- Email: [email protected]
Incorporation of [1-14C]glucosamine by rat intestinal microvillus membrane
736
PRELIMINARY
BIOCHIMICA ET BIOPHYSICA ACTA
NOTES
BBA 71 022
Incorporation of [|-taC]glucosamine by rat intestinal microvillus membrane Synthetic pathways involved in tile production of glycoproteins of the cell surface have not been studied extensively. COOK, LAICO AND EYLaR 1 showed that [14C]glucosamine was incorporated into glycoprotein of a smooth endoplasmic reticulum fraction which contained plasma membranes, and suggested that their data described the biosynthesis of surface membrane glycoprotein. Incorporation of label into an isolated plasma membrane fraction was, however, not established. The small intestine presents a unique opportunity for the study of surface glycoproteins, since nlethods exist for the isolation of highly pure plasma membranes ~. Moreover the intestinal lunfinal surface possesses intense staining properties which are characteristic of glycoproteins a. I f o 4 has shown by electron microscopy that this staining is confined predominantly to a filamentous coat attached to the luminal surface of the microvillus plaslna membrane. This surface coat appears to be part of the surface membrane rather than a layer of mucus, since it respects cell boundaries4, 5 and resists removal by mucolytic agents ~. Since [r-~4Cjglucosamine is an excellent labelled precursor of plasma and tissue glycoproteins~, studies were undertaken to determine whether it might be incorporated into acid-precipitable glycoproteins of intestinal plasma membranes as a preliminary step in the study of their synthesis. The purpose of this communication is to indicate that [I-~4Cjglucosamine is incorporated into, and can be isolated with, microvillus plasma membranes, and to present preliminary evidence which suggests that glycoprotein may be transported to the membrane from a microsomal site. Table I shows the acid-precipitable radioactivity in intestinal subcellular fractions and intestinal contents 6 h following the intraperitoneal injection of [I-HCI glucosamine. At this time interval 3I ?o of the total radioactivity found in the intestinal mucosa was present in the membrane-rich brush border fraction. The specific activity of the brush border fraction was higher than that of any of the reinaining fractions whether expressed per mg of protein or per/xg of hexosamine. Labelling of the brush border fractions cannot, therefore, be due to contamination by other subcellular components. Although tile mucus-rich intestinal content contained approximately one third as much radioactivity as tile intestinal mucosa, the low specific radioactivity of hexosamine in this fraction indicates that it too cannot be responsible for the brush border radioactivity. In order to show that [I-~4CJglucosamine was incorporated into the microvillus plasma membrane, and not into a contaminant of the brush border fraction, purified microvillus membranes were isolated under similar experimental conditions and incorporated radioactivity compared with that of the membrane marker invertase ]3iochim. Biophys. Acla, 15o (i968) 73 (, 738
737
PRELIMINARY NOTES TABLE
I
RADIOACTIVITY SUBCELLULAR
INCORPORATED
INTO ACID-PREClPITABLE
PROTEIN
AND HEXOSAMINE
OF INTESTINAL
FRACTIONS
Male W i s t a r rats, u n f a s t e d , i 5 o - 2 o o g, received i o ffC - I - H C ] g l u c o s a m i n e (New E n g l a n d N uc l e a r, 5 ° t~C/mg) in o.2 ml isotonic saline i n t r a p e r i t o n e a l l y . 6 h l a t e r t h e s m a l l i n t e s t i n e w a s r e m o v e d . I n t e s t i n a l c o n t e n t was o b t a i n e d b y w a s h i n g the i n t e s t i n a l l u m e n w i t h 2o.o tnl i s o t o n i c saline. B r u s h bord ers were i s o l a t e d w i t h o u t purification. O t h e r s u b c e l l u l a r f r a c t i o n s were p r e p a r e d from t h e b r u s h b o r d e r s u p e r n a t a n t by differential c e n t r i f u g a t i o n fol l ow i ng t h e a d d i t i o n of sufficient sucrose to b r i n g the c o n c e n t r a t i o n to o. 3 M. A c i d - p r e c i p i t a b l e f r a c t i o n s were p r e p a r e d by precipit a t i i m w i t h i o % t r i c h l o r o a c e t i c acid a n d 1% p h o s p h o t u n g s t i c acid, followed by lipid e x t r a c t i o n w i t h c h l o r o f o r m m e t h a n o l (i : i, v/v). P r o t e i n a n d h e x o s a m i n e were ( t e t e r m i n e d b y t he m e t h o d s of LowRY et al. s and ALLISON AND SMITH9. P r o t e i n s a n d h e x o s a m i n e s were di s s ol ve d in 0. 4 M N a O H a n d d i s t i l l e d water, r e s p e c t i v e l y , a d d e d to BR.av's TM s o l u t i o n a n d r a d i o a c t i v i t y was det e r m i n e d in a l i q u i d s c i n t i l l a t i o n counter. _
