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Membrane glycoproteins of the rat small intestine
Biochimica et Biophysk'a Acta, 342 (1974) I11-124 .c Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 36663 M E M B R A N E G [ . Y C O P R O T E I N S OF THE RAT SMALL INTESTINE C H E M I C A L C O M P O S I T I O N OF M E M B R A N E G L Y C O P R O T E I N S
Y()UNG S. KIM and JOSE M. PERI)OMO Gastrointestinal Re.~earch Laboratoo', Veterans Administration Hmpital, San Francisco, Calif 94121 aml D~Tulrtment of Medicine, University (~[ Califi)rnia School of Medicine, San Francisco, Calif. 9443 :U.S.A.)
(Received September 3rd, 1973)
SUMMARY Thc glycoprotein and glycolipid content of brush border and submicrosomal membranes from the mucosa of the rat small intestine was investigated. The microsomal preparations were treated to remove the "non-menTbranous" material before analysis. In all of the membranes nearly 80 '~', of the carbohydrate was associated with the protein fraction, and 20 i~,, with the lipid fraction. The carbohydrate of the protein fraction consisted mainly of mannose, galactose, glucose, fucose, glucosamine and galactosamine. With the exception of ribose which was found only in the rough microsomal membrane all the other sugars were present in similar quantities in all the membranes. The sugars in the lipid portion of the membranes consisted primarily of galactose and glucose. Amino sugars associated with the lipid fraction were found in the brush horde," membrane but were nea,'ly absent in the internal membranes. Sialic acid was low in the protein and lipid fractions of all membranes. When sodium dodecylsulfate gel electrophoresis of the membrane protein fractions was performed, many protein and glycoprotein bands were revealed. Each membrane had a different electrophoretic pattern both with respect to protein and to glycoprotein. The data obtained from this study suggest that internal membranes as well as the brush border membranes of the intestinal cells contain glycoproteins and glycolipids.
I NTRODUCTION Evidence has emerged that the plasma membrane of most mammalian cells has at3 externally exposed surface layer of carbohydrate [I 3]. This carbohydrate is associated with glycoproteins and glycolipids [4-8] and may play diverse biological roles such as cell adhesion, recognition, growth regulation and antigenicity [9-12]. Despite the active interest in cell surface glycoproteins, these substances have been studied in detail in only a limited number of cells and tissues [6+7,13 16]. The intestinal cell surface is covered with a carbohydrate-rich glycocalyx and fi'om autoradiographic studies using labelled precursors this glycocalyx appears to be an integral part of the brush border membrane [17, 18].
112 Althot,gh there is evidence that the cell surfitce has externally exposed carbohydrates involved in cell cell interaction, the internal cellular membranes as well may include glycoprotein and glycolipid as structural components [19 21]. A qualitative analysis of mitochondrial membranes and microsomes has shown that these membranes contain hexosamine and sialic acid suggesting that glycoproteins anti glycolipids are present [13, 14, 16]. Detailed carbohydrate analysis of the mitochondrial membrane, however, is not available and lhe liver microsomes p,eparation contained both membranes and vesicular contents [I 3, 14]. In the present study, using a method employed by Meldolesi et al. [22] fo," the preparation of pancreatic vesicles free of secretory contents, membranes fl'om smooth and rough surfaced microsomes were prepared from the mucosa of the rat small intestine. These, together with the brush border membranes, were examined to determine whether the internal and plasma membranes of the intestinal cells contain glycoproteins and glycolipid,s. MATERIALS AND METHODS
Subcellular./)'actionution Male Sprague-Dawley albino rats ~eighing from 2C0 to 250 g were allowed only water for 16 h before sacritice. Rough and smooth surfaced microsomes were prepared from mucosal scrapings of the small intestine by the method reported previously [23]. The following modifications of that method were found to decrease contamination of microsomal membranes by cytosol. A 3-ml layer of 0.5 M sucrose, 15 mM CsCI, was introduced on top of the 1.2 M sucrose, 15 mM CsCI. In addition, the final step of purification included centrifugation of the smooth surfaced microsomes through 5 ml of 0.5 M sucrose-15 mM CsCI. The resulting rough and smooth surfaced microsorrles were separated from non-membranous microsomal components by a modification of the method of Meldolesi et al. [22]. The microsomes were homogenized in 3 ml of 0.17 M NaCI, mixed with 7 ml of 0.2 M N a H C O 3 ( p H 7.8), and sonicated twice at 80 W output for 7.5 s with a probe terminating in a 3-ram diameter tip (Heat Systems-Ultrasonics Co.). During this treatment the temperature of the samples was maintained below 5 C. The sonicated microsomes were centrifuged at 200000 "< ,q for 140 rain and collected as a pellet. A summary of the methods employed for the further treatment of these microsomes is shown in Fig. 1. Total microsome,s" pr~Tmralion
A method [24] exchlding sucrose was also used to prepare total microsomes from 14',,',,, glycerol homogenates of the intestinal mucosa. These microsomal preparations were treated and analyzed in the same way as the rough and smooth microsomes.
Brush border membrane pr~7~arution Brush border membranes were prepared fi'om mucosal scrapings of the small intestine by the method of Forstner et al. [25]. The purified brush border membranes were obtained after removal of the fibrilar core. These membranes possessed sucrase specific activities 20-fold higher than those of the mucosal homogenates, thus indicating a high degree of purification ofthis membrane by the criteria of Forstner et al. [25].
i13 SMOO~! OR ROUGH SURFACED MICROSOMES
Alkaline
treatment,
sonleatlon and eentrlfugatlon
I
Supernat ant
I
Microsomal membranes
(Discarded) Dialysis, PTA precipitation TeA wash (2 X)
Super]alatant (Discarded)
Membrane
ltplds
I IChloroform_methanol extraction (2 X)
Pellet
Me
rane proteins
Fig. I. Summary of the preparation of membrane fractions. Abbreviations are: PTA. phosphotungstic acid; TCA, trichloroacetic acid. The detailed condition of mild "alkaline treatment" of membrane fractions are described under Materials and Methods.
Electron micr(;scopy The rough and s m o o t h microsomes and t h e i r respective m e m b r a n e s were lixcd as a suspension in 0.5 M sucrose I ,;',] OsO4, for 20 h at 4 C. The fixed suspensions were centrifuged at 2 0 0 0 0 / g for 20 min and the pellets d e h y d r a t e d through graded alcohol and e m b e d d c d in Spurr resin. Thin sections wcrc cut using a Sorvall MT2 u l t r a m i c r o t o m e and these were stained with uranyl acetate and lead citrate [26, 27]. Thc stained sections were examined on a R C A E M U - 4 electron microscope.
Lipid e.vtraction of microsomal membranes M i c r o s o m a l and brush b o r d e r m e m b r a n e s were dialyzed overnight against distilled water. The rnembranes were treated with 5 vol. o f cold I':,] p h o s p h o t u n g s t i c acid in 0.5 M HCI. After 20 rain at 4 C the tubes were centrifuged at 5000 / g for 10 rain. The precipitates were resuspended in 20 vol. o f 10'~', trichloroacetic acid and centrifuged at 5000 :: g for 10 rain. This washing was repeated and the tinal pellets were resuspcnded in 3 ml o f methanol. 6 ml o f c h l o r o f o r m were a d d e d to the suspensions and the samples were incubated at 45 :C for 2 h with occasional shaking. The tubes were centrifuged at 10000 ," g for 20 rain and the extraction p r o c e d u r e repeated. The lipid extracts were pooled, washed with 4 ml o f water, and aliquots taken for analysis. The organic solvent insoluble portion was dried in an oven at 37 'C.
Amino acid analysis The rough, smooth and brush b o r d e r m e m b r a n e proteins were solubilized in 0.1 M N a O H , neutralized with 0.1 M HCI and h y d r o l y z e d under N, in 6 M HCI fol
114 22h at I10 C. The hydrolysatewastiltered through glass wool and the HClevaporated at 50 C under vacuum. The dried hydrolysatc was dissolved in 0.2 M citrate buffer (pH 2.2) and the amino acids quantitated using a Model 120C Beckman amino acid analyzer.
