THE JOURNAL OF BIOLOGICAL CHEM~STRV

Vol 252, No. 11, Issue of June 10, pp. 3995-4001, 197’7

Prmted in U S.A.

Intestinal Basement Membrane of Ascaris suum ANALYSIS OF POLYPEPTIDE COMPONENTS*

(Received for publication, October 4, 1976)

CHUNG-HO HUNG, MIKIO OHNO, J. WILLIAM FREYTAG, AND BILLY G. HUDSON+

From the Department of Biochemistry, University of Kansas Medical Center, Kansas City, Kansas 66103

The Ascaris suum intestinal basement membrane was sol-

ubilized on reduction with P-mercaptoethanol in 1% sodium

dodecyl sulfate and 1% sodium dodecyl sulfate, 8 M urea to

the extent of 90% and lOO%, respectively. The reduced mem-

brane consists of at least 17 polypeptides ranging in molecu-

lar weight from 22,500 to greater than 400,000, as deter-

mined by sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis and gel filtration chromatography using 6 M urea

as eluent. Six of these polypeptides contain carbohydrate, as

determined by periodic acid-Schiff stain. Certain polypep-

tides, representing about 8% of the membrane by weight, are

soluble in sodium dodecyl sulfate solutions without reduc-

tion and are not interlinked with each other by disulfide

bonds; these interact in the membrane exclusively through

noncovalent forces. Amino acid and carbohydrate analyses

of the column fractions revealed that certain polypeptides

are collagen-like, being greatly enriched in 4-hydroxypro-

line, hydroxylysine and glycine, and disaccharide units;

whereas others are less collagen-like, being characterized by

the absence of 4-hydroxyproline, low contents of hydroxyly-

sine and glycine, and large contents of glutamic acid and

proline, and enriched in oligosaccharide units. These results

extend the observation from vertebrates to invertebrates

that basement membranes consist of multiple polypeptides

which have compositions of varying degrees of relatedness

to collagen, suggesting a conservation of this structural

characteristic during evolutionary development.

The intestinal basement membrane of the helminth Ascaris suum is classified as a specialized member of the collagen

family of proteins. It exhibits a wide angle x-ray diffraction

pattern characteristic of collagen (2) and contains appreciable

amounts of the amino acids characteristic of collagen (i.e.

hydroxyproline, hydroxylysine, and glycine) (3). The Ascaris membrane is related in amino acid and carbohydrate composi-

tion to that of vertebrate membranes, but it is distinctly

different. These differences are due to a decrease in the con-

tent of hydroxyproline, hydroxylysine, and glycine; an in-

crease in acidic amino acids; and the absence of S-hydroxypro-

line and sialic acid in the Ascaris membrane as compared to

* This work was supported by Grant AM18381 from the National Institutes of Health. A preliminary report of this work was published

(1). $ To whom all correspondence should be addressed.

vertebrate membranes, such as renal glomerular basement

membrane (3).

No information is available regarding the number, molecu-

lar weight, composition, and nature of the interaction of the

polypeptide components of this invertebrate basement mem-

brane. Information of this type has been reported for bovine

renal glomerular basement membrane and shows that it con-

sists of many polypeptide components of varying molecular

weight and composition (4-9) and that certain of these poly-

peptides are cross-linked by disulfide bonds (4, 10) and alde-

hyde-derived collagen cross-links (11).

The purpose of the present research concerning the Ascaris basement membrane was to determine the overall polypeptide

composition, the molecular weight of the polypeptides, and

their mode of interaction; and further, to determine which

polypeptides contain carbohydrate and which contain the

amino acids characteristic of collagen. In a succeeding paper

the chemical nature of the carbohydrate units is reported (12).

EXPERIMENTAL PROCEDURES

Materinls

All electrophoresis chemicals were obtained from Bio-Rad. Schiff s reagent was obtained from Fisher Scientific and P-mercaptoethanol from Eastman. P-Galactosidase, bovine serum albumin, ovalbumin, chymotrypsinogen, calf skin collagen, iPr,PF,l MalNEt, EDTA, and urea were obtained from Sigma. Myosin was prepared by the method of Perry (13). All other chemicals were the best commercially availa- ble and used without further purification.

