried out. The solution was evaporated in high vacuum. The resulting was neutralized with TEA. 2.80 g (0.54 x 10- 2 precipitate was extracted with ethyl acetate, mol) NH2 -Ser(Bzl)-Arg(Tos)-0Me was dis­ and the extract was successively washed with solved in 20 ml DMF, to which equimolar TEA

Polym. 1., Vol. 24, No. 5, 1992 467 Y. HIRANO et a/. was added. The Boc-Tyr-Ile-Gly-NHNH2 solution was mixed with the NH2 -Ser(Bzl)­ C-OH C-NH-)YIGSR) II II Arg(Tos)-0Me solution and stirred at 4oc 0 0 for 24 h, whereby, the pH of the mixture was 1) wsc. C-OH C-NH-)YIGSR) kept alkaline. The mixture was neutralized II 2) NH 2 -YIGSR II with acetic acid, and filtered then the filtrate 0 0 was evaporated in high vacuum. The Boc-Tyr­ PEA PEA-YIGSR Ile-Gly-Ser(Bzl)-Arg(Tos)-OMe was purified by the same method mentioned in step 2. The Figure 2. Scheme of oligopeptide immobilization to PEA resulting solid was recrystallized three times films. from diethylether, purified by column chroma­ tography, and vacuum dried. The protecting for 5 days at 40oC. The -COOH residues group at the C-terminal of Boc-Tyr-Ile-Gly­ locating on the PEA film surface were activated Ser(Bzl)-Arg(Tos)-OMe (2.64 g) was removed at 4°C for 30 min with 4 g water-soluble by saponification with lN-NaOH/MeOH in carbodiimide, (!-ethyl-3-( 3-dimethy lamino­ an ice-bath for 4h. The Boc-Tyr-Ile-Gly­ propyl)-carbodiimide ), dissolved in 400 ml Ser(Bzl)-Arg(Tos)-OH obtained was dissolved phosphate buffer solution (PBS). Then 80 mg in a mixture, thioanisole (6. 7 ml)/1 ,2-ethane­ YIGSR pentapeptide was dissolved in 80ml dithiol (3.3 ml)/m-cresol (0.8 ml)/TFA (37.8 PBS and reacted with the activated PEA films ml), at room temperature, to which TFMSA for 24 h at 4°C. The immobilized films were (5.0 ml) was added in an ice-bath for 1 h, and rinsed with PBS and dodecyl sulfate sodium kept for 1 h at room temperature. 13 Diethyl­ salt solution (SDS) by ultrasonics cleaner ether was added in reaction system, and then and washed with pure water for 72 h. The the precipitate was filtrated, and washed with immobilization condition mentioned above diethylether. The deblocked H-Tyr-Ile-Gly­ was obtained by examining the relation Ser-Arg-OH (YIGSR) was dissolved in a between the amount of YIGSR immobilized small amount of H 2 0 and treated with CM­ and activation time by WSC and reaction time Sephadex C-25 ion exchange column chroma­ with YIGSR, by means of ESCA measure­ tography (ammonium solution form; Pharma­ ments. The surface characterization of the cia Fine Chemicals). The quantity of penta­ YIGSR-immobilized films was carried out by peptide H-Tyr-Ile-Gly-Ser-Arg-OH finally ob­ means of Cis and Nls spectra measured with tained was 1.10 g. The eluates were lyophilized a Shimadzu 750 electron spectroscopy for and dried over P2 0 5 in vacuum. chemical analysis (ESCA) spectrometer using Mg-Ka 1 •2 exciting radiation. The surface com­ Immobilization of YIGSR onto Ethylene-Acry­ position of the immobilized film was tested lic Acid Copolymer Film by the N/C ratio estimated from ESCA. From The schema of surface activation of the NjC ratio obtained, it was confirmed that ethylene-acrylic acid copolymer (PEA) and the YIGSR peptide was really immobilized oligopeptide immobilization are shown in onto PEA film surface. For cell-attachment Figure 2. The ethylene-acrylic acid copolymer tests, the films were stirred in 70% ethanol for pellet (acrylic acid content: 20wt%, lot. No. overnight, and ethanol was displaced with EAA-A510W) contributed from Teijin Co. PBS for I day. The test films were placed in Ltd. was purified in a methanol Soxhlet 24-wells cell culture dish (FALCON). extractor system. PEA films were prepared by casting from 5 wt% THF solution on glass Cell Attachment Activity Tests for YIGSR plates. Finally the films were dried in vacuo A431 epidermoid carcinoma cells and L-929