Neutral sugar analysis The membrane proteins were solubilized in 0.1 M NaOH, quickly neutralized with 0.1 M HCI and hydrolyzed in 0.25 M H2SO4 at 100 C for 24 h in the presence of 400 mg of AG-50 XI2 (H- tbrm) 200-400 mesh (Bio-Rad Labs) which was previously boiled in I M HC1. The N.,-dried membrane lipids were hydrolyzed in a similar fashion except for the omission of the alkaline solubilization step. The free neutral sugars were reduced with NaBH4 and converted to alditol acetates by the method of Kim et al. [28] and these derivatives x~.ere analyzed on a Perkin-Elmer gas liquid chromatograph. Inositol was used as an internal standard and a series of reagent blanks were run simultaneously. Hexosamine h rdroh'sis The membrane proteins were solubilized and neutralized as in the neutral sugars analysis, followed by hydrolysis under N2 in 4 M HCI at 100 ~C for 18 h. The N2-dried membrane lipids were not treated with alkali but were hydrolyzed in the same way. The hydrolysates were filtered, taken to dryness at 50 ~C under vacuum, dissolved in 0.2 M citrate buffer (pH 2.2) and analyzed for their glucosamine and galactosamine contents on a U R-30 resin column using a Model 120C Beckman amino acid analyzer. Sialic acid analysis Membrane proteins and lipids were hydrolyzed in 0.05 M H.~SO4 at 85 C for I h. The hydrolysate was neutralized with a saturated aqueous solution of Ba(OH)2 and the precipitate removed by centrifugation. The supernatant fluid was applied to a 0.5 cm \ 5.0 cm column of AG-50 X-12 (H + form) which was connected on top of a column of similar dimensions filled with AG-I X-8 (formate form, Bio-Rad Labs). The wash from the BaSO4 precipitate was also passed through the columns, followed by an additional wash with 5 ml of water. Sialic acid was eluted from the AG-I X-8 column with 5 ml of 1 M formic acid. Formic acid was removed from the samples by evaporation at 50 C under vacuum. The dry residues were analyzed for sialic acid using the method of Aminoff [29]. Chromogens obtained were scanned from 400 to 600 nm in a spectrophotometer to confirm the identity of the sample with that of authentic N-acetylneuraminic acid. Other chemical analyses Uronic acids were analyzed by the method of Dische [30]. Protein analysis was performed by the method of Lowry et al. [31 ] using bovine serum albumin as standard. Phosphorus was determined on N2-dried lipid fractions by the method of Chen et al. [32]. The amount of phospholipid was estimated by multiplying the phosphorus values by 25.
115
Sodium dodecylsulfate-disc electrophoresis of membranes The membrane proteins were homogenized in 0.10 M Na2CO3 using a manual ground glass grinder. To this suspension an 8-fold excess of sodium dodecylsulfate per mg of protein was added and the mixture was homogenized again. Within the next 2 rain the solution was adjusted to 10'~[i /%mercaptoethanol and incubated at 37 ' C against the upper gel buffer [8] containing 0.1 ',!~isodium dodecylsulfate, ().5,~,i dithiothreitol and 2 M urea. Electrophoresis was performed using a modification of the discontinuous sulfate-borate system described by Neville [33]. The resolving gels (10 cm ~ 0.5 cm) were made at an acrylamide concentration of 7.5",i and a N,N'methylene-bis-acrylamide net cross linkage of 0.45 !',;',. The samples were allowed to enter the stackinggel at0.5 mA/gel and then subjected to 1.5 mA/gel until the bromophenol blue marker moved to within I cm of the bottom of the gel. The following protein standards were employed: catalase (Worthington), thyroglobulin (porcine, Sigma Chemicals), bovine serum albumin (Miles Lab.), glyceraldehyde-3-phosphate dehydrogenase (Boehringer Mannheim), t~-galactosidase (E. coli, Worthirlgton), and chymotrypsinogen A (beef pancreas, Mann Research Lab.). Protein was stained for 2 h in 2.5 '!,,',Coomassie blue solution in 45 '~,, methanol, 10'~.,i acetic acid. The gels were destained in 100,,~imethanol, 1011~iacetic acid solution for 24-48 h. For the localization of carbohydrate the gels were washed free of sodium dodecylsulfate by overnight washing in 40'~ii methanol, 7 ',!;i acetic acid, and a second wash for 4 additional h. The gels were stair.'ed with periodate-Schiff reagents by the method described by Glossmann and Neville [8] except that the gels were oxidized for 4 h. The oxidized gels were washed and stained with Fuchsin for 3 4 h and destained by washing the gels in I o Na2S20~. RESUI.TS
Isolation o.["smooth attd rough surfaced microsomal memhranes In a previous study we have developed a procedure for the effective purification of smooth and rough surfaced microsomes from rat small intestinal mucosa [23]. Since the microsomal fractions consist of membranes and non-membranous components the separation of these components was a prerequisite for the study of the chemical composition of microsomal membranes. The electron photomicrographs shown in Fig. 2 and 3 depict the ultrastructural changes of the smooth and rough microsomes following alkaline treatment and sonication. The vesicular inclusions of both microsomal preparations are absent in the treated samples indicating that essentially all of the osmiophilic material had been released from these microsomes. Protein and phospholipid analysis of the membrane and supernatant fractions are shown in Table I. This treatment removed 45'~,~ of the protein from the microsomes but more than 86",~ of the phospholipid was retained.
Amino acid analysis of membrane proteins Amino acid compositions of the protein fractions from rough, smooth and brush border membranes are shown in Table I I. The anaino acid compositions of these membrane protein fractions were very similar. Aspartic acid, glutamic acid, leucine and glycine were the most abundant amino acids, accounting for about 400., of the residues. Sulfur-containing amino acids were very low. None of the membrane
116
',,
,Z!"
. -...
.
.,,
I / •
3".
..
-,
g
t:ig. 2. I(lectron micrographs of smooth stlrfaccd microsonlCSbefore and after alkaline treatment and ~t~nication. The smooth microsonaes ~A) and smooth membranes (B) were fixed in unbuffered 0.5 M sucrose 1% OsO~ t - 21 000f protein fractions contained hydroxyproline. A small a m o u n t of cysteic acid was present in all three membranes. This would suggest that though the hydrolysis was carried out under N_,, some oxidation of cysteine had taken place.
Carbohydrate analysis of membrane proteins Table III shows the c a r b o h y d r a t e composition of the protein fractions from microsomal and brush border membranes. The smooth m e m b r a n e protein fi'action
117
Fig. 3. Electron micrographs of rough surfaced micrnsomes before and after alkaline Irealmcnl and sonication. Rough microsomes (A) and rough rnembranes IB) were fixed as described in l ig. 2 and Materials and Methods (., 21 (X~)). contained more sugar than either the brush border or the rough m e m b r a n e protein fraction. The sugars present in each were fucose, mannose, galactose, glucose, glucosamine, galactosamine and sialic acid. A peak with an elution time similar to xylose was also present in all m e m b r a n e protein fractions. Ribose was found only in the rough m e m b r a n e suggesting the presence of R N A . Deoxyribose was absent in all m e m b r a n e s analyzed, indicating a lack of nuclear c o n t a m i n a t i o n . G l u c o s a m i n e and galactosamine were present in rough, smoolh and brush border m e m b r a n e protein
IIs
IAI}LF I DISTRIBUTION OF P R O I E I N S AND PHOSPHOLIPIDS AFTER ALKALINE TREATMEN'I AND SONICATION OF R O U G t l AND SMOOTH MICROSOMES Rough and smooth nlicrosomcs were treated with an isotonic NaCI NaHCO3 sohltion followed b~ sonication. Tficse treated nlicrosomes v,ere centrifuged and both supernatant and pellet fractiom were analyzed as described in Materials and Methods. Microsomcs
Rough Smooth
Protein (";,)
Phospholipids ('},,)
Soluble
lnsolublc
Soluble
Insolublc
45.7 47.5
54.3 52.5
12.6 13.7
87.4 86.3
f r a c t i o n s in r a t i o s o f 5.7, 4.0 a n d 2.7, r e s p e c t i v e l y . T h e p r o t e i n f r a c t i o n s f r o m all ot the m e m b r a n e s c o n t a i n e d a s m a l l a m o u n t o f s i a l i c acid : u r o n i c a c i d s w e r e n o t d e t e c t e d . T o e s t a b l i s h t h a t the g l u c o s e f o u n d a s s o c i a t e d w i t h the p r o t e i n p o r t i o n o f the m i c r o s o real m e m b r a n e w a s n o t dtte to a c o n t a m i n a t i o n by s t , c r o s e used in t h e i s o l a t i o n p r o c e d u r e , m i c r o s o m e s w e r e p r e p a r e d in glycerol. M e m b r a n e p r o t e i n s p r e p a r e d in this m a n n e r c o n t a i n e d s i m i l a r g l u c o s e to g a l a c t o s e r a t i o s as in the s u c r o s e p r e p a r a t i o n s . TABLE II AMINO ACID ANALYSIS O1" ROUGII, SMOOTH AND BRUSH BORDER MEMBRANE PROTEINS Rough, smooth and brush border membrane proteins were hydrolyzed tinder N2 in 6 M IICI at 110 C for 22 I1. The hydrolysates were filtered through densely packed glass wool and evaporated under vacuum. Dried hydrolysates were dissolved in 0.2 M citrate buffer (pH 2.2) and analyzed on an amino acid analyzer. The values were not corrected for destruction due to hydrolysis. N.D., not detected. Amino acid
Lys His Arg Cys Asp Thr Ser Glu Pro Gly Ala Val Met lie l.eu Tyr Phe |typ
Moles per I00 amino acid residues Rough
Smooth
Brush border
5.3 1.8 5.3 0.8 10.9 6.0 7.8 I 1.0 5.6 9.2 7.0 6.1 Trace 4.4 9.6 3.3 5.1) N.D.
