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Purification of Plasma Membrane of Guinea Pig Peritoneal Macrophages Eileen Remold-o'donnell
a
a
Department of Biological Chemistry , Harvard Medical School and the Center for Blood Research , Boston, Massachusetts, 02115 Published online: 05 Mar 2007.
To cite this article: Eileen Remold-o'donnell (1977) Purification of Plasma Membrane of Guinea Pig Peritoneal Macrophages, Preparative Biochemistry, 7:6, 441-455, DOI: 10.1080/00327487708065512 To link to this article: http://dx.doi.org/10.1080/00327487708065512
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PREPARATIVE BIOCHEMISTRY, 7(6), 441-455 (1977)
PURIFICATION OF PLASMA MEMBRANE OF GUINEA PIG PERITONEAL MACROPHAGES' Eileen Remold-O'Donnel12 Department of Biological Chemistry, Harvard Medical School and t h e Center f o r BIood Research, Boston, Massachusetts 02115
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ABSTRACT A method using sucrose d e n s i t y gradient c e n t r i f u g a t i o n i s described
f o r t h e p u r i f i c a t i o n of plasma membrane of guinea p i g p e r i t o n e a l exudate macrophages.
Assays f o r compositinn and f o r marker enzyme a c t i v i t i e s have
been modified f o r use with the small amounts o f s u b c e l l u l a r macrophage material.
The plasma membrane was obtained i n 57% y i e l d and uontained 7% of
the p r o t e i n .
The p u r i f i e d plasma membrane f r a c t i o n i s f i v e - f o l d enriched
i n phospholipid t o p r o t e i n r a t i o and contains no contaminating DNA, none of t h e cytoplasmic marker l a c t a t e dehydrogenase, no d e t e c t a b l e mitochondria1 contamination and a low contamination with lysosomal enzymes ( 7 % ) . P u r i f i e d plasma membrane containing 4 mg of p r o t e i n can be prepared from 1 m l of p e l l e t e d macrophages i n a one-day operation. INTRODUCTION I t i s known t h a t macrophages p l a y a major r o l e i n h o s t defense, namely and . ~of tumor c e l l s 5 , 6 ~ 7 , 8 , 9and i n t h e induci n t h e k i l l i n g of b a ~ t e r i a ~ t i o n of t h e immune responselo.
In many of t h e b i o l o g i c a l functions of t h e
macrophage, t h e plasma membrane plays an important r o l e .
In o r d e r t o under-
stand t h e s e functions we w i l l need knowledge o f t h e s t r u c t u r e and a c t i v i t i e s
of macrophage plasma membrane.
Therefore, t h i s study was i n i t i a t e d t o
p u r i f y t h e plasma membrane o f untreated guinea p i g macrophages.
A previous
preparation involved glutaraldehyde-fixed s u r f a c e membranes1
44 1 Copyrisht 01977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming, and recording. or by any information storage and retrieval system, without permission in writing from the publuher.
442
REMOLD-O'DONNELL
MATERIALS a-Naphthylphosphn:e, p-nitrophenyl-2-acetamido-2-deoxy-6-D-glucopryanoside, ATP, cyclic AMP, NADH, N-acetyl-D-glucosamine, UDP-galactose, dithiothreitol, Tris, and bovine serum albumin Fraction V powder were from Sigma Chemical Co. [ 32P]AMF', UDP-[ 3H]galactose, [ 14C]glucose-6-phosphate, [ 3H]ATP. 14C-labeled cyclic AMP, and Liquifluor scintillation solution, NEF-903, were
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from New England Nuclear Corp. [ 3H]ATP was purified by chromatography of 5 mCi of starting material on 15 ml columns of Dowex 50-H+ with water as eluant.
Bio-Solv BBS-3 solubilizer was from Beckman Instruments. AG 1-X8 formate resin and AG 50W-X2 resin (200 to 400 mesh) were from Bio-Rad Laboratories. Hanks' balanced salt solution was from Microbiological Associates, Inc., Bethesda, Md. Sodium caseinate (practical grade, P914) was from Eastman-Kodak. Creatine kinase and creatine phosphate were from Boehringer Mannheim. Salmon sperm DNA was a kind gift of atories).
Dr.
Robert Jackson (Harvard University Biological Labor-
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoli~ chloride
was from Aldrich Chemical Company. 2-Naphthyl-B-D-glucuronidewas from K and K Laboratories, Plainview, New York. p-Nitrophenyl-a-L-fucopyranoside was obtained from Pierce Chemical Company. METHODS Harvest of Macrophages. Macrophages were prepared as described by Nathan
-et a1.12
with minor changes. In short Hartley guinea pigs were injected intra-
peritoneally with 20 ml of sterile 1% sodium caseinate. Four days later the peritoneal cell suspensions were harvested, washed three times in cold Hanks' balanced salt solution, combined and centrifuged at 200xg for 5 min. Homogenization of Macrophages. The washed cell pellet was suspended in ten volumes of cold 5 mM Tris HC1 pH 8.0, 75 mM sucrose and incubated at 4 O for 15 minutes.
(For example, 1 ml of packed cells, approximately 6 x 108 cells were
suspended to 10 ml.)
The hypotonic buffer causes the macrophages to swell but
not to lyse. Cells were disrupted using a motor-driven Teflon pestle (S31 10-el glass homogenlzing vessel, S21 pestle with 0.11
-
0.15 mm clearance,
565 Variable Speed Lab Motor, Tri R Instruments, Jamaica, N . Y . . setting 5.
443
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES approximately 2000 rpm).
C e l l s u s p e n s i o n s w i t h a volume o f 10 m l were sub-
j e c t e d t o 3 t r e a t m e n t s o f 35-40 s e c (approximately 5 p a s s e s ) w i t h t h e homogeni z i n g v e s s e l immersed i n an i c e b a t h .
Immediately a f t e r homogenization,
0.25 volume o f compensating b u f f e r was added which c o n t a i n e d 0.95 M s u c r o s e , 20 mM Tris H C 1 , pH 7.4, 26 mM MgC12, 1 . 5 mM EDTA, 5 mM d i t h i o t h r e i t o l , 0.15 M NaC1, 0.15 M K C 1 .
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S u c r o s e Gradient C e n t r i f u g a t i o n . t o a graduated c y l in d e r .
The l y s e d c e l l s u s p e n s i o n was t r a n s f e r r e d
By adding 1.28 volume o f c o n c e n t r a t e d s u c r o s e s o l u t i o n
(2.30 M s u c r o s e , 10 mM T r i s HC1 pH 7 . 6 , 5 . 3 mM MgCIZ, 0 . 3 mM EDTA, 1 mM d i t h i o t h r e i t o l , 30 mM NaC1, 30 mM KCl), t h e s u c r o s e c o n c e n t r a t i o n o f t h e homogenate was b r o u g h t t o 40.5%.
Using l o n g - t i p p e d P a s t e u r p i p e t s , 4 t o 4.5 m l homogenate
was i n t r o d u c e d under preformed s u c r o s e g r a d i e n t s i n 14 m l c e l l u l o s e n i t r a t e swinging bucket c e n t r i f u g e t u b e s .