468 Polym. J., Vol. 24, No. 5, 1992 Synthesis and Cell-Attachment Activity of YIGSR fibroblast cells were cultured in Dulbecco's with Gly-OMe by DCC and active ester Modified Eagle Medium (DMEM; Nissui procedures, and found that the latter pro­ Seiyaku), and Eagle's MEM (Nissui Seiyaku), cedure is superior with regard to purity, both containing antibiotics Kanamycin (60 purification procedure, and yield. Thus, we mgl- 1) and 10% fetal bovine serum (FBS: carried out the condensation of Boc-Tyr-OSu M.A. Bioproducts, Maryland, U.S.A.), re­ with NH2-Ile-0Me by N-succinimide proce­ spectively. Cell lines used were A431 14 and dure. Further, Boc-Tyr-Ile-NHNH2 derived L-929 15· 16 cells (Riken Gene Bank, Tsukuba, from Boc-Tyr-Ile-OMe was condensed with Japan). A431 and L-929 were trypsinized Gly-OMe by azide procedure, not by DCC from culture dish, washed once in a medium procedure, because of the same reason men­ containing 10% FBS and once in a serum-free tions above. By the liquid-phase procedure medium. The cell density was adjusted to mentioned above, we succeeded in to synthesis 1.88 x 10 5 cells ml- 1 for cell adhesion tests in YIGSR of the order of gram. However, if we serum-free medium. I ml cell suspensions were use peptides synthesizer, pentapeptide obtained added to each of the polymer films placed in would be about 100 mg order in one cycle, and 24-wells in a cell culture dish and kept for may contain impurities. The YIGSR obtained predetermined periods in a humidified in­ was hydrolyzed with 6N-HC1 for 24 hat ll0°C, cubator conditioned to 37°C, 5% C02 and and the product was being assayed by amino 95% air atmosphere. After incubation, the acid analysis. The elemental analyses were unattached cells were removed from the film carried out with Boc-Tyr-Ile-Gly-Ser(Bzl)­ surface by washing with PBS. Adhered cells Arg(Tos)-OMe and H-Tyr-Ile-Gly-Ser-Arg­ were fixed by using glutaraldehyde, stained by OH. The results of amino acid analysis and Giemsa, and by glycerin. The number of cells of elemental analysis on YIGSR synthesized attached to the films was counted. were given in Table I. The result of elemental For cell inhibition test, the starting cell analysis of protected YIGSR suggests that the density was adjusted to 1.88 x 10 5 cells ml- 1 in a serum-free medium. 1 ml cell suspensions Table I. Amino acid analysis and elemental were added to each well, together with YIGSR analysis of YIGSR pentapeptide pentapeptide of different concentrations, and Amino acid analysis .kept in a humidified incubator conditioned to 37°C, 5% C02 and 95% air atmosphere for Count/nmol Ratio 1 h. Besides, 1 ml cell suspensions containing 2 mg of YIGSR pentapeptide were placed in Tyr 18.8 1.00 I I e 19.4 1.03 24-wells cell culture dishes, and kept for Gly 19.9 1.06 predetermined periods at the same conditions Ser 18.0 0.96 as mentioned above. The number of cells Arg 20.3 1.08 attached to the culture dish was counted as a function of incubation time. The cell culture dishes used for cell inhibition test were coated Protected YIGSR Deblocked YIGSR with laminin by adsorption method. Elemental analysis/% Elemental analysis/%

RESULTS AND DISCUSSION Calcd Exptl Calcd Exptl

Pentapeptide Synthesis c 57.9 56.8 52.5 51.2 We have examined condensations of Boc- H 6.7 6.5 7.1 7.2 N 11.8 11.5 18.8 18.0 Tyr with Ile-OMe, and of Boc-Tyr-Ile-OMe

Polym. J., Vol. 24, No. 5, 1992 469 Y. HIRANO et a[.

