Download Regional Differences in Protein Synthesis within the Lens of

Regional differences in protein synthesis
within the lens of the rat
Richard W. Young and H. William Fidhorst
Utilization of 3SS-methionine by cells of the rat lens was studied by combined autoradiographic and chemical techniques. Part of the methionine teas converted to cysteine, glutathione, and taurine. Protein synthesis was essentially restricted to the lens cortex, and appeared to be largely attributable to growth. One day after injection, over 90 per cent of total
lens radioactivity was recovered from the cortex, most of it in water-soluble protein. Eight
labeled protein fractions were separated. Continued peripheral addition of new fibers progressively buried the labeled cells within the body of the lens, so that at 4 weeks the proportion of total radioactivity recovered from the deeper fibers was increased. Insoluble protein
(albuminoid) appeared to be largely derived from the precipitation of certain soluble proteins
in the lens nucleus. Such selective insolubilization may account for the differing patterns of
soluble protein observed in the inner and outer parts of the lens.
Methods
.he ocular lens is unique among mammalian organs in that it is wholly composed of a single, epithelial cell type. Despite continued production of new cells,
older cells are not shed. Instead, they are
incorporated directly into the body of the
lens itself. Thus, it is a population of cells
characterized by growth, rather than renewal.
The protein metabolism of these cells
may be effectively studied with labeled
amino acids. An investigation of the utilization of 3r>S-methionine by cells of the
lens in young adult rats, analyzed by autoradiographic and radiobiochemical techniques, is reported below.
Autoradiography. Female Long-Evans rats, 8
weeks of age, were used. Six animals were injected intraperitoneally with 4 fie per gram of
body weight of 35S-L-methionine in aqueous solution at a concentration of 1.83 me. per milliliter, and a specific activity of 0.65 me. per milligram.* The rats, ranging in weight from 140 to
156 Cm. (mean 150 Cm.), were killed 4, 8, 24,
and 96 hours, 1 and 4 weeks after injection.
The eyes from each animal were fixed in buffered neutral 4 per cent formaldehyde for 2 days.
They were then double-embedded in nitrocellulose-paraffin, sectioned parasagittally at 5 ft, and
prepared for autoradiography by the dipping
technique with Kodak NTB2 liquid emulsion.
Sections were stained with periodic acid-Schiff
(PAS) before coating with emulsion, and hematoxylin after development, or hematoxylin only,
after development of the autoradiograms. The
preparations were developed in Kodak Dektol
for 2 minutes at 17° C. after exposure in a cold,
dry atmosphere for periods ranging from 1 to 3
weeks.
The concentration of silver grains was deter-
From the Department of Anatomy, Center for
the Health Sciences, University of California
at Los Angeles, Los Angeles, Calif. 90024.
This work was supported by United States Public
Health Service Grant NB-03807.
'Radinchemical Centre, Amersham, England.
288
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Volume 5
Number 3
mined in several sections taken at or near the
midsaggital plane at the 1 day and 4 week intervals, using an ocular grid graduated in squares
(each 57 n2 at 1200 x magnification). The number of grains per square was recorded along a
continuous strip, extending across the lens at the
equator (excluding the epithelium and capsule).
The counts from one side were combined with
the corresponding counts on the opposite side,
then averaged. The averages (after subtraction
of background) were then expressed as a percentage of the average concentration in the most
heavily labeled square (to eliminate differences
due to dosage, development, isotope decay, etc.).
Radiobiochemistry. Thirty female Long-Evans
rats, 8 weeks of age, were injected intraperitoneally with 4 y.c per gram of body weight of
35
S-L-methionine in aqueous solution (2 me. per
milliliter) at a specific activity of 0.51 me. per
milligram. The rats were divided into two groups
of 15 each. The animals in group I ranged in
weight from 143 to 170 Gm. (mean 154 Gm.);
those in group II weighed 141 to 171 Gm. (mean
161 Cm.). Group I was killed 1 day after injection; Croup II 27 days later.
The rats were killed individually by an overdose of chloroform; both eyes were enucleated,
and the lenses dissected free of ciliary body,
vitreous, and lens capsule under the dissecting
microscope. The dissections were controlled by
histologic examination of comparably prepared
lenses. In the following procedure, "water" denotes distilled water adjusted to pH 7 with a
minimum of phosphate bufFer. All centrifugation
was carried out at 2,500 r.p.m. in the cold
(4° C.).