n
Fractiou
Honiogenate Brush borders Mitochondria Microsomes Supernatant Intestinal conteut
% of total radioactivity in mucosa
Cou~zts/miu per mg protei~t
Coulzts/min per fig hexosamine
31 x2 17 39
65 ° 223o 444 650 513 iooo
34-o 72.o 39.o 24.0 I7.° 14,3
(EC 3.2.I.26) 2 at several stages of purification. As shown in Fig. I, each stage of membrane purification, indicated by an increment in the specific activity of invertase, is associated with a corresponding increase in specific radioactivity. It is apparent, therefore, that [>14Qglucosamine is incorporated by and remains tightly bound to the plasma membrane during its isolation. ~,
2.0
100
[10000
/
~
Ill
O_
L
1.©~
/
80
C
"8
} 6c
ooo
m ]
.c_
o
g
2
-1000
c
0 Stage of membrane purification
01 3 6 Time (h)
12
24
Fig. I. C o m p a r i s o n of specific a c t i v i t i e s of i n v e r t a s e a n d a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y a t successive s t a g e s of m e m b r a n e purification. I, h o m o g e n a t e ; I1, b r u s h b o r d e r s as i s o l a t e d in T a b l e I; I l I , b r u s h b o r d e r s purified as p r e v i o u s l y describedg; IX', m i c r o v i l l u s m e m b r a n e 2. E x p e r i m e n t a l p r o c e d u r e was t h e s a m e as d e s c r i b e d in T a b l e I. l n v e r t a s e w a s a s s a y e d as d e s c r i b e d b y D A H L Q V I S T 11.
Fig. 2. C h a n g e in specific a c t i v i t y of a c i d - p r e c i p i t a b l e h e x o s a m i n e of i n t e s t i n a l s u b c e l l u l a r f r a c t i o u s w i t h time. [i-14C]Glucosamine, i o ffC, was i n j e c t e d i n t r a p e r i t o n e a l l y a n d d a t a o b t a i n e d as d e s c r i b e d in T a b l e I. R a t s were sacrificed following i n j e c t i o n a t t h e t i m e i n t e r v a l s shown. R e s u l t s s h o w n are for t h e b r u s h b o r d e r f r a c t i o n (A -A), t h e m i c r o s o m a l f r a c t i o n (O O), a n d t h e s u p e r n a t a n t fraction ( © - - © ) .
Biochim. Biophys. Acta, 15 o (I968) 736-738
738
PRELIMINARY NOTES
The specific activity of acid-precipitable hexosamine in the microsomal, supernatant and brush border fractions of the intestinal nmcosa from I to 24 h following [iJ~C~glucosamine injection is shown in Fig. 2. At I h the microsomal specific activity was greater than that of the brush border but diminished rapidly thereafter. The specific activity of the brush border fraction was greatest at 3 h, falling to negligible levels at 24 h. The specific activity of the supernatant hexosamine was also greatest at 3 h, but remained low at all time periods. Early labelling of glycoprotein by the intestinal mucosa, therefore, occurs at an intracellular microsomal site while labelling of the plasma meInbrane is delayed. The microsomal and brush border curves do not conform strictly to that of a precursor product relationship since their periods of maximal specific activity do not coincideE It is possible that the delayed rise of the brush border specific activity curve reflects the transport of glycoprotein from a nlicrosomal to a brush border site. If tiffs is the case, the transport process must require several hours for completion. It seems most likely that glycoprotein is transported in particulate form since the low specific activity of the supernatant hexosamine apparently excludes transport in soluble form. As yet, however, attempts to identify a precursor of membrane glycoprotein in the particulate fractions of the mucosa have not been successful. These results are consistent with the concept that surface glycoproteins of tile intestine are an integral part of the plasma membrane, and indicate that information regarding their synthesis may be obtained by labelling them i~ vivo with [I-~4C glucosamine. This work was supported by Grant No. MA234 o of The Medical Research Council of Canada. The author wishes to express his gratitude to Miss A. PEDNEAt:, and Mrs. G. SAMANlZKfor their excellent technical assistance.
