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Characterization of the Intracellular Proteolytic Enzyme in the Lens*
CHARACTERIZATION OF T H E INTRACELLULAR E N Z Y M E IN T H E L E N S * ANIMA
DEVI,
PROTEOLYTIC
PH.D.
New York was maintained at 0 ° C . as closely as possi ble throughout the process of dissection and homogenization. T h e solutions of the sub strate and inhibitors were freshly prepared just before the experiment. Substrates. Phenylalanine ethyl ester ( P E E ) , tyrosine ethyl ( T E E ) , benzoyl tyrosine ethyl ester ( B T E E ) , lysine ethyl ester ( L E E ) , glycine ethyl ester ( G E E ) were purchased from Sigma and Company. Ben zoyl arginine ethyl ester ( Β Α Ε Ε ) , benzoyl histidine ethyl ester ( B H E E ) , and carbonaphthoxyphenylalanine were kindly sup plied by Dr. E. A. Zeller of Northwestern University, Chicago. Enzymes. Chymotrypsin was purchased from A r m o u r and Company, Chicago. Inhibitors. Ovomucoid, soybean trypsin in hibitor were purchased from Worthington Corporation, New Jersey ; diisopropylfluorophosphate, cetylpyridinium chloride, and p chloro mercuribenzoate were obtained from Fischer Scientific Company, New York.
In 1957 Zeller and Devi, 1 " 3 using a sensi tive manometric method, reported the pres ence of an intracellular proteolytic enzyme in the various parts of the rabbit eye. They also found highest esterase activity in the lens and very feeble activity in the cornea. In this paper the results of an investigation on the characterization of this intracellular en zyme are reported. It will be seen from the results presented in this paper that the intra cellular enzyme as occurs in the lens is very similar in nature to cathepsin C. The method of estimation of this enzyme was based on the liberation of carbon dioxide in a bicarbonate medium by the acid formed from the hydrolysis of synthetic esters by the enzyme and can be represented by the fol lowing equation: C e H 5 C H 2 C H N H 2 C O O C 2 H 5 + E -> C 6 H 5 C H 2 C H N H 2 C O O H + C 2 H 5 O H . T h e conventional manometric method was used to measure the carbon diox ide thus liberated from the reaction mix ture. 4 · 5 T h e reaction velocity, a measure of the enzymic activity, is expressed here in terms of μ-liter of C 0 2 liberated per hour per gm. of the wet tissue. Substrate blanks and en zyme blanks were separately carried out in the complete system without enzyme or sub strate in the reaction mixture.
R E S U L T S AND DISCUSSION
Table 1 shows the experimental conditions employed in the measurement of the esterase activity of the lens homogenate from the rate of hydrolysis of P E E in a bicarbonate me dium. A s can be seen in Table 2, the highest activity was found in the lens. Next to this in activity was the vitreous body, which rep resented approximately 50 percent of the lens activity. T h e retina exhibited only seven or eight percent, whereas cornea was prac-
MATERIALS
Cattle eyes were removed immediately after killing of the animal in the slaughter house and transported in packed ice to the laboratory. Homogenates of the lens and other parts of the eye were prepared in nor mal saline in a Virtis homogenizer within a very short period of time. T h e temperature
TABLE 1 CONDITIONS IN THE MEASUREMENT OF THE ESTERASE ACTIVITY OF LENS HOMOGENATE
1.0 ml. lens homogenate 0.7 ml. NaHCO 3 (0.18M) 0.3 ml. distilled water 1.0 ml. 0.075 M PEE
* From the Department of Ophthalmology, Col lege of Physicians and Surgeons, Columbia Uni versity. Presented before the Eastern Section, New York, November 22, 1958. 29
ANIMA DEVI
30 TABLE 2
DEGRADATION OF PHENYLALANINE ETHYL ESTER BY THE VARIOUS PARTS OF THE STEER EYE
No. of Investi gations 10 10
* * *
Parts of the Eye
Q C 0 2 in /i-liters/ hr/gm of enzyme
Lens Vitreous BodyRetina Parts of Lens Capsule Cornea
850 ± 8 0 408 ± 6 0 60 210 0
^
100
* Only six separate investigations were made with six eyes.