The method initially devised by Krause,1 and
used in a number of subsequent investigations,
was employed for separating the superficial (cortical) and central (nuclear) portions of the lens.
The lenses were collected in a capped centrifuge
tube containing 4 ml. of water, and maintained
in an ice bath. After all the lenses had been dissected, the centrifuge tube was shaken by hand
for 3 minutes. It was then centrifuged for 2.5
hours. The supernatant was taken to 5 ml. with
water (cortical extract). The residue could be
seen under the dissecting microscope to consist
of lens nuclei and a few sedimented free fibers.
This material was transferred to a Potter-Elvehjem homogenizing flask. The centrifuge tube was
then washed twice with the 1.5 ml. of water, the
washes being added to the homogenizing flask.
The residue was next homogenized in the cold
for 5 minutes, then transferred to a capped centrifuge tube. The homogenizing flask was then
washed with 1.4 ml. of water; the wash was
added to the homogenate, and the homogenate
centrifuged for 1.5 hours. The supernatant was
then taken to 5 ml. with water (nuclear extract).
Regional differences in protein synthesis 289
The homogenizing flask was then washed again
with 0.8 ml. of water, and the washings added to
the residue, which was suspended by gentle shaking and stored at 4° C. overnight, to assure the
complete extraction of water-soluble material. It
was then centrifuged for 2 hours, and the supernatant (albuminoid wash) taken to 2 ml. with
water. The residue was lyophilized, then taken
up as a suspension in 10 ml. of chloroform:
methanol (2:1), and incubated overnight at
4° C. This solution was centrifuged for 0.5 hours,
after the addition of 0.2 volumes of methanol
to sediment the residue. The supernatant was removed, and the extraction repeated with 5 ml.
of chloroform:methanol (lipid extract). The residue was dried overnight in a vacuum desiccator,
taken up as a suspension in 9 ml. of 6N HC1, and
hydrolyzed under reflux for 20 hours at 107° C.
The hydrolysate was then taken to 10 ml. with
water (albuminoid hydrolysate).
Four aliquots from each fraction were plated
on preweighed copper planchets. The planchets
were reweighed after drying, the difference in
weight representing the weight of the sample. The
weight of the hydrolysate obtained in this manner included the weight of the associated HC1.
Accordingly, the net weight of the hydrolysate
was obtained by subtracting from the gross weight
the weight of a comparable volume of 6N HC1
solution. Radioactivity was analyzed in a thin
end-window, gas-flow Geiger-Muller counter.
Counts were corrected for background, for selfabsorption (using a constant activity sodium 35 Ssulfate correction curve), and for isotope decay.
The means and standard errors were averaged.
Four 10 fi\. aliquots of the aqueous extracts were
taken for a microbiuret determination of protein
concentration,* using a bovine serum albumin
standard. The four values were averaged.
Continuous flow electrophoresis was performed
at 4° C. on S & S 598 paper curtains in a Misco
electrochromatography chamber, \ at 774 V., 4
to 5 ma., in pH 8.6 veronal buffer, ionic strength
0.019, using a 3.8 mm. wide internal wick. Aliquots of 1 ml. from the cortical and nuclear extracts were run for 18 to 19 hours. The curtains
were stained with bromphenol blue after heat
fixation (110° C. for 30 minutes). The unbound
dye and nonprotein sample constituents were
then removed by prolonged washing in 6 per
cent acetic acid. After analysis by autoradiography
with x-ray film, a 3 cm. wide horizontal strip
was cut from the curtain near its lower edge. The
amount of protein-bound dye on the strip was
then measured with a densitometer equipped with
an external galvanometer, \ using a 510 m/i nar"Coleman Instruments, Maywood, 111.
fMicrochemical Specialties, Berkeley, Calif.
JPhotovolt model 425, Photovolt Corporation, New York.
N. Y.
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Investigative Ophthalmology
Juno 1966
290 Young and Fulhorst
Fig. 1. Outer cortical zone at the equator of the lens of an 8-week-old rat killed 1 day after
injection of Hr>S-methionme. Silver grains are concentrated at the periphery, in the region of
lens fiber formation. (Autoradiogram, hematoxylin, x200.)
row-band interference filter. The strips were then
assayed for radioactivity in a chromatogram strip
counter. Areas under the dye-binding and radioactivity curves were determined by planimetry.
