The plasma membrane of the rabbit lens
cortical fiber
I. Isolation, characterization, and biosynthesis of two
membrane intrinsic polypeptides
Mihir Bagchi, Patricia Ann Gordon, Jose Ramon Alcald, and Harry Maisel
Rabbit lens cortical fiber plasma membrane polypeptides were isolated and two membrane
intrinsic proteins were characterized by SDS-PAGE. A polypeptide with a molecular weight of
26 kilodaltons is the major constituent of the plasma membrane. The molecular weight and
antigenic properties of the other polypeptide studied are similar to polypeptides of cortical
water-soluble a-crystallin. Biosynthesis of these two polypeptides was studied. After initially
high rates, synthesis of both of these polypeptides decreased considerably and maintained a
steady rate for the rest of the culture time.
Key words: lens, plasma membrane,membrane intrinsic polypeptides,
organ culture, biosynthesis, SDS-PAGE, crystallins
he cortical fiber cells of the lens constitute
a good source of plasma membranes because
they have a very high ratio of surface to volume. l The technique for their isolation is also
relatively simple. 2 Thus several investigators
have isolated and partially characterized
bovine, chicken, and human lens cortical
fiber cell plasma membranes. 1 " 10 We have
studied rabbit lens cortical fiber cell plasma
membrane polypeptides and have also monitored the synthesis of two detergent solubilized (intrinsic) membrane polypeptides in
the organ cultured lens. It has been found
that a polypeptide with molecular weight of
26 kilodaltons (K) is the predominant polypeptide constituent of the bovine lens plasma
membrane.2 Another polypeptide, which has
electrophoretic mobility and antigenic properties similar to a-crystallin, is also present in
the plasma membrane.2 In organ culture of
lenses these two polypeptides continue to be
synthesized. During the first 12 hr of culture
the rates of synthesis for these two polypeptides were higher than at 24 hr and thereafter
remained stable for another day. The synthetic rate of the a-crystallin-like polypeptide appears to be higher than that of the 26
K polypeptide.
Materials and methods
From the Department of Anatomy, Wayne State University School of Medicine, Detroit, Mich.
This study was supported by NIH grants EY-01848 (MB)
andEY-01855(HM).
A preliminary report of this work was presented at the
1978 ARVO spring meeting in Sarasota, Fla.
Submitted for publication July 24, 1978.
Reprint requests: Dr. Mihir Bagchi, Department of
Anatomy, Wayne State University School of Medicine, Detroit, Mich. 48201.
562
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Organ culture of intact rabbit lens. Lenses
from freshly decapitated New Zealand white rabbits (approximately 2 kg) were cultured in Medium-199 (Grand Island Biological Co., Grand Island, N. Y.)'1 supplemented with 10% rabbit
serum (M-199S) in a closed system.12> 13 The lenses
were cultured at 33.8° ± 1° C. in M-199S having a
pH of 7.2 and an osmolarity of 310 mOsm. The
lenses were- evaluated at six different intervals (6,
0146-0404/79/060562+08300.80/0 © 1979 Assoc. for Res. in Vis. and Ophthal., Inc.
Volume J8
Number 6
12, 18, 24, 30, and 48 hr), and in each experiment
they were exposed to 3 H-leucine (specific activity
62 CI/mM; Schwarz/Mann) at 50 /u,Ci/m] in minimal essential medium lacking unlabeled leucine
(osmolarity 310 mOsm, pH 7.2) for the last 6 hr of
incubation. At least 12 lenses were pooled for each
time period studied.
Isolation of lens cortical fiber cell plasma membrane polypeptides. Following culture, the capsule and the adhering epithelium were removed,
and cortex and nucleus were separated mechanically at 4° C. The cortical fiber mass was
weighed and homogenized in 9 vol (w/v) of 50 mM
Tris-HCl, 5 mM MgCl2, 10 mM 2-mercaptoethanol, at pH 7.4. Nuclear fibers and epithelium
were retained. The plasma membranes were extracted essentially by the method of Alcala et al., 2
but in some cases discontinuous sucrose gradient
centrifugation2 was also employed. By electron
microscopy it was found that the former method
resulted in a much higher yield of plasma membranes, which appeared to be less contaminated
with nonmembranous material.
Electron microscopy. Membrane-rich pellets
were fixed in 4% glutaraldehyde in cacodylate buffer and postfixed in 1% osmium. Thin, sections
were examined under a Philips-201 electron microscope (Mahwah, N. J.).