Department of Medici~ze, Uldversity of Toronto, Toronto [VesterJz Hospital, Toro~to 2-B (Ca~ada)
GOl~t)ON G. I:ORSTNER
G. M. W . COOK, M. T. LAIcO AND }L. i t . }2~YLAR,Proc. Natl. Acad. Sci. U.S., 54 (I965) 247, G. FORSTNER, S. SABTESIN AND 14. J. ISSELBACHER, Biochem. J., lO6 (I968) 381. A. RAMBOURG,M. NEUTRA AND C. P. LGBLOND, Anal. Record, 154 (1966) 4 I. S. [To, J. Cell Biol., 27 {I9651 475T. M. MUKHERJEE AND A. VV'YNN WILLIAMS, ./. Cell Biol., 34 (1967) 447. M. R. Sm,:TLAR, J. C. CApPS AND D. L. HERN, t3iochim. Biophys. Acla, 83 (19641 93. D. B. ZILVERSM1T, C. ]~NTENMAN AND ~'I. C. FISHLER, .[. Gen. Physiol., 26 (1943) 325 . (2). H . LOWRY, N. J. ROSEBROU~iH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 9 D. J. ALLISON AND Q. "]'. SMITH, A~za[. Biochem., 13 (19651 51o. IO G. A. BRAY, A n a l . Biochem., I (I96o) 279. i1 A. ])AHLQVIST,Anal. Biochem., 7 (1964) T8. 1 2 3 4 5 6 7 8
Received February i9th, i968
Biochim. Biophys. Acta, i5o (1908) 736-738
PRELIMINARY
BIOCHIMICA ET BIOPHYSICA ACTA
NOTES
BBA 71 022
Incorporation of [|-taC]glucosamine by rat intestinal microvillus membrane Synthetic pathways involved in tile production of glycoproteins of the cell surface have not been studied extensively. COOK, LAICO AND EYLaR 1 showed that [14C]glucosamine was incorporated into glycoprotein of a smooth endoplasmic reticulum fraction which contained plasma membranes, and suggested that their data described the biosynthesis of surface membrane glycoprotein. Incorporation of label into an isolated plasma membrane fraction was, however, not established. The small intestine presents a unique opportunity for the study of surface glycoproteins, since nlethods exist for the isolation of highly pure plasma membranes ~. Moreover the intestinal lunfinal surface possesses intense staining properties which are characteristic of glycoproteins a. I f o 4 has shown by electron microscopy that this staining is confined predominantly to a filamentous coat attached to the luminal surface of the microvillus plaslna membrane. This surface coat appears to be part of the surface membrane rather than a layer of mucus, since it respects cell boundaries4, 5 and resists removal by mucolytic agents ~. Since [r-~4Cjglucosamine is an excellent labelled precursor of plasma and tissue glycoproteins~, studies were undertaken to determine whether it might be incorporated into acid-precipitable glycoproteins of intestinal plasma membranes as a preliminary step in the study of their synthesis. The purpose of this communication is to indicate that [I-~4Cjglucosamine is incorporated into, and can be isolated with, microvillus plasma membranes, and to present preliminary evidence which suggests that glycoprotein may be transported to the membrane from a microsomal site. Table I shows the acid-precipitable radioactivity in intestinal subcellular fractions and intestinal contents 6 h following the intraperitoneal injection of [I-HCI glucosamine. At this time interval 3I ?o of the total radioactivity found in the intestinal mucosa was present in the membrane-rich brush border fraction. The specific activity of the brush border fraction was higher than that of any of the reinaining fractions whether expressed per mg of protein or per/xg of hexosamine. Labelling of the brush border fractions cannot, therefore, be due to contamination by other subcellular components. Although tile mucus-rich intestinal content contained approximately one third as much radioactivity as tile intestinal mucosa, the low specific radioactivity of hexosamine in this fraction indicates that it too cannot be responsible for the brush border radioactivity. In order to show that [I-~4CJglucosamine was incorporated into the microvillus plasma membrane, and not into a contaminant of the brush border fraction, purified microvillus membranes were isolated under similar experimental conditions and incorporated radioactivity compared with that of the membrane marker invertase ]3iochim. Biophys. Acla, 15o (i968) 73 (, 738
