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Control of lens glycolysis
BIOCHIMICA ET BIOPHYSICA ACTA
547
BBA 25 810 CONTROL OF LENS GLYCOLYSIS MARJORIE F. LOU AND JIN H. KINOSHITA Howe Laboratory of Ophthalmology, Harvard Medical School, and the Massachusetts Eye and Ear Infirmary, Boston, Mass. (U.S.A.)
(Received January 9th, 1967)
SUMMARY
I. The calf-lens phosphoiructokinase (ATP: I)-Iructose-6-phosphate I-phosphotransferase, EC 2.7.I.II ) was isolated and purified 86 fold. I t was found that ATP inhibits lens phosphofructokinase at a concentration above 0.25 mM. Cyclic AMP was the most effective in releasing the inhibition of phosphofructokinase b y glucose 6-phosphate. 2. The calf-lens hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1 ) is susceptible to inhibition by low levels of glucose 6-phosphate. The inhibitory effect of glucose 6-phosphate can be partially counteracted b y increasing concentrations of inorganic phosphate. 3. The normal content of ATP and glucose 6-phosphate in the lens are at levels where considerable degree of inhibition would be exerted on the two enzymes, hexokinase and phosphofructokinase. 4. The levels of the glycolytic intermediates in the lens were measured after incubation under aerobic conditions at 5.5, IO and 15 mM glucose. Increasing the availability of glucose raised the intracellular glucose, but the levels of sugar phosphates and the production of lactate remained unchanged. 5- Increasing the availability of glucose did, however, stimulate the lens sorbitol pathway. Increasing levels of sorbitol were found in the lenses incubated at the 3 different concentrations of glucose.
INTRODUCTION Studies on the control mechanisms of glycolysis in a number of tissues have indicated that one of the rate-limiting reactions appears to occur at the phosphofructokinase (ATP:D-fructose-6-phosphate I-phosphotransferasej EC 2.7.1.11 ) step. The control is effected by the ability of the reaction product to inhibit the enzyme. The inhibition of phosphofructokinase b y excess ATP has been demonstrated in heart 1, muscle ~ and brain 3. This inhibition is released b y the addition of AMP and several other metabolites. Other sites and factors m a y be involved in the control of glycolysis. For example, R o s e ANI) O'CoNNELLa found t h a t the rate of glucose utilization in human erythrocytes depends upon the rate of the hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.I) step which is governed b y the removal of glucose Biochim. Biophys: Acta, 141 (1967) 547-559
548
M. F. LOU, J. H. KINOSHITA
6-phosphate, an effective inhibitor of hexokinase. In Ehrlich ascites tumor cell, Wtr 5 demonstrated that orthophosphate is the major rate-limiting factor for glycolysis. In the lens, glycolysis is the principal source of biological energy required for the maintenance of its function (for review see ref. 6). Thus it is of considerable interest to understand how glycolysis is regulated in the lens. It has been demonstrated that the rate of glycolysis is increased in rabbit-lens extract when hexokinase is added to the reaction mixture or when glucose is replaced by glucose 6-phosphate as the substrate~, 8. These observations were later supported by the demonstration that the lactate production from glucose 6-phosphate was considerably higher than that from glucose in extracts of rabbit, calf and rat lenses s, and that the activity of lens hexokinase is the lowest among all the enzymes of the glycolytic pathway 1°. These findings all suggest that the hexokinase reaction is the rate-limiting step. Nevertheless the rate of glucose utilization by the intact lens is less than that of the hexokinase reaction, therefore other regulatory factors may be involved. In this paper evidence is presented suggesting that both the hexokinase and phosphofructokinase reactions participate in controlling lens glycolysis. MATERIALS
Calf eyes were obtained from a local abattoir soon after slaughtering, and the lenses removed and frozen until needed. The rabbit lenses were obtained by removing them carefully from rabbit eyes soon after the young animals had been killed by air embolism. Aqueous humor samples were withdrawn before the laboratory animals were killed. The calf aqueous humor was obtained after the eyes arrived at the laboratory. DPNH, TPNH, nucleotides and sugar phosphates were obtained from Sigma Company, while uniformly ~*C-labeled glucose was obtained from New England Nuclear. All enzymes used in the determination of the glycolytic lens intermediates were products of Boehringer. METHODS
Determination of hexokinase activity Hexokinase assay was carried out according to the modification of the method of SHERMANI0. Lens homogenate was prepared by homogenizing IO calf lenses in a IO ml mixture of 0.2 M potassium phosphate buffer (pH 7.2), I mM versene and I mM fl-mercaptoethanol. The homogenate was dialyzed against IO times its volume of the same buffer solution for 3 h and then centrifuged at IOOOO × g for IO rain. The supernatant fluid was used for the source of hexokinase. The assay mixture contained in a final volume of 0.75 ml: 0.66 mM of [14C~glucose with specific activity of 1.77"1o5 counts/min per t~mole; 3-3 mM ATP; 6.6 mM MgC1,; 3.2 mM NaF; 6.6 mM mercaptoethanol; 66 mM Tris-HC1 buffer (pH 7.8) and aliquots of the dialyzed supernatant fluids. The mixture was allowed to incubate at 37 ° for 15 min. The reaction was stopped by immediately applying 50/A of each sample to a DEAE-cellulose paper (1.5 cm × 14 cm). Methods of separation and the measurement of the reaction product [14C]glucose 6-phosphate were similar to the technique described for [I~C]galactose I-phosphate~L Biochim. Biophys. Aaa, x4x (x967) 547-559
LENS GLYCOLYSIS
549
Purification of phosphofructokinase All operations were performed in the cold room at 5 ° except where otherwise mentioned. The general pattern of the purification of phosphofructokinase was that devised by MANSOURt,TM. Step ~: preparation of lens homogenate, 30 calf lenses were homogenized in 200 ml mixture of IO mM Tris-HC1 buffer (pH 8.0) ; 2 mM versene; 5 mM fl-mercaptoethanol; 50 mM MgSO~ and 0.5 mM ATP in a TenBroeck homogenizer and centrifuged at IOOOO × g for 2o rain to remove insoluble material. The supernatant fluid was saved. Step 2 : ethanolfractionation. To a 5o-ml solution of the lens extract, kept at --8 ° in a salt-ice bath, 4.35 ml of 95 % ethanol (--20 °) were added in a dropwise fashion with continuous stirring. The resulting mixture containing 8 % ethanol was gently stirred for 30 min after which it was centrifuged at IOOOO × g for IO min and the precipitate discarded. To the supernatant fluid, 3.1 ml of the cold ethanol were added, which raised the ethanol concentration to 13 %. The mixture was stirred for 30 min and the precipitate recovered after centrifugation contained the enzyme. The precipitate was taken up in IO ml of the same buffer mixture used for the lens homogenization. Step 3: column chromatography. DEAE-cellulose powder was washed and prepared for use by a modification of the procedure of SOBER AND PETERSON12. A freshly made column (3 cm × 24 cm) of DEAE-cellulose was equilibrated with a solution consisting of IO mM Tris-HC1 buffer (pH 8.0); 20 mM MgSO4; 5 mM fi-mercaptoethanol; o.I mM ATP and o.oi mM fructose 1,6-diphosphate in the cold room for an overnight period. The ethanol fraction usually containing 22 mg of protein was applied to the column after which the column was washed with o.oi M Tris buffer until the absorbance of eluates at 280 m/~ was less than o.I absorbance units. The first buffer was followed by a series of Tris buffers varying in concentration from o.o5 M to 0.3 M. Using a stepwise elution technique, fractions of io ml were collected at a flow rate of approx. I ml/min. The fractions containing phosphofructokinase activity were collected with the 0.2 M buffer. A purification of 86-fold was achieved at this stage. Step 4: ammonium sulfate fractionation. The pooled active fractions of the enzyme was precipitated by adding solid (NH4)~SO 4 to give a concn, of 40 %. In this form the enzyme was stable and could be stored for a week. The precipitate was taken up by the Tris-buffer mixture containing the stabilizing agents used for column equilibration. This preparation of the enzyme was used for all the kinetic studies. Table I indicates the overall purification procedure for lens phosphofructokinase.
Determination of phosphofructokinase activity For the enzyme assay, an aliquot of the enzyme preparation was added to a reaction mixture consisting of: 50 mM Tris buffer (pH 7.4) ; o.o12 mM D P N H ; 0.2 mM MgC12; o. i mM ATP; I mM fructose 6-phosphate; triose phosphate isomerase; glycerol phosphate dehydrogenase and aldolase in a total volume of I.O ml. The reaction was run at room temperature over a 6 min period. The enzymes present in the reaction mixture rapidly converted the product formed, fructose 1,6-diphosphate, to glycerol phosphate which was estimated by the disappearance of D P N H fluorometfically. A Farrand fluorometer model A-2 was used for this purpose. Biochim. Biophys. ~4cta, 141 (i967) 547-559
55 °
M.F.
LOU, J. H. KINOSHITA
TABLE I PURIFICATION
OF PHOSPHOFRUCTOKINAS~
FROM
C A L F L]~NS
One phosphofructokinase activity u n i t is the a m o u n t of enzyme t h a t catalyzes the formation of I ffmole of p r o d u c t per min. Details are presented in the t e x t under METHODS.
Steps
Vol.