Preparation of Basement Membrane

The membrane was prepared by the method previously described (3) and by a modification of this method. This modification involves (a) placing the dissected intestines directly into an ice-cold solution of 0.85% NaCl containing 10 rnM EDTA, 10 rnM iPr,PF, 25 rnM MalNEt followed by sonic disruption in this solution; and (6) wash- ing the insoluble membrane released on sonication with the same ice-cold solutions.

Electrophoresis

Two systems of sodium dodecyl sulfate-polyacrylamide gel electro- phoresis were employed for the determination of membrane polypep- tide composition and molecular weight.

Systmn 1 - Electrophoresis was performed essentially as previ- ously described (4) on 5% acrylamide gels (9 cm) containing 0.1% sodium dodecyl sulfate. Gel columns were stored at room tempera- ture for 12 to 16 h and prerun for 30 min prior to the application of

’ The abbreviations used are: iPr,PF, diisopropyl fluorophosphate; MalNEt, N-ethylmaleimide; PAS, periodic acid-Schiff; TEMED, N,N,N’,N’-tetramethylethylene diamine.

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3996 Basement Membrane Pol.ypeptides

protein samples. Electrophoresis was performed with a current of 8 mA/tube for 4l12 h, at which time the tracking dye had migrated about 8.5 cm.

Gels were stained for protein by fixing in 40% isopropyl alcohol for 1 h and then staining in a solution of 0.05% Coomassie blue, 10% acetic acid, 10% isopropyl alcohol for 6 h. Gels were then washed with several changes of 10% acetic acid over a period of 2 to 3 days. Duplicate gels were stained for carbohydrate using PAS reagents as described by Fairbanks et nl. (14). Spectrophotometric scanning of the Coomassie blue-stained gels and the PAS-stained gels was per- formed at 650 nm and 550 nm, respectively, using a Gilford spectro- photometer equipped with a scanning accessory.

Samples of membrane were prepared for electrophoresis by incu- bating 3 mg of membrane in 1 ml of 1% sodium dodecyl sulfate, 2% p- mercaptoethanol, 0.1 M phosphate buffer (pH 7.0) overnight at 37” The insoluble material was removed by centrifugation and the su- pernatant solution adjusted to 15% (v/v) glycerol, and then 25 ~1 of 0.05% bromphenol blue was added. Aliquots (10 to 50 ~1) of this solution were then applied to gel columns.

System 2 - Electrophoresis was performed on 5% acrylamide gels as described for System 1 except that 8 M urea was included in the gel columns and membrane samples. In order to obtain good polymeriza- tion of gels, freshly prepared urea solutions (no longer than 3 h after preparation) were absolutely required. After the addition of urea to ihe acrylamide solution, the pH was readjusted to 7.0 with concen- trated phosphoric acid and the mixture was deaerated. Conditions for electrophoresis and staining procedures were identical to those described in System 1 above.

For the molecular weight determination of membrane polypep- tides observed by electrophoresis using Systems 1 and 2, the posi- tions of the protein bands were measured and their mobilities were calculated relative to the mobility of bromphenol blue. Their appar- ent molecular weights were estimated by comparison of relative mobility with a calibration line of log molecular weight versus rela- tive mobility. A calibration line was constructed for both Systems 1 and 2 using the following standard proteins of known molecular weights: myosin (212,000) (151, P-galactosidase (135,000) (161, bovine serum albumin (69,000) (171, ovalbumin (43,000) (181, and chymo- trypsinogen (25,700) (17).

Soluhility ofBasement Membrane

Membrane suspension (1 mgiml) was incubated in 1% sodium dodecyl sulfate, 0.1 M phosphate, pH 7.0, with and without 2% p- mercaptoethanol overnight at 37” with shaking. The insoluble mate- rial was removed by centrifugation and several aliquots of the super- natant solution were analyzed for protein using the method of Geiger and Bessman (19). Bovine serum albumin was used as a reference to construct a standard curve. Solubility of membranes in 8 M urea, 1% sodium dodecyl sulfate, 0.1 M phosphate, pH 7.0, with and without p- mercaptoethanol was also determined by this same procedure.