737
PRELIMINARY NOTES TABLE
I
RADIOACTIVITY SUBCELLULAR
INCORPORATED
INTO ACID-PREClPITABLE
PROTEIN
AND HEXOSAMINE
OF INTESTINAL
FRACTIONS
Male W i s t a r rats, u n f a s t e d , i 5 o - 2 o o g, received i o ffC - I - H C ] g l u c o s a m i n e (New E n g l a n d N uc l e a r, 5 ° t~C/mg) in o.2 ml isotonic saline i n t r a p e r i t o n e a l l y . 6 h l a t e r t h e s m a l l i n t e s t i n e w a s r e m o v e d . I n t e s t i n a l c o n t e n t was o b t a i n e d b y w a s h i n g the i n t e s t i n a l l u m e n w i t h 2o.o tnl i s o t o n i c saline. B r u s h bord ers were i s o l a t e d w i t h o u t purification. O t h e r s u b c e l l u l a r f r a c t i o n s were p r e p a r e d from t h e b r u s h b o r d e r s u p e r n a t a n t by differential c e n t r i f u g a t i o n fol l ow i ng t h e a d d i t i o n of sufficient sucrose to b r i n g the c o n c e n t r a t i o n to o. 3 M. A c i d - p r e c i p i t a b l e f r a c t i o n s were p r e p a r e d by precipit a t i i m w i t h i o % t r i c h l o r o a c e t i c acid a n d 1% p h o s p h o t u n g s t i c acid, followed by lipid e x t r a c t i o n w i t h c h l o r o f o r m m e t h a n o l (i : i, v/v). P r o t e i n a n d h e x o s a m i n e were ( t e t e r m i n e d b y t he m e t h o d s of LowRY et al. s and ALLISON AND SMITH9. P r o t e i n s a n d h e x o s a m i n e s were di s s ol ve d in 0. 4 M N a O H a n d d i s t i l l e d water, r e s p e c t i v e l y , a d d e d to BR.av's TM s o l u t i o n a n d r a d i o a c t i v i t y was det e r m i n e d in a l i q u i d s c i n t i l l a t i o n counter. _
n
Fractiou
Honiogenate Brush borders Mitochondria Microsomes Supernatant Intestinal conteut
% of total radioactivity in mucosa
Cou~zts/miu per mg protei~t
Coulzts/min per fig hexosamine
31 x2 17 39
65 ° 223o 444 650 513 iooo
34-o 72.o 39.o 24.0 I7.° 14,3
(EC 3.2.I.26) 2 at several stages of purification. As shown in Fig. I, each stage of membrane purification, indicated by an increment in the specific activity of invertase, is associated with a corresponding increase in specific radioactivity. It is apparent, therefore, that [>14Qglucosamine is incorporated by and remains tightly bound to the plasma membrane during its isolation. ~,
2.0
100
[10000
/
~
Ill
O_
L
1.©~
/
80
C
"8
} 6c
ooo
m ]
.c_
o
g
2
-1000
c
0 Stage of membrane purification
01 3 6 Time (h)
12
24
Fig. I. C o m p a r i s o n of specific a c t i v i t i e s of i n v e r t a s e a n d a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y a t successive s t a g e s of m e m b r a n e purification. I, h o m o g e n a t e ; I1, b r u s h b o r d e r s as i s o l a t e d in T a b l e I; I l I , b r u s h b o r d e r s purified as p r e v i o u s l y describedg; IX', m i c r o v i l l u s m e m b r a n e 2. E x p e r i m e n t a l p r o c e d u r e was t h e s a m e as d e s c r i b e d in T a b l e I. l n v e r t a s e w a s a s s a y e d as d e s c r i b e d b y D A H L Q V I S T 11.