Extraction with 10 per cent trichloroacetic acid
(TCA) was used to determine the partition of
radioactivity between protein and nonprotein
constituents in two aliquots from each of the
three aqueous extracts. An equal volume of
20 per cent cold TCA was added, the solution
incubated at 4° C. for 15 minutes, the supernatant obtained by centrifugation, and the residue
(precipitate) washed three times with cold 10
per cent TCA. The combined supernatants (representing the nonprotein fraction) were then extracted three times with equal volumes of ether
(which removed most of the TCA, but none of
the radioactivity), taken to 2 ml. with water, and
two 0.1 ml. aliquots of each plated, weighed,
and counted in the C-M counter. The TCA precipitates were washed twice with ethanol, then
three times with ether, and hydrolyzed with 6N
HC1.
The free amino acid fraction of the aqueous
extracts obtained at the 1 day interval was prepared by ultrafiltration through dialysis tubing
under vacuum in the cold. This fraction, the albuminoid hydrolysates, and the hydrolysates of
the TCA precipitates were lyophilized, then taken
up in a small volume of water or 80 per cent
ethanol, and analyzed by thin-layer chromatography (TLC). Aliquots were spotted on 20 by
20 cm. glass plates coated with a 250 fi layer of
silica gel G.° Butanol (normal):acetic acid;water
•Brinknwnn Instruments, Westbury, N. Y,
(3:1:1) was used for development in the first
dimension, and phenol:water (4:1) in the second
dimension. The plates were sprayed with ninhydrin (0.2 per cent in n-butanol), and the spots
identified by comparison with standard maps derived from running known reagents under comparable conditions. Radioactivity was detected by
apposition of Kodak Blue Brand Medical x-ray
film for periods ranging from 4 days to 1 month.
Results
Autoradiography. Some labeling was observed in all ocular structures. Most heavily reactive at the early intervals were the
lens, corneal epithelium, retina, and ciliary
epithelium. At later intervals (1 to 4
weeks), the lens exhibited a greater retention of labeling than these other tissues.
In the lens, at 4 and 8 hours after injection, the autoradiographic reaction was
almost entirely restricted to the cortex,
within which it was particularly heavy in
the more superficial fibers. The most intense labeling occurred at the equator, in
the region of lens fiber information.
Deeper in the lens, the reaction decreased
progressively, and was negligible over the
center of the lens. The lens epithelium was
moderately labeled. The reaction was
weaker, but significant, over the capsule.
There was little apparent change at 1
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Volume 5
Number 3
Regional differences in protein synthesis 291
Fig. 2. A, lens of a 12-week-old rat killed 28 days after injection of 35S-methionine. Peripheral
addition of lens fibers during the 4 week interval has buried the heavily labeled fibers within
the lens body, yielding a reaction band (arrow) which outlines the limits of the lens as it existed at 8 weeks. The black areas over the lens nucleus ate artifacts. (Autoradiogram, PASheniatoxylin. x20.) B, details of the equatorial region of the same lens. Note the progressive
decline in labeling intensity in new lens fibers at the periphery. {x50.)
day (Fig. 1). By 4 days, the first few of
the most superficial lens fibers in the equatorial region were labeled somewhat less
intensely than the immediately subjacent
fibers, in which labeling was greatest.
From 1 through 4 weeks, a continuation of
this process had yielded a complete, superficial coating of progressively less radioactive lens fibers, thickest at the equator,
and thinnest at the poles (Fig. 2). Capsular labeling appeared not to have decreased, but the epithelium was now only
slightly reactive at the exposures used. The
central portion of the lens nucleus remained unlabeled.
An analysis of the concentration of silver
grains along an equatorial axis extending
from the center of the lens to the periphery
at 1 day and 4 weeks is given in Fig. 3.
It is evident that most of the bound radioactivity ascribed to the lens nucleus at 4
weeks had actually been utilized by cells
initially located in the cortex. These labeled fibers were subsequently buried by
the peripheral apposition of more superficial lens fibers. As a result, an increased
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292 Young and Fulhorst
Invcsligativc Oiththalmology
June 1966
proportion of radioactivity was recovered
in the second or "nuclear" extract, as described below.