6.0 1.4 4.4 1.2 I I. I 6.5 7.2 9.8 5.0 7.9 7.5 7.2 N.I). 5.1 10.5 3.0 5.4 N.D.
6. I 1.4 5.7 0.7 10.4 5.7 6.9 12.3 5.4 10.9 8.3 5.5 Trace 3.8 9.3 2.8 3.9 N.D.
119
"FABLE II1 CARBOHYI)RATE COMPOSITION MEMBRANE PROTEINS
OF
ROUGH,
SMOOTH,
AND
BRUSH
BORDER
M e m b r a n e p r o t e i n s were a n a l y z e d for n e u t r a l s u g a r s a f t e r t h e i r c o n v e r s i o n to a l d i t o l a c e t a t e s by gas l i q u i d c h r o m a t o g r a p h y as d e s c r i b e d by K i m et al. [28]. H e x o s a m i n e s were q u a n t i t a t e d f r o m their I tCI h y d r o l y s a t e s by using the U R - 3 I ) c o l u m n o f a n a m i n o acid a n a l y z e r . T h e sialic acid c o n t e n t ',,,'as d e t e r m i n e d using the m e t h o d o f A m i n o f f [29]. U r o n i c acid a n a l y s i s ,,,,'as d o n e by the t e c h n i q u e develo p e d by D i s c h e [30]. M o r e d e t a i l s o f the m e t h o d o l o g y are d e s c r i b e d in the text. R e s u l t s s h o ~ n are m e a n s . S.D. o f 6 to 9 p r e p a r a t i o n s . N . D . , not detected. Carbohydrate
{nmoles.mg of membrane protein) Rough
Fucose Ribose Mannose Galactose Glucose Glucosamine Galactosamine Sialic acid Uronic acids
91 : 115 • 128 . 99 -. 190 : 149 26-. 13 . N.D.
26 62 28 31 84 48 9 4
Smooth
Brush border
106 ~. Trace 160 • 165 • 314 154 39 18 N.I).
9O • N.D. 89 • 95 : 153 . 78 29 r 12 • N.D.
45 45 48 103 56 15 7
20 52 29 66 13 10 3
Carhohydrate anal rsi.s of membrane lipids" Carbohydrate compositions of the lipid fractions fi'om rough, smooth and brush border membranes are shown in Table IV. Mannose and fucose were measurable in the lipid fraction, but were present to a lesser degrec than galactose and glucosc, the major sugar constitucnts of the membrane lipids. The brush border membrane lipids also had a high content of galactose and glucose but, in contrast to thc microsomal membranes, glucosamine and galactosamine were measurable. A peak corresponding to xylose was also prcscnt in thc lipid fi'action. T A B L E IV CARBOHYDRATE COMPOSITION M E M B R A N E I.IF'IDS
OF
ROUGH,
SMOOTH,
AND
BRUSH
BORDER
R o u g h , s m o o t h a n d b r u s h b o r d e r i;lenabrane lipids ,*ere h y d r o l y z e d u n d e r the c o n d i t i o n s d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s a n d the a n a l y s i s p e r f o r m e d as in T a b l e 111. R e s u l t s show.n are m e a n s .: S.D. o f 4 to 9 p r e p a r a t i o n s . Traces, - (l.9 nrnoles p e r nag o f p r o t e i n . Carbohydrate
(nmoles;mg of membrane protein) Rough
I:ucose Mannose (ialactosc Glucose Glucosamine Galactosamine Sialic a c i d
12-6 • 105 • 54 ; Trace Trace 4 j
6 5 51 28
0
Smooth
Brush border
9 • 3-. 89 • 79 . Trace Trace 3 .
3 5 38 48 7 5 3
I 1 II 34
I
I 1 14 26 I 0 I
120
.S',;dium clodecyL~ul./alc'-~e/ eleclrol~hore.~i.~ o~ mc'mhram" prolein.~ Figs 4 a n d 5 s h o w the d i s c o n t i n u o u s gel e l e c l r o p h o r e t i c p a t t e r n s o f r o u g h a n d
smooth surti~ced microsomal membranes and the brush border membrane proteins at'ter Coonlassie blue (Fig. 4) and periodate Schifl" staining (Fig. 5). The bands revealed by bolh staining techniques in all three membrane fractions showed rather
distinct patterns. At least thirty protein bands were observed in the rough surfaced microsomes
A
B i
C
A
B
C
m
i i
~
o
m
1 =-"
.• i~¸ 2-=',
..._
~c 1
2 i•:i
•4 " " "
n
4g 1
•
i
6---J
• . i~:i¸
m
6-'-"
Fig. 4. Sodlure dodecylsulfate-disc gel electrophoresis o1 membrane proteins. These membranes were prepared as described in Fig. I except that the phosphotungstic acid precipitation step was omitted. The solubilized membranes of the rough surfaced microsomes (A), tile smooth microsomes (B) and the brush border nlembranes tC) were applied to each gel in the amount of 525, 16~ and 45/~g of protein, respectively, and stained with Coomassie blue stain as described under Materials and Methods. The electrophoretic mobilities of the standard molecular weight markers are shov,n as follows: I, thyroglobulin: 2, /,~-galactosidasc: 3, bovine serum albumin; 4, catalase: 5, glyceraldehyde-3-phosphate dehydrogenase; 6, chymotrypsinogen. Bromophcnol blue dye front is marked with tantalum ~.ire. I:ig. 5. Sodium dodecylsulFate.-disc gel electrophoresis of membrane gl}coproteins. These membranes ~,ere prepared as in Fig. 4. The solubilized membranes of the rough surfaced microsomes (A), the smooth microsomes (13) and the brush bodrer membranes IC) were applied to each gel in the amount of 2.63 rag, g40 Hg and gg5/tg of protein, respectively and stained with pcriodate-SchiFF's reagent as described under Materials and Methods. The electrophoretic mobilities of the standard molecular weight markers are shown as related to Gel A.