Gel Filtration Chromatography ofReduced Membrane

Sepharose CL-4B was packed to a height of 85 cm in a Pharmacia K261100 glass column (2.6 x 100 cm). The column was equilibrated with deaerated and nitrogen-flushed 6 M urea, 0.1% P-mercaptoetha- nol, 0.1 M PO,, pH 6.0. The membrane sample for column fractiona- tion was prepared by incubating 55 mg of whole membrane in 11.0 ml of deareated 8 M urea, 0.1 M PO, (pH 6.01, 4% P-mercaptoethanol for 24 h at 37” with constant shaking under a nitrogen barrier. The sample (10 ml) was diluted to 6 M urea prior to application to the column and eluted with equilibrating buffer under a hydrostatic pressure head of 25 cm at a flow rate of 10 ml/h. Fractions of 2.2 ml were collected. The protein content of the column fractions was measured as a function of the absorbance at 650 nm, using the calorimetric procedure of Geiger and Bessman (191, and by measure- ment of the absorbance at 280 nm. The polypeptides in the column fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of a given polypeptide in a fraction, measured in terms of its absorbance at 650 nm, was calculated by multiplying the absorbance value for total protein content by the relative amount (%) of that polypeptide in the fraction. The relative amount was determined from the electrophoresis results in which the polyacrylamide gels were scanned spectrophotometrically and the relative peak areas measured from the recording.

Fractions containing certain polypeptides were pooled, dialyzed exhaustively against distilled water at 4”, lyophilized, and analyzed for amino acids and neutral monosaccharides. Fractions eluting

before void volume of column and after column volume were treated in the same manner and used as controls in carbohydrate determina- tions. Electrophoresis was conducted by incubating 50 ~1 of sample fractions with 50 ~1 of 2% sodium dodecyl sulfate, 2% P-mercaptoeth- anal, 0.1 M PO, (pH 7.0) overnight at 27”. All 100 pl of sample was applied to 5% polyacrylamide gels as previously described (System 1).

The column was calibrated with S-carboxymethylated standard proteins using the same experimental conditions. Serum albumin (5 mg), ovalbumin (4 mg), and acid-soluble calf skin collagen (10 mg) were reduced and alkylated in 10 ml of 8 M urea, 0.5 M Tris, pH 8.5, by the method of Mann and Fish (20). The molecular weights of collagen components are: u chain 95,000 (21, 22), p component (di- mer, 190,000), and y component (trimer, 285,000).

Chemical Analyses

For amino acid analyses, samples were hydrolyzed with glass- distilled 6 N HCl under reduced pressure in sealed tubes at 105” for 24 h. The amino acid contents of the hydrolysates were determined on a Beckman 121 HP analyzer by the procedure of Guire et al. (23).

Neutral monosaccharides were identified and quantitated as alditol acetates by gas-liquid chromatography essentially as described by Kim et al. (241, but a different column packing was used. Sugars were released from the protein by hydrolysis with Dowex 50-X12 resin and 0.25 N H,SO, for 24 h at loo”, then reduced and acetylated. The chromatography was performed on a single column (2 mm x 6 ft) of 3% SP2340 (Supelco) on Supelcoport (80 to 120 mesh) with nitrogen as carrier gas at a flow rate of 20 mlimin in a Hewlett-Packard 5931A gas chromatograph. Temperature was programmed from 180 to 225” at a rate of l”/min. This column packing is far more stable than the 3% ECNSS-M customarily used in this analysis and gives excellent resolution of alditol acetates.

RESULTS

Solubility - The membrane is soluble to the extent of 8% in

1% sodium dodecyl sulfate, 0.1 M phosphate buffer (pH 7.0)

with or without 8 M urea. On reduction with 2% P-mercapto-

ethanol, its solubility is increased to 90% in this buffer without

urea and to 100% with 8 M urea.

Electrophoresis ~ Membrane was analyzed by polyacryl-

amide gel electrophoresis using a sodium dodecyl sulfate/phos-

phate buffer (System 1). It consists of several polypeptide

components as shown by the electrophoretic pattern presented

in Fig. 1, Gel A. To assess whether this multiplicity of compo-

nents was generated artifactually by proteolytic degradation

during the isolation procedure, the membrane was isolated

with and without proteolytic inhibitors under conditions used

previously for glomerular basement membrane (9). The pat-

tern for membrane prepared at O-3” in the absence of exoge-

nous protease inhibitors is identical to that of membrane

prepared at O-3” using inhibitors (EDTA, MalNEt, and

iPr,PF), Gel B, and at 27” without these inhibitors, Gel C.