Radiobiochemistry. Autoradiographic examination of the 35S-L-methionine solution, separated by two-dimensional TLC,
demonstrated the presence of several
SILVER GRAIN
CONCENTRATION
Fig. 3. Distribution of silver grains in autoradiograms of lenses from rats sacrificed 1 day (above)
and 28 days (below) after injection of 35S-methionine. Grain concentration was analyzed in a zone
extending from the equatorial surface (left) to
the center of the lens nucleus (C, arrow). The
shaded area under the curve represents the approximate proportions of total radioactivity recovered in the cortical extract at each interval
(cf. Tables I and II). Note that most of the
radioactivity recovered in the nuclear extract at
28 days was present in the cortex at 1 day.
weakly labeled contaminants. Analysis of
one-dimensional separations in the strip
counter indicated that these represented
less than 3 per cent of the total radioactivity.
Histologic examination of lenses dissected by the procedures used in this part
of the study revealed that adherence of the
lens epithelium to the capsule resulted in
removal of the epithelium during decapsulation. Consequently, the following results
are based on analysis of the lens proper,
free of both capsule and epithelium.
The residue of the first or "cortical" extract was also examined histologically, in
5 M paraffin sections stained by a variety of
procedures. This material indicated that
loss of protein from the lens nuclei was
negligible, and that the cortical extract
contained only the outermost lens fibers.
The distribution of radioactivity among
the several lens fractions isolated from
animals killed 1 day after injection is given
in Table I. The major portion (by weight)
of the lens material is soluble in water, and
most of this material is protein. The second most prominent fraction is water-insoluble protein, termed albuminoid. (The
albuminoid weight includes the water
taken up during peptide bond hydrolysis.)
The total amount of water-soluble protein
recovered in the cortical and nuclear extracts was roughly comparable. Subsequent overnight extraction of the residue
yielded only a small, additional amount of
water-soluble protein. Lipid constitutes a
minor fraction of the lens material.
Most of the radioactivity was recovered
Table I. Distribution of radioactivity among different lens constituents 1 day
after administration of 3fiS-methionine
Weight
(ma.)
Protein
Lens fraction
Counts per minute
(mean +. S.E.)
Per cent
total c.p.m.
Per cent c.p.m.
in protein
Cortical extract
Nuclear extract
Albuminoid wash
Lipid extract
Albuminoid hydrolysate
Total
178.7
121.6
17.1
5.3
123.7
446.4
138.0
112.0
12.4
—
—
262.4
995,965 ± 3705
58,615+ 480
5,998 ± 64
479 ± 11
17,290+ 166
1,078,347 ± 3740
92.4
5.4
0.6
<0.1
1.6
100.0
80.2
77.4
70.2
—
—
—
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Regional differences in protein synthesis 293
Volume 5
Number 3
Table II. Distribution of radioactivity among different lens constituents 28 days
after administration of 3fiS-methionine
Weight
(ma.)
Protein
Lens fraction
Counts per minute
(mean ± S.E.)
Per cent
total c.p.m.
Per cent c.p.m.
in protein
Cortical extract
Nuclear extract
Albuminoid wash
Lipicl extract
Albuminoid hydrolysate
Total
170.6
187.0
28.2
6.7
177.7
570.2
132.7
147.7
15.4
1,025,846 ± 3589
327,945+1130
27,742+ 187
1,361 + 19
49,545+ 305
1,432,439 + 3782
71.6
22.9
2.0
<0.1
3.5
100.0
89.9
90.8
89.9
—
—
—
—
295.8
in the aqueous extract of the superficial fibers (92.4 per cent). Only 5.4 per cent
occurred in the subsequent aqueous extract of the lens nuclei, and less than 1 per
cent in the albuminoid wash. Labeling of
the lipid fraction was negligible. The insoluble protein contained veiy little 3r>S, accounting for only 1.6 per cent of the total
radioactivity in the lens.
Extraction of the aqueous extracts with
10 per cent TCA removed some of the
water-soluble radioactivity. This fraction,
considered to represent nonprotein, ranged
from 20 to 30 per cent of the total.