SDS-PAGE and molecular weight determinations. Sodium dodecyl sulfate—polyacrylamide gel
electro phore sis (SDS-PAGE) was performed with
both disc and slab gels.2 Membrane pellets were
dissolved in 2% SDS solutions at 37° C for 2 hr.
Molecular weight estimations were carried out according to the method of Weber and Osborn. 14
The standard markers consisted of ribonuclease A
(13.7 K), chymotrypsinogen (25 K), ovalbumin
(45 K), aldolase (158 K) (all obtained from Pharmacia Fine Chemicals, Piscataway, N. J.), plus
myoglobin (17.2 K), cytochrome C (12.4 K), lysozyme (14.4 K), catalase (60 K), and phosphorylase-a (92 K) (from Sigma Chemical Co., St.
Louis, Mo.).
Scanning. Gels were stained with either amidoblack or Coomassie blue (G-250), and the relative
density of the stains was determined with a Canalco (Elkhart, Ind.) integrating densitometer at
530 nM.
Isolation of radioactive bands. Stained gels
were sectioned and incubated with hydrogen peroxide (30%) overnight at 60° C, following which 10
ml of Aquasol (New England Nuclear, Boston,
Mass.) were added and the amount of label was
monitored with a Beckman liquid scintillation
counter (LS-3133P) (Palo Alto, Calif.).
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Plasma membrane of lens corticalfibers.I.
563
-45000
54-
32-
-25000
1-
-17600
B
M
Fig. 1. SDS-PAGE of cortical fiber cell plasma
membrane (slab gel). A = Plasma membrane isolated by sucrose density gradient centrifugation;
B = same obtained by urea treatment. M, C, & O
are molecular weight markers; they are, respectively, myoglobin, chymotrypsinogen, and ovalbumin. Arabic numerals (1 to 6) indicate number
of the polypeptide band, the first being nearest to
the front of the gel.
Leucine pool size determination. Pool sizes of
free intracellular leucine were determined with an
automated amino-acid analyzer (Modified Phoenix
A. A. Analyzer).15
Immunological identification. The analytical
immunodiffusion technique 2 was utilized to determine the immunological similarity of band 2
polypeptide (membrane SDS-PAGE preparation)
to rabbit soluble a-crystallin with the use of antisera to lens total soluble proteins and to acrystallin.
Results
Fig. 1 shows a 5.13% polyacrylamide-1%
SDS slab gel. The banding patterns of the
membrane pellets obtained either by sucrose
density gradient centrifugation (A) or by the
urea extraction method (B) were similar. Fig.
2 presents electron micrographs of membrane pellets isolated by sucrose density
gradient (A) and from the urea-insoluble pellet (B), As is apparent from the micrographs,
the urea treatment yielded cleaner mem-
564 Bagchi et at.
Invest. Ophthalmol. Visual Sci.
June 1979
Fig. 2. Electron micrograph of the cortical fiber cell plasma membrane. A, Obtained by
sucrose density gradient. B, Same by urea treatment.
brane preparations. Both membrane preparations showed abundantpentalaminar membrane structures (gap junctions). In most of
these studies urea treatment was used to obtain plasma membranes. 16 Fig. 3 shows a typical disc gel (5.13% polyacrylamide-1% SDS)
preparation of the plasma membrane fraction
stained with Coomassie blue. The solid line
represents the relative abundance of stain in
each band, and the dotted line indicates the
amount of radioactive label present in each of
the gel slices. It is evident from the densitometric scanning of the stained gel that about
50% of lens membrane polypeptides were
present in band 3 and about 13% in band 2.
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The molecular weight of band 2 was calculated as 21,000 daltons (21 K) and that of band
3 as 26,000 daltons (26 K). A polypeptide
with 26 K molecular weight is also reported
to be the predominant polypeptide of the
bovine, chicken, and human lens cortical
fiber plasma membranes.'" 3 - 8- ">• !7- 18 Bloemendal1 has named this polypeptide MP-26,
It has been claimed that MP-26 is the main
constituent of plasma-membrane gap junctions, 1 ' 4' 19 but no definitive experiments
have been done to demonstrate that the
MP-26 is specific gap junction polypeptide.
The other polypeptide (20 K) had about the
same molecular weight as a water-soluble
Volume 18
Number 6
Plasma membrane of lens cortical fibers. 1.