Since the solubility of the membrane is increased from 90 to

100% in the sodium dodecyl sulfate buffer with the inclusion of

8 M urea, its total polypeptide constituents could be analyzed

by sodium dodecyl sulfate electrophoresis by using 8 M urea in

both sample and polyacrylamide gels (System 2). Also, the

inclusion of urea would be expected to cause dissociation of

components which might be incompletely dissociated in so-

dium dodecyl sulfate alone. As shown in Gel D, Fig. 1, the

pattern with urea is basically the same as that without urea

(Gels A to C), except that mobilities are reduced and resolu-

tion of two polypeptides (VZZZ and IX) is greatly improved.

Polyacrylamide gels of membrane electrophoresed using

System 1 and System 2 were scanned spectrophotometrically

to detect precisely polypeptide components and to identify

those which contain carbohydrate. The gel scans of membrane

electrophoresed without urea are shown in Fig. 2. At least 17

polypeptides were detected by Coomassie blue stain (Fig. 24 1,

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Basement Membrane Polypeptides 3997

FIG. 1. Analysis of basement membrane polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membrane was analyzed in sodium dodecyl sulfate without urea (System 11, Gels A, B, and C, and with 8 M urea in both the sample and the gel column (System 21, Gel D. Membrane was isolated at O-3” (Gels A andD) and 27” (Gel C) without proteolytic inhibitors and at O-3” with 10 rnM EDTA, 25 rnM MalNEt, 10 rnM iPr,PF (Gel B). Approximately 30 Fg of membrane was applied to gels. Numeral designation of the poly- peptides in Gels A, B, and C is based on both visual inspection and spectrophotometric scans of gels (Fig. 2). Polypeptides in Gel D are tentatively designated (- - -).

of which at least five stain positive for carbohydrate (Fig. 2Z3).

The gel scans of membrane electrophoresed with the inclusion

of 8 M urea in sample and polyacrylamide gel are shown in

Fig. 3. The pattern of those polypeptides which penetrate the

gel (VZZZ to XVZZ) is closely related to that obtained without

urea (Fig. 2A); thus, the components are tentatively desig-

nated by the numbers used in Fig. 2A. The improved resolu-

tion of polypeptideg VIII and IX using urea (Fig. 3A) permits

the identification of both components as glycoproteins based

on the scan of PAS-stained gels (Fig. 3B).

The apparent molecular weights of the polypeptides ob-

served by both electrophoretic systems were determined from

their relative mobilities in comparison to that of standard

proteins. The calibration line, constructed from the standards,

is similar for both System 1 and 2 (Fig. 4). Molecular weight

values of the membrane polypeptides are summarized in Table

I. The values, determined by both systems, for a given poly-

peptide agree within 10% except for polypeptides XII, XIII,

XVI, and XVII which differ by 24 to 35%. These large differ-

ences are clearly evident by comparing the positions of these

ool-

A

B

MIGRATION DISTANCE fcml

FIG. 2. Spectrophotometric scans of polyacrylamide gels of mem- brane analyzed in sodium dodecyl sulfate without urea (System 1). A (upper), gel was stained for protein with Coomassie blue (sample size was 30 pg of membrane); B (lower), gel was stained for carbohydrate with PAS reagents (sample size was 30 pg of membrane). Numerals give migration position of polypeptides designated in Fig. 1.

polypeptides relative to that of IX and X in Figs. 2A and 3A.

Gel Filtration Chromatography - The polypeptide composi-

tion of the membrane was also determined by gel filtration

chromatography using 6 M urea, 1% 2-mercaptoethanol as

eluent. This solvent was chosen because: (a) the membrane

remains completely soluble on dilution from 8 to 6 M urea; (b)

urea might cause further dissociation of components in the

absence of sodium dodecyl sulfate, and (c) column fractions

could be analyzed by electrophoresis by the addition of sodium

dodecyl sulfate prior to the electrophoresis. The elution pro-

file, given in Fig. 5, shows the presence of multiple polypep-

tides that range in molecular weight from 20,000 to greater

than 400,000. A substantial amount of material elutes in the

void volume of the column and has a molecular weight greater

than 400,000. The column fractions were subsequently ana-

lyzed by sodium dodecyl sulfate-gel electrophoresis to deter-

mine the identity of the polypeptides in each of the respective

fractions. The results are presented in Fig. 6. The identity and

distribution of polypeptides in the column fractions is consist-

ent with that predicted on the basis of that determined by

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Basement Membrane Polypeptides