The g r a d i e n t s had been made from 3.1 m l each
of 40.0 and 34.0 w/w% s u c r o s e s o l u t i o n s which had been made by a d d i t i o n t o s o l i d s u c r o s e o f 10 mM T r i s HC1, pH 7 . 6 , 5 . 3 mM MgC12, 0 . 3 mM EDTA, 1 mM d i t h i o t h r e i t o l , 30 mM NaC1, 30 mM KC1.
The t u b e s were f i l l e d by l a y e r i n g 20% s u c r o s e i n t h e same
b u f f e r on t o p o f t h e g r a d i e n t s and were c e n t r i f u g e d a t 40,000 rpm f o r 6 h o u r s i n a SW 40 T i r o t o r (Beckman I n s t r u m e n t s , P a l o A l t o , C a l i f . ) .
40 t o 34 w/w%
g r a d i e n t s were chosen on t h e b a s i s o f e a r l i e r experiments u s i n g 40 t o 24% linear gradients.
I n t h e s e experiments t h e plasma membrane was found a s a
s i n g l e band i n t h e g r a d i e n t a t a s u c r o s e c o n c e n t r a t i o n o f 33.5% ( d e n s i t y = 1 . 1 4 ) . The y i e l d and r e p r o d u c i b i l i t y were improved w i t h t h e method f i n a l l y a d a p t e d . F r a c t i o n s ( u s u a l l y 18-21) were c o l l e c t e d dropwise from t h e bottom t h e tube.
of
The r e f r a c t i v e i n d i c e s were measured and c o n v e r t e d t o d e n s i t i e s .
The p e l l e t s were combined and suspended i n s u c r o s e b u f f e r by g e n t l e u s e o f a Teflon p e s t l e homogenizer. DNA and RNA E x t r a c t i o n .
The DNA and RNA e x t r a c t i o n method i s a m o d i f i -
c a t i m o f t h e Schmidt-Thannhauser p r o c e d u r e 1 3 .
Bovine serum albumin (375 pg)
was added a s a c a r r i e r t o 0.30 m l o r l e s s o f t h e s u b c e l l u l a r f r a c t i o n s . DNA, RNA and p r o t e i n were p r e c i p i t a t e d by a d d i t i o n o f 2.0 m l c o l d 0.24 M H C 1 0 4
and c o l l e c t e d by c e n t r i f u g a t i o n .
The p e l l e t s were washed two t i m e s w i t h
REMOLD-o ' DONNELL
444
2 . 0 m l o f c o l d 0 . 2 0 M HC104 and t h e RNA was s e p a r a t e d from t h e p e l l e t by adding 0.80 m l o f 0.30 M KOH a t 37", mixing v i g o r o u s l y on a Vortex mixer and i n c u b a t i n g a t 37' f o r 60 minutes w i t h o c c a s i o n a l v i g o r o u s mixing.
After
c o o l i n g on i c e , 0.80 m l c o l d 0.50MHC104 was added t o p r e c i p i t a t e p r o t e i n and DNA.
Following c e n t r i f u g a t i o n , t h e s u p e r n a t a n t s were q u a n t i t a t i v e l y t r a n s -
f e r r e d f o r t h e RNA a s s a y .
DNA was e x t r a c t e d from t h e p e l l e t s by adding 0.60
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m l o f 0.60 M HC104 and i n c u b a t i n g f o r 15 minutes a t 90" w i t h o c c a s i o n a l
vigorous mixing.
The t u b e s were c o o l e d t o room t e m p e r a t u r e and f o l l o w i n g
centrifugation t h e supernatants transferred t o t e s t tubes,
The p e l l e t s were
e x t r a c t e d a second time w i t h an a d d i t i o n a l 0.50 m l o f 0.60 M HClO4. DNA Assay.
DNA was a s s a y e d by a v a r i a t i o n 1 4 o f t h e p r o c e d u r e o f
B u r t o n l 5 > l 6 . The DNA r e a g e n t , which was f r e s h l y p r e p a r e d f o r each a s s a y , c o n t a i n e d 1.8 g diphenylamine i n 100 m l r e d i s t i l l e d a c e t i c a c i d w i t h 1.5 m l concentrated s u l f u r i c a c id .
To each e x t r a c t ( 1 . 1 ml) was added 1.70 m l
r e a g e n t followed by 0.10 m l o f an aqq?ous s o l u t i o n o f a c e t a l d e h y d e p e r ml). was r e a d .
(1.6 mg
Following o v e r n i g h t i n c u b a t i o n a t room t e m p e r a t u r e , t h e OD600
nm
The r e a d i n g s were compared t o t h o s e o f s t a n d a r d d i l u t i o n s o f
salmon sperm DNA ( c o n c e n t r a t i o n determined by phosphate a n a l y s i s ) which had been s u b j e c t e d t o t h e h o t HC104 e x t r a c t i o n p r o c e d u r e .
RNA Assay.
To 1 . 6 m l o f t h e n e u t r a l i z e d e x t r a c t 1.6 m l o r c i n o l r e a g e n t
was added c o n s i s t i n g o f 100 m l 1 2 M HC1, 20 mg FeC13.6H20 and 600 mg o r c i n o l ( r e c r y s t a l l i z e d from benzene:
petroleum e t h e r ) .
Following i n c u b a t i o n a t
100' f o r 30 minutes, t h e OD600 nm was r e a d and compared t o t h a t o f s t a n d a r d d i l u t i o n s o f c h i c k embryo RNA which had been s u b j e c t e d t o t h e KOH e x t r a c tion step. P r o t e i n Determination.
P r o t e i n c o n c e n t r a t i o n was measured by a modifi-
c a t i o n o f t h e method o f Lowry Phospholipid Determination.
GIa. F r a c t i o n s o f 0.10 m l o r less were e x t r a c t e d
u s i n g a m o d i f i c a t i o n o f t h e method o f H a j r a
el8. Ch1oroform:methanol
( l : l , 0.9 ml) and 0 . 0 2 m l 1 M HC1 were added and t h e m i x t u r e d i s p e r s e d w i t h a Vortex mixer.
An a d d i t i o n a l 0 . 3 m l chloroform was added, followed by
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
445
0.3 ml 2 M KC1 in 1 M H2SO4. Following mixing and centrifuging, the clear lower organic phase was transferred to test tubes and allowed to evaporate to dryness at room temperature. The phospholipids were converted to inorganic phosphate and phosphate measured using the methods of Ames and Dubinlg. Adenylate Cyclase Assay. Adenylate cyclase activity was measured by a modification20 of the method of Krishna
The gZ1.