Units/ml Total units
Protein (mg/ml)
Specific activity
Yield
Lens h o m o g e n a t e E t h a n o l fraction (8-13 °/o) DEAf-cellulose chromatography (NHa)~SO4 (o.4o %)
2oo 3° 35 5
o.33 o.63 0.29 o.64
47.5 21. 5 0. 5 1.8
o.oo 7 o.o 3 o,6 o. 4
IOO 28.1 15.2 4.8
66.6 18.8 io.2 3 .2
Measurement of intermediates of the glycolysis in rabbit lens Young rabbit lenses weighing 200 ± IO mg were incubated in IO ml TC-I99 medium 1~ with varying glucose concentrations of 5.5, IO and I5 raM. The tonicity of the incubation medium was 300 mosM and was equilibrated with 95 % air and 5 % CO2. After incubation at 37 ° for 24 h, the lens was removed from the culture tube and homogenized in 4 ml of o.6 M HCIO~. After centrifugation, the supernatant fluid was neutralized with 5 M K O H and the KC10~ removed b y centrifugation. Aliquots of the clear supernatant fluid were then assayed for each intermediate. Lactate was determined b y the method of BARKER AND SU'MMERSON14'. Glucose, sugar phosphates and adenine nucleotides were determined b y the modified method of LowRY et aN n. Sugars and sorbitol of the incubated lens were determined b y gasliquid chromatography, the methods of which are previously described 11. RESULTS
Properties of ~hos~hofructokinase Similar to the findings of MANSOUR, WAKID AND SPROUSE 16, the presence of ATP fructose 1,6-diphosphate, Mg 2+ and mercaptoethanol was found necessary to 16!
12
0.5
~,g
~
0.2
~
~
b ~ Q2
~
~
~
~
~ o.~
~
~
~
~"
~
~~
,o~
q
~
~
~
~-
V [ ~ - ~ - ~ ~o-~
Fig. I. p H optimum for lens phosphofructo~nase. In the p H range 6.o-7.5, 5o mM phosphate buffers were used; for the p H range of 7.3-8.6, 5o mM Tris buffers; p H 7.o, 5o mM imidazole buffer and p H 9-5, 5 o mM carbonate-bicarbonate buEer. Fig. 2. Lineweaver-Burk plot for phosphofructokinase with fructose 6-phosphate. The p H of the reaction is 8.0 and the A T P concentration is constant at o.I m ~ .
Biochim, Biophys. Acta, 141 (I967) 547-559
LENS GLYCOLYSIS
551
stabilize the lens phosphofructokinase during the purification procedures. In the presence of these stabilizing agents, the enzyme was able to withstand temperatures at 37 °. However, after DEAE-cellulose column chromatography, the active fractions collected were unstable even in the presence of these agents. A 50 % loss was incurred during the overnight storage at 4 ° . However, it was found that the enzyme precipitated b y (NH4)2SO4 could be stored up to a week without appreciable loss of activity. As shown in Fig. I the optimum p H of lens phosphofructokinase was 8. Michaelis-Menten constants of 19o and 7 ~M were determined for fructose 6-phosphate and ATP respectively. The plot of the reciprocal of initial velocity against the reciprocal of the fructose 6-phosphate concentrations reveals a typical substrate curve (Fig. 2). In contrast, the Lineweaver-Burk plot for ATP shows that at high concentrations the velocity of the reaction is markedly decreased indicating substrate inhibition (Fig. 3). The inhibition of phosphofructokinase begins to occur above 0.25 mM ATP. Above this concentration ATP becomes increasingly effective as an inhibitor of
O.e 0.[
~ 0.4 ~
0,4~
~
. . ~ , . . 1 "'~
~ , ~3 .........................
~-~-~,,'...~,I~, .................... " .................... m
o- ~ '
~,
•
~, ©
~o2 q
~7
~_~.e ,.;'............
.... ,..-,,~""" ~.....
~
~
o_~,
o.6'
~
~::,. o2
~b
2b
~/[A~P] ~o-~
3b
_
_
~
4o
°o~-
I
~o
1
4o
~.
~o
~/~r~_l~o-~
Fig. 3- I n h i b i t i o n of lens p h o s p h o f r u c t o k i n a s e by ATI ~. A double reciprocal plot of reaction velocity versus concentration of s u b s t r a t e ATP is given. Details of the e n z y m e assay are described in MEn,tOIlS. The velocity is expressed as /,moles of fructose 1,6-diphosphate formed per minute. The a r r o w p o i n t s to o.25 raM, the concentration above which ATP begins to inhibit the lens phosphofructokinase. Fig. 4- The effect of p H and fructose 6-phosphate on the ATP inhibition of phosphofructokinase. The a s s a y conditions are the same as those in MI~THODS, except t h a t fructose 6-phosphate varied from I mM to o.I mM in 5 ° mM imidazole buffer (pH 7) or in 5 ° mM Tris buffer (pH 8).
the reaction. At 5 mM, ATP inhibits phosphofructokinase activity b y 5 ° %. The inhibitory effect of ATP on the lens phosphofructokinase is independent of the level of fructose 6-phosphate or of p H (Fig. 4). Thus, the shape of the curve is unaffected b y different levels of fructose 6-phosphate or b y a difference of I p H unit. This is in contrast to UYEDA AND RACKER2 who reported that the magnitude of ATP inhibition was p H dependent in rabbit-muscle phosphofructokinase. The ATP inhibition is more pronounced at p H ' s lower than 9, which is consistentwith the observation OfMANSOUR1 who found that ATP inhibition was less effective at alkaline pH's. However, in contrast with our findings, MANSOUR found that inhibition of heart phosphofructokinase was less pronounced with increasing concentrations of the second substrate, fructose 6-phosphate. The effects of other compounds were also studied (Table II). At tile same concentration of ATP, which produced 5 ° % inhibition, GTP caused only a 26 % effect. B$ochim. Biophys..4cta, 14i (t967) 547-559
552
M. F. LOU, J. H. KINOSHITA
TABLE II INHIBIt?ION
OF PHOSP/-IOFRUCTOKINASE
E x p e r i m e n t a l c o n d i t i o n s are t h e s a m e as d e s c r i b e d in Fig. I. I n all cases t h e c o m p o u n d s t e s t e d were a d d e d t o a r e a c t i o n m i x t u r e c o n t a i n i n g o.I mM ATP.
Compound added
COnch.
Activity
Inhibition
CraM) 0
(%) 7.6
Citrate
Phosphoenolpyruvate GTP
Sor bito l
0.5 2.5 5.0 0.5 2. 5 0.5 2.5 5.o 50.0
6.6 5.8 5.8 5.8 7.0 5.6 3.6 2.8 7.6
-
-
I3 24 24 -8 26.3 52.7 63.o --
TABLE III R~L~ASE
OF
INORGANIC
ATP
INHIBITION
OF P H O S P H O F R U C T O K I N A S E
BY NUCLEOTIDES
AND
PHOSPHATE
C o n d i t i o n s of t h e e x p e r i m e n t were i d e n t i c a l as t h a t d e s c r i b e d in Fig. z. I n a l l cases, l e ns phos phof r u c t o k i n a s e a c t i v i t y was i n h i b i t e d b y 5 mM ATP. The a c t i v i t y w i t h o . i mM A T P is t h e uni n h i b i t e d rate. T h e a c t i v i t y is e x p r e s s e d as t h e c h a n g e in a b s o r b a n c e pe r rain.
A TP (raM)
Compound added
O.I
O
5.0 5.0 5.o 5.o 5.0 5.o 5.o 5.o 5.0 5.0 5.0 5,0 5.0 5,o 5,0 5,o 5.0 5,o 5.0 5.0 5,0 5.0 5.0 5.0 5.o
o ADP ADP ADP 3'-AMP 3'-AMP 3'-AMP Cyclic A M P Cyclic A M P Cyclic A M P 5'-AMP 5 "-AMP 5'-AMP dAMP dAMP dAMP CMP CMP CMP UMP UMP UMP GMP GMP GMP P~ Pt Pt
5.0 5.o 5.0
Conch. (mM)
A ctivity
Activity (%)
I 1.0
IOO
5.3 7.0 8.o 6.8 6.0 7.o 6.9 9.o
48 64 73 62 55 64 63 82 115 lO9 59 69 68 6z 74 73 63 73 68 68 73 68 64 73 69 66 76 66
o.i o.5 i.o 0.o 5 o. 5 I.O o.I 0.5 I.O o.I 0.5 1.o o.I 0. 5 I.o o.I o.5 I.o o.I 0. 5 I.O o.I o. 5 I.O o.~ o.5
I2.O 6.4 7.5 7.4 6.8 8. I 8.o 6.9 8.0 7.4 7.5 8.o 7.4 7.0 8.0 7.5 7,3 8.4
r.o
7.3
12.7
Biochim. Biophys. Acta, 141 {I967) 547-559
LENS GLYCOLYSIS
553
Citrate, at concentration above 2. 5 raM, produced a 24% inhibition. However, phosphoenolpyruvate had no effect.
Release of A TP inhibition Various nucleotides and inorganic phosphate are known to release the ATP inhibition of phosphofructokinase. As summarized in Table III, the most effective nucleotide in releasing ATP inhibition is 3',5'-cyclic AMP. The 50% inhibited phosphofructokinase activity produced by 5 mM ATP was completely released in the presence of 0.5 mM cyclic AMP. However, at the same concentration, ADP, AMP derivatives and inorganic phosphate were much less effective, overcoming the inhibition by only 15-25 %. The plot of the reciprocal of the cyclic AMP concentration against the reciprocal of the reaction velocity is given in Fig. 5. At o.I mM ATP, the lens phosphofructokinase is not inhibited. However, under these conditions increasing cyclic AMP concentrations appear to stimulate the phosphofructokinase reaction as shown in the graph. 0.8
.,,, ~ ....................~...........
~ ~,.:o................... 0.6
-
,,,~,.,, ............... .... ~,,,."~
.........