121 with the apparent molecular weights varying from 18 000 to about 280 000. There were 5 major protein bands with the apparent molecular weights of 133 000, I 19 000, 110 000. 101 000, 69 000 and 42 000 in the rough surfaced microsomes. Periodic acidSchiff staining of the duplicate gel (Fig. 5) showed 2 major broad bands, 4 narrow bands, one of which moved with the tracking dye. The apparent molecular weights of the broad bands were I10 000 and 40 000. Two narrow bands had the apparent molecular weights of 87 000 and 78 000: one of the narrow bands barely penetrated the running gel and the other migrated with the tracking dye. The protein constitt.ents of the smooth surfaced microsomal membranes as visualized by the Coomassie stain were resolved into 28 protein bands with the apparent molecular weight varying from 25 000 to 290000 (Fig. 4). There were 5 major protein bands with the apparent molecular weights of 200000, 130000. 119 000. 83 000 and 25 000 in this membrane fraction. When a duplicate gel was stained with pcriodate Schiff reagent for the localization of carbohydrate, 4 broad bands with the apparent molecular weight 180 000, 110 000, 72 000 and 50 000 and two narrow bands were seen. In addition, using either staining method, bands were observed on the top of the resolving gel and coincident with the bromophenol blue tracking dye. Gel electrophoresis of brush border membrane proteins yielded 16 Coomassie blue staining protein bands (Fig. 4), the most intensely stained bands having the apparent molecular weights of 110000, 95 000, 85 000, 75 000, 39 000 and 30000. Periodate Schiff staining (Fig. 5) yielded 3 broad bands with the apparent molecular weights of 120 000, 95 000 and 75 000. A narrow band moved with the tracking dye and another band barely penetrated the resolving gel. A considerable amount of periodate-Schiff staining material was observed in the stacking gel. DISCUSSION
Smooth and rough microsomes were prepared from the mucosa of the rat small intestine and treated as others have for the preparation of pancreatic secretory vesicles free of their contents [22]. The result of this approach is that the nonmembranous microsomal material has apparently been released from the intestinal microsomes as evidenced from morphological and chemical analysis. When these membranes and the brush border mcmbranes were examined for their carbohydrate content, all of the sugars commonly associated with glycoproteins were found to be present in the three membrane fractions. The smooth membrane protein fraction contained more carbohydrate than the brush border membrane fraction. Comparison between the carbohydrate contents of the protein fractions of the smooth and rough membranes is difficult since the detection of ribose in the rough membrane suggests the possibility of the presence of RNA which may be ribosomal in origin. Ribosomal contamination of the protein fraction would result in an apparent reduction in the carbohydrate to protein ratio. 801t,,i of the carbohydrate was associated with the protein fraction. In all of the membranes, glucosamine was the predominant amino sugar: galactosamine was also detected, but at lower levels. The high glucosamine to galactosamine ratio in membranes observed here has also been reported elsewhere [34], Mannose, galactose and fucose accounted for most of the neutral hexoses in the protein fraction of the membranes. Mannose, which is not present in mucin, a secretory product of the intestinal mucosa, was a major constituent
122 of the membrane glycoprotems. The fucose content of the internal membrane protein was much higher than that previously reported for mernbrane preparations of other tissues [34, 35]. Glucose, a sugar normally associated with glycogen, glycolipids and collagen, was a major constituent of the proteins of all of the membranes. These membranes had been extensively extracted with chloroform-methanol to remove the lipid, and hydroxyproline, an amino acid abundant in collagen, was not detected by amino acid analysis. Others [13-15] have previously detected glucose in membranes and recently Parodi et al. [36] have investigated a glucose transferring system in liver which involves a membrane protein acceptor. Hudson and Spiro [37, 38] have found that basement membrane proteins contain glucose but, in contrast to the findings rcported here. the basement membrane protein was rich in hydroxyprolinc. In thc present study, the glucose seems to be associated with a protein of the membranes since this sugar is precipitated with phosphotungstic and trichloroacetic acids. However, glucose may also be a part of the glucolipid described by' Parodi el al. [36] which was not extracted with chloroform-nlethanol (2:1, v:v) and was co-precipitated with the glucose-containing protein by trichloroacetic acid. A peak corresponding to xylose was tbund in the gas chromatographic analysis of the membrane proteins. This sugar is apparently not associated with the membranes since it was nearly absent in membranes prepared in glycerol. 20",, of the carbohydrate in the membranes ~as extracted into the lipid phase. The neutral sugars associated with the glycolipids of the three membrane preparations were qualitatively the same but were quantitatively distinct. The brush border fraction had less carbohydrate than the internal membranes. The fueose and galactose contents of the brush border membrane lipid were significantly lower than the iaternal membranes. Since the amount of galactose in lipids is generally higher than glucose, and the presence of fucose suggests a completed carbohydrate structure, the brush bordm lipid may be in a state of partial completion or may have been subject to degradation by enzymes present m the intestinal lumen. There seems to be an unusual relationship hetwcen fucose and hexosamine in these membranes. Fucose in glycolipids is normally associated with hexosaminecontaining lipids [39]. In the present study, however, fucose was highest in the internal membranes, which had only trace amounts of glucosamine and galactosamine. While the brush border contained higher amounts of these amino sugars, the fuco~;e content was very low. These results indicate tha! glycolipids devoid of hexosami~:e but containing fucose exist in the internal membranes but that such lipids may be absent in thc plasma membrane. Sialic acid was found in both lipid and protein flactions from all of the membranes but in relatively small amounts. Although it is possible that siali¢ acid may have been lost during the preparation of the mcmbrane samples, these results suggest that most of the mcmbrane glycoproteins and glycolipids are either Iov+ in or devoid of sialic acid. Sodium dodecylsulfate--polyacrylamide gel etectrophoresis revealed that the membranes consist of a variety of proteins, several of which contain carbohydrate. There was little similarity of the protein from the difl'erent membrane fractions, although the high degree of heterogeneity made comparison ot protein bands difficult. Glycoprotein bands were in general broad and fewer in number than the protein bands in all three membrane fractions cxamined. Furthermore, some of the periodateSchiff staining material remained in the stacking gel, indicating a high moleculat
123 weight of some membrane glycoproteins. On staining with Coomassie blue and periodate Schiff reagents, all three membrane fractions showed a fast moving band which ran coincident with the tracking dye. The same mobility was observed in the absence of the dye, ruling out the possibility that thc dyc was inducing some artifact in the pattern. A similar fraction has been reported by others [40-42] and has been attributed to either lipids or artifacts [43, 44]. From the data presented in this study, it appears that internal membranes such as microsomal membranes as well as brush border membranes conlain glycoproteins of unusual carbohydratc composition. A C K N O W L E I ) G M ENTS