60 p 1 assay con-
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tained 0.9 to 1.0 mM [3H]-ATP (2,fk3H), 25 cpm per pmole, 25 mM Tris-HC1 pH 7.6, 5 mM MgC12, 0.2 mM EDTA, 30 mM KC1, 2 mM cyclic AMP, 9mM creatine phosphate, 0 . 5 mg of creatine kinase per ml, 10 mM NaF and 25 to 40 ~1 of the fraction being assayed. Lactate Dehydrogenase Assay. Lactate dehydrogenase was assayed by measuring spectrophotometrically the decrease of NADH concentration. To start the reaction, 2.5 ml of a buffer-substrate solution (0.30 mM NADH, 1.1 mM sodium pyruvate in 50 mM potassium phosphate pH 7.3 at room temperature) was added to aliquots of 20 to 50 ~1 of the fractions. After 20 minutes at room temperature the
nm was read, subtracted from the
starting OD366 nm and converted to nmoles NADH oxidized. Succinate Dehydrogenase Assay.
Succinate-2-(p-iodophenyl)-3-(p-
nitrophenyl)-5-phenyltetrazolium-reductase activity was measured by the
method of PenningtonZ2 incorporating the modification of a 30 min preincubation at 40 as described by Porteous and Clarkz3. B-Glucuronidase Assay. 6-Glucuronidase was assayed using a modification of the method of GreenbergZ4. A buffer-substrate solution consisting of 0.5 mM 2-naphthyl-B-D-glucuronide, 0.12 v/v% Triton X-100 in 100 mM sodium acetate pH 5.3 was briefly equilibrated at 37'.
To start the assay, 0.4 ml
was added to 50-100 111 of the fractions being assayed which were then incubated for 10 min at 37'.
The reaction was terminated by the addition of
2.0 ml of a 4"-solution of 0.15 M Tris brought to pH 12 with NaOH.
Fluor-
escence was read in an Aminco spectrophotofluorometer using activation wavelength 348 nm, fluorescence wavelength 412 nm. To determine blank fluor-
446
REMOLD-O'DONNELL
escence, buffer blanks were assayed in parallel; to standardize each assay, the fluorescence o f a series of 0-naphthol solutions was read. N-acetyl-0-D-glucosaminidase Assay. The fractions to be assayed (40 ~1
or less) were incubated for 30 min at 37' in a volume of 0.40 ml containing 0.25
M sodium acetate pH
5.0 and 1.5 mM p-nitrophenyl-2-acetamido-2-deoxy-0-
D-glucopyranoside and 0.10 v/v% Triton X-100 according to the method of Carlsen and Piercez5. The reaction was stopped by adding 1.6 ml of 0.10 M
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sodium glycinate pH 10.6. a-L-Fucosidase Assay. a-L-Fucosidase was assayed using a modification of the method of Carlsen and Pierce25 (see previous assay).
The fractions
to be assayed (100 ~1 o r less) were incubated for 30 min at 37" in a volume of 0.40 ml containing 0.50 M sodium acetate pH 5.0,
1.5 mM p-nitrophenyl-a-
L-fucopyranoside and 0.10 v/v% Triton X-100. Galactosyl Transferase Assay. UDP-galactose: N-acetyl-D-glucosamine galactosyl transferase activity was assayed by a modification o f the method of Whitehead
s.26. The 70 u1 incubation mixture contained 60 mM caco-
dylate buffer pH 6.5,
65 mM N-acetyl-D-glucosamine, 0.05 mM [ 3H]UDP-galactose
(D-gala~tose-~H) with 25 to 30 cpm per pmole. 7 mM MnC12, 0.03% Triton X-100, 2 mg per ml bovine serum albumin, 1 mM ATP and 20 pl o r less of the fractions
being assayed. Reactions were incubated for 30 min at 37" and stopped by freezing in a bath of Dry Ice and acetone. The mixtures were diluted with 0.5 ml water at 4 " , immediately filtered under reduced pressure through Dowex
1-formate columns (0.5 x 3 cm) and the eluate plus a 1 ml wash collected and counted in a toluene-Liquifluor-Biosolv s o l u b i l i z e r - s c i n t i l l a t i o n mixture (0.86:0.04:0.10). For each fraction a separate blank incubation was run in parallel in which N-acetyl-D-glucosamine was omitted. RESULTS AND DISCUSSION Homogenization and Fractionation. Casein-elicited peritoneal exudate cells were allowed to swell in hypotonic basic buffer and homogenized as described. Compensating buffer was immediately added
to
restore isotonicity.
thereby stabilizing intracellular organelles. Examination of stained
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
447
(Wright's stain) preparations showed that the procedure caused rupture of 90% or more of the cells while maintaining to a great extent the intactness of the nuclei. The homogenate in 40.5% sucrose (a solution less dense than
nuclei, lysosomes and mitochondria, but more dense than membranes) was layered under continuous sucrose gradients and centrifuged. Upon centrifugation a pellet formed. Some material entered the gradient and moved upward. A single, clearly-visible band found at the interface formed by the top of the
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gradient and the 20% sucrose contained the plasma membrane as indicated below. Distribution of Markers. Fig. 1 shows the distribution of adenylate cyclase which has been shown to be a plasma menbrane componentz7 (lower panel), of protein (middle panel) and of the lysosomal enzyme 5-glucuronidase (upper panel).
A
large fraction of the adenylate cyclase activity was found in a
band at a density of 1.14 constituting the plasma membrane fraction. A smaller part which has found in the pellet, presumably due to sticking of the membrane to more dense cellular elements and to incompletely ruptured cells, was not further characterized. Of the protein, 20-30% was found in the pellet fraction; 50-60% remained in the region where the homogenate was introduced (fractions 1 through 5 or 6).
In 3 experiments, the pooled plasma
membrane banding fractions contained an average of 57 f 1% of the adenylate cyclase activity and 7 t 1% of the cell protein. Most of the activity of the lysosomal enzyme B-glucuronidase (upper panel) was found in the pellet; little was found in the plasma membrane banding fractions. Pooled plasma membrane fractions which contained 57 f 1% of the adenylate cyclase activity contained 8
f
1% of the 8-glucuronidase. The
distribution of the additional lysosomal enzymes N-acetyl-6-D-glucosaminidase and a-L-fucosidasewas also assayed and found to be similar as can be seen in Fig. 2 . Fig. 3 demonstrates the distribution of succinate dehydrogenase, a marker for mitochondria, the cytoplasmic enzyme lactate dehydrogenase, phospholipid as a general membrane marker and as a reference, adenylate cyclase. Succinate dehydrogenase was mainly found in the pellet and in a wide
REMOLD-O'DONNELL
448
301
B- GLUCURON/L!AS€
20 10
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0
30 20 10
o , , l
, , , , , ,
P 2 4 6
I
,
8 10 12 14 16 18 20 FRACTJON
FIGURE 1
D i s t r i b u t i o n of adenylate cyclase a c t i v i t y , p r o t e i n and 6-glucuronidase a c t i v i t y following f r a c t i o n a t i o n on sucrose d e n s i t y gradients. The lower panel shows adenylate cyclase p l o t t e d on t h e o r d i n a t e a s t h e percent of t o t a l recovered a c t i v i t y i n each gradient f r a c t i o n . Recovery of adenylate cyclase i n t h i s preparation i s 8 2 % ; t o t a l recovered a c t i v i t y i s 920 pmoles per min per gradient. The d i s t r i b u t i o n of p r o t e i n i s shown i n t h e middle panel. Recovery o f p r o t e i n is 99%; t o t a l recovered p r o t e i n is 9 . 3 mg per gradient. The upper panel shows t h e d i s t r i b u t i o n of 6glucuronidase. Recovery of 6-glucuronidase i s 104%; t o t a l recovered a c t i v i t y i s 170 nmoles per min per g r a d i e n t . Fraction P i n d i c a t e s t h e p e l l e t f r a c t i o n . Fractions 1 t o 6 represent t h e a r e a a t t h e bottom of t h e centrifuge tube where t h e 40.5% sucrose s o l u t i o n containing t h e macrophage homogenate was layered.
a r e a of t h e gradient with a density g r e a t e r than t h a t of t h e plasma membrane. The cytoplasmic enzyme l a c t a t e dehydrogenase was found primarily i n f r a c t i o n s 1 t o 5 , i . e . its p o s i t i o n was unchanged following c e n t r i f u g a t i o n a s was predictable f o r a s o l u b l e p r o t e i n i n 40.5% sucrose.