(3.4 rn
b ..~ \: Q2
l
2
__
I
4
~
I~
6
~/[~,li~m~,~o'~
8
1 - -
10
Fig. 5- Release of AT1~ inhibition of p h o s p h o f r u c t o k i n a s e b y 3',5'-cyclic AMP. A double-reciprocal plot of reaction v e l o c i t y versus c o n c e n t r a t i o n of 3',5'-cyclic A M P is given. T h e A T P c o n c e n t r a t i o n w a s either o.I or 0.2 5 m M while t h e c o n c e n t r a t i o n of fructose 6 - p h o s p h a t e w a s c o n s t a n t a t 5 ° ffM. T h e p H w a s m a i n t a i n e d a t 7.0 w i t h imidazole buffer. Velocities are e x p r e s s e d as t h e / z m o l e s of p r o d u c t fructose d i p h o s p h a t e f o r m e d per min.
When phosphofructokinase is inhibited by ATP (2.5 mM), the stimulation of the reaction velocity with increasing concentrations of cyclic AMP is more pronounced. These results indicate that when ATP is present at levels below the inhibitory range, cyclic AMP appears to be an activator of lens phosphofructokinase. When phosphofructokinase is inhibited by ATP, cyclic AMP counteracts the inhibitory effect of the trinucleotide, in addition to activating the enzyme.
Hexokinase inhibition study Due to the extremely low hexokinase activity in lens and the instability of the enzyme, we were unable to purify the enzyme to any significant extent. Consequently, a dialyzed calf-lens homogenate was used as the source of the enzyme. Glucose 6-phosphate, the product of the reaction exerts a pronounced inhibitory effect on hexokinase. There is a progressive decrease in hexokinase activity with increasing concentrations of glucose 6-phosphate. A concentration of o.o133 mM of Biochim. Biophys. Acta, 141 (1967) 5 4 7 - 5 5 9
554
M.F.
LOU, J .
H. K I N O S H I T A
glucose 6-phosphate produces a I8 % inhibition as indicated in Table IV. At a concentration IO times higher, the inhibition is 55 %. As shown in Table V, ADP, the other reaction product of hexokinase, is not inhibitory. In contrast, the studies with purified ascites-tumor hexokinase 2 as well as brain hexokinase 2~ revealed that A D P appears to be a competitive inhibitor of ATP. Galactose x-phosphate is of interest because of its possible importance in cataracts of galactosemia 6. It did, however, exert no inhibition on lens hexokinase (Table V). The inhibition of hexokinase by glucose 6-phosphate is released partially by inorganic phosphate. Table VI presents data on the effect of phosphate concentration TABLE
IV
INHIBITION OF LENS HEXOKINASE BY GLUCOSE a-PHOSPHATE T h e r e a c t i o n m i x t u r e u s e d in d e t e r m i n i n g h e x o k i n a s e a c t i v i t y is d e s c r i b e d in t h e METHODS.
Glucose 6-phosphate (M × 10 -5) 0
1.33 3-33 6.67 IO.O 13.3 33"3
TABLE
A ctivity (l~mole/min × i o -~)
Inhibition (%)
2.2 1.8 1.6 1.4 1.2 I.O 0.8
-18 27 36 45 55 64
V
EFFECT OF A D P
AND GALACTOSE I-PHOSPHATE ON HEXOKINASE ACTIVITY
T h e c o n d i t i o n s of t h e a s s a y a r e g i v e n in t h e METHODS.
Compound added
O ADP ADP GaI-I-P
TABLE
ConcH. (raM)
Activity (Ixmole/min × ~o -~)
Activity (%)
o.33 1.33 o.67
0.86 o.75 o.94 o.So
IOO 87 iio 93
VI
EFFECT OF INORGANIC PHOSPHATE ON THE INHIBITION OF HEXOKINASE BY GLUCOSE 6-PHOSPHATE T h e c o n d i t i o n s of t h e a s s a y a r e g i v e n in t h e METHODS.
Glucose 6-phosphate
Inorganic phosphate
Hexokinase activity
(raM)
(raM)
(k~mole/min X zO-3)
Activity (%)
1.7 0. 4 o.5 o.6 o.8 o.9
IOO 24 29 35 47 53
o 0.33 0.33 0.33 0.33 o.33
o o 1.33 3-33 6.67 13. 3
Biochim. Biophys. Acta, ~41 (1967) 5 4 7 - 5 5 9
LENS GLYCOLYSIS
555
on the glucose 6-phosphate inhibition of hexokinase. Under no condition is recovery of activity complete. When hexokinase activity is 75 % inhibited by glucose 6-phosphate, a level of phosphate ion of 13 mM lowered the inhibition to 24 %. In comparison UYEDA and RACKER2 have reported that when brain hexokinase is 5o % inhibited, the presence of IO mM inorganic phosphate released the inhibition to 24 %; while at 75 % inhibition of red cell hexokinase, IO mM inorganic phosphate lowered the inhibition to 32 %. In many respects the hexokinase of the lens resembles that of the red cell in its susceptibility to glucose 6-phosphate inhibition and to the degree of the release of inhibition by inorganic phosphate. The glucose 6-phosphate inhibitory effect on hexokinase is only slightly reversed by high concentrations of ATP. For example, the inhibition of hexokinase by glucose 6-phosphate is at the 55 % level with o.o67 mM ATP. When the ATP concentration is raised to o.67 mM, a substantial inhibition of 48 % is still observed. Thus, the trinucleotide is less effective than phosphate ion in counteracting the glucose 6-phosphate inhibition of hexokinase. No active ATPase is present in this preparation, but it must be kept in mind that the results are based on experiments with crude lens extracts. The normal glucose content in the aqueous h u m o r and the lens of rabbit, rat and calf
In Table VII the data on the concentrations of glucose and glucose 6-phosphate in the aqueous humor and the lens are given along with the lens-ATP content. Since phosphorylated derivatives do not readily penetrate biological membranes, it is reasonable to assume that all the glucose 6-phosphate is intracellular. From the results given in Table Vii, the concentration of glucose 6-phosphate can be estimated to be o.I mM in the lens. This level of glucose 6-phosphate, as indicated in Table IV, should produce at least a 50 % inhibition of hexokinase. The ATP concentration appears high in the lens. The rat lens has an ATP concentration of IO mM while the rabbit and calf lenses are well above I raM. These concentrations should be sufficient to exert marked inhibition of lens phosphofructokinase. TABLE VII GLUCOSE, GLUCOSE 6-PHOSPHATE AND A T P CO~TE~T IN VARmUS Lt~NSF~S S a m p l e s of a q u e o u s h u m o r were w i t h d r a w n from a n a e s t h e s i z e d r a t s a n d r a b b i t s a f t e r w h i c h t h e lenses were r e m o v e d , w e i g h e d a n d i m m e d i a t e l y h o m o g e n i z e d in o.6 M cold HCIO 4. A q u e o u s h u m o r was r e m o v e d from t h e calf eyes a f t e r t h e y a r r i v e d a t t h e l a b o r a t o r y . The r a t l e ns a n a l y z e d w e i g h e d a p p r o x . 25 rag, r a b b i t lens, 200 m g a n d calf lens, 800 rag. The r e s u l t s are g i v e n as t h e m e a n ± s t a n d a r d error of t h e m e a n of a t l e a s t i 2 t i s s u e s a m p l e s . T h e v a l u e s for l e ns w a t e r us e d t o c o n v e r t t h e d a t a to m i l l i m o l a r c o n c e n t r a t i o n s were d e t e r m i n e d in s e p a r a t e e x p e r i m e n t s a n d were 62°/o for r a t lens a n d 6 7 % for r a b b i t a n d calf lenses. T h e m o l a r i t y of i n t r a c e l l u l a r glucose w a s c a l c u l a t e d c o r r e c t i n g t h e c o n t e n t of lens glucose for t h e e x t r a c e l l u l a r space of t h e lens. F or t h i s p u r p o s e m a n n i t o l space was d e t e r m i n e d for each t y p e of lens iv.
Lens A T P ttmole/g lens
Rat Rabbit Calf
7.2 2~_i.i 3.1 ~- 0.5 2.3 ± 0.3
mM
12 4.6 3.4
Lens glucose 6-phosphate
Lens glucose
mt~mole/g lens m M
ttmole/g lens
mM
.4 queous humor glucose (mM)
0.67 -~ 0.04 0.59 ± 0.05 0.54 ± 0.06
0.55 0.45 0.65
6.1 ± 0.5 4.8 ~- 0.3 2.2 ~ 0.3
87,6 ~- 0.5 119 ~- 0.6 8i ~ 0. 5
o.I4 o.17 o.12
Biochim. Biophys. Acta, 141 (I967) 547-559
556
M.F.
LOU, J. H. KINOSHITA
TABLE VIII THE
INCUBATION
O F R A B B I T LI~NS I N VARXZING C O N C I ~ N T R A T I O N S
OF GLUCOSE
The r a b b i t lens was incubated for 24-h period in 95 % air and 5 % COy The total a m o u n t of lactate produced during the incubation is given. The other intermediates are those found in the lens after incubation. All values are given as/xmoles per gram of lens. The lens glucose, sorbitol and fructose were determined b y gas liquid c h r o m a t o g r a p h y 11. The p h o s p h o r y l a t e d intermediates were determined enzymaticaIly 15. The results are averages of at least 6 lenses.
Glucose in medium (mM) 5.5
Lens glucose
o.75
Sorbitol Fructose
2. 7
Glc-6-P
Fru-6-P
Fru-z,6-P 2 Triose phosphate
A TP
Total lactate produced
1.2
O.lO 5
0.028
o.15o
0.75
3.1
22o
io
1.9o
io.i
2.o
o.~45
o.o38
o.i75
o.85
3.o
235
15
4.30
16.8
3.3
o.13o
0.033
o.i6o
o.6o
2.9
235
Another interesting fact is that there is a significant quantity of free glucose in the lens I~. The extracellular space of the lens, estimated by mannitol, is 6- 7 % based on lens weight or IO % on lens water is. A correction for the glucose in the lens extracellular space can be made assuming that the glucose level is the same in this space as in the aqueous humor. Even after this correction, an appreciable amount of free glucose appears intracellularly. The low glucose value for calf aqueous humor is explained b y the difference between time of the sacrifice of the animal and the sampling of the aqueous humor. With the laboratory animals no such delay is encountered.