We wish to thank most sincerely Mr Noel Taylor for his great assistance in the preparation of the electron micrographs. We gratefully acknowledge the technical assistance of Mrs Lillian Remer and Mr Frank Fearney. We are also indebted to Dr James S. Whitehead for his assistance in preparing the manuscript. REFERENCI~S 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Bcnnct, II. S. (19631 J. Ilistochcm. Cytochem. 11, 14-23 Martincz-Palonlo, A. (1970) Int. Rev. Cytol. 29, 29-75 Nicolson, G. I.. and Singer, S. J. (1971) Proc. Natl. Acad. Sci. U.S. 68, 942 945 Winzler, R. J. ( 19691 in Red Cell Membrane Structure and Function. (Jamieson, G. A. and Greenwalt, 17. J., eds) pp 157-171, I.ippincott, Philadelphia Hakomori, S. and Strycharz, G. D. (19681 Biochemistry 7, 1279-1286 l_cnard, J. {19701 Biochemistry 9, 1129-1132 Marchcsi, V. T. and Andrc,,,,s, E. P. (1971) Science 174, 1247-1248 Gk)ssman, H. and Neville, Jr, D. (1971) J. Biol. Chem. 246, 6339.6346 Roseman, S. {1970) Chem. Phys. Lipids 5, 270 297 Roth, S., Mc(iuire, E. J. and Roscman, S. I1971) J. Cell Biol. 51, 525 535 Burger, M. M. and Goldberg, A. (19671 Proc. Natl. Acad. Sci. (J.S. 57, 359-366 lnbar, M. and Sachs, L. (1969) Proc. Natl. Aead. Sci. U.S. 63, 1418-1425 l i, Y. T., I.i, S. C. and Shetlar, M. R. (19651 Cancer Rcs. 25, 1225-1231 Yamashina, 1., lzumi, K. and Naka, H. ~1964) J. Biochem. Tokyo 55, 652 658 Pepper, D. S. and Jamieson, G. A. (19691 Biochemistry 8, 3362 3368 Bosmann, H. B., Myers, M. W., Dehond, D,. Ball, R. and Case, K. R. (19721J. Cell Biol. 55, 147 160 Ito, S. (1965) J. Cell Biol. 27, 4752,91 Rambourg, A., Neutra, M. and I_eblond, G. 17. (.1966) Anat. Rcc. 154, 41-72 Stock, T. L. (19721 in Membrane Research (Fox, F. C., ed.), pp. 71--93, Academic Press, New York Marchesi, V. 1., Segrest, J. P. and Kahane, I. (19721 in Membrane Research (Fo×, F. C., ed.), pp. 41-51, Academic Press, New York Nicolson, G. L. (19721 in Membrane Research (Fox, F. C,. ed.), pp. 53 70, Academic Press, Ne~, York Meldolesi, J., Jamieson, J. D. and Palade, G. (1971) J. Cell Biol. 49, 109-129 Kim, Y. S., Perdomo, J. and Nordberg, J. (19711 J. Biol. Chem. 246, 5466 5476 Kim, Y. S., Birtwhistle, W. and Kim, Y. W. (1972) J. Clin. Invest. 51, 1419 1430 Forstner, G. G., Sabesin, S. M. and Isselbacher, K. J. (19681 Biochem. J. 106, 381 390 Watson, M. I . (1958)J. Biophys. Biochem. Cytol. 4, 4752,77 Re~,nolds, D. S. {1963) J. Ceil Biol. 17, 208 212 Kim, J. H.. Shome, B., Liao, T-H. and Pierce, J. (i. (1967) Anal. Biochem. 2(I, 258 274 Aminolf, I). (19611 Biochem. J. 81, 384-392 I)ische, Z. (19471J. Biol. Chem. 167, 189-198
124 31 Lowry, O. H., R o s e n b r o u g h , N. J.. Farr, A. L. and Randall, R. J. (1951) J. I'liol. ( ' h e m . 193,265.275 32 Chen, P. S., Taribara, T. Y. a n d Wagner, H. (1956) Anal. Chenl. 28, 1756 1758 33 Neville, Jr, 1). M. (1971) J. Biol. C h e m . 246, 6328 6334 34 Miyajima. N., Tomikav, a, M., Kawasaki, T. and Y a m a s h i n a , I. (1969) J. Biochem. T o k y o 66, 711 732 35 Itelgeland, L., Christensen, T. B. and Janson, 1. L. 11972) Biochim. Biophys. Acta 286, 62-71 36 Parodi, A. G., Behrens, N. H., Leloir, I.. F. and Carminatti, It. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3268 3272 37 Iludson, B. G. and Spiro, R. G. (1972) J. Biol. C h e m . 247, 4229-4238 38 H u d s o n , B. G. and Spiro. R. (L (1972) J. Biol. C h e m . 247, 4239 4247 39 Hakomori, S. 11971) in T h e D y n a m i c Structure of (_'ell M e m b r a n e s (llolzl Wallach, 1). F. and Fisher, H., eds), pp. 65 96, Springer, New York 40 Lenaz. G.. Itaard, N. I-.., Silnlan, H. I. and Green, D. I(. (1968) Arch. l~iochem. Biophys. 128. 293 -303 41 Laico, M. T., Ruoslahti, 15. 1., Papcrmaster, 1). S. and Drcycr, W. ,1. (1970) Proc. Natl. Acad. Sci. U.S. 67, 120.[27 42 Yu, B. P. and Masoro, E. J. (1970) 13iochcnfist)'y 9, 2909 2917 43 Guidotti, (i. (1972) in A n n u . Re',. Biochem. (Snell, I-., ed.) 41, 731-752 44 Dreyer, %'. J., Papcr)'nastcr, D. S. and K u h n , II. (1972) Ann. N.Y. Acad. Sci. 195, 61 74
BBA 36663 M E M B R A N E G [ . Y C O P R O T E I N S OF THE RAT SMALL INTESTINE C H E M I C A L C O M P O S I T I O N OF M E M B R A N E G L Y C O P R O T E I N S
Y()UNG S. KIM and JOSE M. PERI)OMO Gastrointestinal Re.~earch Laboratoo', Veterans Administration Hmpital, San Francisco, Calif 94121 aml D~Tulrtment of Medicine, University (~[ Califi)rnia School of Medicine, San Francisco, Calif. 9443 :U.S.A.)
(Received September 3rd, 1973)
SUMMARY Thc glycoprotein and glycolipid content of brush border and submicrosomal membranes from the mucosa of the rat small intestine was investigated. The microsomal preparations were treated to remove the "non-menTbranous" material before analysis. In all of the membranes nearly 80 '~', of the carbohydrate was associated with the protein fraction, and 20 i~,, with the lipid fraction. The carbohydrate of the protein fraction consisted mainly of mannose, galactose, glucose, fucose, glucosamine and galactosamine. With the exception of ribose which was found only in the rough microsomal membrane all the other sugars were present in similar quantities in all the membranes. The sugars in the lipid portion of the membranes consisted primarily of galactose and glucose. Amino sugars associated with the lipid fraction were found in the brush horde," membrane but were nea,'ly absent in the internal membranes. Sialic acid was low in the protein and lipid fractions of all membranes. When sodium dodecylsulfate gel electrophoresis of the membrane protein fractions was performed, many protein and glycoprotein bands were revealed. Each membrane had a different electrophoretic pattern both with respect to protein and to glycoprotein. The data obtained from this study suggest that internal membranes as well as the brush border membranes of the intestinal cells contain glycoproteins and glycolipids.
I NTRODUCTION Evidence has emerged that the plasma membrane of most mammalian cells has at3 externally exposed surface layer of carbohydrate [I 3]. This carbohydrate is associated with glycoproteins and glycolipids [4-8] and may play diverse biological roles such as cell adhesion, recognition, growth regulation and antigenicity [9-12]. Despite the active interest in cell surface glycoproteins, these substances have been studied in detail in only a limited number of cells and tissues [6+7,13 16]. The intestinal cell surface is covered with a carbohydrate-rich glycocalyx and fi'om autoradiographic studies using labelled precursors this glycocalyx appears to be an integral part of the brush border membrane [17, 18].
112 Althot,gh there is evidence that the cell surfitce has externally exposed carbohydrates involved in cell cell interaction, the internal cellular membranes as well may include glycoprotein and glycolipid as structural components [19 21]. A qualitative analysis of mitochondrial membranes and microsomes has shown that these membranes contain hexosamine and sialic acid suggesting that glycoproteins anti glycolipids are present [13, 14, 16]. Detailed carbohydrate analysis of the mitochondrial membrane, however, is not available and lhe liver microsomes p,eparation contained both membranes and vesicular contents [I 3, 14]. In the present study, using a method employed by Meldolesi et al. [22] fo," the preparation of pancreatic vesicles free of secretory contents, membranes fl'om smooth and rough surfaced microsomes were prepared from the mucosa of the rat small intestine. These, together with the brush border membranes, were examined to determine whether the internal and plasma membranes of the intestinal cells contain glycoproteins and glycolipid,s. MATERIALS AND METHODS
Subcellular./)'actionution Male Sprague-Dawley albino rats ~eighing from 2C0 to 250 g were allowed only water for 16 h before sacritice. Rough and smooth surfaced microsomes were prepared from mucosal scrapings of the small intestine by the method reported previously [23]. The following modifications of that method were found to decrease contamination of microsomal membranes by cytosol. A 3-ml layer of 0.5 M sucrose, 15 mM CsCI, was introduced on top of the 1.2 M sucrose, 15 mM CsCI. In addition, the final step of purification included centrifugation of the smooth surfaced microsomes through 5 ml of 0.5 M sucrose-15 mM CsCI. The resulting rough and smooth surfaced microsorrles were separated from non-membranous microsomal components by a modification of the method of Meldolesi et al. [22]. The microsomes were homogenized in 3 ml of 0.17 M NaCI, mixed with 7 ml of 0.2 M N a H C O 3 ( p H 7.8), and sonicated twice at 80 W output for 7.5 s with a probe terminating in a 3-ram diameter tip (Heat Systems-Ultrasonics Co.). During this treatment the temperature of the samples was maintained below 5 C. The sonicated microsomes were centrifuged at 200000 "< ,q for 140 rain and collected as a pellet. A summary of the methods employed for the further treatment of these microsomes is shown in Fig. 1. Total microsome,s" pr~Tmralion
A method [24] exchlding sucrose was also used to prepare total microsomes from 14',,',,, glycerol homogenates of the intestinal mucosa. These microsomal preparations were treated and analyzed in the same way as the rough and smooth microsomes.
Brush border membrane pr~7~arution Brush border membranes were prepared fi'om mucosal scrapings of the small intestine by the method of Forstner et al. [25]. The purified brush border membranes were obtained after removal of the fibrilar core. These membranes possessed sucrase specific activities 20-fold higher than those of the mucosal homogenates, thus indicating a high degree of purification ofthis membrane by the criteria of Forstner et al. [25].
i13 SMOO~! OR ROUGH SURFACED MICROSOMES
Alkaline
treatment,
sonleatlon and eentrlfugatlon
I
Supernat ant
I
Microsomal membranes
(Discarded) Dialysis, PTA precipitation TeA wash (2 X)
Super]alatant (Discarded)
Membrane
ltplds
I IChloroform_methanol extraction (2 X)
Pellet
Me
rane proteins
Fig. I. Summary of the preparation of membrane fractions. Abbreviations are: PTA. phosphotungstic acid; TCA, trichloroacetic acid. The detailed condition of mild "alkaline treatment" of membrane fractions are described under Materials and Methods.