Phospholipid was
mainly found i n t h e p e l l e t and t h e plasma membrane f r a c t i o n .
Thw, t h e
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
ap
40
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30
20 10
0
so 40
30
10
0
30
FRACTION
FIGURE 2 Distribution of the lysosomal enzymes, @-glucuronidase. N-acetyl-B-Dglucosaminidase and a-L-fucosidase following fractionation using 4024% sucrose gradients. @-glucuronidase, i.e., percent of total recovered @-glucuronidase activity in each fraction is plotted on the ordinate of the second lowest panel. (For comparison adenylate cyclase activity distribution in this preparation is shown on the lowest panel.) Percent of total recovered N-acetyl-@-D-glucosaminidase activity is shown in the third lowest panel and of a-L-fucosidase in the upper panel.
phospholipid to protein ratio was increased 5-fold in the plasma membrane relative to homogenates. Distribution of phospholipid was not identical to that of adenylate cyclase, indicating that other cellular membranes have a fractionation pattern different from that of plasma membrane.
449
REMOLD-O'DONNELL
450
30
20 10
-
n
LACTATE DEHYOROGENASE
"t Downloaded by [Tufts University] at 14:17 03 December 2014
20
be 20 10 0
AOENYLATE CYCLASE
P 2 4 6 8 10 12 14 16 (820 22
FRACTION
FIGURE 3
Distribution of phospholipid, lactate dehydrogenase and succinate dehydrogenase following fractionation. Phospholipid, i.e.. percent of total recovered phospholipid, in each fraction is plotted on the ordinate of the second lowest panel. @denylate cyclase distribution in this preparation is shown on the lowest panel.) Percent of total lactate dehydrogenase activity is plotted on the ordinate of the third panel. Percent of total succinate dehydrogenase activity [succinate-2-(p-iodophenyl)-3-(pnitropheny1)-5-phenyltetrazolium-reductase activity] is plotted on the ordinate of the upper panel.
Fig. 4 shows the distribution of galactosyl transferase, RNA and DNA
in reference to adenylate cyclase. Most of the RNA and DNA was found in the pellet, although low levels of RNA were also present in the plasma membrane fraction, possibly an artifact of the orcinol assay. Whereas glycosyl transferases are generally considered to be components of the Golgi appara-
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
451
GALACTOSYL TRANSFERASE 30 20 10
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0
FRACTION
FIGURE 4 D i s t r i b u t i o n of DNA, RNA (orcinol p o s i t i v e m a t e r i a l ) and g a l a c t o s y l t r a n s f e r a s e a c t i v i t y following f r a c t i o n a t i o n using continuous sucrose gradients.
t u s z 8 , recent findings i n d i c a t e t h a t t h e s e enzymes a r e present on t h e c e l l surface o f various mammalian c e l l s z 9 , such a s
lymphocyte^^^ and f i b r o b l a s t s 3 1 .
Since g a l a c t o s y l t r a n s f e r a s e c o f r a c t i o n a t e s with t h e plasma membrane, two p o s s i b i l i t i e s have t o be considered: 1. t h a t t h e plasma membrane f r a c t i o n s contain elements o f t h e Golgi apparatus o r 2 . t h a t g a l a c t o s y l t r a n s f e r a s e
i s a macrophage plasma membrane component.
452
REMOLWI'DONNELL
From these experiments it can be concluded that the plasma membrane prepared as described contains 7% of the cell protein, 57% of the plasma membrane marker adenylate cyclase, no mitochondria1 components, no lactate dehydrogenase, no DNA, little RNA and a 5-fold increased phospholipid to protein ratio. Furthermore, the contamination with lysosomal enzymes is low (less than 8%).
Galactosyl transferase was found to be present in the
plasma membrane fraction.
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Purity of the Cell Preparation. Although these peritoneal exudate macrophages are contaminated with 10-25% polymorphonuclear leukocytes, the latter were resistant to homogenization. When exudate homogenates which contained 90% o r more ruptured cells were stained with Wright's stain, the remaining intact cells included a large percent of polymorphonuclear leukocytes. The resistance o f polymorphonuclear leukocytes to homogenization was verified by the fractionation pattern of M a s e activity, a plasma membrane marker of polymorphonuclear leukocytes which is not detectable in caseinelicited macrophages of guinea pigs32*33. A large fraction of AMF'ase activity was found in the pellet fraction which contains the unbroken cells. In 3 experiments the pellet fraction which contained 10 activity contained 52
+
?
8% of the particulate AMF'ase.
1% of adenylate cyclase Thus, the contribution
of polymorphonuclear leukocytes to the plasma membrane preparation is only half that indicated by the differential cell count, i.e. 5-12%. In conclusion, a purification method for macrophage plasma membrane was developed which is relatively simple, requires exudate cells from approximately 6 animals and can be completed by one researcher within a day.
Fol-
lowing homogenization, the plasma membrane is in contact with other subcellular elements for only 2 hours. During this time the homogenates are held near O o , at pH 7 . 6 in concentrated sucrose. These conditions minimize lysosomal enzyme release and activity. Furthermore, maximum precautions have been taken to prevent alterations of plasma membrane during purification. Thus, a simple rapid method i.s now available f o r preparing macrophage plasma membrane in high purity and good yield.
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
453
ACKNOWLEDGEMENTS I wish t o thank D r . John David, D r . Guido G u i d o t t i and D r . Manfred
L. Karnovsky, i n whose l a b o r a t o r i e s t h i s work was performed, f o r s u p p o r t , d i s c u s s i o n and h e l p f u l s u g g e s t i o n s .
I am g r a t e f u l a l s o t o Dr. Robert
Jackson, Dr. Steven C l a r k e , Dr. Eva Neer and D r . Heinz Remold f o r suggest i o n s a n d d i s c u s s i o n , and t o Judy Shaw f o r p e r f o r m i n g t h e a - L - f u c o s i d a s e
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assays.
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After 3 days culture of guinea pig peritoneal exudate cells, when only macrophages remain on the dishes, the ecto-AMPase activity was found to be non-detectable, i.e., less than 0.8 nmoles x mg-l x min-l
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(E. Remold-O'Donnell, unpublished experiments).