Incubation of the lens with different levels of glucose The effect of increasing concentrations of glucose on the levels of various glycolytic intermediates was studied with rabbit lenses incubated at 5.5, IO and 15 mM glucose (Table VIII). As the results indicate, increasing the level of glucose raises the intracellular glucose. Correcting for the extracellular space, the concentrations of intracellular glucose are 0.5, 2.0 and 5.5 mM after the lenses are incubated for 2 4 h in the three different levels of glucose. Even though the intracellular glucose level is increased the production of lactate is not affected. Furthermore, the levels of the phosphorylated intermediates of the early stages of the glycolytic mechanism are also not markedly altered. It thus appears that the rate of glycolysis in the intact lens is not stimulated by raising the glucose supply. The sorbitol pathway in the lens is another means by which glucose is metabolized (for review see ref. 6). Aldose reductase, the first enzyme of this pathway, competes with hexokinase for glucose. Because of the high Km Of aldose reductase, most of the glucose is phosphorylated and only a limited amount reduced to sorbitol at physiological levels of glucose. At 5.5 mM of glucose in the external medium the lens hexokinase appears saturated. Raising the concentration above this level does not seem to significantly increase glycolysis but it does stimulate the sorbitol pathway. At Io and 15 mM glucose, considerable amounts of sorbitol are found in the lens. Increases in fructose are also observed but at lower levels than sorbitol. These results are similar to those previously observed in our laboratory 1~. Biochim. Biophys. Acta, 14i (1967) 547-559
LENS GLYCOLYSIS
557
DISCUSSION The aerobic phase of glucose metabolism in the lens appears to play a limited role in energy production. I t has been estimated that 33 % of total ATP produced through the metabolism of glucose is due to respiration, assuming all the oxygen consumed by the lens is utilized for ATP synthesis ~°,21. KINOSHITA, KERN AND MEROLA1:~ found in calf lens that the energy requiring mechanisms, cation transport and protein synthesis, are maintained equally well anaerobically as they are aerobically. Consistent with these findings are those of KERN 2~ who found that anoxia has no effect on the amino acid transport mechanism in the calf lens. In the rabbit lens, KINSEY AND REDDYz:~ observed that the activity of the amino acid transport mechanism was reduced 27 ~o b y anoxia. In other studies with rabbit lens, BECKERz4 states that the accumulation of rubidium is relatively little affected b y dinitrophenol, fluoroacetate, cyanide or anoxia. Thus, the observations on rat, rabbit and calf lenses indicate that this ocular tissue depends primarily on anaerobic glycolysis for its supply of biological energy. Because an active aerobic phase of glucose metabolism does not exist in the lens, the principal end product of glucose metabolism is lactic acid. In a dense tissue such as the lens, elimination of lactate b y diffusion into the intraocular fluids m a y become a problem if the production of this acid is unduly high. Consequently, the rate of the glycolytic mechanism must be so poised that it is sufficient to supply the necessary amounts of ATP, but not excessively rapid to cause deleterious p H effects. The precise control of lens glycolysis seems to reside with the subtle properties of the two enzymes, hexokinase and phosphofructokinase. The activity of phosphofructokinase is regulated b y the concentration of ATP, while the level of glucose 6-phosphate governs the lens hexokinase activity. The normal intracellular composition of the lens is uniquely arranged to take advantage of the sensitivity of these two enzymes. Thus the normal concentrations of ATP and glucose 6-phosphate are sufficiently high to be well within the inhibitory range of the lens phosphofructokinase and hexokinase, respectively. When the phosphofructokinase reaction is inhibited b y ATP an increase in fructose 6-phosphate results. The equilibrium of the isomerase reaction favors the conversion of fructose 6-phosphate to glucose 6-phosphate. Therefore, when adequate amounts of ATP are available in the lens, the inhibition of the phosphofructokinase reaction leads to a build-up of glucose 6-phosphate, the factor that governs the hexokinase reaction. On the other hand if the level of ATP were reduced, the phosphofructokinase reaction would be accelerated, decreasing the glucose 6-phosphate level which in turn would stimulate the hexokinase reaction. Under these circumstances, the overall rate of glycolysis would be increased. Other important factors in controlling glycolysis are cyclic AMP and phosphate ions. Variations in the levels of these substances also regulate the degree of activity of the phosphorylating enzymes. Since the lens has a very low concentration of hexokinase, the capacity to phosphorylate glucose is limited. The inhibitory effect of glucose 6-phosphate further limits the phosphorylation mechanism and consequently the overall rate of glycolysis. The lens hexokinase appears as sensitive to low levels of glucose 6-phosphate as is the red-cell hexokinase. At o.I and o.oi mM glucose 6-phosphate, the lens hexokinase was inhibited b y 55 % and ~8 % respectively. At o.I mM glucose 6-phosphate, inhiBiochim. Biophys. ~lcta, 141 (I967) 547-559
558
M.F.
LOU, J. H. KINOSHITA
bition of hexokinase of ascites tumor was 2o %, mouse brain, 21% and red cells, 57 % (see ref. 2). Because the inhibitory controls are so effective, simply increasing the,intracellular glucose concentration does not increase lactate production. Under these conditions, the levels of glucose 6-phosphate and other phosphorylated intermediates are not significantly altered. These findings suggest that the controlling factors of lens glycolysis are manifested at the hexokinase step. In view of the evidence, the hexokinase reaction does appear to serve as the 'pacemaker' of lens glucose metabolism as initially described by PIRIE ~. Although increasing the level of glucose in the medium does not seem to affect the rate of glycolysis, it does seem to stimulate the relatively active sorbitol pathway in the lens ~,~. A marked accumulation of sorbitol and significant increases in fructose occur when the glucose level is raised above 5-5 raM. Increases have been previously observed in the lenses of diabetic rats 26. Although the aerobic phase of glucose metabolism is limited in the lens, a definite Pasteur effect is demonstrable~2, es. The results of the effect of anaerobiosis on lens metabolism will be reported later. ACKNOWLEDGEMENTS
This work was supported by the U.S. Atomic Energy Commission Contract AT (3o-1) 1368 and the U.S. Public Health Service for the Career Development Award S-K3-I7O82 and grant NB 06090. The authors wish to express their appreciation to Mr. LORENZO MEROLA AND Mr. BILL TUNG for their technical assistance.
REFERENCES I 2 3 4
5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22
T. E. MANSOOR, J. Biol. Chem., 238 (1963) 2285. K. UYEDA AND E, RACKER, J. Biol. Chem., 240 (I965) 4682. J. V. 1DASSONNEAUAND O. I-~. LOWRY, Biochem. Biophys. Res. Commun., I (1962) io. I. A. R o s e AND E. L. O'CoNNELL, J. Biol. Chem., 239 (1964) 12. R. W o , J. Biol. Chem., 24o (1965) 2827. J. H. KINOSHITA, Invest. Ophthalmol., 4 (1965) 619. H. GREEN AND S. A. SOLOMON, Arch. Ophthalmol., 61 (1959) 616. R. WECKERS AND H. SOLLMANN, Arch. Int. Med. Exp., 13 (1938) 483. R. VA~ HEYNINanN, Exptl. Eye Res., 9 (1965) 298. J. R. SI-IER~AN, Anal. Biochem., 5 (1963) 548. S. ]7IAYMAN, M. F. L o o , L. O. MEROLA AND J. H. KINOSHITA, Biochim. Biophys. Acta, 128 (1966) 474. FI. A. SOEER AND E, A. PETERSON, J. Am. Chem. Soc., 78 (1956) 751. J. H. KINOSHITA, H. L. KERN AND L. O. MEROLA, Biochim. Biophys. Acta, 47 (1961) 458. S. B. BARKER AND W. PI. SUMMERSON, J. Biol. Chem., 138 (1941) 535O. H. LowRY, J. V. PASSONNEAU, F. X. HASSELBERGER AND D. W. SCHULZ, J. Biol. Chem., 239 (1964) 18. T. E. MANSOUR, N. WAKID AND H. M. SPROUSE, J . Biol. Chem., 241 (1966) 1512. J. F. KUCK, Invest. Ophthalmol., 5 (1966) 65. R. THOFT AND J. H. •INOSHITA, Exptl. Eye Res., 4 (1965) 287. J. H. KINOSI-III"A, S. FUTTERMAN, K. SATOH AND L. O. MEROLA, Biochim. Biophys. Acta, 74 (1963) 340. O. IZLIEFELD AND O. HOCKWlN, Arch. Ophthalmol., 162 (196o) 346. T. SIPPEL, Invest. Ophthalmol., I (1962) 385 . H. KEEN, Invest. Ophthalmol., I (1962) 368.
Biochim. Biophys. Acta, I41 (1967) 547-559
LENS GLYCOLYSIS 23 24 25 26 27 28
V. B. A. R. A. R.
559
E. I~INSt~Y AND D. V, N. REDDY, lnvesl. Ophthalmol., 2 (1962) 229. BECKER, Invest. Ophthalmol., I (1962) 502. PIRIE, Bibliotheca ophthalmol,, 49 (1957) 287. VAN HEYNINGEN, Nature, 184 (1959) 184. SoLs AI~O R, K. CRANE, J. Biol. Chem., 206 (1954) 925. V&N HE~CNINGEN, Biochem. J . , 96 (1965) 419.