Electron micr(;scopy The rough and s m o o t h microsomes and t h e i r respective m e m b r a n e s were lixcd as a suspension in 0.5 M sucrose I ,;',] OsO4, for 20 h at 4 C. The fixed suspensions were centrifuged at 2 0 0 0 0 / g for 20 min and the pellets d e h y d r a t e d through graded alcohol and e m b e d d c d in Spurr resin. Thin sections wcrc cut using a Sorvall MT2 u l t r a m i c r o t o m e and these were stained with uranyl acetate and lead citrate [26, 27]. Thc stained sections were examined on a R C A E M U - 4 electron microscope.
Lipid e.vtraction of microsomal membranes M i c r o s o m a l and brush b o r d e r m e m b r a n e s were dialyzed overnight against distilled water. The rnembranes were treated with 5 vol. o f cold I':,] p h o s p h o t u n g s t i c acid in 0.5 M HCI. After 20 rain at 4 C the tubes were centrifuged at 5000 / g for 10 rain. The precipitates were resuspended in 20 vol. o f 10'~', trichloroacetic acid and centrifuged at 5000 :: g for 10 rain. This washing was repeated and the tinal pellets were resuspcnded in 3 ml o f methanol. 6 ml o f c h l o r o f o r m were a d d e d to the suspensions and the samples were incubated at 45 :C for 2 h with occasional shaking. The tubes were centrifuged at 10000 ," g for 20 rain and the extraction p r o c e d u r e repeated. The lipid extracts were pooled, washed with 4 ml o f water, and aliquots taken for analysis. The organic solvent insoluble portion was dried in an oven at 37 'C.
Amino acid analysis The rough, smooth and brush b o r d e r m e m b r a n e proteins were solubilized in 0.1 M N a O H , neutralized with 0.1 M HCI and h y d r o l y z e d under N, in 6 M HCI fol
114 22h at I10 C. The hydrolysatewastiltered through glass wool and the HClevaporated at 50 C under vacuum. The dried hydrolysatc was dissolved in 0.2 M citrate buffer (pH 2.2) and the amino acids quantitated using a Model 120C Beckman amino acid analyzer.
Neutral sugar analysis The membrane proteins were solubilized in 0.1 M NaOH, quickly neutralized with 0.1 M HCI and hydrolyzed in 0.25 M H2SO4 at 100 C for 24 h in the presence of 400 mg of AG-50 XI2 (H- tbrm) 200-400 mesh (Bio-Rad Labs) which was previously boiled in I M HC1. The N.,-dried membrane lipids were hydrolyzed in a similar fashion except for the omission of the alkaline solubilization step. The free neutral sugars were reduced with NaBH4 and converted to alditol acetates by the method of Kim et al. [28] and these derivatives x~.ere analyzed on a Perkin-Elmer gas liquid chromatograph. Inositol was used as an internal standard and a series of reagent blanks were run simultaneously. Hexosamine h rdroh'sis The membrane proteins were solubilized and neutralized as in the neutral sugars analysis, followed by hydrolysis under N2 in 4 M HCI at 100 ~C for 18 h. The N2-dried membrane lipids were not treated with alkali but were hydrolyzed in the same way. The hydrolysates were filtered, taken to dryness at 50 ~C under vacuum, dissolved in 0.2 M citrate buffer (pH 2.2) and analyzed for their glucosamine and galactosamine contents on a U R-30 resin column using a Model 120C Beckman amino acid analyzer. Sialic acid analysis Membrane proteins and lipids were hydrolyzed in 0.05 M H.~SO4 at 85 C for I h. The hydrolysate was neutralized with a saturated aqueous solution of Ba(OH)2 and the precipitate removed by centrifugation. The supernatant fluid was applied to a 0.5 cm \ 5.0 cm column of AG-50 X-12 (H + form) which was connected on top of a column of similar dimensions filled with AG-I X-8 (formate form, Bio-Rad Labs). The wash from the BaSO4 precipitate was also passed through the columns, followed by an additional wash with 5 ml of water. Sialic acid was eluted from the AG-I X-8 column with 5 ml of 1 M formic acid. Formic acid was removed from the samples by evaporation at 50 C under vacuum. The dry residues were analyzed for sialic acid using the method of Aminoff [29]. Chromogens obtained were scanned from 400 to 600 nm in a spectrophotometer to confirm the identity of the sample with that of authentic N-acetylneuraminic acid. Other chemical analyses Uronic acids were analyzed by the method of Dische [30]. Protein analysis was performed by the method of Lowry et al. [31 ] using bovine serum albumin as standard. Phosphorus was determined on N2-dried lipid fractions by the method of Chen et al. [32]. The amount of phospholipid was estimated by multiplying the phosphorus values by 25.
115
Sodium dodecylsulfate-disc electrophoresis of membranes The membrane proteins were homogenized in 0.10 M Na2CO3 using a manual ground glass grinder. To this suspension an 8-fold excess of sodium dodecylsulfate per mg of protein was added and the mixture was homogenized again. Within the next 2 rain the solution was adjusted to 10'~[i /%mercaptoethanol and incubated at 37 ' C against the upper gel buffer [8] containing 0.1 ',!~isodium dodecylsulfate, ().5,~,i dithiothreitol and 2 M urea. Electrophoresis was performed using a modification of the discontinuous sulfate-borate system described by Neville [33]. The resolving gels (10 cm ~ 0.5 cm) were made at an acrylamide concentration of 7.5",i and a N,N'methylene-bis-acrylamide net cross linkage of 0.45 !',;',. The samples were allowed to enter the stackinggel at0.5 mA/gel and then subjected to 1.5 mA/gel until the bromophenol blue marker moved to within I cm of the bottom of the gel. The following protein standards were employed: catalase (Worthington), thyroglobulin (porcine, Sigma Chemicals), bovine serum albumin (Miles Lab.), glyceraldehyde-3-phosphate dehydrogenase (Boehringer Mannheim), t~-galactosidase (E. coli, Worthirlgton), and chymotrypsinogen A (beef pancreas, Mann Research Lab.). Protein was stained for 2 h in 2.5 '!,,',Coomassie blue solution in 45 '~,, methanol, 10'~.,i acetic acid. The gels were destained in 100,,~imethanol, 1011~iacetic acid solution for 24-48 h. For the localization of carbohydrate the gels were washed free of sodium dodecylsulfate by overnight washing in 40'~ii methanol, 7 ',!;i acetic acid, and a second wash for 4 additional h. The gels were stair.'ed with periodate-Schiff reagents by the method described by Glossmann and Neville [8] except that the gels were oxidized for 4 h. The oxidized gels were washed and stained with Fuchsin for 3 4 h and destained by washing the gels in I o Na2S20~. RESUI.TS
Isolation o.["smooth attd rough surfaced microsomal memhranes In a previous study we have developed a procedure for the effective purification of smooth and rough surfaced microsomes from rat small intestinal mucosa [23]. Since the microsomal fractions consist of membranes and non-membranous components the separation of these components was a prerequisite for the study of the chemical composition of microsomal membranes. The electron photomicrographs shown in Fig. 2 and 3 depict the ultrastructural changes of the smooth and rough microsomes following alkaline treatment and sonication. The vesicular inclusions of both microsomal preparations are absent in the treated samples indicating that essentially all of the osmiophilic material had been released from these microsomes. Protein and phospholipid analysis of the membrane and supernatant fractions are shown in Table I. This treatment removed 45'~,~ of the protein from the microsomes but more than 86",~ of the phospholipid was retained.
Amino acid analysis of membrane proteins Amino acid compositions of the protein fractions from rough, smooth and brush border membranes are shown in Table I I. The anaino acid compositions of these membrane protein fractions were very similar. Aspartic acid, glutamic acid, leucine and glycine were the most abundant amino acids, accounting for about 400., of the residues. Sulfur-containing amino acids were very low. None of the membrane
116
',,
,Z!"
. -...
.
.,,
I / •
3".
..