tically free from esterase activity. The ab sence of esterace activity in cornea might be due to the difficulty of preparing a homogenate of it as cornea is a very tough material and resists homogenization. This point needs further investigation. As the main object of this communication is to characterize the lens enzyme, and once it is known that the highest enzymic activity is found in lens, only lens homogenate was subsequently used in all experiments unless otherwise mentioned. Table 3 shows the liberation of C 0 2 ex pressed in μ-liters per hour by one gm. of the wet tissue in a reaction mixture of total volume of 3.0 ml. containing different sub strates. Although lens enzyme is capable of catalyzing the hydrolysis of tyrosine ethyl ester ( T E E ) , 6 phenylalanine ethyl ester and very slightly benzoyl arginine ethyl ester, it will be seen that the best and ideal substrate for this study is phenylalanine ethyl ester ( P E E ) . 7 The hydrolysis of P E E by lens enTABLE 3 DEGRADATION OF VARIOUS SYNTHETIC AMINO ACID ESTERS BY STEER LENS HOMOGENATE
Substrate Used
Percent Degradation
Phenylalanine ethyl ester ( P E E ) Tyrosine ethyl ester ( T E E ) Benzoyl tyrosine ethyl ester ( B T E E ) Benzoyl arginine ethyl ester (BAEE) Benzoyl histidine ethyl ester ( B H E E ) Lysine ethyl ester ( L E E ) Glycine ethyl ester ( G E E ) Carbonaphthoxyphenylalanine
80-90 80-90 80-90 20 nil nil nil 10
TIME
IN MINUTES
Fig. 1 (Devi). Degradation of phenylalanine ethyl ester by lens homogenate.
zyme as can be seen in Figure 1 is propor tional to the time of incubation up to 15 to 20 minutes ; in other words, follows the zero order reaction, then attains the first order kinetics. This is because the initial substrate concentration was high and practically all of the enzyme combined with it and remained so for a considerable part of the reaction, but later on the substrate concentration dropped and the course of the reaction followed first order kineics. The possibility of product in hibition was excluded by the fact that the addition of further substrate at this point (after 15 minutes) restored the original re action velocity. The velocity of reaction also shows a linear increase with the increase of enzyme concentration (fig. 2). It should be recalled that P E E has also been shown to be the best and the ideal substrate for chymotrypsin and intracellular protease. This sug-
ii M
MGMS. OF HOMOGENATE /rn OF THE REACTION MIXTURE
Fig. 2. (Devi). Effect of enzyme concentration on the activity of the steer lens homogenate.
31
P R O T E O L Y T I C ENZYME IN L E N S
^
^s Fig. 3 (Devi). Effect of substrate concentration (L. B. method) on the degradation of P E E by steer lens homogenate.
gests that the lens enzyme is identical with chymotrypsin. Both enzymes have the same pH optima (6.8) in the hydrolysis of P E E and both are stable when standing at room temperature for 72 hours. The Michaelis Menton constant (Km) was determined at pH 6.8 after first ascer taining that the rate of reaction at this pH was proportional to time, until 80 percent of the substrate had been cleaved. The reaction velocity under the conditions of the experi ment was a function of the substrate con centration. Figure 3 shows the data thus obtained. Applying the conventional Lineweaver- Bark equation 1 _ Km V
1
1
Vmax
where Km is Michaelis and Menton constant,
v is over-all velocity of the enzymatic reac tion, Vmax is the maximum velocity, and [S] is the substrate concentration, the Km value (calculated by the extrapolation of the linear portion of the plot) is 3.0 X 103 which is of the same order as that obtained for chymo trypsin. Table 4 shows the effects of various in hibitors of proteolytic enzymes on the ester ase activity of the lens homogenate. The maximum inhibition of the esterase activity was found with diisopropylfluorophosphate. With soybean trypsin inhibitor and cetylpyridinium chloride, only 40 to SO percent of the esterase activity was suppressed. Ovomucoid blocks only 28 percent of the activity, while p-chloromercuribenzoate blocks only 24 percent. The same inhibitors, at the same concentration, inhibit to the same extent the esterase activity of crystalline chymotrypsin. The concentration of chymotrypsin used throughout these studies was adjusted to give esterase comparable to the lens activity. As can be seen from Table 4, both enzymes under the same experimental conditions suffer the same degree of inhibition of ester ase activity. These results clearly indicate the similarity of the lens enzyme with chy motrypsin. The question arises whether this evolution of C 0 2 by the lens in the reaction media is due to the high content of lactic acid in the lens. As mentioned before, the enzyme blank in each experiment is substracted from the main reaction before the Q values are calcu lated.