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Purification of Plasma Membrane of Guinea Pig Peritoneal Macrophages Eileen Remold-o'donnell
a
a
Department of Biological Chemistry , Harvard Medical School and the Center for Blood Research , Boston, Massachusetts, 02115 Published online: 05 Mar 2007.
To cite this article: Eileen Remold-o'donnell (1977) Purification of Plasma Membrane of Guinea Pig Peritoneal Macrophages, Preparative Biochemistry, 7:6, 441-455, DOI: 10.1080/00327487708065512 To link to this article: http://dx.doi.org/10.1080/00327487708065512
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PREPARATIVE BIOCHEMISTRY, 7(6), 441-455 (1977)
PURIFICATION OF PLASMA MEMBRANE OF GUINEA PIG PERITONEAL MACROPHAGES' Eileen Remold-O'Donnel12 Department of Biological Chemistry, Harvard Medical School and t h e Center f o r BIood Research, Boston, Massachusetts 02115
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ABSTRACT A method using sucrose d e n s i t y gradient c e n t r i f u g a t i o n i s described
f o r t h e p u r i f i c a t i o n of plasma membrane of guinea p i g p e r i t o n e a l exudate macrophages.
Assays f o r compositinn and f o r marker enzyme a c t i v i t i e s have
been modified f o r use with the small amounts o f s u b c e l l u l a r macrophage material.
The plasma membrane was obtained i n 57% y i e l d and uontained 7% of
the p r o t e i n .
The p u r i f i e d plasma membrane f r a c t i o n i s f i v e - f o l d enriched
i n phospholipid t o p r o t e i n r a t i o and contains no contaminating DNA, none of t h e cytoplasmic marker l a c t a t e dehydrogenase, no d e t e c t a b l e mitochondria1 contamination and a low contamination with lysosomal enzymes ( 7 % ) . P u r i f i e d plasma membrane containing 4 mg of p r o t e i n can be prepared from 1 m l of p e l l e t e d macrophages i n a one-day operation. INTRODUCTION I t i s known t h a t macrophages p l a y a major r o l e i n h o s t defense, namely and . ~of tumor c e l l s 5 , 6 ~ 7 , 8 , 9and i n t h e induci n t h e k i l l i n g of b a ~ t e r i a ~ t i o n of t h e immune responselo.
In many of t h e b i o l o g i c a l functions of t h e
macrophage, t h e plasma membrane plays an important r o l e .
In o r d e r t o under-
stand t h e s e functions we w i l l need knowledge o f t h e s t r u c t u r e and a c t i v i t i e s
of macrophage plasma membrane.
Therefore, t h i s study was i n i t i a t e d t o
p u r i f y t h e plasma membrane o f untreated guinea p i g macrophages.
A previous
preparation involved glutaraldehyde-fixed s u r f a c e membranes1
44 1 Copyrisht 01977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming, and recording. or by any information storage and retrieval system, without permission in writing from the publuher.
442
REMOLD-O'DONNELL
MATERIALS a-Naphthylphosphn:e, p-nitrophenyl-2-acetamido-2-deoxy-6-D-glucopryanoside, ATP, cyclic AMP, NADH, N-acetyl-D-glucosamine, UDP-galactose, dithiothreitol, Tris, and bovine serum albumin Fraction V powder were from Sigma Chemical Co. [ 32P]AMF', UDP-[ 3H]galactose, [ 14C]glucose-6-phosphate, [ 3H]ATP. 14C-labeled cyclic AMP, and Liquifluor scintillation solution, NEF-903, were
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from New England Nuclear Corp. [ 3H]ATP was purified by chromatography of 5 mCi of starting material on 15 ml columns of Dowex 50-H+ with water as eluant.
Bio-Solv BBS-3 solubilizer was from Beckman Instruments. AG 1-X8 formate resin and AG 50W-X2 resin (200 to 400 mesh) were from Bio-Rad Laboratories. Hanks' balanced salt solution was from Microbiological Associates, Inc., Bethesda, Md. Sodium caseinate (practical grade, P914) was from Eastman-Kodak. Creatine kinase and creatine phosphate were from Boehringer Mannheim. Salmon sperm DNA was a kind gift of atories).
Dr.
Robert Jackson (Harvard University Biological Labor-
2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoli~ chloride
was from Aldrich Chemical Company. 2-Naphthyl-B-D-glucuronidewas from K and K Laboratories, Plainview, New York. p-Nitrophenyl-a-L-fucopyranoside was obtained from Pierce Chemical Company. METHODS Harvest of Macrophages. Macrophages were prepared as described by Nathan
-et a1.12
with minor changes. In short Hartley guinea pigs were injected intra-
peritoneally with 20 ml of sterile 1% sodium caseinate. Four days later the peritoneal cell suspensions were harvested, washed three times in cold Hanks' balanced salt solution, combined and centrifuged at 200xg for 5 min. Homogenization of Macrophages. The washed cell pellet was suspended in ten volumes of cold 5 mM Tris HC1 pH 8.0, 75 mM sucrose and incubated at 4 O for 15 minutes.
(For example, 1 ml of packed cells, approximately 6 x 108 cells were
suspended to 10 ml.)
The hypotonic buffer causes the macrophages to swell but
not to lyse. Cells were disrupted using a motor-driven Teflon pestle (S31 10-el glass homogenlzing vessel, S21 pestle with 0.11
-
0.15 mm clearance,
565 Variable Speed Lab Motor, Tri R Instruments, Jamaica, N . Y . . setting 5.
443
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES approximately 2000 rpm).
C e l l s u s p e n s i o n s w i t h a volume o f 10 m l were sub-
j e c t e d t o 3 t r e a t m e n t s o f 35-40 s e c (approximately 5 p a s s e s ) w i t h t h e homogeni z i n g v e s s e l immersed i n an i c e b a t h .
Immediately a f t e r homogenization,
0.25 volume o f compensating b u f f e r was added which c o n t a i n e d 0.95 M s u c r o s e , 20 mM Tris H C 1 , pH 7.4, 26 mM MgC12, 1 . 5 mM EDTA, 5 mM d i t h i o t h r e i t o l , 0.15 M NaC1, 0.15 M K C 1 .
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S u c r o s e Gradient C e n t r i f u g a t i o n . t o a graduated c y l in d e r .
The l y s e d c e l l s u s p e n s i o n was t r a n s f e r r e d
By adding 1.28 volume o f c o n c e n t r a t e d s u c r o s e s o l u t i o n
(2.30 M s u c r o s e , 10 mM T r i s HC1 pH 7 . 6 , 5 . 3 mM MgCIZ, 0 . 3 mM EDTA, 1 mM d i t h i o t h r e i t o l , 30 mM NaC1, 30 mM KCl), t h e s u c r o s e c o n c e n t r a t i o n o f t h e homogenate was b r o u g h t t o 40.5%.
Using l o n g - t i p p e d P a s t e u r p i p e t s , 4 t o 4.5 m l homogenate
was i n t r o d u c e d under preformed s u c r o s e g r a d i e n t s i n 14 m l c e l l u l o s e n i t r a t e swinging bucket c e n t r i f u g e t u b e s .