-,
g
t:ig. 2. I(lectron micrographs of smooth stlrfaccd microsonlCSbefore and after alkaline treatment and ~t~nication. The smooth microsonaes ~A) and smooth membranes (B) were fixed in unbuffered 0.5 M sucrose 1% OsO~ t - 21 000f protein fractions contained hydroxyproline. A small a m o u n t of cysteic acid was present in all three membranes. This would suggest that though the hydrolysis was carried out under N_,, some oxidation of cysteine had taken place.
Carbohydrate analysis of membrane proteins Table III shows the c a r b o h y d r a t e composition of the protein fractions from microsomal and brush border membranes. The smooth m e m b r a n e protein fi'action
117
Fig. 3. Electron micrographs of rough surfaced micrnsomes before and after alkaline Irealmcnl and sonication. Rough microsomes (A) and rough rnembranes IB) were fixed as described in l ig. 2 and Materials and Methods (., 21 (X~)). contained more sugar than either the brush border or the rough m e m b r a n e protein fraction. The sugars present in each were fucose, mannose, galactose, glucose, glucosamine, galactosamine and sialic acid. A peak with an elution time similar to xylose was also present in all m e m b r a n e protein fractions. Ribose was found only in the rough m e m b r a n e suggesting the presence of R N A . Deoxyribose was absent in all m e m b r a n e s analyzed, indicating a lack of nuclear c o n t a m i n a t i o n . G l u c o s a m i n e and galactosamine were present in rough, smoolh and brush border m e m b r a n e protein
IIs
IAI}LF I DISTRIBUTION OF P R O I E I N S AND PHOSPHOLIPIDS AFTER ALKALINE TREATMEN'I AND SONICATION OF R O U G t l AND SMOOTH MICROSOMES Rough and smooth nlicrosomcs were treated with an isotonic NaCI NaHCO3 sohltion followed b~ sonication. Tficse treated nlicrosomes v,ere centrifuged and both supernatant and pellet fractiom were analyzed as described in Materials and Methods. Microsomcs
Rough Smooth
Protein (";,)
Phospholipids ('},,)
Soluble
lnsolublc
Soluble
Insolublc
45.7 47.5
54.3 52.5
12.6 13.7
87.4 86.3
f r a c t i o n s in r a t i o s o f 5.7, 4.0 a n d 2.7, r e s p e c t i v e l y . T h e p r o t e i n f r a c t i o n s f r o m all ot the m e m b r a n e s c o n t a i n e d a s m a l l a m o u n t o f s i a l i c acid : u r o n i c a c i d s w e r e n o t d e t e c t e d . T o e s t a b l i s h t h a t the g l u c o s e f o u n d a s s o c i a t e d w i t h the p r o t e i n p o r t i o n o f the m i c r o s o real m e m b r a n e w a s n o t dtte to a c o n t a m i n a t i o n by s t , c r o s e used in t h e i s o l a t i o n p r o c e d u r e , m i c r o s o m e s w e r e p r e p a r e d in glycerol. M e m b r a n e p r o t e i n s p r e p a r e d in this m a n n e r c o n t a i n e d s i m i l a r g l u c o s e to g a l a c t o s e r a t i o s as in the s u c r o s e p r e p a r a t i o n s . TABLE II AMINO ACID ANALYSIS O1" ROUGII, SMOOTH AND BRUSH BORDER MEMBRANE PROTEINS Rough, smooth and brush border membrane proteins were hydrolyzed tinder N2 in 6 M IICI at 110 C for 22 I1. The hydrolysates were filtered through densely packed glass wool and evaporated under vacuum. Dried hydrolysates were dissolved in 0.2 M citrate buffer (pH 2.2) and analyzed on an amino acid analyzer. The values were not corrected for destruction due to hydrolysis. N.D., not detected. Amino acid
Lys His Arg Cys Asp Thr Ser Glu Pro Gly Ala Val Met lie l.eu Tyr Phe |typ
Moles per I00 amino acid residues Rough
Smooth
Brush border
5.3 1.8 5.3 0.8 10.9 6.0 7.8 I 1.0 5.6 9.2 7.0 6.1 Trace 4.4 9.6 3.3 5.1) N.D.
6.0 1.4 4.4 1.2 I I. I 6.5 7.2 9.8 5.0 7.9 7.5 7.2 N.I). 5.1 10.5 3.0 5.4 N.D.
6. I 1.4 5.7 0.7 10.4 5.7 6.9 12.3 5.4 10.9 8.3 5.5 Trace 3.8 9.3 2.8 3.9 N.D.
119
"FABLE II1 CARBOHYI)RATE COMPOSITION MEMBRANE PROTEINS
OF
ROUGH,
SMOOTH,
AND
BRUSH
BORDER
M e m b r a n e p r o t e i n s were a n a l y z e d for n e u t r a l s u g a r s a f t e r t h e i r c o n v e r s i o n to a l d i t o l a c e t a t e s by gas l i q u i d c h r o m a t o g r a p h y as d e s c r i b e d by K i m et al. [28]. H e x o s a m i n e s were q u a n t i t a t e d f r o m their I tCI h y d r o l y s a t e s by using the U R - 3 I ) c o l u m n o f a n a m i n o acid a n a l y z e r . T h e sialic acid c o n t e n t ',,,'as d e t e r m i n e d using the m e t h o d o f A m i n o f f [29]. U r o n i c acid a n a l y s i s ,,,,'as d o n e by the t e c h n i q u e develo p e d by D i s c h e [30]. M o r e d e t a i l s o f the m e t h o d o l o g y are d e s c r i b e d in the text. R e s u l t s s h o ~ n are m e a n s . S.D. o f 6 to 9 p r e p a r a t i o n s . N . D . , not detected. Carbohydrate
{nmoles.mg of membrane protein) Rough
Fucose Ribose Mannose Galactose Glucose Glucosamine Galactosamine Sialic acid Uronic acids
91 : 115 • 128 . 99 -. 190 : 149 26-. 13 . N.D.
26 62 28 31 84 48 9 4
Smooth
Brush border
106 ~. Trace 160 • 165 • 314 154 39 18 N.I).
9O • N.D. 89 • 95 : 153 . 78 29 r 12 • N.D.
45 45 48 103 56 15 7
20 52 29 66 13 10 3
Carhohydrate anal rsi.s of membrane lipids" Carbohydrate compositions of the lipid fractions fi'om rough, smooth and brush border membranes are shown in Table IV. Mannose and fucose were measurable in the lipid fraction, but were present to a lesser degrec than galactose and glucosc, the major sugar constitucnts of the membrane lipids. The brush border membrane lipids also had a high content of galactose and glucose but, in contrast to thc microsomal membranes, glucosamine and galactosamine were measurable. A peak corresponding to xylose was also prcscnt in thc lipid fi'action. T A B L E IV CARBOHYDRATE COMPOSITION M E M B R A N E I.IF'IDS
OF
ROUGH,
SMOOTH,
AND
BRUSH
BORDER
R o u g h , s m o o t h a n d b r u s h b o r d e r i;lenabrane lipids ,*ere h y d r o l y z e d u n d e r the c o n d i t i o n s d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s a n d the a n a l y s i s p e r f o r m e d as in T a b l e 111. R e s u l t s show.n are m e a n s .: S.D. o f 4 to 9 p r e p a r a t i o n s . Traces, - (l.9 nrnoles p e r nag o f p r o t e i n . Carbohydrate
(nmoles;mg of membrane protein) Rough
I:ucose Mannose (ialactosc Glucose Glucosamine Galactosamine Sialic a c i d
12-6 • 105 • 54 ; Trace Trace 4 j
6 5 51 28
0
Smooth
Brush border
9 • 3-. 89 • 79 . Trace Trace 3 .