TABLE 4 INHIBITION OF ESTERASE ACTIVITY OF THE LENS HOMOGENATE BY DIFFERENT INHIBITORS OF PROTEOLYTIC ENZYME
Chymotrypsin Inhibitors
Ovomucoid Soybean trypsin inhibitor Diisopropyl-fluorophosphate Cetylpyridinium chloride p-Chloromercuribenzoate
Lens
Q C 0 2 in juL/gm./ hr.XlO 6
Inhibition
Q C 0 2 in juL/gm./hr.
Inhibition
45.2 32.6 26.7 16 29 35
26 41 65 38 25
580 420 300 128 261 380
28 48 79.3 44.5 24
32
ANIMA DEVI
It seems obvious, especially from the study of the stability, specificity of these two en zymes, similarity of K m values, p H optima, and the degree of inhibition of their esterase activity by different inhibitors, that the lens enzyme is in all probability similar in nature to chymotrypsin. The primary function of an extracellular protease (for example, trypsin and chymo trypsin) is to hydrolyze protein. However, the function of the lens enzyme is not clear. While it is similar in substrate specificity to that of chymotrypsin, it does not appear to function as an extracellular protease owing to the high concentration of protein in the lens. It is suggested, therefore, that its func tion may be one of synthesis rather than degradation since intracellular proteases (for example, cathepsin C) have been shown
to synthesize higher peptides by transpeptidation. Perhaps this lens enzyme at this high concentration is required for maintaining the physical and chemical state of the protein in such a way that lens transparency is secured at all times of the lens' life. SUMMARY
1. An intracellular proteolytic enzyme has been detected in the steer lens which is very similar to cathepsin C. 2. The enzyme is highly inhibited by diisopropylfluorophosphate, ovomucoid, and a less degree of inhibition was found in the case of cetylpyridinium chloride, soybean trypsin inhibitor, and p-chloromercuribenzoate. 630 West
168th Street
(32).
REFERENCES
1. Zeller, E. A., Devi, A., and Carbon, J. A.: Fed. Proc, 15:395, 19S6. 2. Zeller, E. A., and Devi, A. : Nat. meet. Am. Chem. Soc., Sept., 1956. 3. : Am. J. Ophth., 44:281 (Nov. Pt. II) 1957. 4. Parks, R. E., Jr., and Plaut, G. W. E.: J. Biol. Chem., 203:755, 1953. 5. Jandorf, B. J.: J. Pharm. Exper. Therapy, 98:77-84, 1950, Fed. Proc, 9:156, 1950. 6. Jansen, E. F., Curl, A. L., and Balls, A. K.: J. Biol. Chem., 189:671, 1951. 7a. Kaufman, S., and Neurath, H.: Arch. Biochem., 21:437, 1949. 7b. Goldenberg, H., and Goldenberg, V.: Arch. Biochem., 29:156, 1950.
OSMOTIC PRESSURE MEASUREMENTS OF INTRAOCULAR F L U I D S BY A N I M P R O V E D C R Y O S C O P I C M E T H O D * PHYSIOLOGIC S I G N I F I C A N C E RELATIVE TO AQUEOUS H U M O R DYNAMICS M I R Y A M Z.
KASS,+
P H . D . , AND HARRY GREEN,*
PH.D.
Philadelphia, Pennsylvania It has been generally assumed that the bulk flow of aqueous humor, from the pos* From the Research Department, Wills Eye Hospital, and the Department of Biochemistry, Graduate School of Medicine, University of Penn sylvania. This investigation was supported in part by a grant-in-aid from the National Council to Combat Blindness (Grant No. G-206). Presented before the Eastern Section, November 22, 1958, New York. t Address after September 1, 1959: Rehnvot, Israel (57 Yaakov Street). t Present address: Biochemistry Researcli Section, Smith Kline and French Laboratories, 1530 Spring Garden Street, Philadelphia 30, Pennsylvania.
terior chamber through the pupillary space into the anterior chamber and out at the angle, is maintained by an osmotic pump mechanism activated by an osmotic pressure gradient increasing from the plasma to the posterior chamber fluid. In experimental sup port of this concept are the many osmotic pressure measurements of the aqueous humor and plasma made in the rabbit, man, and other animal species, recently reviewed by Levene. 1 Because of the relative ease of re moving aqueous humor from the anterior chamber in sufficient amounts for analysis,
DEVI,
PROTEOLYTIC
PH.D.