Table II presents comparable data derived from animals killed 28 days after injection. The amount of radioactivity was
greater (by about 25 per cent). In the
nuclear fraction, the percentage of total
radioactivity had increased markedly, constituting 22.9 per cent of the total (compared to 5.4 per cent at 1 day). About 10
per cent of the water-soluble radioactivity
was extracted with TCA, and was therefore ascribed to nonprotein. The proportion of total radioactivity occurring in the
albuminoid had increased over the 1 day
figure (3.5 per cent compared to 1.6 per
cent).
Two-dimensional TLC separations of the
free amino acid fraction of the lens, 1 day
after injection, yielded up to 30 ninhydrinpositive spots. No effort was made to systematically identify all of these. However,
the distribution appeared to be similar to
that previously described in the rat- and
other mammalian lenses.3' ' The largest
spots were due to reduced glutathione
(GSH), taurine, and glutamic acid.
Methione was present in small amounts.
No cysteine, cystine, or oxidized glutathione were detected.
Several radioactive constituents were
revealed by autoradiographic analysis of
Gly
(TluSer
Fig. 4. Map of the thin-layer chromatographic
separation of albuminoid hydrolysate. The origin
is shown by a black dot. The direction of the
second development (phenol:water) is indicated
by an arrow. P, phenylalanine; L, I, leucine, isoleucine; T, tyrosine; M, methionine (radioactive);
V, valine; Pro, proline; A, alanine; Gly, glycine;
Th, threonine; Ser, serine; GA, glutamic acid;
AA, aspartic acid; H, histidine; C, cysteine (radioactive); Ar, arginine; Ly, lysine. Cystine was not
detected, perhaps due to its destruction during
hydrolysis.
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294
Investigative Ophthalmology
June 1966
Young and Fulhorst
Table III. Distribution of protein and radioactivity at 1 day (cortex) and
28 days (nucleus) after injection of 8r'S-methionine
Protein fraction
Source
Protein"
Cortex
Nucleus
Radioactivity!
Cortex
Nucleus
a
P,
P*
&
Pt
1 ft
y<
Y*
32.2
22.1
8.7
6.3
15.8
8.9
7.3
6.8
4.8
9.9
5.0
7.1
14.5
24.2
11.7
14.7
24.7
20.7
1.6
2.4
27.0
9.9
5.2
6.9
3.1
6.0
1.6
5.1
20.6
29.5
16.2
19.5
"Per cent of total protein-bound dye.
tPer cent of total radioactivity. The variations in per cent protein/per cent radioactivity probably reflect differing proportions of sulfur-containing amino acids in the several protein fractions.
these separations. The labeled components
have been identified as methionine (including sulfone-sulfoxide), taurine, GSH,
and three additional spots with relatively
low Rt values in both solvents, tentatively
identified as cystathionine, cysteic acid,
and cysteinesulfinic acid.
Autoradiographic analysis of TLC separations of the hydrolysates of water-soluble and water-insoluble proteins in both
experimental groups indicated that some
of the 35S had been incorporated into protein as a constituent of cysteine, as well as
in methionine. A typical map of the albuminoid hydrolysate is given in Fig. 4.
Eight water-soluble protein fractions
were resolved by continuous flow electrophoresis of cortical and nuclear extracts
from both groups of rats. Two additional
fractions, one with high anodal and the
other high cathodal mobility, temporarily
bound the dye, then faded and disappeared in the staining bath. The extracts
also contained several components, probably nonprotein in nature, which fluoresced
in ultraviolet light before, but not after
staining.5
The pattern of distribution of the eight
stable protein fractions* differed in the
inner and outer portions of the lens in a
similar fashion in both groups of animals
"Classically, the soluble lens proteins have been divided
into three groups (a, /?, and 7). In paper strip electrophoresis (pH 8.6) rat lenses yield four fractions, the
middle two of which are considered to be ft components.
By comparing densitometric analysis of such strips with
similarly analyzed continuous flow separations, the eight
fractions separated by the latter technique have been
classified.
(Fig. 5). The a component (highest net
negative charge) and the /?2 fraction were
stronger in the cortex; /?1} /3;,, and /?5 were
low and similar in both cortex and nucleus. Both y components (lowest net
negative charge) were stronger in the nucleus of the lens. The relative distribution
of total soluble protein among these fractions is given in Table III.
One day after injection, there was very
little radioactivity in the soluble proteins
of the more centrally located lens fibers.