B

OOL- ”

2 4 6 8 10

MIGRATION DISTANCE r<rn,

FIG. 3. Spectrophotometric scans of polyacrylamide gels of mem- brane analyzed in sodium dodecyl sulfate with 8 M urea in sample and gels (System 2). A (upper), gel was stained for protein with Coomassie blue (sample size was 30 pg of membrane). B (loluer), gel was stained for carbohydrate with PAS reagents (sample size was 30 pg of membrane). Numerals designate components.

sodium dodecyl sulfate-gel electropnoresis (Fig. 21, i.e. the

elution sequence is polypeptide I ---) XVII. This indicates that

dissociation of reduced membrane polypeptides is the same

with 6 M urea or 1% sodium dodecyl sulfate. The relative

amounts of the various polypeptides were determined by com-

bining the electrophoresis and gel filtration results. The calcu-

lated distribution of polypeptides is shown on the elution

profile in Fig. 5 and the amounts are given in Table I. Their

molecular weight values, as determined by comparison of

their distribution coefficients to that of standard proteins (Fig.

51, are given in Table I.

The amino acid and carbohydrate compositions of column

fractions that consist of certain polypeptides were determined

and the results given in Table II. The results show similarities

and distinct differences with regard to the amino acids charac-

teristic of collagen and the distribution of carbohydrate units.

All fractions contain hydroxylysine and half-cystine in vary-

ing proportions and Fractions D and E are void in hydroxypro-

1 01 02 03 04 05 06 07 08 09 10

Relattve MoblIlly

FIG. 4. Calibration lines for the determination of molecular weights of membrane polypeptide by sodium dodecyl sulfate-poly- acrylamide gel electrophoresis. O-0, calibration line for electro- phoresis in sodium dodecyl sulfate without urea (System 1); O-0, calibration line for electrophoresis with 8 M urea in sample and gel (System 2). The standard proteins and their molecular weights are: A, myosin (212,000); B, P-galactosidase (135,000); C, serum albumin (69,000); D, ovalbumin (43,000); and E, chymotrypsinogen (25,700).

1.. A

Lb

\

‘L. d

\

‘T

-2

Fraction Number

FIG. 5. Standard calibration curve (A) and elution profile (B) of reduced membrane fractionated on Sepharose CL4B. Column size was 2.6 x 85 cm. Elution was performed with 6 M urea, 0.1% /3- mercaptoethanol, 0.1 M PO,, pH 7, at room temperature. A flow rate of 10 ml/h was maintained and fractions of 2.2 ml were collected. Aliquots were taken from each fraction and analyzed for protein by the method of Geiger and Bessman (19) (. ) and absorbance at 280 nm (-), and for polypeptide composition by sodium dodecyl sul- fate-polyacrylamide gel electrophoresis without urea (System 1). The calculated distribution of polypeptides as determined by protein analysis of A (i5,, and sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis(-.-) is shown in B. Numerals give location of compo- nents designated in Fig. 2. The standard proteins and their molecu- lar weights are: a, y chain (285,000); b, p chains (190,000); c, 01 chains (95,000) of calf skin collagen; d, serum albumin (68,000); e, ovalbu- min (43,000); and f, P-mercaptoethanol.

r B

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Basement Membrane Polypeptides 3999

TABLE I

Summary of molecular weight, carbohydrate staining, and relative amount of membrane polypeptides

Sodium dodecyl sulfate electrophoresis Gel filtration chromatography

Polypqi$ *urn- Without urea With 8 M urea (I

I - -

PAS stain” Molecular weight’ PAS staind Molecular

weight’ Molecular weight’ Relative amountu

I II

III

IV V

VI VII

VIII IX

X

XI XII

XIII XIV

+ >400,000 + 400,000

365,000

350,000 + 330,000

320,000 300,000

I + 240,000 235,000

220,000

215,000 135,000

+ 115,000 96,000

r I

” + >320,000

IV

*+ 320,000

+ 245,000 + 230,000

220,000

215,000 178,000

+ 145,000 96,000

I

t

h >450,000 40.3

III

IV

I

R 260,000 19.1

VII VIII

h 150,000 14.6

XI XII 100,000 1.9

XIII 70,000 14.8 XIV \

xv 77,000 80,500 XVI 48,500 65,000 h 35,000 9.2

XVII 22,500 33,000 XVII

o Polypeptides are designated in Figs. 2A and 3A. in Fig. 4. b Polypeptides which stained positive with PAS in Fig. 2B. f Calculated from elution position and calibration line in Fig. 5, B