3 5 38 48 7 5 3
I 1 II 34
I
I 1 14 26 I 0 I
120
.S',;dium clodecyL~ul./alc'-~e/ eleclrol~hore.~i.~ o~ mc'mhram" prolein.~ Figs 4 a n d 5 s h o w the d i s c o n t i n u o u s gel e l e c l r o p h o r e t i c p a t t e r n s o f r o u g h a n d
smooth surti~ced microsomal membranes and the brush border membrane proteins at'ter Coonlassie blue (Fig. 4) and periodate Schifl" staining (Fig. 5). The bands revealed by bolh staining techniques in all three membrane fractions showed rather
distinct patterns. At least thirty protein bands were observed in the rough surfaced microsomes
A
B i
C
A
B
C
m
i i
~
o
m
1 =-"
.• i~¸ 2-=',
..._
~c 1
2 i•:i
•4 " " "
n
4g 1
•
i
6---J
• . i~:i¸
m
6-'-"
Fig. 4. Sodlure dodecylsulfate-disc gel electrophoresis o1 membrane proteins. These membranes were prepared as described in Fig. I except that the phosphotungstic acid precipitation step was omitted. The solubilized membranes of the rough surfaced microsomes (A), tile smooth microsomes (B) and the brush border nlembranes tC) were applied to each gel in the amount of 525, 16~ and 45/~g of protein, respectively, and stained with Coomassie blue stain as described under Materials and Methods. The electrophoretic mobilities of the standard molecular weight markers are shov,n as follows: I, thyroglobulin: 2, /,~-galactosidasc: 3, bovine serum albumin; 4, catalase: 5, glyceraldehyde-3-phosphate dehydrogenase; 6, chymotrypsinogen. Bromophcnol blue dye front is marked with tantalum ~.ire. I:ig. 5. Sodium dodecylsulFate.-disc gel electrophoresis of membrane gl}coproteins. These membranes ~,ere prepared as in Fig. 4. The solubilized membranes of the rough surfaced microsomes (A), the smooth microsomes (13) and the brush bodrer membranes IC) were applied to each gel in the amount of 2.63 rag, g40 Hg and gg5/tg of protein, respectively and stained with pcriodate-SchiFF's reagent as described under Materials and Methods. The electrophoretic mobilities of the standard molecular weight markers are shown as related to Gel A.
121 with the apparent molecular weights varying from 18 000 to about 280 000. There were 5 major protein bands with the apparent molecular weights of 133 000, I 19 000, 110 000. 101 000, 69 000 and 42 000 in the rough surfaced microsomes. Periodic acidSchiff staining of the duplicate gel (Fig. 5) showed 2 major broad bands, 4 narrow bands, one of which moved with the tracking dye. The apparent molecular weights of the broad bands were I10 000 and 40 000. Two narrow bands had the apparent molecular weights of 87 000 and 78 000: one of the narrow bands barely penetrated the running gel and the other migrated with the tracking dye. The protein constitt.ents of the smooth surfaced microsomal membranes as visualized by the Coomassie stain were resolved into 28 protein bands with the apparent molecular weight varying from 25 000 to 290000 (Fig. 4). There were 5 major protein bands with the apparent molecular weights of 200000, 130000. 119 000. 83 000 and 25 000 in this membrane fraction. When a duplicate gel was stained with pcriodate Schiff reagent for the localization of carbohydrate, 4 broad bands with the apparent molecular weight 180 000, 110 000, 72 000 and 50 000 and two narrow bands were seen. In addition, using either staining method, bands were observed on the top of the resolving gel and coincident with the bromophenol blue tracking dye. Gel electrophoresis of brush border membrane proteins yielded 16 Coomassie blue staining protein bands (Fig. 4), the most intensely stained bands having the apparent molecular weights of 110000, 95 000, 85 000, 75 000, 39 000 and 30000. Periodate Schiff staining (Fig. 5) yielded 3 broad bands with the apparent molecular weights of 120 000, 95 000 and 75 000. A narrow band moved with the tracking dye and another band barely penetrated the resolving gel. A considerable amount of periodate-Schiff staining material was observed in the stacking gel. DISCUSSION
Smooth and rough microsomes were prepared from the mucosa of the rat small intestine and treated as others have for the preparation of pancreatic secretory vesicles free of their contents [22]. The result of this approach is that the nonmembranous microsomal material has apparently been released from the intestinal microsomes as evidenced from morphological and chemical analysis. When these membranes and the brush border mcmbranes were examined for their carbohydrate content, all of the sugars commonly associated with glycoproteins were found to be present in the three membrane fractions. The smooth membrane protein fraction contained more carbohydrate than the brush border membrane fraction. Comparison between the carbohydrate contents of the protein fractions of the smooth and rough membranes is difficult since the detection of ribose in the rough membrane suggests the possibility of the presence of RNA which may be ribosomal in origin. Ribosomal contamination of the protein fraction would result in an apparent reduction in the carbohydrate to protein ratio. 801t,,i of the carbohydrate was associated with the protein fraction. In all of the membranes, glucosamine was the predominant amino sugar: galactosamine was also detected, but at lower levels. The high glucosamine to galactosamine ratio in membranes observed here has also been reported elsewhere [34], Mannose, galactose and fucose accounted for most of the neutral hexoses in the protein fraction of the membranes. Mannose, which is not present in mucin, a secretory product of the intestinal mucosa, was a major constituent
122 of the membrane glycoprotems. The fucose content of the internal membrane protein was much higher than that previously reported for mernbrane preparations of other tissues [34, 35]. Glucose, a sugar normally associated with glycogen, glycolipids and collagen, was a major constituent of the proteins of all of the membranes. These membranes had been extensively extracted with chloroform-methanol to remove the lipid, and hydroxyproline, an amino acid abundant in collagen, was not detected by amino acid analysis. Others [13-15] have previously detected glucose in membranes and recently Parodi et al. [36] have investigated a glucose transferring system in liver which involves a membrane protein acceptor. Hudson and Spiro [37, 38] have found that basement membrane proteins contain glucose but, in contrast to the findings rcported here. the basement membrane protein was rich in hydroxyprolinc. In thc present study, the glucose seems to be associated with a protein of the membranes since this sugar is precipitated with phosphotungstic and trichloroacetic acids. However, glucose may also be a part of the glucolipid described by' Parodi el al. [36] which was not extracted with chloroform-nlethanol (2:1, v:v) and was co-precipitated with the glucose-containing protein by trichloroacetic acid. A peak corresponding to xylose was tbund in the gas chromatographic analysis of the membrane proteins. This sugar is apparently not associated with the membranes since it was nearly absent in membranes prepared in glycerol. 20",, of the carbohydrate in the membranes ~as extracted into the lipid phase. The neutral sugars associated with the glycolipids of the three membrane preparations were qualitatively the same but were quantitatively distinct. The brush border fraction had less carbohydrate than the internal membranes. The fueose and galactose contents of the brush border membrane lipid were significantly lower than the iaternal membranes. Since the amount of galactose in lipids is generally higher than glucose, and the presence of fucose suggests a completed carbohydrate structure, the brush bordm lipid may be in a state of partial completion or may have been subject to degradation by enzymes present m the intestinal lumen. There seems to be an unusual relationship hetwcen fucose and hexosamine in these membranes. Fucose in glycolipids is normally associated with hexosaminecontaining lipids [39]. In the present study, however, fucose was highest in the internal membranes, which had only trace amounts of glucosamine and galactosamine. While the brush border contained higher amounts of these amino sugars, the fuco~;e content was very low. These results indicate tha! glycolipids devoid of hexosami~:e but containing fucose exist in the internal membranes but that such lipids may be absent in thc plasma membrane. Sialic acid was found in both lipid and protein flactions from all of the membranes but in relatively small amounts. Although it is possible that siali¢ acid may have been lost during the preparation of the mcmbrane samples, these results suggest that most of the mcmbrane glycoproteins and glycolipids are either Iov+ in or devoid of sialic acid. Sodium dodecylsulfate--polyacrylamide gel etectrophoresis revealed that the membranes consist of a variety of proteins, several of which contain carbohydrate. There was little similarity of the protein from the difl'erent membrane fractions, although the high degree of heterogeneity made comparison ot protein bands difficult. Glycoprotein bands were in general broad and fewer in number than the protein bands in all three membrane fractions cxamined. Furthermore, some of the periodateSchiff staining material remained in the stacking gel, indicating a high moleculat
123 weight of some membrane glycoproteins. On staining with Coomassie blue and periodate Schiff reagents, all three membrane fractions showed a fast moving band which ran coincident with the tracking dye. The same mobility was observed in the absence of the dye, ruling out the possibility that thc dyc was inducing some artifact in the pattern. A similar fraction has been reported by others [40-42] and has been attributed to either lipids or artifacts [43, 44]. From the data presented in this study, it appears that internal membranes such as microsomal membranes as well as brush border membranes conlain glycoproteins of unusual carbohydratc composition. A C K N O W L E I ) G M ENTS
We wish to thank most sincerely Mr Noel Taylor for his great assistance in the preparation of the electron micrographs. We gratefully acknowledge the technical assistance of Mrs Lillian Remer and Mr Frank Fearney. We are also indebted to Dr James S. Whitehead for his assistance in preparing the manuscript. REFERENCI~S 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
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