However, all proteins derived from the
more superficial fibers of the cortex showed
appreciable activity at this interval. Over
25 per cent of the soluble protein radioactivity was in the (32 fraction, almost 25
per cent in the a component, and about
36 per cent in the two y fractions. The
remaining four (3 fractions, constituting
about 25 per cent of the total protein, contained less than 12 per cent of the total
protein radioactivity. No evidence of a
minor, highly labeled fraction was obtained.0
Of particular interest was the distribution of radioactivity in the nuclear fraction
at 4 weeks, in the light of the above evidence that these proteins are synthesized
in the cortex. The nuclear pattern of radioactivity, like that of the proteins with
which it was associated, differed significantly from that of the cortex. The a and
/?2 fractions together contained only about
30 per cent, and the remaining /? components about 20 per cent of the total activ-
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Volume 5
Number 3
Regional differences in protein synthesis 295
Fig. 5. Continuous flow electrophoresis of soluble lens proteins. The anodal pole is on the
left. A, curtain showing distribution of separated proteins in the cortical extract (1 day
group). Note the heavy staining of the a fraction (left), fi» fraction (center, arrow), and the
two y fractions (right). B, autoradiogram of the separation, depicting the distribution of radioactivity among the several protein components. C, curtain showing the protein pattern obtained from the nuclear extract (4 week group). There is a marked predominance of the y
fractions (arrow). D, the corresponding autoradiogram. There are cortical-nuclear differences
in the distribution of protein and protein-bound S35.
ity. Thus, the two y fractions comprised
roughly one-half of the total activity associated with the nuclear water-soluble
proteins. Since most of the radioactivity
contained in the nucleus at 4 weeks was
present in the cortex at 1 day, these data
are presented for comparison (Fig. 5 and
Table III). (The cortical patterns of protein and radioactivity at 4 weeks were
similar to those at 1 day.)
Discussion
Hf
'S, injected as a component of methionine, was subsequently recovered from
the lens in cysteine, glutathione, and
taurine, as well as in methionine. Evi-
dently, cells of the lens are capable of
converting methionine to cysteine which,
in turn, is used in the synthesis of protein
and the tripeptide, glutathione, and in part
is converted to taurine.2' 7 3
Protein synthesis in the lens is almost
entirely restricted to the outer, cortical
cell layers. 0 ' ao1 Over 90 per cent of total
lenticular radioactivity was localized in
the superficial fibers at 1 day, most of it
in protein. Continued peripheral addition
of new lens fibers progressively buried the
heavily labeled cells which were present
near the surface at 1 day. During succeeding days, the rate of 35S incorporation
dropped sharply, as the level of circulat-
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296 Young and Fulhorst
ing amino acid 35S decreased, as shown
by the rapid decline in silver grain concentration at the lens periphery in the
older animals. As a consequence, at 4
weeks a higher proportion of total radioactivity was recovered in the nuclear protein fraction.
Short-term radioisotope studies have
suggested that there is a rapid turnover
of lens proteins.9-12'13 However, in the
current material, more protein radioactivity
was recovered from the lens 4 weeks after
a single injection of labeled methionine
than at 1 day after injection. A significant
decrease in activity might be expected,
despite the continued availability of very
low levels of amino acid 35S, if any considerable breakdown of protein had taken
place within the 27-day interval. The lens
alone, among several initially labeled ocular structures, retained appreciable concentrations of radioactivity at later intervals. Autoradiograms of lenses from rats
killed as late as 11 weeks after injection
of 3H methionine failed to show any significant depletion of the radioactivity incorporated in the first few days.14 A relatively low level of protein turnover is
certainly not excluded by these data, since
radioactivity liberated by degradation of
lens proteins might be reutilized in situ
in the synthesis of new protein. Nevertheless, the findings appear to indicate that
protein synthesis in the lens during the
period studied is largely attributable to
growth.
Such growth is accompanied by a net
production of albuminoid, which occurs
predominantly in the lens nucleus.1':5> 1C
There appear to be two possible mechanisms of albuminoid formation:
1. Direct synthesis from amino acid precursors. Lerman and associates17 apparently observed some direct incorporation
of labeled amino acids into the albuminoid
of rat lenses in vitro. About 2 per cent of
total protein radioactivity was recovered
in the albuminoid fraction 1 day after
injection in the current work. This figure
appears too low, and the interval too long
7»ijcs
lhjc Ophthalmology
June 1966
after injection, to be ascribed with certainty to direct synthesis.