(- Calculated from relative mobility in Fig. 2A and calibration line and A, respectively.

in Fig. 4. o Calculated from relative peak areas presented in Fig. 5B.

d Polypeptides which stained positive with PAS in Fig. 3B. h Unresolved polypeptides.

( Calculated from relative mobility in Fig. 3A and calibration line

SM A B C n E x

I I I I I I I 60 70 80 90 100 110 120

FRACTION NUMBER

D E

FIG. 6 (left). Analysis of fractions from Sepharose CL-4B column insoluble membrane fractions by sodium dodecyl sulfate-gel electro- (Fig. 5) by sodium dodecyl sulfate-gel electrophoresis. Numerals phoresis. A, reduced whole membrane; B, reduced sodium dodecyl designate column fraction analyzed and letters designate fractions sulfate-insoluble membrane fraction; C and E, reduced sodium dode- that were analyzed for amino acid and carbohydrate compositions. cyl sulfate-soluble membrane fraction; and D, unreduced sodium SM, starting material. dodecyl sulfate-soluble membrane fraction. Numerals identify poly-

FIG. 7 (right). Analysis of sodium dodecyl sulfate-soluble and peptide designated in Figs. 1 and 2.

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4000 Basement Membrane Pol.ypeptides

line. Fraction C, consisting mainly of polypeptides VIII and

IX, is most collagen-like in that it has the largest proportion of

hydroxyproline, hydroxylysine, and glycine. In contrast, Frac-

tion D, consisting of mainly XIII, and E, consisting mainly of

XIV to XVII, are least collagen-like in that they contain the

least amount of glycine and hydroxylysine, are void of hydrox-

yproline, and contain the largest proportion of glutamic acid

and proline. Both the disaccharide and oligosaccharide units of

the membrane (12) are present in all fractions, but in varying

amounts; the relative amount of disaccharide unit to oligosac-

charide unit is largest in Fraction C and lowest in Fraction E.

Mode oflnteraction between Polypeptides - The partial solu-

bility of the membrane, 8% by weight, in sodium dodecyl

sulfate in the absence of a reducing agent clearly represents a

separation of the membrane into two fractions, sodium dodecyl

sulfate-soluble and -insoluble fractions, which are held to-

gether exclusively by noncovalent forces in the native mem-

TABLE II

Amino acid and carboh,ydrate composition of column fractions

component

Amino acid 4-Hydroxy-

proline”

Aspartic acid Threonine

Serine

Glutamic acid Proline

Glycine

Alanine

Half-cystine

Valine

Methionine

Isoleucine

Leucine

Tyrosine

Phenylalanine

Hydroxylysine

Lysine

Histidine

Arginine

Monosaccharide

Fucose

Mannose Gala&se

Glucose’

Disaccharideioligo-

saccharide”

23.8 18.2 43.8

87.9 101.8 63.6

50.8 57.3 32.9

55.9 43.4 42.9

115.4 127.7 111.4

63.5 93.9 98.9

165.9 150.6 256.9

66.7 68.9 73.2

5.4 8.5 6.4 44.6 49.6 32.5

3.0 5.5 2.3

31.6 34.7 27.0

66.3 61.1 65.7

21.0 23.9 18.8

22.2 19.7 19.8

17.6 10.4 18.4

33.5 25.8 17.8

28.5 23.7 14.9

96.2 75.2 52.7

3.4 3.3 1.8

4.6 3.7 2.6

18.2 11.8 16.7

13.3 8.9 15.6

2.911 2.411 ‘31 1.611 0.511’

Column fraction”

A B C D E

reszduellOO0 amino acid rescdues

0 0

61.7 69.0

51.7 43.5

79.3 77.4

174.7 146.5

131.7 122.1

134.2 101.1

59.1 62.5

34.4 35.3

44.5 38.0

4.6 8.7

21.8 23.4

37.6 38.5

28.4 25.7

12.6 19.6

4.6 3.4

16.4 38.0

34.8 46.2

67.6 100.8

2.0 7.1

2.0 7.5

5.0 9.0

3.1 - 11

” Column fractions are those designated in Fig. 6. Their polypep-

tide compositions are: A, >99% I; B, 38% II + III and 62% IV-VII; C,

75% VIII + IX and 25% X-XII; D, 93% XIII and 7% XIV-XVII; E, 12%

XIII and 88% XIV-XVII, as determined from spectrophotometric

scans of gels in Fig. 6.