New York was maintained at 0 ° C . as closely as possi ble throughout the process of dissection and homogenization. T h e solutions of the sub strate and inhibitors were freshly prepared just before the experiment. Substrates. Phenylalanine ethyl ester ( P E E ) , tyrosine ethyl ( T E E ) , benzoyl tyrosine ethyl ester ( B T E E ) , lysine ethyl ester ( L E E ) , glycine ethyl ester ( G E E ) were purchased from Sigma and Company. Ben zoyl arginine ethyl ester ( Β Α Ε Ε ) , benzoyl histidine ethyl ester ( B H E E ) , and carbonaphthoxyphenylalanine were kindly sup plied by Dr. E. A. Zeller of Northwestern University, Chicago. Enzymes. Chymotrypsin was purchased from A r m o u r and Company, Chicago. Inhibitors. Ovomucoid, soybean trypsin in hibitor were purchased from Worthington Corporation, New Jersey ; diisopropylfluorophosphate, cetylpyridinium chloride, and p chloro mercuribenzoate were obtained from Fischer Scientific Company, New York.
In 1957 Zeller and Devi, 1 " 3 using a sensi tive manometric method, reported the pres ence of an intracellular proteolytic enzyme in the various parts of the rabbit eye. They also found highest esterase activity in the lens and very feeble activity in the cornea. In this paper the results of an investigation on the characterization of this intracellular en zyme are reported. It will be seen from the results presented in this paper that the intra cellular enzyme as occurs in the lens is very similar in nature to cathepsin C. The method of estimation of this enzyme was based on the liberation of carbon dioxide in a bicarbonate medium by the acid formed from the hydrolysis of synthetic esters by the enzyme and can be represented by the fol lowing equation: C e H 5 C H 2 C H N H 2 C O O C 2 H 5 + E -> C 6 H 5 C H 2 C H N H 2 C O O H + C 2 H 5 O H . T h e conventional manometric method was used to measure the carbon diox ide thus liberated from the reaction mix ture. 4 · 5 T h e reaction velocity, a measure of the enzymic activity, is expressed here in terms of μ-liter of C 0 2 liberated per hour per gm. of the wet tissue. Substrate blanks and en zyme blanks were separately carried out in the complete system without enzyme or sub strate in the reaction mixture.
R E S U L T S AND DISCUSSION
Table 1 shows the experimental conditions employed in the measurement of the esterase activity of the lens homogenate from the rate of hydrolysis of P E E in a bicarbonate me dium. A s can be seen in Table 2, the highest activity was found in the lens. Next to this in activity was the vitreous body, which rep resented approximately 50 percent of the lens activity. T h e retina exhibited only seven or eight percent, whereas cornea was prac-
MATERIALS
Cattle eyes were removed immediately after killing of the animal in the slaughter house and transported in packed ice to the laboratory. Homogenates of the lens and other parts of the eye were prepared in nor mal saline in a Virtis homogenizer within a very short period of time. T h e temperature
TABLE 1 CONDITIONS IN THE MEASUREMENT OF THE ESTERASE ACTIVITY OF LENS HOMOGENATE
1.0 ml. lens homogenate 0.7 ml. NaHCO 3 (0.18M) 0.3 ml. distilled water 1.0 ml. 0.075 M PEE
* From the Department of Ophthalmology, Col lege of Physicians and Surgeons, Columbia Uni versity. Presented before the Eastern Section, New York, November 22, 1958. 29
ANIMA DEVI
30 TABLE 2
DEGRADATION OF PHENYLALANINE ETHYL ESTER BY THE VARIOUS PARTS OF THE STEER EYE
No. of Investi gations 10 10
* * *
Parts of the Eye
Q C 0 2 in /i-liters/ hr/gm of enzyme
Lens Vitreous BodyRetina Parts of Lens Capsule Cornea
850 ± 8 0 408 ± 6 0 60 210 0
^
100
* Only six separate investigations were made with six eyes.