Fig. 2. C h a n g e in specific a c t i v i t y of a c i d - p r e c i p i t a b l e h e x o s a m i n e of i n t e s t i n a l s u b c e l l u l a r f r a c t i o u s w i t h time. [i-14C]Glucosamine, i o ffC, was i n j e c t e d i n t r a p e r i t o n e a l l y a n d d a t a o b t a i n e d as d e s c r i b e d in T a b l e I. R a t s were sacrificed following i n j e c t i o n a t t h e t i m e i n t e r v a l s shown. R e s u l t s s h o w n are for t h e b r u s h b o r d e r f r a c t i o n (A -A), t h e m i c r o s o m a l f r a c t i o n (O O), a n d t h e s u p e r n a t a n t fraction ( © - - © ) .
Biochim. Biophys. Acta, 15 o (I968) 736-738
738
PRELIMINARY NOTES
The specific activity of acid-precipitable hexosamine in the microsomal, supernatant and brush border fractions of the intestinal nmcosa from I to 24 h following [iJ~C~glucosamine injection is shown in Fig. 2. At I h the microsomal specific activity was greater than that of the brush border but diminished rapidly thereafter. The specific activity of the brush border fraction was greatest at 3 h, falling to negligible levels at 24 h. The specific activity of the supernatant hexosamine was also greatest at 3 h, but remained low at all time periods. Early labelling of glycoprotein by the intestinal mucosa, therefore, occurs at an intracellular microsomal site while labelling of the plasma meInbrane is delayed. The microsomal and brush border curves do not conform strictly to that of a precursor product relationship since their periods of maximal specific activity do not coincideE It is possible that the delayed rise of the brush border specific activity curve reflects the transport of glycoprotein from a nlicrosomal to a brush border site. If tiffs is the case, the transport process must require several hours for completion. It seems most likely that glycoprotein is transported in particulate form since the low specific activity of the supernatant hexosamine apparently excludes transport in soluble form. As yet, however, attempts to identify a precursor of membrane glycoprotein in the particulate fractions of the mucosa have not been successful. These results are consistent with the concept that surface glycoproteins of tile intestine are an integral part of the plasma membrane, and indicate that information regarding their synthesis may be obtained by labelling them i~ vivo with [I-~4C glucosamine. This work was supported by Grant No. MA234 o of The Medical Research Council of Canada. The author wishes to express his gratitude to Miss A. PEDNEAt:, and Mrs. G. SAMANlZKfor their excellent technical assistance.
Department of Medici~ze, Uldversity of Toronto, Toronto [VesterJz Hospital, Toro~to 2-B (Ca~ada)
GOl~t)ON G. I:ORSTNER
G. M. W . COOK, M. T. LAIcO AND }L. i t . }2~YLAR,Proc. Natl. Acad. Sci. U.S., 54 (I965) 247, G. FORSTNER, S. SABTESIN AND 14. J. ISSELBACHER, Biochem. J., lO6 (I968) 381. A. RAMBOURG,M. NEUTRA AND C. P. LGBLOND, Anal. Record, 154 (1966) 4 I. S. [To, J. Cell Biol., 27 {I9651 475T. M. MUKHERJEE AND A. VV'YNN WILLIAMS, ./. Cell Biol., 34 (1967) 447. M. R. Sm,:TLAR, J. C. CApPS AND D. L. HERN, t3iochim. Biophys. Acla, 83 (19641 93. D. B. ZILVERSM1T, C. ]~NTENMAN AND ~'I. C. FISHLER, .[. Gen. Physiol., 26 (1943) 325 . (2). H . LOWRY, N. J. ROSEBROU~iH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 9 D. J. ALLISON AND Q. "]'. SMITH, A~za[. Biochem., 13 (19651 51o. IO G. A. BRAY, A n a l . Biochem., I (I96o) 279. i1 A. ])AHLQVIST,Anal. Biochem., 7 (1964) T8. 1 2 3 4 5 6 7 8
Received February i9th, i968
Biochim. Biophys. Acta, i5o (1908) 736-738