Biochim. Biophys. Aeta, 141 (I967 547-559
547
BBA 25 810 CONTROL OF LENS GLYCOLYSIS MARJORIE F. LOU AND JIN H. KINOSHITA Howe Laboratory of Ophthalmology, Harvard Medical School, and the Massachusetts Eye and Ear Infirmary, Boston, Mass. (U.S.A.)
(Received January 9th, 1967)
SUMMARY
I. The calf-lens phosphoiructokinase (ATP: I)-Iructose-6-phosphate I-phosphotransferase, EC 2.7.I.II ) was isolated and purified 86 fold. I t was found that ATP inhibits lens phosphofructokinase at a concentration above 0.25 mM. Cyclic AMP was the most effective in releasing the inhibition of phosphofructokinase b y glucose 6-phosphate. 2. The calf-lens hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1 ) is susceptible to inhibition by low levels of glucose 6-phosphate. The inhibitory effect of glucose 6-phosphate can be partially counteracted b y increasing concentrations of inorganic phosphate. 3. The normal content of ATP and glucose 6-phosphate in the lens are at levels where considerable degree of inhibition would be exerted on the two enzymes, hexokinase and phosphofructokinase. 4. The levels of the glycolytic intermediates in the lens were measured after incubation under aerobic conditions at 5.5, IO and 15 mM glucose. Increasing the availability of glucose raised the intracellular glucose, but the levels of sugar phosphates and the production of lactate remained unchanged. 5- Increasing the availability of glucose did, however, stimulate the lens sorbitol pathway. Increasing levels of sorbitol were found in the lenses incubated at the 3 different concentrations of glucose.
INTRODUCTION Studies on the control mechanisms of glycolysis in a number of tissues have indicated that one of the rate-limiting reactions appears to occur at the phosphofructokinase (ATP:D-fructose-6-phosphate I-phosphotransferasej EC 2.7.1.11 ) step. The control is effected by the ability of the reaction product to inhibit the enzyme. The inhibition of phosphofructokinase b y excess ATP has been demonstrated in heart 1, muscle ~ and brain 3. This inhibition is released b y the addition of AMP and several other metabolites. Other sites and factors m a y be involved in the control of glycolysis. For example, R o s e ANI) O'CoNNELLa found t h a t the rate of glucose utilization in human erythrocytes depends upon the rate of the hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.I) step which is governed b y the removal of glucose Biochim. Biophys: Acta, 141 (1967) 547-559
548
M. F. LOU, J. H. KINOSHITA
6-phosphate, an effective inhibitor of hexokinase. In Ehrlich ascites tumor cell, Wtr 5 demonstrated that orthophosphate is the major rate-limiting factor for glycolysis. In the lens, glycolysis is the principal source of biological energy required for the maintenance of its function (for review see ref. 6). Thus it is of considerable interest to understand how glycolysis is regulated in the lens. It has been demonstrated that the rate of glycolysis is increased in rabbit-lens extract when hexokinase is added to the reaction mixture or when glucose is replaced by glucose 6-phosphate as the substrate~, 8. These observations were later supported by the demonstration that the lactate production from glucose 6-phosphate was considerably higher than that from glucose in extracts of rabbit, calf and rat lenses s, and that the activity of lens hexokinase is the lowest among all the enzymes of the glycolytic pathway 1°. These findings all suggest that the hexokinase reaction is the rate-limiting step. Nevertheless the rate of glucose utilization by the intact lens is less than that of the hexokinase reaction, therefore other regulatory factors may be involved. In this paper evidence is presented suggesting that both the hexokinase and phosphofructokinase reactions participate in controlling lens glycolysis. MATERIALS
Calf eyes were obtained from a local abattoir soon after slaughtering, and the lenses removed and frozen until needed. The rabbit lenses were obtained by removing them carefully from rabbit eyes soon after the young animals had been killed by air embolism. Aqueous humor samples were withdrawn before the laboratory animals were killed. The calf aqueous humor was obtained after the eyes arrived at the laboratory. DPNH, TPNH, nucleotides and sugar phosphates were obtained from Sigma Company, while uniformly ~*C-labeled glucose was obtained from New England Nuclear. All enzymes used in the determination of the glycolytic lens intermediates were products of Boehringer. METHODS
Determination of hexokinase activity Hexokinase assay was carried out according to the modification of the method of SHERMANI0. Lens homogenate was prepared by homogenizing IO calf lenses in a IO ml mixture of 0.2 M potassium phosphate buffer (pH 7.2), I mM versene and I mM fl-mercaptoethanol. The homogenate was dialyzed against IO times its volume of the same buffer solution for 3 h and then centrifuged at IOOOO × g for IO rain. The supernatant fluid was used for the source of hexokinase. The assay mixture contained in a final volume of 0.75 ml: 0.66 mM of [14C~glucose with specific activity of 1.77"1o5 counts/min per t~mole; 3-3 mM ATP; 6.6 mM MgC1,; 3.2 mM NaF; 6.6 mM mercaptoethanol; 66 mM Tris-HC1 buffer (pH 7.8) and aliquots of the dialyzed supernatant fluids. The mixture was allowed to incubate at 37 ° for 15 min. The reaction was stopped by immediately applying 50/A of each sample to a DEAE-cellulose paper (1.5 cm × 14 cm). Methods of separation and the measurement of the reaction product [14C]glucose 6-phosphate were similar to the technique described for [I~C]galactose I-phosphate~L Biochim. Biophys. Aaa, x4x (x967) 547-559
LENS GLYCOLYSIS
549
Purification of phosphofructokinase All operations were performed in the cold room at 5 ° except where otherwise mentioned. The general pattern of the purification of phosphofructokinase was that devised by MANSOURt,TM. Step ~: preparation of lens homogenate, 30 calf lenses were homogenized in 200 ml mixture of IO mM Tris-HC1 buffer (pH 8.0) ; 2 mM versene; 5 mM fl-mercaptoethanol; 50 mM MgSO~ and 0.5 mM ATP in a TenBroeck homogenizer and centrifuged at IOOOO × g for 2o rain to remove insoluble material. The supernatant fluid was saved. Step 2 : ethanolfractionation. To a 5o-ml solution of the lens extract, kept at --8 ° in a salt-ice bath, 4.35 ml of 95 % ethanol (--20 °) were added in a dropwise fashion with continuous stirring. The resulting mixture containing 8 % ethanol was gently stirred for 30 min after which it was centrifuged at IOOOO × g for IO min and the precipitate discarded. To the supernatant fluid, 3.1 ml of the cold ethanol were added, which raised the ethanol concentration to 13 %. The mixture was stirred for 30 min and the precipitate recovered after centrifugation contained the enzyme. The precipitate was taken up in IO ml of the same buffer mixture used for the lens homogenization. Step 3: column chromatography. DEAE-cellulose powder was washed and prepared for use by a modification of the procedure of SOBER AND PETERSON12. A freshly made column (3 cm × 24 cm) of DEAE-cellulose was equilibrated with a solution consisting of IO mM Tris-HC1 buffer (pH 8.0); 20 mM MgSO4; 5 mM fi-mercaptoethanol; o.I mM ATP and o.oi mM fructose 1,6-diphosphate in the cold room for an overnight period. The ethanol fraction usually containing 22 mg of protein was applied to the column after which the column was washed with o.oi M Tris buffer until the absorbance of eluates at 280 m/~ was less than o.I absorbance units. The first buffer was followed by a series of Tris buffers varying in concentration from o.o5 M to 0.3 M. Using a stepwise elution technique, fractions of io ml were collected at a flow rate of approx. I ml/min. The fractions containing phosphofructokinase activity were collected with the 0.2 M buffer. A purification of 86-fold was achieved at this stage. Step 4: ammonium sulfate fractionation. The pooled active fractions of the enzyme was precipitated by adding solid (NH4)~SO 4 to give a concn, of 40 %. In this form the enzyme was stable and could be stored for a week. The precipitate was taken up by the Tris-buffer mixture containing the stabilizing agents used for column equilibration. This preparation of the enzyme was used for all the kinetic studies. Table I indicates the overall purification procedure for lens phosphofructokinase.
Determination of phosphofructokinase activity For the enzyme assay, an aliquot of the enzyme preparation was added to a reaction mixture consisting of: 50 mM Tris buffer (pH 7.4) ; o.o12 mM D P N H ; 0.2 mM MgC12; o. i mM ATP; I mM fructose 6-phosphate; triose phosphate isomerase; glycerol phosphate dehydrogenase and aldolase in a total volume of I.O ml. The reaction was run at room temperature over a 6 min period. The enzymes present in the reaction mixture rapidly converted the product formed, fructose 1,6-diphosphate, to glycerol phosphate which was estimated by the disappearance of D P N H fluorometfically. A Farrand fluorometer model A-2 was used for this purpose. Biochim. Biophys. ~4cta, 141 (i967) 547-559
55 °
M.F.
LOU, J. H. KINOSHITA
TABLE I PURIFICATION
OF PHOSPHOFRUCTOKINAS~
FROM
C A L F L]~NS
One phosphofructokinase activity u n i t is the a m o u n t of enzyme t h a t catalyzes the formation of I ffmole of p r o d u c t per min. Details are presented in the t e x t under METHODS.
Steps
Vol.
