f
Factors limiting glycolysis in the
rat lens
John W. Patterson and Kenneth W. Bunting
Glycolysis has been studied in extracts of rat lens with different concentrations of ATP and
AMP as cofactors and various intermediates as substrates.
With optimal concentrations of cofactors, lactate formation from glucose approximates that
obtained with equivalent concentrations of pyruvate.
The activity found in extracts is much greater than that observed for the intact lens, so that
an enzyme as such is not the limiting factor in lens glycolysis.
With varying cofactor concentrations, steps associated with different enzymes may be limiting. The step associated with hexokinase, 3-phosphoglyceric kinase, or glijceraldehyde phosphate dehydrogenase may be limiting under appropriate circumstances.
A-
A,
tions and conclusions. The major differences are with respect to the factors that
limit glycolysis.
. n extensive study of glycolysis in the
rabbit lens was reported in a series of
papers by Green, Bocher, and Leopold in
1955.1'3 This study reviewed previous work
and presented new evidence which demonstrated the presence of the classical glycolytic pathway in the lens. The authors
concluded that glycolysis was limited by
hexokinase, the level of inorganic phosphate, and the availability of phosphate
"acceptor."
The purpose of this article is to present
data concerning glycolysis in the rat lens.
The results are consistent with the data
obtained in previous studies. However, new
observations lead to different interpreta-
Methods
Glycolysis was studied by mixing 0.1 c.c. of
substrate solution with 0.1 c.c. of lens extract, incubating for one hour, and assaying for glucose
disappearance and lactate formation.
The basic medium for preparing the enzyme
and substrate solutions contained the following:
Tris-HCl buffer, 50 mM.; ethylenediamine tetracetate (EDTA), 2 mM.; MgCl. • 6H:O, lOmM.; and
KH.PO4, 2, 4, or 20 mM. The pH was adjusted
to 7.4.
The substrates were prepared in double concentrations so that the final concentration was 5.6
mM. for hexose derivatives and 11.1 mM. for
triose derivatives. When appropriate, adenosinetriphosphate (ATP), adenosine diphosphate (ADP),
adenosinemonophosphate (AMP), and reduced
diphosphopyridinedinucleotide (DPNH) were incorporated in the substrate solution. DPNH was
used in 10 mM. concentration with the others varying as indicated. These substances were obtained
commercially as follows: glucose, C (Nutritional
Biochemicals); glucose-6-phosphate disodium salt,
G-6-P (Sigma); fructose diphosphate sodium salt,
FDP (Sigma); 3-phosphoglyceric acid tricyclohexyl-ammonium salt, 3-PGA (Boehringer);
From the Department of Physiology, Vanderbilt
University, School of Medicine, Nashville, Term.
This research was supported in part by Research
Grant A-2954 from the National Institute of
Arthritis and Metabolic Diseases, United States
Public Health Service, Bethesda, Md.
Presented at the Annual Meeting of the Association for Research in Ophthalmology, June, 1963,
Atlantic City, N. J.
612
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Volume 2
Number 6
_A
V
i
pyruvate sodium salt, Pyr. (Matheson, Coleman
and Bell); ATP disodium salt (Sigma); ADP
sodium salt (Sigma); AMP sodium salt (Sigma),
and DPNH (Sigma, preweighed vials). The substrate solution was adjusted to pH 7.4 with a
Beckman pH meter and wanned at 37° C. before the addition of the lens extract.
The lens extract was prepared from the lenses
of young 26 to 37-day-old male Sprague-Dawley
rats. The rats were decapitated, the eyes removed, corneas incised with scalpel and scissors.
The lenses were extruded, rolled on tissue paper,
and weighed after being dropped into a tared
homogenizer tube containing basic medium
chilled in ice water. The cold homogenate was
centrifuged at 3,000 r.p.m. for 10 minutes, the
supernatant decanted into a chilled tube, and the
extract used immediately. The concentration was
planned to give a final value very close to 10 per
cent, or approximately one lens per 0.2 c.c. total
volume in each incubation tube. All calculations
were adjusted to a final concentration of 10 per
cent lens extract.
The incubation was carried out in 10 by 75 mm.
test tubes supported in a constant temperature
water bath set at 37° C. The medium was exposed to air. One hour after the addition of the
enzyme extract, the reaction was terminated by
precipitating the proteins with Zn2SOj-Ba(OH)..
The supernatant was used for assay. Glucose was
determined by the glucose oxidase method,4 and
lactate by the method of Barker and Summerson.5
Results
The effects of varying the concentration
of phosphate, ATP, AMP, and ATP in the
presence of AMP are shown in Figs. 1 to 4.