The g r a d i e n t s had been made from 3.1 m l each
of 40.0 and 34.0 w/w% s u c r o s e s o l u t i o n s which had been made by a d d i t i o n t o s o l i d s u c r o s e o f 10 mM T r i s HC1, pH 7 . 6 , 5 . 3 mM MgC12, 0 . 3 mM EDTA, 1 mM d i t h i o t h r e i t o l , 30 mM NaC1, 30 mM KC1.
The t u b e s were f i l l e d by l a y e r i n g 20% s u c r o s e i n t h e same
b u f f e r on t o p o f t h e g r a d i e n t s and were c e n t r i f u g e d a t 40,000 rpm f o r 6 h o u r s i n a SW 40 T i r o t o r (Beckman I n s t r u m e n t s , P a l o A l t o , C a l i f . ) .
40 t o 34 w/w%
g r a d i e n t s were chosen on t h e b a s i s o f e a r l i e r experiments u s i n g 40 t o 24% linear gradients.
I n t h e s e experiments t h e plasma membrane was found a s a
s i n g l e band i n t h e g r a d i e n t a t a s u c r o s e c o n c e n t r a t i o n o f 33.5% ( d e n s i t y = 1 . 1 4 ) . The y i e l d and r e p r o d u c i b i l i t y were improved w i t h t h e method f i n a l l y a d a p t e d . F r a c t i o n s ( u s u a l l y 18-21) were c o l l e c t e d dropwise from t h e bottom t h e tube.
of
The r e f r a c t i v e i n d i c e s were measured and c o n v e r t e d t o d e n s i t i e s .
The p e l l e t s were combined and suspended i n s u c r o s e b u f f e r by g e n t l e u s e o f a Teflon p e s t l e homogenizer. DNA and RNA E x t r a c t i o n .
The DNA and RNA e x t r a c t i o n method i s a m o d i f i -
c a t i m o f t h e Schmidt-Thannhauser p r o c e d u r e 1 3 .
Bovine serum albumin (375 pg)
was added a s a c a r r i e r t o 0.30 m l o r l e s s o f t h e s u b c e l l u l a r f r a c t i o n s . DNA, RNA and p r o t e i n were p r e c i p i t a t e d by a d d i t i o n o f 2.0 m l c o l d 0.24 M H C 1 0 4
and c o l l e c t e d by c e n t r i f u g a t i o n .
The p e l l e t s were washed two t i m e s w i t h
REMOLD-o ' DONNELL
444
2 . 0 m l o f c o l d 0 . 2 0 M HC104 and t h e RNA was s e p a r a t e d from t h e p e l l e t by adding 0.80 m l o f 0.30 M KOH a t 37", mixing v i g o r o u s l y on a Vortex mixer and i n c u b a t i n g a t 37' f o r 60 minutes w i t h o c c a s i o n a l v i g o r o u s mixing.
After
c o o l i n g on i c e , 0.80 m l c o l d 0.50MHC104 was added t o p r e c i p i t a t e p r o t e i n and DNA.
Following c e n t r i f u g a t i o n , t h e s u p e r n a t a n t s were q u a n t i t a t i v e l y t r a n s -
f e r r e d f o r t h e RNA a s s a y .
DNA was e x t r a c t e d from t h e p e l l e t s by adding 0.60
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m l o f 0.60 M HC104 and i n c u b a t i n g f o r 15 minutes a t 90" w i t h o c c a s i o n a l
vigorous mixing.
The t u b e s were c o o l e d t o room t e m p e r a t u r e and f o l l o w i n g
centrifugation t h e supernatants transferred t o t e s t tubes,
The p e l l e t s were
e x t r a c t e d a second time w i t h an a d d i t i o n a l 0.50 m l o f 0.60 M HClO4. DNA Assay.
DNA was a s s a y e d by a v a r i a t i o n 1 4 o f t h e p r o c e d u r e o f
B u r t o n l 5 > l 6 . The DNA r e a g e n t , which was f r e s h l y p r e p a r e d f o r each a s s a y , c o n t a i n e d 1.8 g diphenylamine i n 100 m l r e d i s t i l l e d a c e t i c a c i d w i t h 1.5 m l concentrated s u l f u r i c a c id .
To each e x t r a c t ( 1 . 1 ml) was added 1.70 m l
r e a g e n t followed by 0.10 m l o f an aqq?ous s o l u t i o n o f a c e t a l d e h y d e p e r ml). was r e a d .
(1.6 mg
Following o v e r n i g h t i n c u b a t i o n a t room t e m p e r a t u r e , t h e OD600
nm
The r e a d i n g s were compared t o t h o s e o f s t a n d a r d d i l u t i o n s o f
salmon sperm DNA ( c o n c e n t r a t i o n determined by phosphate a n a l y s i s ) which had been s u b j e c t e d t o t h e h o t HC104 e x t r a c t i o n p r o c e d u r e .
RNA Assay.
To 1 . 6 m l o f t h e n e u t r a l i z e d e x t r a c t 1.6 m l o r c i n o l r e a g e n t
was added c o n s i s t i n g o f 100 m l 1 2 M HC1, 20 mg FeC13.6H20 and 600 mg o r c i n o l ( r e c r y s t a l l i z e d from benzene:
petroleum e t h e r ) .
Following i n c u b a t i o n a t
100' f o r 30 minutes, t h e OD600 nm was r e a d and compared t o t h a t o f s t a n d a r d d i l u t i o n s o f c h i c k embryo RNA which had been s u b j e c t e d t o t h e KOH e x t r a c tion step. P r o t e i n Determination.
P r o t e i n c o n c e n t r a t i o n was measured by a modifi-
c a t i o n o f t h e method o f Lowry Phospholipid Determination.
GIa. F r a c t i o n s o f 0.10 m l o r less were e x t r a c t e d
u s i n g a m o d i f i c a t i o n o f t h e method o f H a j r a
el8. Ch1oroform:methanol
( l : l , 0.9 ml) and 0 . 0 2 m l 1 M HC1 were added and t h e m i x t u r e d i s p e r s e d w i t h a Vortex mixer.
An a d d i t i o n a l 0 . 3 m l chloroform was added, followed by
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
445
0.3 ml 2 M KC1 in 1 M H2SO4. Following mixing and centrifuging, the clear lower organic phase was transferred to test tubes and allowed to evaporate to dryness at room temperature. The phospholipids were converted to inorganic phosphate and phosphate measured using the methods of Ames and Dubinlg. Adenylate Cyclase Assay. Adenylate cyclase activity was measured by a modification20 of the method of Krishna
The gZ1.
60 p 1 assay con-
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tained 0.9 to 1.0 mM [3H]-ATP (2,fk3H), 25 cpm per pmole, 25 mM Tris-HC1 pH 7.6, 5 mM MgC12, 0.2 mM EDTA, 30 mM KC1, 2 mM cyclic AMP, 9mM creatine phosphate, 0 . 5 mg of creatine kinase per ml, 10 mM NaF and 25 to 40 ~1 of the fraction being assayed. Lactate Dehydrogenase Assay. Lactate dehydrogenase was assayed by measuring spectrophotometrically the decrease of NADH concentration. To start the reaction, 2.5 ml of a buffer-substrate solution (0.30 mM NADH, 1.1 mM sodium pyruvate in 50 mM potassium phosphate pH 7.3 at room temperature) was added to aliquots of 20 to 50 ~1 of the fractions. After 20 minutes at room temperature the
nm was read, subtracted from the
starting OD366 nm and converted to nmoles NADH oxidized. Succinate Dehydrogenase Assay.