’ 3-Hydroxyproline is absent in all fractions.

’ Values are corrected for the amount of monosaccharide that

arises from column packing.

” Value is undetermined because value was small in comparison

to the correction value for the amount of glucose arising from column

packing.

” Values were calculated on the basis of 1 glucose residue per

disaccharide unit and 1 mannose residue per oligosaccharide unit

(12). ’ Value calculated assuming glucose content is equal to hydroxyly-

sine content.

brane. The identity of the polypeptides comprising these two

membrane fractions was determined by sodium dodecyl sul-

fate-polyacrylamide gel electrophoresis. As shown in Fig. 7,

the sodium dodecyl sulfate-insoluble fraction consists of only

12 (I to XI, XIII) of the 17 polypeptides found in the whole

membrane (compare Gel A with R); whereas, the soluble

fraction consists of the remaining 5 polypeptides (XII, XIV to

XVII) and is devoid of all those comprising the insoluble

fraction except for a proportion of polypeptides VIII and IX

(compare Gels A and C).

The polypeptides comprising the sodium dodecyl sulfate-

insoluble fraction appear to be held together in part by inter-

chain disulfide bonds because this fraction is only soluble in

sodium dodecyl sulfate on reduction with 2-mercaptoethanol.

In contrast, those in the sodium dodecyl sulfate-soluble frac-

tion are not cross-linked by disulfide bonds because the pat-

terns (Fig:. 7) obtained by electrophoresis of the unreduced

(Gel E) and reduced (Gel D) fractions are very similar.

Thus, polypeptides VIII, IX, XII, and XIV to XVII compris-

ing the sodium dodecyl sulfate-soluble fraction, are held to-

gether with polypeptides I to XI and XIII, comprising the

sodium dodecyl sulfate-insoluble fraction, exclusively through

noncovalent interactions. Certain, if not all, of the polypep-

tides in the sodium dodecyl sulfate-insoluble fraction are held

together by interchain disulfide bonds.

DISCUSSION

The Ascaris intestinal basement membrane is solubilized on

reduction with P-mercaptoethanol in 1% sodium dodecyl sul-

fate and 1% sodium dodecyl sulfate, 8 M urea to the extent of

90% and lOO%, respectively. Determination of its polypeptide

composition and molecular weight by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis, with and without the in-

clusion of 8 M urea in the gel system, showed it to consist of at

least 17 polypeptides ranging in molecular weight from 22,500

to greater than 400,000. Six of these polypeptides contain

carbohydrate as determined by PAS stain. The molecular

weight values determined with and without urea in the sys-

tem agree within 10% for most of the polypeptides, but certain

of those of lower molecular weights (XII, XIII, XVI, and XVII)

differ by 24 to 35%. The reason for these differences is not clear

because standard proteins gave similar calibration lines with

and without urea.

The polypeptide composition and molecular weight were

also determined by gel filtration chromatography using 6 M

urea as eluent. The reduced membrane, being completely

soluble in this solvent, consists of multiple polypeptides rang-

ing in molecular weight from 20,000 to greater than 400,000

with approximately 40% of the material having a molecular

weight in excess of 400,000. A comparison of the distribution of

polypeptides obtained from the column profile to that obtained

by sodium dodecyl sulfate-gel electrophoresis indicates that

urea did not cause further dissociation of components than was

obtained with sodium dodecyl sulfate. This conclusion was also

borne out by the similar electrophoretic patterns obtained for

membrane electrophoresed with and without urea (Figs. 2 and

3). For certain polypeptides, such as VIII, IX, and XIII, the

molecular weight values determined by gel filtration chroma-

tography differ greatly from the values determined by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (Table I).