565
Fig. 3. SDS-PAGE of cortical fiber cell plasma membrane. Solid line indicates the relative
abundance of the stain in each band and dotted line indicates CPM/slice in the gel. Each point
represents one slice of the gel.
a
Fig. 4. SDS-PAGE of water-soluble a-crystallin
obtained by Ultrogel gel filtrations on the left and
total plasma-membrane polypeptide profile on the
right. a-Crystallin and band 2 of the plasma membrane have similar electrophoretic mobility.
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Fig. 5. Band 2 of the plasma membrane was isolated and run with a-crystallin in a 10% polyacrylamide-1% SDS disc gel. Both band 2 and
a-crystallin showed two bands with similar electrophoretic mobility.
Invest. Ophthalmol. Visual Sci.
June 1979
566 Bagchi et al.
3
SF
B2
Fig. 6. Analytical immunodiffusion reactions of
band 2 (B2) of the plasma membrane, watersoluble proteins (SF), a-crystallin isolated by gel
filtration, with antisera to the a-crystallin. All
three proteins gave a single confluent precipitin
band, indicating identity.
a-crystallin polypeptide. The presence of
crystallins has been demonstrated in chicken,
bovine, and human cortical fiber cell plasma
membranes. 2 ' 3j 5l 7l l0 Further experiments
were performed to partially characterize this
band. Fig. 4 shows two disc gels (1% SDS5.13% polyacrylamide), one of total membrane polypeptides and the other of watersoluble a-crystallin obtained by the ultrogel
(ACA/24, LKB, Rockville, Md.) gel-filtration
technique (Bagchi, in preparation). Band 2 of
the membrane preparation and a-crystallin
polypeptides have similar electrophoretic
mobilities. Band 2 was isolated2 and run with
a-crystallin in a 10% polyacrylamide-1%
SDS gel. Under these circumstances acrystallin separates into its two main subunits a A and acB.l Fig. 5 clearly shows that the
membrane isolated band 2 polypeptides also
separated into two bands with electrophoretic mobilities similar to a A and a B . Analytical immunodiffusion was performed with
anti-a antiserum, and as is evident from Fig.
6, soluble a-crystallin obtained by gel filtration, total water-soluble proteins (CS,, see
Bagchi and Gordon15), and band 2 of the
membrane preparation all give a single, confluent precipitin band, indicating identity. It
seems likely that a major constituent of this
band contained polypeptides with the antigenic properties of a-crystallin.
The specific activity of the intracellular
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H-leucine pool of incubated lenses was determined at intervals corresponding to those
used in measuring polypeptide synthesis.
Since the specific activity of the 3 H-leucine
pool varied, the cpm/mg of the polypeptides
was expressed in terms of the specific activity
of the 3 H-leucine pool at the end of the initial
6 hr incubation (for further details see Bagchi
and Gordon15). Fig. 7 depicts the rates of synthesis of bands 2 and MP-26; both polypeptides showed their highest rate of synthesis
during the initial 12 hr, decreasing considerably by 18 hr and remaining stable over the
next 30 hr. It was found that the rate of synthesis of band 2 and MP-26 was similar for
the first 6 hr or culture and that thereafter
the rate of synthesis of band 2 was greater
than that of MP-26. Both band 2 and MP-26
showed a wide range of synthetic activity
when evaluated at the first 6 hr of culture (six
experiments). But in each experiment, when
12 lenses were pooled, the synthetic activities of band 2 and MP-26 were nearly
identical. At least two experiments were performed for the remainder of the time periods
studied, and the synthetic rate of band 2
or MP-26 showed statistically insignificant
variation (Student's t test). As indicated
above, band 2 of the membrane preparation
had antigenic and electrophoretic characteristics similar to those of soluble a-crystallin polypeptides; Fig. 8 compares the
synthetic activity of band 2 with that of
a-crystallin polypeptides obtained by gel filtration of the total water-soluble proteins and
SDS-PAGE. Band 2 shows a relatively higher
synthetic rate compared to that of a-crystallin
polypeptide.
Discussion
The polypeptide composition of the lens
cortical fiber cell plasma-membrane has been
described for several mammalian species and
for the chicken. l"3> 9l 16) 18 Some progress has
also been made in identifying polypeptides of
the lens cortical fiber plasma membranes that
may be associated with defined membrane
regions. 4 ' 5 ' 17 However, not enough is known
to assign specific physiological functions to
these pepudes.