Succinate-2-(p-iodophenyl)-3-(p-
nitrophenyl)-5-phenyltetrazolium-reductase activity was measured by the
method of PenningtonZ2 incorporating the modification of a 30 min preincubation at 40 as described by Porteous and Clarkz3. B-Glucuronidase Assay. 6-Glucuronidase was assayed using a modification of the method of GreenbergZ4. A buffer-substrate solution consisting of 0.5 mM 2-naphthyl-B-D-glucuronide, 0.12 v/v% Triton X-100 in 100 mM sodium acetate pH 5.3 was briefly equilibrated at 37'.
To start the assay, 0.4 ml
was added to 50-100 111 of the fractions being assayed which were then incubated for 10 min at 37'.
The reaction was terminated by the addition of
2.0 ml of a 4"-solution of 0.15 M Tris brought to pH 12 with NaOH.
Fluor-
escence was read in an Aminco spectrophotofluorometer using activation wavelength 348 nm, fluorescence wavelength 412 nm. To determine blank fluor-
446
REMOLD-O'DONNELL
escence, buffer blanks were assayed in parallel; to standardize each assay, the fluorescence o f a series of 0-naphthol solutions was read. N-acetyl-0-D-glucosaminidase Assay. The fractions to be assayed (40 ~1
or less) were incubated for 30 min at 37' in a volume of 0.40 ml containing 0.25
M sodium acetate pH
5.0 and 1.5 mM p-nitrophenyl-2-acetamido-2-deoxy-0-
D-glucopyranoside and 0.10 v/v% Triton X-100 according to the method of Carlsen and Piercez5. The reaction was stopped by adding 1.6 ml of 0.10 M
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sodium glycinate pH 10.6. a-L-Fucosidase Assay. a-L-Fucosidase was assayed using a modification of the method of Carlsen and Pierce25 (see previous assay).
The fractions
to be assayed (100 ~1 o r less) were incubated for 30 min at 37" in a volume of 0.40 ml containing 0.50 M sodium acetate pH 5.0,
1.5 mM p-nitrophenyl-a-
L-fucopyranoside and 0.10 v/v% Triton X-100. Galactosyl Transferase Assay. UDP-galactose: N-acetyl-D-glucosamine galactosyl transferase activity was assayed by a modification o f the method of Whitehead
s.26. The 70 u1 incubation mixture contained 60 mM caco-
dylate buffer pH 6.5,
65 mM N-acetyl-D-glucosamine, 0.05 mM [ 3H]UDP-galactose
(D-gala~tose-~H) with 25 to 30 cpm per pmole. 7 mM MnC12, 0.03% Triton X-100, 2 mg per ml bovine serum albumin, 1 mM ATP and 20 pl o r less of the fractions
being assayed. Reactions were incubated for 30 min at 37" and stopped by freezing in a bath of Dry Ice and acetone. The mixtures were diluted with 0.5 ml water at 4 " , immediately filtered under reduced pressure through Dowex
1-formate columns (0.5 x 3 cm) and the eluate plus a 1 ml wash collected and counted in a toluene-Liquifluor-Biosolv s o l u b i l i z e r - s c i n t i l l a t i o n mixture (0.86:0.04:0.10). For each fraction a separate blank incubation was run in parallel in which N-acetyl-D-glucosamine was omitted. RESULTS AND DISCUSSION Homogenization and Fractionation. Casein-elicited peritoneal exudate cells were allowed to swell in hypotonic basic buffer and homogenized as described. Compensating buffer was immediately added
to
restore isotonicity.
thereby stabilizing intracellular organelles. Examination of stained
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
447
(Wright's stain) preparations showed that the procedure caused rupture of 90% or more of the cells while maintaining to a great extent the intactness of the nuclei. The homogenate in 40.5% sucrose (a solution less dense than
nuclei, lysosomes and mitochondria, but more dense than membranes) was layered under continuous sucrose gradients and centrifuged. Upon centrifugation a pellet formed. Some material entered the gradient and moved upward. A single, clearly-visible band found at the interface formed by the top of the
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gradient and the 20% sucrose contained the plasma membrane as indicated below. Distribution of Markers. Fig. 1 shows the distribution of adenylate cyclase which has been shown to be a plasma menbrane componentz7 (lower panel), of protein (middle panel) and of the lysosomal enzyme 5-glucuronidase (upper panel).
A
large fraction of the adenylate cyclase activity was found in a
band at a density of 1.14 constituting the plasma membrane fraction. A smaller part which has found in the pellet, presumably due to sticking of the membrane to more dense cellular elements and to incompletely ruptured cells, was not further characterized. Of the protein, 20-30% was found in the pellet fraction; 50-60% remained in the region where the homogenate was introduced (fractions 1 through 5 or 6).
In 3 experiments, the pooled plasma
membrane banding fractions contained an average of 57 f 1% of the adenylate cyclase activity and 7 t 1% of the cell protein. Most of the activity of the lysosomal enzyme B-glucuronidase (upper panel) was found in the pellet; little was found in the plasma membrane banding fractions. Pooled plasma membrane fractions which contained 57 f 1% of the adenylate cyclase activity contained 8
f
1% of the 8-glucuronidase. The
distribution of the additional lysosomal enzymes N-acetyl-6-D-glucosaminidase and a-L-fucosidasewas also assayed and found to be similar as can be seen in Fig. 2 . Fig. 3 demonstrates the distribution of succinate dehydrogenase, a marker for mitochondria, the cytoplasmic enzyme lactate dehydrogenase, phospholipid as a general membrane marker and as a reference, adenylate cyclase. Succinate dehydrogenase was mainly found in the pellet and in a wide
REMOLD-O'DONNELL
448
301
B- GLUCURON/L!AS€
20 10
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0
30 20 10
o , , l
, , , , , ,
P 2 4 6
I
,
8 10 12 14 16 18 20 FRACTJON
FIGURE 1
D i s t r i b u t i o n of adenylate cyclase a c t i v i t y , p r o t e i n and 6-glucuronidase a c t i v i t y following f r a c t i o n a t i o n on sucrose d e n s i t y gradients. The lower panel shows adenylate cyclase p l o t t e d on t h e o r d i n a t e a s t h e percent of t o t a l recovered a c t i v i t y i n each gradient f r a c t i o n . Recovery of adenylate cyclase i n t h i s preparation i s 8 2 % ; t o t a l recovered a c t i v i t y i s 920 pmoles per min per gradient. The d i s t r i b u t i o n of p r o t e i n i s shown i n t h e middle panel. Recovery o f p r o t e i n is 99%; t o t a l recovered p r o t e i n is 9 . 3 mg per gradient. The upper panel shows t h e d i s t r i b u t i o n of 6glucuronidase. Recovery of 6-glucuronidase i s 104%; t o t a l recovered a c t i v i t y i s 170 nmoles per min per g r a d i e n t . Fraction P i n d i c a t e s t h e p e l l e t f r a c t i o n . Fractions 1 t o 6 represent t h e a r e a a t t h e bottom of t h e centrifuge tube where t h e 40.5% sucrose s o l u t i o n containing t h e macrophage homogenate was layered.
a r e a of t h e gradient with a density g r e a t e r than t h a t of t h e plasma membrane. The cytoplasmic enzyme l a c t a t e dehydrogenase was found primarily i n f r a c t i o n s 1 t o 5 , i . e . its p o s i t i o n was unchanged following c e n t r i f u g a t i o n a s was predictable f o r a s o l u b l e p r o t e i n i n 40.5% sucrose.