Optimal results for glucose uptake and
lactate formation are obtained with 20 mM.
phosphate, 4 mM. ATP, and 2 mM. AMP.
The normal level of inorganic phosphate
in lens in 2 mM. This is an adequate level
if it is continually regenerated. If the needs
of the system must be supplied, then 11
mM. of phosphate would be required for
5.5 mM. of glucose. The use of a 20 mM.
concentration insures an adequate supply.
However, 4 mM. is highly effective (Fig.
6)
The normal concentration of ATP as determined by the firefly lantern extract
method" is 2.5 mM based on total lens
weight and 4 mM. based on lens water.
With the addition of 0.5 mM. ATP, glucose
uptake is depressed. At higher concentrations, however, it is stimulated. Depression
GUjcolysis in rat lens
613
GLUCOSE
UPTAKE
LACTATE
FORMED
Fig. 1. The effect of ATP on glucose uptake and
lactate formation by lens extract in basic medium
containing 2 mM. phosphate.
Fig. 2. The effect of ATP on glucose uptake and
lactate formation by lens extract in basic medium
containing 20 mM. phosphate.
00
1
"* GLUCOSE
UPTAKE
80
-
60
- • LACTATE
FORMED
40
ATP 2.5 mM
Pi 20 mM
20
1
1
1
I
Fig. 3. The effect of AMP on glucose uptake and
lactate formation by lens extract in basic medium
containing 2.5 mM. ATP and 20 mM. phosphate.
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614 Patterson and Bunting
Investigative Ophthalmology
December 1963
Fig. 4. The effect of ATP on glucose uptake and
lactate formation by lens extract in basic medium
containing 2 mM. AMP and 20 mM. phosphate.
1
r
I
I
|
8
1
ATP
|
4mM 1 0-6-P
Pyr + OPNH
1
1
r Q
4 mM I 8-6-P
2mM L P»r + DPNH
ATP
1
Pi 20 mM
a |
G-6-P |
Pyr + DPNH
I
|
i
i
i
Fig. 5. Lactate formation from glucose, glucose-6phosphate, and pyruvate by lens extract in basic
medium containing 20 mM. phosphate and ATP
and AMP where indicated.
of activity following ATP addition has also
been observed in other systems, such as
with phosphofructokinase7 and cation
transport in the lens.s
The addition of AMP as a phosphate
"acceptor" might be expected to favor
lactate formation rather than glucose uptake. Both, however, are stimulated and the
activities parallel each other. The use of
ADP as an "acceptor" produced less stimulation than AMP. Thus, with G-6-P as subtrate, in the presence of 2.5 mM. ATP and
4.0 mM. phosphate, the lactate formed was
62 and 47 /xg for AMP and ADP, respectively. This concurs with earlier reports.1
The limiting step in a series of reactions
may be determined by assaying for the
relative concentration of different metabolic intermediates, and determining which
ones are accumulated under different conditions. This approach has the advantages
of being applicable to a whole organ and
of not interfering with normal changes in
cofactor concentration and enzyme activity.
It does require the use of a variety of
microassays for different intermediates. A
second and simpler approach is to determine the rate of formation of the final
product when different intermediates are
added as substrates. Under these circumstances the experimental conditions cannot
be uniform and interpretations must be
more guarded.
In this study, the second of these methods was used to approximate the limiting
step in glycolysis in the lens. The results
apply to lenses obtained from young rats
under the particular conditions employed.
Lactate formation with glucose, G-6-P,
and pyruvate as substrates is shown in
Fig. 5. The amount of lactate formed from
pyruvate serves as a standard for maximum
activity inasmuch as it represents the last
step in the series of reactions. This reaction
is independent of the concentration of
adenine nucleotides. Without the addition
of cofactors, glucose and G-6-P yield relatively small amounts of lactate. The yield
is increased in the presence of 4 mM. ATP
and increased to a level similar to that obtained with pyruvate after the further addition of 2 mM. AMP.
With glucose as a substrate in the above
three situations, the glucose uptake was
43, 83, and 110 fig per hour, respectively.
The highest value equals 5.5 mg. per gram
of lens per hour—a value much higher than
the 1 to 2 mg. per gram per hour observed
with whole lenses.9-10 The rate of glucose
uptake under optimal conditions is slightly
greater than the rate of lactate formation
from pyruvate.