Units/ml Total units
Protein (mg/ml)
Specific activity
Yield
Lens h o m o g e n a t e E t h a n o l fraction (8-13 °/o) DEAf-cellulose chromatography (NHa)~SO4 (o.4o %)
2oo 3° 35 5
o.33 o.63 0.29 o.64
47.5 21. 5 0. 5 1.8
o.oo 7 o.o 3 o,6 o. 4
IOO 28.1 15.2 4.8
66.6 18.8 io.2 3 .2
Measurement of intermediates of the glycolysis in rabbit lens Young rabbit lenses weighing 200 ± IO mg were incubated in IO ml TC-I99 medium 1~ with varying glucose concentrations of 5.5, IO and I5 raM. The tonicity of the incubation medium was 300 mosM and was equilibrated with 95 % air and 5 % CO2. After incubation at 37 ° for 24 h, the lens was removed from the culture tube and homogenized in 4 ml of o.6 M HCIO~. After centrifugation, the supernatant fluid was neutralized with 5 M K O H and the KC10~ removed b y centrifugation. Aliquots of the clear supernatant fluid were then assayed for each intermediate. Lactate was determined b y the method of BARKER AND SU'MMERSON14'. Glucose, sugar phosphates and adenine nucleotides were determined b y the modified method of LowRY et aN n. Sugars and sorbitol of the incubated lens were determined b y gasliquid chromatography, the methods of which are previously described 11. RESULTS
Properties of ~hos~hofructokinase Similar to the findings of MANSOUR, WAKID AND SPROUSE 16, the presence of ATP fructose 1,6-diphosphate, Mg 2+ and mercaptoethanol was found necessary to 16!
12
0.5
~,g
~
0.2
~
~
b ~ Q2
~
~
~
~
~ o.~
~
~
~
~"
~
~~
,o~
q
~
~
~
~-
V [ ~ - ~ - ~ ~o-~
Fig. I. p H optimum for lens phosphofructo~nase. In the p H range 6.o-7.5, 5o mM phosphate buffers were used; for the p H range of 7.3-8.6, 5o mM Tris buffers; p H 7.o, 5o mM imidazole buffer and p H 9-5, 5 o mM carbonate-bicarbonate buEer. Fig. 2. Lineweaver-Burk plot for phosphofructokinase with fructose 6-phosphate. The p H of the reaction is 8.0 and the A T P concentration is constant at o.I m ~ .
Biochim, Biophys. Acta, 141 (I967) 547-559
LENS GLYCOLYSIS
551
stabilize the lens phosphofructokinase during the purification procedures. In the presence of these stabilizing agents, the enzyme was able to withstand temperatures at 37 °. However, after DEAE-cellulose column chromatography, the active fractions collected were unstable even in the presence of these agents. A 50 % loss was incurred during the overnight storage at 4 ° . However, it was found that the enzyme precipitated b y (NH4)2SO4 could be stored up to a week without appreciable loss of activity. As shown in Fig. I the optimum p H of lens phosphofructokinase was 8. Michaelis-Menten constants of 19o and 7 ~M were determined for fructose 6-phosphate and ATP respectively. The plot of the reciprocal of initial velocity against the reciprocal of the fructose 6-phosphate concentrations reveals a typical substrate curve (Fig. 2). In contrast, the Lineweaver-Burk plot for ATP shows that at high concentrations the velocity of the reaction is markedly decreased indicating substrate inhibition (Fig. 3). The inhibition of phosphofructokinase begins to occur above 0.25 mM ATP. Above this concentration ATP becomes increasingly effective as an inhibitor of
O.e 0.[
~ 0.4 ~
0,4~
~
. . ~ , . . 1 "'~
~ , ~3 .........................
~-~-~,,'...~,I~, .................... " .................... m
o- ~ '
~,
•
~, ©
~o2 q
~7
~_~.e ,.;'............
.... ,..-,,~""" ~.....
~
~
o_~,
o.6'
~
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~b
2b
~/[A~P] ~o-~
3b
_
_
~
4o
°o~-
I
~o
1
4o
~.
~o
~/~r~_l~o-~
Fig. 3- I n h i b i t i o n of lens p h o s p h o f r u c t o k i n a s e by ATI ~. A double reciprocal plot of reaction velocity versus concentration of s u b s t r a t e ATP is given. Details of the e n z y m e assay are described in MEn,tOIlS. The velocity is expressed as /,moles of fructose 1,6-diphosphate formed per minute. The a r r o w p o i n t s to o.25 raM, the concentration above which ATP begins to inhibit the lens phosphofructokinase. Fig. 4- The effect of p H and fructose 6-phosphate on the ATP inhibition of phosphofructokinase. The a s s a y conditions are the same as those in MI~THODS, except t h a t fructose 6-phosphate varied from I mM to o.I mM in 5 ° mM imidazole buffer (pH 7) or in 5 ° mM Tris buffer (pH 8).
the reaction. At 5 mM, ATP inhibits phosphofructokinase activity b y 5 ° %. The inhibitory effect of ATP on the lens phosphofructokinase is independent of the level of fructose 6-phosphate or of p H (Fig. 4). Thus, the shape of the curve is unaffected b y different levels of fructose 6-phosphate or b y a difference of I p H unit. This is in contrast to UYEDA AND RACKER2 who reported that the magnitude of ATP inhibition was p H dependent in rabbit-muscle phosphofructokinase. The ATP inhibition is more pronounced at p H ' s lower than 9, which is consistentwith the observation OfMANSOUR1 who found that ATP inhibition was less effective at alkaline pH's. However, in contrast with our findings, MANSOUR found that inhibition of heart phosphofructokinase was less pronounced with increasing concentrations of the second substrate, fructose 6-phosphate. The effects of other compounds were also studied (Table II). At tile same concentration of ATP, which produced 5 ° % inhibition, GTP caused only a 26 % effect. B$ochim. Biophys..4cta, 14i (t967) 547-559
552
M. F. LOU, J. H. KINOSHITA
TABLE II INHIBIt?ION
OF PHOSP/-IOFRUCTOKINASE
E x p e r i m e n t a l c o n d i t i o n s are t h e s a m e as d e s c r i b e d in Fig. I. I n all cases t h e c o m p o u n d s t e s t e d were a d d e d t o a r e a c t i o n m i x t u r e c o n t a i n i n g o.I mM ATP.
Compound added
COnch.
Activity
Inhibition
CraM) 0
(%) 7.6
Citrate
Phosphoenolpyruvate GTP
Sor bito l
0.5 2.5 5.0 0.5 2. 5 0.5 2.5 5.o 50.0
6.6 5.8 5.8 5.8 7.0 5.6 3.6 2.8 7.6
-
-
I3 24 24 -8 26.3 52.7 63.o --
TABLE III R~L~ASE
OF
INORGANIC
ATP
INHIBITION
OF P H O S P H O F R U C T O K I N A S E
BY NUCLEOTIDES
AND
PHOSPHATE
C o n d i t i o n s of t h e e x p e r i m e n t were i d e n t i c a l as t h a t d e s c r i b e d in Fig. z. I n a l l cases, l e ns phos phof r u c t o k i n a s e a c t i v i t y was i n h i b i t e d b y 5 mM ATP. The a c t i v i t y w i t h o . i mM A T P is t h e uni n h i b i t e d rate. T h e a c t i v i t y is e x p r e s s e d as t h e c h a n g e in a b s o r b a n c e pe r rain.
A TP (raM)
Compound added
O.I
O
5.0 5.0 5.o 5.o 5.0 5.o 5.o 5.o 5.0 5.0 5.0 5,0 5.0 5,o 5,0 5,o 5.0 5,o 5.0 5.0 5,0 5.0 5.0 5.0 5.o
o ADP ADP ADP 3'-AMP 3'-AMP 3'-AMP Cyclic A M P Cyclic A M P Cyclic A M P 5'-AMP 5 "-AMP 5'-AMP dAMP dAMP dAMP CMP CMP CMP UMP UMP UMP GMP GMP GMP P~ Pt Pt
5.0 5.o 5.0
Conch. (mM)
A ctivity
Activity (%)
I 1.0
IOO
5.3 7.0 8.o 6.8 6.0 7.o 6.9 9.o
48 64 73 62 55 64 63 82 115 lO9 59 69 68 6z 74 73 63 73 68 68 73 68 64 73 69 66 76 66
o.i o.5 i.o 0.o 5 o. 5 I.O o.I 0.5 I.O o.I 0.5 1.o o.I 0. 5 I.o o.I o.5 I.o o.I 0. 5 I.O o.I o. 5 I.O o.~ o.5
I2.O 6.4 7.5 7.4 6.8 8. I 8.o 6.9 8.0 7.4 7.5 8.o 7.4 7.0 8.0 7.5 7,3 8.4
r.o
7.3
12.7
Biochim. Biophys. Acta, 141 {I967) 547-559
LENS GLYCOLYSIS
553
Citrate, at concentration above 2. 5 raM, produced a 24% inhibition. However, phosphoenolpyruvate had no effect.
Release of A TP inhibition Various nucleotides and inorganic phosphate are known to release the ATP inhibition of phosphofructokinase. As summarized in Table III, the most effective nucleotide in releasing ATP inhibition is 3',5'-cyclic AMP. The 50% inhibited phosphofructokinase activity produced by 5 mM ATP was completely released in the presence of 0.5 mM cyclic AMP. However, at the same concentration, ADP, AMP derivatives and inorganic phosphate were much less effective, overcoming the inhibition by only 15-25 %. The plot of the reciprocal of the cyclic AMP concentration against the reciprocal of the reaction velocity is given in Fig. 5. At o.I mM ATP, the lens phosphofructokinase is not inhibited. However, under these conditions increasing cyclic AMP concentrations appear to stimulate the phosphofructokinase reaction as shown in the graph. 0.8
.,,, ~ ....................~...........
~ ~,.:o................... 0.6
-
,,,~,.,, ............... .... ~,,,."~
.........