Phospholipid was
mainly found i n t h e p e l l e t and t h e plasma membrane f r a c t i o n .
Thw, t h e
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
ap
40
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30
20 10
0
so 40
30
10
0
30
FRACTION
FIGURE 2 Distribution of the lysosomal enzymes, @-glucuronidase. N-acetyl-B-Dglucosaminidase and a-L-fucosidase following fractionation using 4024% sucrose gradients. @-glucuronidase, i.e., percent of total recovered @-glucuronidase activity in each fraction is plotted on the ordinate of the second lowest panel. (For comparison adenylate cyclase activity distribution in this preparation is shown on the lowest panel.) Percent of total recovered N-acetyl-@-D-glucosaminidase activity is shown in the third lowest panel and of a-L-fucosidase in the upper panel.
phospholipid to protein ratio was increased 5-fold in the plasma membrane relative to homogenates. Distribution of phospholipid was not identical to that of adenylate cyclase, indicating that other cellular membranes have a fractionation pattern different from that of plasma membrane.
449
REMOLD-O'DONNELL
450
30
20 10
-
n
LACTATE DEHYOROGENASE
"t Downloaded by [Tufts University] at 14:17 03 December 2014
20
be 20 10 0
AOENYLATE CYCLASE
P 2 4 6 8 10 12 14 16 (820 22
FRACTION
FIGURE 3
Distribution of phospholipid, lactate dehydrogenase and succinate dehydrogenase following fractionation. Phospholipid, i.e.. percent of total recovered phospholipid, in each fraction is plotted on the ordinate of the second lowest panel. @denylate cyclase distribution in this preparation is shown on the lowest panel.) Percent of total lactate dehydrogenase activity is plotted on the ordinate of the third panel. Percent of total succinate dehydrogenase activity [succinate-2-(p-iodophenyl)-3-(pnitropheny1)-5-phenyltetrazolium-reductase activity] is plotted on the ordinate of the upper panel.
Fig. 4 shows the distribution of galactosyl transferase, RNA and DNA
in reference to adenylate cyclase. Most of the RNA and DNA was found in the pellet, although low levels of RNA were also present in the plasma membrane fraction, possibly an artifact of the orcinol assay. Whereas glycosyl transferases are generally considered to be components of the Golgi appara-
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
451
GALACTOSYL TRANSFERASE 30 20 10
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0
FRACTION
FIGURE 4 D i s t r i b u t i o n of DNA, RNA (orcinol p o s i t i v e m a t e r i a l ) and g a l a c t o s y l t r a n s f e r a s e a c t i v i t y following f r a c t i o n a t i o n using continuous sucrose gradients.
t u s z 8 , recent findings i n d i c a t e t h a t t h e s e enzymes a r e present on t h e c e l l surface o f various mammalian c e l l s z 9 , such a s
lymphocyte^^^ and f i b r o b l a s t s 3 1 .
Since g a l a c t o s y l t r a n s f e r a s e c o f r a c t i o n a t e s with t h e plasma membrane, two p o s s i b i l i t i e s have t o be considered: 1. t h a t t h e plasma membrane f r a c t i o n s contain elements o f t h e Golgi apparatus o r 2 . t h a t g a l a c t o s y l t r a n s f e r a s e
i s a macrophage plasma membrane component.
452
REMOLWI'DONNELL
From these experiments it can be concluded that the plasma membrane prepared as described contains 7% of the cell protein, 57% of the plasma membrane marker adenylate cyclase, no mitochondria1 components, no lactate dehydrogenase, no DNA, little RNA and a 5-fold increased phospholipid to protein ratio. Furthermore, the contamination with lysosomal enzymes is low (less than 8%).
Galactosyl transferase was found to be present in the
plasma membrane fraction.
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Purity of the Cell Preparation. Although these peritoneal exudate macrophages are contaminated with 10-25% polymorphonuclear leukocytes, the latter were resistant to homogenization. When exudate homogenates which contained 90% o r more ruptured cells were stained with Wright's stain, the remaining intact cells included a large percent of polymorphonuclear leukocytes. The resistance o f polymorphonuclear leukocytes to homogenization was verified by the fractionation pattern of M a s e activity, a plasma membrane marker of polymorphonuclear leukocytes which is not detectable in caseinelicited macrophages of guinea pigs32*33. A large fraction of AMF'ase activity was found in the pellet fraction which contains the unbroken cells. In 3 experiments the pellet fraction which contained 10 activity contained 52
+
?
8% of the particulate AMF'ase.
1% of adenylate cyclase Thus, the contribution
of polymorphonuclear leukocytes to the plasma membrane preparation is only half that indicated by the differential cell count, i.e. 5-12%. In conclusion, a purification method for macrophage plasma membrane was developed which is relatively simple, requires exudate cells from approximately 6 animals and can be completed by one researcher within a day.
Fol-
lowing homogenization, the plasma membrane is in contact with other subcellular elements for only 2 hours. During this time the homogenates are held near O o , at pH 7 . 6 in concentrated sucrose. These conditions minimize lysosomal enzyme release and activity. Furthermore, maximum precautions have been taken to prevent alterations of plasma membrane during purification. Thus, a simple rapid method i.s now available f o r preparing macrophage plasma membrane in high purity and good yield.
PLASMA MEMBRANE OF PERITONEAL MACROPHAGES
453
ACKNOWLEDGEMENTS I wish t o thank D r . John David, D r . Guido G u i d o t t i and D r . Manfred
L. Karnovsky, i n whose l a b o r a t o r i e s t h i s work was performed, f o r s u p p o r t , d i s c u s s i o n and h e l p f u l s u g g e s t i o n s .
I am g r a t e f u l a l s o t o Dr. Robert
Jackson, Dr. Steven C l a r k e , Dr. Eva Neer and D r . Heinz Remold f o r suggest i o n s a n d d i s c u s s i o n , and t o Judy Shaw f o r p e r f o r m i n g t h e a - L - f u c o s i d a s e
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assays.
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After 3 days culture of guinea pig peritoneal exudate cells, when only macrophages remain on the dishes, the ecto-AMPase activity was found to be non-detectable, i.e., less than 0.8 nmoles x mg-l x min-l
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(E. Remold-O'Donnell, unpublished experiments).