Table II. Surface composition of YIGSR-immobi1ized PEA film compared to PEA film

Elemental ratio/% Designation 0/C NjC

PEA 7.7 0 PEA-YIGSR 13.2 5.0

Nls ESCA spectra, respectively, for PEA and PEA-YIGSR. Obviously, Cis of the PEA­

Binding energy /eV YIGSR film exhibits a shoulder at 289- 29i eV, characteristic to carbon atom of the (a) amid bound. The Nis spectrum of PEA­ YIGSR is remarkable, in contrast to the absence of peak for PEA. N/C ratios of PEA and PEA-YIGSR film estimated from ESCA spectra were shown in Table II. As mentioned earlier, the PEA sample is a copolymer com­ posed of 91.1 mol% ethylene and 8.9mol% acrylic acid. If we assume that the surface composition is almost the same as the bulk composition, then the elemental ratio 0/C(%) of PEA surface is calculated as 8.0(%). v Comparing this value with the experimental value 7.7(%), we may conclude that the 410 405 400 395 surface composition is almost the same as the Binding energy /eV bulk composition. (b) On the other hand, for PEA-YIGSR, the Figure 3. ESCA spectra of PEA and PEA-YIGSR films. experimental values are: 0/C = i3.2% and (a). C1s spectra; (b), N1s spectra. N/C=5.0%. If we designate the mol% immobilization of YIGSR to the whole film sample tested may contain 0.5 mol H 20 per surface as x, then the experimental values mole pentapeptide as the water of crystalliza­ 0/C= i3.2% and N/C=5.0 led to x=ca. 3% tion, and deblocked YIGSR may contain and x=ca. 2%, respectively. Namely, YIGSR I mol H 2 0 per mole pentapeptide. If we take pentapeptide is immobilized to about 2.5% of into consideration the water of crystallization, the film surface. This figure indicates that the experimental values agree with the theo­ 28 mol% of the whole acrylic acid units is retical values. The results obtained from reacted with YIGSR. amino acid analysis and elemental analysis indicate that the YIGSR pentapeptide was Cell Attachment Activity of YIGSR-Immobi­ really synthesized. lized PEA Film Figures 4 and 5, illustrate the % cell attach­ Characterization of YIGSR-Immobilized PEA­ ment to PEA and PEA-YIGSR films for A431 Film and L-929, respectively, at the concentration Figures 3(a) and 3(b) illustrate Cis and of 1.0 x I 05 cells/em 2 . The error bars represent

470 Polym. J., Vol. 24, No.5, 1992 Synthesis and Cell-Attachment Activity of YIGSR

(%) (%)

fi':iJPEA !::. A4.31 L-929 OOPEA-YIGSR +• +• 0 L-929 c c Q) Q) E E .c .c 0 0 0 0 +• +• +- ..... 0 0

(j) Q) 0 .,__ ....0 0 0 (/1. * 0 ml) Time Peptide concentration

Figure 4. Percent of L-929 cells adhered onto PEA and Figure 6. Inhibition effects of YIGSR pentapeptide PEA-YIGSR films, in the absence of serum. toward L-929 and A431 as functions of YIGSR concentration. Incubation time, 1 h. (%) 100 A4.31 fi':iJPEA rnPEA-YIGSR +- c 1::. A4.31 Q) 0 L-929 E +• .c c 0 Q) 0 E +- .c +- 0 0 0 +• +- (j) 0 0 .... (j) 0 ....0 * 0 (/1. 30 60 Time 120 180(minl

Figure 5. Percent of A431 cells adhered onto PEA and Time PEA-YIGSR films, in the absence of serum. Figure 7. Inhibition effects of YIGSR pentapeptide toward L-929 and A431 cells as functions of time. the standard errors for five different experi­ YIGSR concentration, 2 mg ml- 1 . ments (N = 5), each containing 20 measuring points. As a whole, numerical values of % PEA. Such rather low values of % cell cell attachment are rather uniform, and the attachment for PEA-YIGSR may originate number of points showing the maximum and from that the degree of YIGSR immobiliza­ minimum values of error bar are only 10%. tion is low as 2.5 mol% of the film surface. So the results are regarded as significant. For L-929 cell, about 50% of the cell was adhered Inhibition Effect of YIGSR Pentapeptide for to PEA-YIGSR, almost independent of in­ L-929 and A431 Cells cubation time (30-180min), compared with More distinct means to evaluate the inter­ the figure 30% to PEA. For A431 cell, % cell actions between the pentapeptide and cells is adhesion to both PEA-YIGSR and PEA were the cell inhibition test. In Figure 6, the % cell lower than those for L-929, and somewhat attachments of L-929 and A431 are plotted depended on the incubation time, and at against the YIGSR concentration. The % cell 180 min, the % of cell adhesion to PEA­ attachment of L-929 dramatically decreases YIGSR (ca. 50%) was about twice that to with increasing peptide concentration and