This suggests anomalous behavior of these polypeptides in

sodium dodecyl sulfate analogous to that observed for a poly-

peptide isolated from glomerular basement membrane (7) and

collagen chains (25).

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Basement Membrane Polypeptides 4001

Chemical analyses of column fractions that contain certain

polypeptides revealed that most, if not all, contain hydroxyly-

sine in varying proportions. Polypeptides, VIII or IX or both,

(M,. = 150,000) are the most collagen-like, being characterized

by t,he presence of the largest content of hydroxyproline, hy-

droxylysine, and glycine. In contrast, polypeptide XIII (Mr =

70,000) and possibly XIV to XVII are least collagen-like, being

characterized by the absence of hydroxyproline and the pres-

ence of the least content of hydroxylysine and glycine and the

largest of glutamic acid and proline. The disaccharide and

oligosaccharide units are constituents of both the collagen-like

(VIII and IX) and less collagen-like polypeptide (XIII), but the

proportion of the disaccharide unit relative to the oligosaccha-

ride unit is greater in the more collagen-like polypeptides.

The more collagen-like polypeptides appear to be linked

with the less collagen-like polypeptide (XIII) and probably to

the high molecular weight components (I to VIII) by disulfide

bonds in the native membrane. This conclusion is based on the

finding that these components, representing 92% of membrane

by weight, are soluble in denaturants, sodium dodecyl sulfate

and urea, only after reduction with P-mercaptoethanol. Cer-

tain of the membrane polypeptides (XII, XIV to XVII), repre-

senting the remainder of the weight, are soluble in sodium

dodecyl sulfate without reduction and are not interlinked with

each other by disulfide bonds; therefore, these interact in the

membrane exclusively through noncovalent forces. A substan-

tial amount of the reduced membrane (40%) exists as very

high molecular weight, (>400,000, components I to III) in both

sodium dodecyl sulfate and urea. These may represent aggre-

gates of the more collagen-like and less collagen-like peptides

that are covalently cross-linked by bonds other than disulfide,

such as the lysyl-derived cross-links present in vertebrate

collagen and basement membranes (11).

The multiplicity of components observed presumably re-

flects the true nature of the membrane in which each of these

polypeptides is a distinct integral membrane component. Al-

ternatively, the multiplicity could be partly due to the associa-

tion of nonmembrane polypeptides that are derived from cellu-

lar constituents during membrane isolation. In this event,

those few polypeptides that are soluble in sodium dodecyl

sulfate or urea without cleavage of disulfide bonds would be

the likely candidates (XII, XIV to XVII). The additional possi-

bility that the multiple polypeptides are generated artifac-

tually during membrane isolation by the action of tissue-

associated proteases is unlikely because the polypeptide com-

position of membrane prepared at O-3” or 27” without specific

enzyme inhibitors is identical to that membrane prepared at

O-3” in the presence of EDTA, iPr,PF, and MalNEt (Fig. 1). A

similar result was obtained with glomerular basement mem-

brane except that a single polypeptide was degraded in the

absence of iPr,PF (9).

The results of this study extend the observation from verte-

brates to invertebrates that basement membranes consist of

multiple polypeptides which have compositions of varying de-

grees of relatedness to collagen (less collagen-like to more

collagen-like) (5, 7, 8). This suggests a conservation of this

structural characteristic during evolutionary development.

Recent studies (7, 8) are consistent with the proposal (5, 26)

that the relatedness reflects the proportion of collagen-like to

non-collagen segments in each polypeptide. The identification

of multiple collagen components of varying sizes from pepsin

digests of vertebrate basement membrane (27-29) further sup-

ports this proposal.

The less collagen-like polypeptides may be related to the

polar extension peptide of procollagen, which has a small

amount of collagen-like sequence and large amounts of glu-

tamic acid (30). It is conceivable that these are derived biosyn-

thetically by cleavage of a large procollagen-like component

and are retained as part of the membrane structure. It is

possible that the function of these more polar polypeptides is to

interact with surface components of cells positioned onto the

basement membrane or serve in the alignment of the more

collagen-like polypeptides, or both.

1.

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REFERENCES

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C H Hung, M Ohno, J W Freytag and B G Hudsoncomponents.

Intestinal basement membrane of Ascaris suum. Analysis of polypeptide

1977, 252:3995-4001.J. Biol. Chem. 

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