(3.4 rn
b ..~ \: Q2
l
2
__
I
4
~
I~
6
~/[~,li~m~,~o'~
8
1 - -
10
Fig. 5- Release of AT1~ inhibition of p h o s p h o f r u c t o k i n a s e b y 3',5'-cyclic AMP. A double-reciprocal plot of reaction v e l o c i t y versus c o n c e n t r a t i o n of 3',5'-cyclic A M P is given. T h e A T P c o n c e n t r a t i o n w a s either o.I or 0.2 5 m M while t h e c o n c e n t r a t i o n of fructose 6 - p h o s p h a t e w a s c o n s t a n t a t 5 ° ffM. T h e p H w a s m a i n t a i n e d a t 7.0 w i t h imidazole buffer. Velocities are e x p r e s s e d as t h e / z m o l e s of p r o d u c t fructose d i p h o s p h a t e f o r m e d per min.
When phosphofructokinase is inhibited by ATP (2.5 mM), the stimulation of the reaction velocity with increasing concentrations of cyclic AMP is more pronounced. These results indicate that when ATP is present at levels below the inhibitory range, cyclic AMP appears to be an activator of lens phosphofructokinase. When phosphofructokinase is inhibited by ATP, cyclic AMP counteracts the inhibitory effect of the trinucleotide, in addition to activating the enzyme.
Hexokinase inhibition study Due to the extremely low hexokinase activity in lens and the instability of the enzyme, we were unable to purify the enzyme to any significant extent. Consequently, a dialyzed calf-lens homogenate was used as the source of the enzyme. Glucose 6-phosphate, the product of the reaction exerts a pronounced inhibitory effect on hexokinase. There is a progressive decrease in hexokinase activity with increasing concentrations of glucose 6-phosphate. A concentration of o.o133 mM of Biochim. Biophys. Acta, 141 (1967) 5 4 7 - 5 5 9
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H. K I N O S H I T A
glucose 6-phosphate produces a I8 % inhibition as indicated in Table IV. At a concentration IO times higher, the inhibition is 55 %. As shown in Table V, ADP, the other reaction product of hexokinase, is not inhibitory. In contrast, the studies with purified ascites-tumor hexokinase 2 as well as brain hexokinase 2~ revealed that A D P appears to be a competitive inhibitor of ATP. Galactose x-phosphate is of interest because of its possible importance in cataracts of galactosemia 6. It did, however, exert no inhibition on lens hexokinase (Table V). The inhibition of hexokinase by glucose 6-phosphate is released partially by inorganic phosphate. Table VI presents data on the effect of phosphate concentration TABLE
IV
INHIBITION OF LENS HEXOKINASE BY GLUCOSE a-PHOSPHATE T h e r e a c t i o n m i x t u r e u s e d in d e t e r m i n i n g h e x o k i n a s e a c t i v i t y is d e s c r i b e d in t h e METHODS.
Glucose 6-phosphate (M × 10 -5) 0
1.33 3-33 6.67 IO.O 13.3 33"3
TABLE
A ctivity (l~mole/min × i o -~)
Inhibition (%)
2.2 1.8 1.6 1.4 1.2 I.O 0.8
-18 27 36 45 55 64
V
EFFECT OF A D P
AND GALACTOSE I-PHOSPHATE ON HEXOKINASE ACTIVITY
T h e c o n d i t i o n s of t h e a s s a y a r e g i v e n in t h e METHODS.
Compound added
O ADP ADP GaI-I-P
TABLE
ConcH. (raM)
Activity (Ixmole/min × ~o -~)
Activity (%)
o.33 1.33 o.67
0.86 o.75 o.94 o.So
IOO 87 iio 93
VI
EFFECT OF INORGANIC PHOSPHATE ON THE INHIBITION OF HEXOKINASE BY GLUCOSE 6-PHOSPHATE T h e c o n d i t i o n s of t h e a s s a y a r e g i v e n in t h e METHODS.
Glucose 6-phosphate
Inorganic phosphate
Hexokinase activity
(raM)
(raM)
(k~mole/min X zO-3)
Activity (%)
1.7 0. 4 o.5 o.6 o.8 o.9
IOO 24 29 35 47 53
o 0.33 0.33 0.33 0.33 o.33
o o 1.33 3-33 6.67 13. 3
Biochim. Biophys. Acta, ~41 (1967) 5 4 7 - 5 5 9
LENS GLYCOLYSIS
555
on the glucose 6-phosphate inhibition of hexokinase. Under no condition is recovery of activity complete. When hexokinase activity is 75 % inhibited by glucose 6-phosphate, a level of phosphate ion of 13 mM lowered the inhibition to 24 %. In comparison UYEDA and RACKER2 have reported that when brain hexokinase is 5o % inhibited, the presence of IO mM inorganic phosphate released the inhibition to 24 %; while at 75 % inhibition of red cell hexokinase, IO mM inorganic phosphate lowered the inhibition to 32 %. In many respects the hexokinase of the lens resembles that of the red cell in its susceptibility to glucose 6-phosphate inhibition and to the degree of the release of inhibition by inorganic phosphate. The glucose 6-phosphate inhibitory effect on hexokinase is only slightly reversed by high concentrations of ATP. For example, the inhibition of hexokinase by glucose 6-phosphate is at the 55 % level with o.o67 mM ATP. When the ATP concentration is raised to o.67 mM, a substantial inhibition of 48 % is still observed. Thus, the trinucleotide is less effective than phosphate ion in counteracting the glucose 6-phosphate inhibition of hexokinase. No active ATPase is present in this preparation, but it must be kept in mind that the results are based on experiments with crude lens extracts. The normal glucose content in the aqueous h u m o r and the lens of rabbit, rat and calf
In Table VII the data on the concentrations of glucose and glucose 6-phosphate in the aqueous humor and the lens are given along with the lens-ATP content. Since phosphorylated derivatives do not readily penetrate biological membranes, it is reasonable to assume that all the glucose 6-phosphate is intracellular. From the results given in Table Vii, the concentration of glucose 6-phosphate can be estimated to be o.I mM in the lens. This level of glucose 6-phosphate, as indicated in Table IV, should produce at least a 50 % inhibition of hexokinase. The ATP concentration appears high in the lens. The rat lens has an ATP concentration of IO mM while the rabbit and calf lenses are well above I raM. These concentrations should be sufficient to exert marked inhibition of lens phosphofructokinase. TABLE VII GLUCOSE, GLUCOSE 6-PHOSPHATE AND A T P CO~TE~T IN VARmUS Lt~NSF~S S a m p l e s of a q u e o u s h u m o r were w i t h d r a w n from a n a e s t h e s i z e d r a t s a n d r a b b i t s a f t e r w h i c h t h e lenses were r e m o v e d , w e i g h e d a n d i m m e d i a t e l y h o m o g e n i z e d in o.6 M cold HCIO 4. A q u e o u s h u m o r was r e m o v e d from t h e calf eyes a f t e r t h e y a r r i v e d a t t h e l a b o r a t o r y . The r a t l e ns a n a l y z e d w e i g h e d a p p r o x . 25 rag, r a b b i t lens, 200 m g a n d calf lens, 800 rag. The r e s u l t s are g i v e n as t h e m e a n ± s t a n d a r d error of t h e m e a n of a t l e a s t i 2 t i s s u e s a m p l e s . T h e v a l u e s for l e ns w a t e r us e d t o c o n v e r t t h e d a t a to m i l l i m o l a r c o n c e n t r a t i o n s were d e t e r m i n e d in s e p a r a t e e x p e r i m e n t s a n d were 62°/o for r a t lens a n d 6 7 % for r a b b i t a n d calf lenses. T h e m o l a r i t y of i n t r a c e l l u l a r glucose w a s c a l c u l a t e d c o r r e c t i n g t h e c o n t e n t of lens glucose for t h e e x t r a c e l l u l a r space of t h e lens. F or t h i s p u r p o s e m a n n i t o l space was d e t e r m i n e d for each t y p e of lens iv.
Lens A T P ttmole/g lens
Rat Rabbit Calf
7.2 2~_i.i 3.1 ~- 0.5 2.3 ± 0.3
mM
12 4.6 3.4
Lens glucose 6-phosphate
Lens glucose
mt~mole/g lens m M
ttmole/g lens
mM
.4 queous humor glucose (mM)
0.67 -~ 0.04 0.59 ± 0.05 0.54 ± 0.06
0.55 0.45 0.65
6.1 ± 0.5 4.8 ~- 0.3 2.2 ~ 0.3
87,6 ~- 0.5 119 ~- 0.6 8i ~ 0. 5
o.I4 o.17 o.12
Biochim. Biophys. Acta, 141 (I967) 547-559
556
M.F.
LOU, J. H. KINOSHITA
TABLE VIII THE
INCUBATION
O F R A B B I T LI~NS I N VARXZING C O N C I ~ N T R A T I O N S
OF GLUCOSE
The r a b b i t lens was incubated for 24-h period in 95 % air and 5 % COy The total a m o u n t of lactate produced during the incubation is given. The other intermediates are those found in the lens after incubation. All values are given as/xmoles per gram of lens. The lens glucose, sorbitol and fructose were determined b y gas liquid c h r o m a t o g r a p h y 11. The p h o s p h o r y l a t e d intermediates were determined enzymaticaIly 15. The results are averages of at least 6 lenses.
Glucose in medium (mM) 5.5
Lens glucose
o.75
Sorbitol Fructose
2. 7
Glc-6-P
Fru-6-P
Fru-z,6-P 2 Triose phosphate
A TP
Total lactate produced
1.2
O.lO 5
0.028
o.15o
0.75
3.1
22o
io
1.9o
io.i
2.o
o.~45
o.o38
o.i75
o.85
3.o
235
15
4.30
16.8
3.3
o.13o
0.033
o.i6o
o.6o
2.9
235
Another interesting fact is that there is a significant quantity of free glucose in the lens I~. The extracellular space of the lens, estimated by mannitol, is 6- 7 % based on lens weight or IO % on lens water is. A correction for the glucose in the lens extracellular space can be made assuming that the glucose level is the same in this space as in the aqueous humor. Even after this correction, an appreciable amount of free glucose appears intracellularly. The low glucose value for calf aqueous humor is explained b y the difference between time of the sacrifice of the animal and the sampling of the aqueous humor. With the laboratory animals no such delay is encountered.