Polym. J., Vol. 24, No. 5, 1992 471 Y. HIRANO et a{.

levels off to ca. 20% at 1.0 mg ml- 1 . With Martin, and M. Dubois-Dalcq, J. Cell. Biochem., respect to A431 cell, the % cell attachment at 27, 317 (1985). 3. J. E. Engel, E. Odermatt, A. Engel, J. Madri, H. 1.0 mg ml- 1 is 30%. Such results indicate that Rhode, and R. Timpl, J. Mol. Bioi., 150, 97 (1981). YIGSR pentapeptide inhibits 80 and 70% cell 4. M. Sasaki, H. K. Kleinman, H. Huber, R. Dutzmann, attachment of L-929 and A431, respectively, andY. Yamada, J. Bioi. Chern., 263, 16536 (1988). to the laminin-coated culture dish at 2.0 5. M. Sasaki, S. Kato, K. Kohno, G. R. Martin, and Y. Yamada, Proc. Nat!. Acad. Sci. U.S.A., 84, 935 mg ml- 1 YIGSR concentration. (1987). Figure 7 shows the % cell attachments of 6. M. Sasaki and Y. Yamada, J. Bioi. Chern., 262, L-929 and A431 against the incubation time at 17111 (1987). 2 mg ml- 1 peptide concentration. As obvious 7. Y. Iwamoto, F. A. Robey, J. Graf, M. Sasaki, H. K. from the figure, % cell attachment of L-929 Kleinman, Y. Yamada, and G. R. Martin, Science, 238, 1132 (1987). drastically decreases at initial stage and leveled 8. J. Graf, R. C. Ogel, F. A. Robey, M. Sasaki, off to 70% at 60 min, however, that of A431 G. R. Martin, Y. Yamada, and H. K. Kleinman, gradually decreases with incubation time and Biochemistry, 26, 6896 (1987). reaches to 30% (i.e., 70% inhibition) at 9. J. Graf, Y. Iwamoto, M. Sasaki, G. R. Martin, H. K. Kleinman, F. A. Robey, and Y. Yamada, Cell, 180min. 48, 989 (1987). 10. J. Murata, I. Saiki, I. Azuma, and N. Nishi, Int. Acknowledgments. The authors wish to Bioi. Macromol., 11, 97 (1998). thank Messrs Kazuhiro Ami, Keiji Okumura, II. N. Izumiya, T. Kato, H. Aoyanagi, and M. Waki, "Basis and Experiments for Peptide Synthesis" (in and Hiroaki Minami, Osaka Institute of Japanese), Maruzen, Tokyo, 1987, p 50. Technology, for their assistance in the 12. N. Izumiya, T. Kato, H. Aoyanagi, and M. Waki, experiments. This work was supported by the "Basis and Experiments for Peptide Synthesis" (in Japanese), Maruzen, Tokyo, 1987, p 97. Grant-in-Aid to A.N. for Scientific Research 13. H. Yajima, N. Fujii, H. Ogawa, and H. Kawatani, (No. 01604026) on Priority Area from the J. Chern. Soc., Chern. Commun., 107 (1974). Ministry of Education, Science, and Culture 14. D. J. Giard, S. A. Aaronson, G. J. Todaro, P. of Japan. Arnstein, J. H. Kersey, H. Dosik, and W. P. Parks, J. Nat!. Cancer Inst., 51, 1417 (1973). 15. H. Katsuta, T. Takaoka, K. Mitamura, I. Kawada, REFERENCES H. Kuwahara, and S. Kawahara, Jpn. J. Exp. Med., 29, 191 (1959). I. J. R. Couchman, M. Hiiiik, D. A. Rees, and R. 16. H. Katsuta and T. Takaoka, "Nutritional Require­ Timpl, J. Cell Bioi., 96, 177 (1983). ments of Cultured Cells," Japan Scientific Societies 2. H. K. Kleinman, F. B. Cannon, G. W. Laurie, J. R. Press, Tokyo, Japan, 1978, p 257. Hassell, M. Aumailley, V. P. Terranova, G. R.

472 Polym. J., Vol. 24, No. 5, 1992