tically free from esterase activity. The ab sence of esterace activity in cornea might be due to the difficulty of preparing a homogenate of it as cornea is a very tough material and resists homogenization. This point needs further investigation. As the main object of this communication is to characterize the lens enzyme, and once it is known that the highest enzymic activity is found in lens, only lens homogenate was subsequently used in all experiments unless otherwise mentioned. Table 3 shows the liberation of C 0 2 ex pressed in μ-liters per hour by one gm. of the wet tissue in a reaction mixture of total volume of 3.0 ml. containing different sub strates. Although lens enzyme is capable of catalyzing the hydrolysis of tyrosine ethyl ester ( T E E ) , 6 phenylalanine ethyl ester and very slightly benzoyl arginine ethyl ester, it will be seen that the best and ideal substrate for this study is phenylalanine ethyl ester ( P E E ) . 7 The hydrolysis of P E E by lens enTABLE 3 DEGRADATION OF VARIOUS SYNTHETIC AMINO ACID ESTERS BY STEER LENS HOMOGENATE
Substrate Used
Percent Degradation
Phenylalanine ethyl ester ( P E E ) Tyrosine ethyl ester ( T E E ) Benzoyl tyrosine ethyl ester ( B T E E ) Benzoyl arginine ethyl ester (BAEE) Benzoyl histidine ethyl ester ( B H E E ) Lysine ethyl ester ( L E E ) Glycine ethyl ester ( G E E ) Carbonaphthoxyphenylalanine
80-90 80-90 80-90 20 nil nil nil 10
TIME
IN MINUTES
Fig. 1 (Devi). Degradation of phenylalanine ethyl ester by lens homogenate.
zyme as can be seen in Figure 1 is propor tional to the time of incubation up to 15 to 20 minutes ; in other words, follows the zero order reaction, then attains the first order kinetics. This is because the initial substrate concentration was high and practically all of the enzyme combined with it and remained so for a considerable part of the reaction, but later on the substrate concentration dropped and the course of the reaction followed first order kineics. The possibility of product in hibition was excluded by the fact that the addition of further substrate at this point (after 15 minutes) restored the original re action velocity. The velocity of reaction also shows a linear increase with the increase of enzyme concentration (fig. 2). It should be recalled that P E E has also been shown to be the best and the ideal substrate for chymotrypsin and intracellular protease. This sug-
ii M
MGMS. OF HOMOGENATE /rn OF THE REACTION MIXTURE
Fig. 2. (Devi). Effect of enzyme concentration on the activity of the steer lens homogenate.
31
P R O T E O L Y T I C ENZYME IN L E N S
^
^s Fig. 3 (Devi). Effect of substrate concentration (L. B. method) on the degradation of P E E by steer lens homogenate.
gests that the lens enzyme is identical with chymotrypsin. Both enzymes have the same pH optima (6.8) in the hydrolysis of P E E and both are stable when standing at room temperature for 72 hours. The Michaelis Menton constant (Km) was determined at pH 6.8 after first ascer taining that the rate of reaction at this pH was proportional to time, until 80 percent of the substrate had been cleaved. The reaction velocity under the conditions of the experi ment was a function of the substrate con centration. Figure 3 shows the data thus obtained. Applying the conventional Lineweaver- Bark equation 1 _ Km V
1
1
Vmax
where Km is Michaelis and Menton constant,
v is over-all velocity of the enzymatic reac tion, Vmax is the maximum velocity, and [S] is the substrate concentration, the Km value (calculated by the extrapolation of the linear portion of the plot) is 3.0 X 103 which is of the same order as that obtained for chymo trypsin. Table 4 shows the effects of various in hibitors of proteolytic enzymes on the ester ase activity of the lens homogenate. The maximum inhibition of the esterase activity was found with diisopropylfluorophosphate. With soybean trypsin inhibitor and cetylpyridinium chloride, only 40 to SO percent of the esterase activity was suppressed. Ovomucoid blocks only 28 percent of the activity, while p-chloromercuribenzoate blocks only 24 percent. The same inhibitors, at the same concentration, inhibit to the same extent the esterase activity of crystalline chymotrypsin. The concentration of chymotrypsin used throughout these studies was adjusted to give esterase comparable to the lens activity. As can be seen from Table 4, both enzymes under the same experimental conditions suffer the same degree of inhibition of ester ase activity. These results clearly indicate the similarity of the lens enzyme with chy motrypsin. The question arises whether this evolution of C 0 2 by the lens in the reaction media is due to the high content of lactic acid in the lens. As mentioned before, the enzyme blank in each experiment is substracted from the main reaction before the Q values are calcu lated.