Incubation of the lens with different levels of glucose The effect of increasing concentrations of glucose on the levels of various glycolytic intermediates was studied with rabbit lenses incubated at 5.5, IO and 15 mM glucose (Table VIII). As the results indicate, increasing the level of glucose raises the intracellular glucose. Correcting for the extracellular space, the concentrations of intracellular glucose are 0.5, 2.0 and 5.5 mM after the lenses are incubated for 2 4 h in the three different levels of glucose. Even though the intracellular glucose level is increased the production of lactate is not affected. Furthermore, the levels of the phosphorylated intermediates of the early stages of the glycolytic mechanism are also not markedly altered. It thus appears that the rate of glycolysis in the intact lens is not stimulated by raising the glucose supply. The sorbitol pathway in the lens is another means by which glucose is metabolized (for review see ref. 6). Aldose reductase, the first enzyme of this pathway, competes with hexokinase for glucose. Because of the high Km Of aldose reductase, most of the glucose is phosphorylated and only a limited amount reduced to sorbitol at physiological levels of glucose. At 5.5 mM of glucose in the external medium the lens hexokinase appears saturated. Raising the concentration above this level does not seem to significantly increase glycolysis but it does stimulate the sorbitol pathway. At Io and 15 mM glucose, considerable amounts of sorbitol are found in the lens. Increases in fructose are also observed but at lower levels than sorbitol. These results are similar to those previously observed in our laboratory 1~. Biochim. Biophys. Acta, 14i (1967) 547-559
LENS GLYCOLYSIS
557
DISCUSSION The aerobic phase of glucose metabolism in the lens appears to play a limited role in energy production. I t has been estimated that 33 % of total ATP produced through the metabolism of glucose is due to respiration, assuming all the oxygen consumed by the lens is utilized for ATP synthesis ~°,21. KINOSHITA, KERN AND MEROLA1:~ found in calf lens that the energy requiring mechanisms, cation transport and protein synthesis, are maintained equally well anaerobically as they are aerobically. Consistent with these findings are those of KERN 2~ who found that anoxia has no effect on the amino acid transport mechanism in the calf lens. In the rabbit lens, KINSEY AND REDDYz:~ observed that the activity of the amino acid transport mechanism was reduced 27 ~o b y anoxia. In other studies with rabbit lens, BECKERz4 states that the accumulation of rubidium is relatively little affected b y dinitrophenol, fluoroacetate, cyanide or anoxia. Thus, the observations on rat, rabbit and calf lenses indicate that this ocular tissue depends primarily on anaerobic glycolysis for its supply of biological energy. Because an active aerobic phase of glucose metabolism does not exist in the lens, the principal end product of glucose metabolism is lactic acid. In a dense tissue such as the lens, elimination of lactate b y diffusion into the intraocular fluids m a y become a problem if the production of this acid is unduly high. Consequently, the rate of the glycolytic mechanism must be so poised that it is sufficient to supply the necessary amounts of ATP, but not excessively rapid to cause deleterious p H effects. The precise control of lens glycolysis seems to reside with the subtle properties of the two enzymes, hexokinase and phosphofructokinase. The activity of phosphofructokinase is regulated b y the concentration of ATP, while the level of glucose 6-phosphate governs the lens hexokinase activity. The normal intracellular composition of the lens is uniquely arranged to take advantage of the sensitivity of these two enzymes. Thus the normal concentrations of ATP and glucose 6-phosphate are sufficiently high to be well within the inhibitory range of the lens phosphofructokinase and hexokinase, respectively. When the phosphofructokinase reaction is inhibited b y ATP an increase in fructose 6-phosphate results. The equilibrium of the isomerase reaction favors the conversion of fructose 6-phosphate to glucose 6-phosphate. Therefore, when adequate amounts of ATP are available in the lens, the inhibition of the phosphofructokinase reaction leads to a build-up of glucose 6-phosphate, the factor that governs the hexokinase reaction. On the other hand if the level of ATP were reduced, the phosphofructokinase reaction would be accelerated, decreasing the glucose 6-phosphate level which in turn would stimulate the hexokinase reaction. Under these circumstances, the overall rate of glycolysis would be increased. Other important factors in controlling glycolysis are cyclic AMP and phosphate ions. Variations in the levels of these substances also regulate the degree of activity of the phosphorylating enzymes. Since the lens has a very low concentration of hexokinase, the capacity to phosphorylate glucose is limited. The inhibitory effect of glucose 6-phosphate further limits the phosphorylation mechanism and consequently the overall rate of glycolysis. The lens hexokinase appears as sensitive to low levels of glucose 6-phosphate as is the red-cell hexokinase. At o.I and o.oi mM glucose 6-phosphate, the lens hexokinase was inhibited b y 55 % and ~8 % respectively. At o.I mM glucose 6-phosphate, inhiBiochim. Biophys. ~lcta, 141 (I967) 547-559
558
M.F.
LOU, J. H. KINOSHITA
bition of hexokinase of ascites tumor was 2o %, mouse brain, 21% and red cells, 57 % (see ref. 2). Because the inhibitory controls are so effective, simply increasing the,intracellular glucose concentration does not increase lactate production. Under these conditions, the levels of glucose 6-phosphate and other phosphorylated intermediates are not significantly altered. These findings suggest that the controlling factors of lens glycolysis are manifested at the hexokinase step. In view of the evidence, the hexokinase reaction does appear to serve as the 'pacemaker' of lens glucose metabolism as initially described by PIRIE ~. Although increasing the level of glucose in the medium does not seem to affect the rate of glycolysis, it does seem to stimulate the relatively active sorbitol pathway in the lens ~,~. A marked accumulation of sorbitol and significant increases in fructose occur when the glucose level is raised above 5-5 raM. Increases have been previously observed in the lenses of diabetic rats 26. Although the aerobic phase of glucose metabolism is limited in the lens, a definite Pasteur effect is demonstrable~2, es. The results of the effect of anaerobiosis on lens metabolism will be reported later. ACKNOWLEDGEMENTS
This work was supported by the U.S. Atomic Energy Commission Contract AT (3o-1) 1368 and the U.S. Public Health Service for the Career Development Award S-K3-I7O82 and grant NB 06090. The authors wish to express their appreciation to Mr. LORENZO MEROLA AND Mr. BILL TUNG for their technical assistance.
REFERENCES I 2 3 4
5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21 22
T. E. MANSOOR, J. Biol. Chem., 238 (1963) 2285. K. UYEDA AND E, RACKER, J. Biol. Chem., 240 (I965) 4682. J. V. 1DASSONNEAUAND O. I-~. LOWRY, Biochem. Biophys. Res. Commun., I (1962) io. I. A. R o s e AND E. L. O'CoNNELL, J. Biol. Chem., 239 (1964) 12. R. W o , J. Biol. Chem., 24o (1965) 2827. J. H. KINOSHITA, Invest. Ophthalmol., 4 (1965) 619. H. GREEN AND S. A. SOLOMON, Arch. Ophthalmol., 61 (1959) 616. R. WECKERS AND H. SOLLMANN, Arch. Int. Med. Exp., 13 (1938) 483. R. VA~ HEYNINanN, Exptl. Eye Res., 9 (1965) 298. J. R. SI-IER~AN, Anal. Biochem., 5 (1963) 548. S. ]7IAYMAN, M. F. L o o , L. O. MEROLA AND J. H. KINOSHITA, Biochim. Biophys. Acta, 128 (1966) 474. FI. A. SOEER AND E, A. PETERSON, J. Am. Chem. Soc., 78 (1956) 751. J. H. KINOSHITA, H. L. KERN AND L. O. MEROLA, Biochim. Biophys. Acta, 47 (1961) 458. S. B. BARKER AND W. PI. SUMMERSON, J. Biol. Chem., 138 (1941) 535O. H. LowRY, J. V. PASSONNEAU, F. X. HASSELBERGER AND D. W. SCHULZ, J. Biol. Chem., 239 (1964) 18. T. E. MANSOUR, N. WAKID AND H. M. SPROUSE, J . Biol. Chem., 241 (1966) 1512. J. F. KUCK, Invest. Ophthalmol., 5 (1966) 65. R. THOFT AND J. H. •INOSHITA, Exptl. Eye Res., 4 (1965) 287. J. H. KINOSI-III"A, S. FUTTERMAN, K. SATOH AND L. O. MEROLA, Biochim. Biophys. Acta, 74 (1963) 340. O. IZLIEFELD AND O. HOCKWlN, Arch. Ophthalmol., 162 (196o) 346. T. SIPPEL, Invest. Ophthalmol., I (1962) 385 . H. KEEN, Invest. Ophthalmol., I (1962) 368.
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V. B. A. R. A. R.
559
E. I~INSt~Y AND D. V, N. REDDY, lnvesl. Ophthalmol., 2 (1962) 229. BECKER, Invest. Ophthalmol., I (1962) 502. PIRIE, Bibliotheca ophthalmol,, 49 (1957) 287. VAN HEYNINGEN, Nature, 184 (1959) 184. SoLs AI~O R, K. CRANE, J. Biol. Chem., 206 (1954) 925. V&N HE~CNINGEN, Biochem. J . , 96 (1965) 419.
Biochim. Biophys. Aeta, 141 (I967 547-559