The two polypeptide species common to all
Volume 18
Number 6
Plasma membrane of lens cortical fibers. 1. 567
50
50 r
40
40
30
a
| 30
Band 2
Bond 2
20
20
\
10
a Crystallin
o
10
«Band3
o
6
12
18
24
30
36
42
6
12
18 24 30
Time (hrs)
36
42
48
48
Time (hrs)
Fig. 7. Time course of incorporation of 3H-leucine
into plasma membrane polypeptides band 2 and
MP-26. Lenses were exposed to isotope 6 hr prior
to isolation of the protein. Each point represents
data collected from at least 12 lenses (pooled). Bars
(6 hr) represent standard deviations.
lens fiber plasma membranes studied thus far
are MP-26 and a crystallin polypeptide. In
mammalian species the latter is a-crystallin;
in birds it is 8-crystallin.3 The possible physiological and evolutionary significance of
these differences is of some interest.
Experiments were performed in our laboratory to study the synthetic activity of these
two membrane polypeptides in the organcultured lens. It was hoped that their synthetic activity might give us some insight into
their possible functions. It was found that
both proteins, MP-26 and band 2, after an
initial higher rate of synthesis, maintain a
stable synthetic rate throughout the culture
period. Hunter and Segal20 proposed that
membrane polypeptide synthesis is often associated with the regulatory mechanisms
used by the cells in order to adjust their
transport systems. In the lens, maintenance
of intercellular transport is extremely important, since the organ is not vascularized. The
physiological significance of the steady-state
synthesis of the MP-26 and band 2 by the
cortical fiber cells is not clear.
MP-26 has been strongly implicated as the
main constituent of plasma-membrane gap
junctions. ** 4l 21 Vermorken et al. 22 stated that
the molecular mechanism of gap-junction assembly may rely either on de novo synthesis
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0
Fig. 8. Time course of incorporation of 3H-leucine
into band 2 of the plasma membrane and watersoluble a-crystallin lenses were exposed to isotope
6 hr prior to isolation of the proteins. Each point
represents data collected from at least 12 lenses
(pooled). Bars (6 hr) represent standard deviations.
of membrane constituents or on lateral displacement within the plane of the plasma
membrane of a pre-existing pool of junctional
constituents. It was also reported 22 that
MP-34, a membrane polypeptide of 34 K molecular weight, can be synthesized in a cellfree system with cortical fiber cell polysomes.
In those experiments synthesis of MP-26
could not be detected. Our organ culture experiments clearly showed a steady rate of
synthesis of MP-26 polypeptides throughout
the culture period. Further experiments are
needed to explore the physiological role of
MP-26. Since gap junctions are thought to
facilitate the intercellular transport of ions,
amino acids, nucleotides, and other biologically important materials, 23 these structures
may be of great significance in the metabolic
economy of the lens cortex.
Band 2 is the other polypeptide whose synthetic activity was evaluated. Band 2 has a
molecular weight and antigenic properties
similar to those of the water soluble acrystallin, but it is isolated together with the
rest of the membrane polypeptides, which
are water- and urea-insoluble. It is important
to note that this polypeptide becomes water
soluble when the membrane is dissociated
with a detergent such as SDS. Even though
this polypeptide comprises only 13% of the
total membrane protein, it has a relatively
high synthetic rate compared to other mem-
568 Bagchi et al.
brane polypeptides. This band also has a significantly higher synthetic rate than the
water-soluble a-crystallin. However, it is
known that a-crystallins are a group of proteins having the same polypeptide composition and antigenic properties.24' 25 So it is
possible that only one species of these proteins is synthesized, and posttranslational
modifications give rise to different protein
species. Spector et al.26> 27 showed that one
species of a-crystallin, referred to as HL protein (highly labeled protein), has a synthetic
rate that is 3 to 4 times higher than total
a-crystallin. The HL protein comprises only
10% of the total water-soluble a-crystallins.
Studies in progress using DEAE column
chromatography and iso-electric focusing are
designed to compare our band 2 polypeptide
with the HL protein.
Since both band 2 and MP-26 polypeptides
showed the highest synthetic rates and a significant range of variation in their synthetic
activities during the first 6 hr of culture, it is
possible that freshly isolated lenses require
some time in the incubation medium before
steady-state conditions are re-established. It
is not clear at this time why the synthetic rate
of band 2 is higher than that of MP-26 after an
initial similar rate of synthesis. After the first
12 hr of culture both band 2 and MP-26
showed decreased rates of synthesis and then
maintained a stable rate of synthesis for the
rest of the incubation period.
We thank Drs. V. N. Reddy, H. Rothstein, and A.
Weinsieder for critically reading this manuscript and
Mr. B. Chakrapani for performing the free leucine estimation in Dr. V. N. Reddy's laboratory.
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