TABLE 4 INHIBITION OF ESTERASE ACTIVITY OF THE LENS HOMOGENATE BY DIFFERENT INHIBITORS OF PROTEOLYTIC ENZYME
Chymotrypsin Inhibitors
Ovomucoid Soybean trypsin inhibitor Diisopropyl-fluorophosphate Cetylpyridinium chloride p-Chloromercuribenzoate
Lens
Q C 0 2 in juL/gm./ hr.XlO 6
Inhibition
Q C 0 2 in juL/gm./hr.
Inhibition
45.2 32.6 26.7 16 29 35
26 41 65 38 25
580 420 300 128 261 380
28 48 79.3 44.5 24
32
ANIMA DEVI
It seems obvious, especially from the study of the stability, specificity of these two en zymes, similarity of K m values, p H optima, and the degree of inhibition of their esterase activity by different inhibitors, that the lens enzyme is in all probability similar in nature to chymotrypsin. The primary function of an extracellular protease (for example, trypsin and chymo trypsin) is to hydrolyze protein. However, the function of the lens enzyme is not clear. While it is similar in substrate specificity to that of chymotrypsin, it does not appear to function as an extracellular protease owing to the high concentration of protein in the lens. It is suggested, therefore, that its func tion may be one of synthesis rather than degradation since intracellular proteases (for example, cathepsin C) have been shown
to synthesize higher peptides by transpeptidation. Perhaps this lens enzyme at this high concentration is required for maintaining the physical and chemical state of the protein in such a way that lens transparency is secured at all times of the lens' life. SUMMARY
1. An intracellular proteolytic enzyme has been detected in the steer lens which is very similar to cathepsin C. 2. The enzyme is highly inhibited by diisopropylfluorophosphate, ovomucoid, and a less degree of inhibition was found in the case of cetylpyridinium chloride, soybean trypsin inhibitor, and p-chloromercuribenzoate. 630 West
168th Street
(32).
REFERENCES
1. Zeller, E. A., Devi, A., and Carbon, J. A.: Fed. Proc, 15:395, 19S6. 2. Zeller, E. A., and Devi, A. : Nat. meet. Am. Chem. Soc., Sept., 1956. 3. : Am. J. Ophth., 44:281 (Nov. Pt. II) 1957. 4. Parks, R. E., Jr., and Plaut, G. W. E.: J. Biol. Chem., 203:755, 1953. 5. Jandorf, B. J.: J. Pharm. Exper. Therapy, 98:77-84, 1950, Fed. Proc, 9:156, 1950. 6. Jansen, E. F., Curl, A. L., and Balls, A. K.: J. Biol. Chem., 189:671, 1951. 7a. Kaufman, S., and Neurath, H.: Arch. Biochem., 21:437, 1949. 7b. Goldenberg, H., and Goldenberg, V.: Arch. Biochem., 29:156, 1950.
OSMOTIC PRESSURE MEASUREMENTS OF INTRAOCULAR F L U I D S BY A N I M P R O V E D C R Y O S C O P I C M E T H O D * PHYSIOLOGIC S I G N I F I C A N C E RELATIVE TO AQUEOUS H U M O R DYNAMICS M I R Y A M Z.
KASS,+
P H . D . , AND HARRY GREEN,*
PH.D.
Philadelphia, Pennsylvania It has been generally assumed that the bulk flow of aqueous humor, from the pos* From the Research Department, Wills Eye Hospital, and the Department of Biochemistry, Graduate School of Medicine, University of Penn sylvania. This investigation was supported in part by a grant-in-aid from the National Council to Combat Blindness (Grant No. G-206). Presented before the Eastern Section, November 22, 1958, New York. t Address after September 1, 1959: Rehnvot, Israel (57 Yaakov Street). t Present address: Biochemistry Researcli Section, Smith Kline and French Laboratories, 1530 Spring Garden Street, Philadelphia 30, Pennsylvania.
terior chamber through the pupillary space into the anterior chamber and out at the angle, is maintained by an osmotic pump mechanism activated by an osmotic pressure gradient increasing from the plasma to the posterior chamber fluid. In experimental sup port of this concept are the many osmotic pressure measurements of the aqueous humor and plasma made in the rabbit, man, and other animal species, recently reviewed by Levene. 1 Because of the relative ease of re moving aqueous humor from the anterior chamber in sufficient amounts for analysis,