2. Insolubilization of initially soluble
proteins. Albuminoid accumulates deep
within the lens, in a microenvironment in
which protein synthesis is negligible. Factors which lower the rate of soluble protein synthesis have little or no effect on
albuminoid accumulation.18"20 The observed increase in the proportion of total
protein radioactivity recovered in albuminoid over the 4-week period studied
is also consistent with the view that albuminoid is formed by the insolubilization
of soluble protein. Additional experiments,
reported separately,14 strongly support
this contention.
Albuminoid formation by preferential
insolubilization of certain soluble protein
fractions could contribute to the differences in the patterns of protein1- -1"-5 and
protein-bound 35S in the inner and outer
parts of the lens. Such selective precipitation of soluble protein fractions in the lens
nucleus would have the effect of leaving
a residual pattern of soluble nuclear proteins which differs from that present in the
more superficial fibers.
The technical assistance of Mrs. Mirdza Berzins
is gratefully acknowledged.
REFERENCES
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lenticular proteins to their chemical composition, Arch. Ophth. 10: 788, 1933.
2. Dardenne, U., and Kirsten, C : Presence and
metabolism of amino acids in young and old
lenses, Exper. Eye Res. 1: 415, 1962.
3. Malatesta, C : Studio cromatografico degli
amino-acidi liberi nei liquidi endoculari, nel
cristallino e nel sangue di bue, Boll. d'Oculistica 31: 762, 1952.
4. Recldy, D. V. N., and Kinsey, V. E.: Studies
on the crystalline lens. IX. Quantitative
analysis of free amino acids and related compounds, INVEST. OPHTH. 1: 635, 1962.
5. Wood, D. C : An electrophoretic study of
soluble lens proteins from different species,
Am. J. Ophth. 51: 305, 1961.
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protein, INVEST. OPHTH. 3: 517, 1964.
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Volume 5
Number 3
Regional differences in protein synthesis 297
7. Mandel, P., Dardenne, U., and Lessinger, A.:
Incorporation et degradation de la methionine par le cristallin de bovides, Compt. rend.
Acad. sc. 245: 985, 1957.
8. Kinsey, V. E., and Merriam, F. C.: Studies
on the crystalline lens. II. Synthesis of glutathione in the normal and cataractous rabbit lens, Arch. Ophth. 44: 370, 1950.
9. Waley, S. C : Metabolism of amino acids in
the lens, Biochetn. J. 91: 576, 1964.
10. Lessinger, A., and Mandel, P.: Incorporation
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11. Ilanna, C : Changes in DNA, RNA, and protein synthesis in the developing lens, INVEST.
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on the crystalline lens. III. Incorporation of
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13. Weber, D.: Uber die mittlere Lebensdauer
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16. Dische, Z., Borenfreund, E., and Zelmenis,
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lens protein of normal, galactose and xylosefed rats, Am. J. Ophth. 51: 1012, 1961.
Dische, Z., Borenfreund, E., and Zelmenis,
C : Proteins and protein synthesis in rat
lenses with galactose cataract, Arch. Ophth.
55: 633, 1956.
Dische, Z., Elliott, J., Pearson, E., and Merriam, G. R.: Changes in proteins and protein
synthesis: In tryptophane deficiency and radiation cataracts of rats, Am. J. Ophth. 47:
368, 1959.
Dische, Z., and Zelmenis, C : Nutritional
and endocrine influence on the synthesis of
albuminoid in rat lenses. I. The eftect of restricted diet and L-thyroxine on the albuminoid formation, Am. J. Ophth. 48: 500,
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Morner, C. T.: Untersuchung der Proteinsubstanzen in den leichtbrechenden Medien des
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Bjork, I.: Chromatographic separation of
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Francois, J., and Rabaey, M.: On the existence of an embryonic lens protein, Arch.
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Kinoshita, J. H., and Merola, L. O.: The
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Kleifeld, O., Hockwin, O., and Fuchs, R.:
Untersuchungen uber wasserloslichen Eiweisse in Kern und Rinde der Linsen alter
und junger Tiere und menschlicher klarer
und getriibter Linsen, Deutsche Ophth.
Cesellschaft, Bericht. 60: 108, 1956.
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