If all of the glucose uptake were in-
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Volume 2
Number 6
Glycolysis in rat lens 615
Pl
GLUCOSE |
4 mM
6-6-P |
1
TOP
3-PGA + OPNH
ATP
2.5 mf
AMP
2.0 mN
GLUCOSE
S-6-P
FDP
|
3-PGA + DPNH
1
i
|
|
1
Fig. 6. Lactate formation from glucose, glucose6-phosphate, fructose diphosphate, and 3-phosphoglyceric acid by lens extract in basic medium
containing 4 mM. phosphate and ATP and AMP
where indicated.
stantaneous, it would provide a G-6-P concentration of 3 mM. The fact that the concentration of G-6-P is 5.6 mM. accounts
for the slightly higher yield of lactate from
G-6-P, when compared with glucose in the
presence of ATP and AMP. Experiments
in which the G-6-P concentration was 4.4
mM. showed a lactate formation below
that obtained with glucose.
In experiments in which glucose, G-6-P,
FDP, and 3-PGA are used as substrates
(Fig. 6), the yield of lactate is much
greater with 3-PGA than with any of the
others, and is of the same order of magnitude as is obtained with pyruvate. This
suggests that in the presence of ATP a
limiting step in glycolysis is associated with
one of the enzymes between FDP and 3PGA. The production of lactate from FDP
in the presence of ATP was greater with
AMP than with ADP, the yields being 52
and 44 fig, respectively. This confirms
earlier work.3
Discussion
The capacity of the enzymes of the lens
to convert glucose to lactate is greater
than is required by the whole lens. Therefore, the enzymes as such are not likely to
limit metabolism.
Metabolism is limited by the interplay of
cofactors on the enzymes, and different
steps may be limiting under different conditions. In other tissues, limitations have
been associated, depending on the specific
conditions, with hexokinase, phosphofructokinase, or glyceraldehydephosphate
dehydrogenase.11"13
In this study, three situations were considered: (1) with no cofactors, (2) with
ATP added in the presence of low and
high phosphate concentrations, and (3)
with ATP and AMP added in the presence
of low and high phosphate concentrations.
In the first situation, the step associated
with hexokinase appears to be limiting.
This is supported by the fact that the
formation of lactate from glucose is regularly about half of that obtained from G-6P. This block is relieved by raising the
concentration of ATP to physiologic levels.
In the second situation, the step associated with 3-phosphoglyceric kinase appears to be limiting. Lactate formation is
low with glucose, G-6-P, and FDP as substrates and high with 3-PGA and pyruvate
as substrates. Therefore, one of the steps
between FDP and 3-PGA seems to be
limiting. Aldolase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase, or 3-phosphoglyceric kinase
could be limiting. The fact that the block
is removed by AMP and that the substrates
which produce ADP are slightly more
active suggests that the step associated
with a phosphate "acceptor" is limiting. The
situation is the same with low and high
phosphate concentrations.
In the third situation, the step associated with glyceraldehyde phosphate
dehydrogenase appears to be limiting.
With low phosphate concentrations there
seems to be a block between FDP and 3PGA. This is removed by using higher
concentrations of phosphate. Therefore, the
step requiring phosphate as a substrate is
probably limiting.
In the intact lens, the normal levels of
phosphate, ATP, and AMP are at levels
where a change in concentration would be
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616
Patterson and Bunting
expected to result in a change in glycolytic
activity. It is interesting to speculate as to
how these changes may control glycolysis
to meet the metabolic needs of the lens—
first by limiting the over-all reaction and
second by producing specific blocks which
may favor side reactions stemming from
the main glycolytic pathway.
REFERENCES
1. Green, H. G., Bocher, C. A., and Leopold,
I. H.: Anaerobic carbohydrate metabolism of
the crystalline lens, I. Clucose and glucose-6phosphate, Am. J. Ophth. 39: (Part II), 106,
1955.
2. Green, H. G., Bocher, C. A., and Leopold,
I. H.: Anaerobic carbohydrate metabolism
of the crystalline lens, II. Fructose diphosphate, Am. J. Ophth. 39: (Part II), 113,
1955.
3. Green, H. G., Bocher, C. A., and Leopold, I.
H.: Anaerobic carbohydrate metabolism of
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phosphoglycerate, and phosphoenol pyruvate,
Am. J. Ophth. 40: (Part II), 237, 1955.
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Biochem. J. 66: Proc. 12, 1957.
Investigative Ophthalmology
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9. Kuck, J. F. R., Jr.: Glucose metabolism and
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10. Farkas, T. G., Ivory, R. F., and Patterson, J.
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