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Intracellular distribution of vitamin A esterase activity in rat liver
Intracellular Distribution of Vitamin A Esterase Activity in Rat Liver’
From the Department of Biochemistry and Nutrition, and the Laboratories of the Allan Hancock Foundation, University of Southern California, Los Angeles, California’ Received
January
19, 1954
INTRODUCTION McGugan and Laughland (1)) in 1952, successfully demonstrated the presence of an enzyme in the rat liver homogenates capable of hydrolyzing vitamin A acetate. Ganguly and Deuel (2) recently confirmed this observation, and stated that the activity is quantitatively localized in the microsomal fraction of the rat liver homogenate. Almost simultaneously, Krause and Powell (3) reported that the activity in relation to vitamin A acetate is not concentrated in any particular fraction, but is widely distributed in the various fractions of rat liver homogenate obtained by differential centrifugation. In view of this latter report it was felt necessary to publish this short communication. EXPERIMENTAL Crystalline vitamin A acetate dissolved in n-hexane was chromatographed through an alumina column, and was eluted with 4% acetone in light petroleum (b.p. 60-70°C.) to remove any vitamin A alcohol present (4). To suitable portions of this chromatographed material, 5 g. of G 1020 (Atlas Powder Co.)’ and 200 mg. 1 This work was supported by a grant from the Nutrition Foundation, Inc., of New York. * Present address: Senior Research Fellow of the National Institute of Science of India, Depart.ment of Biochemistry, Indian Institute of Science, Bangalore, India. 8 Contribution No. 358 of the Department of Biochemistry and Nutrition, School of Medicine, and the Allan Hancock Foundation, University of South&n California. 4 Chemically known as polyoxyethylene-6-sorbitol monolaurate, and kindly contributed by the Atlas Powder Co., Wilmington, Delaware. 186
VITAMIN
A ESTERASE ACTIVITY
187
of cu-tocopherol were added, and the solvent was evaporated off; 20 mg. of ascorbic acid was now added to the residue, which was then dispersed in 100 ml. of water in a Waring blendor. Growing male rats of the University of Southern California strain were depleted of vitamin A following the U.S.P. procedure (5), and were then maintained on the U.S.P. vitamin A-free diet with additional supplements of 15-20 I.U. of vitamin A per week until used for the experiment. They were put under light Nembutal anesthesia, and were bled to death by heart puncture. The residual blood in the liver was perfused with normal saline. The liver was chilled in crushed ice, weighed, minced with a pair of scissors, and then homogenized with the help of a PotterElvehjem homogenizer (6) in 0.25 M sucrose solution, using the ratio of 1 g. liver tissue and 2 ml. of sucrose solution. The homogenate w-as filtered through a flannel, a portion of the whole homogenate was kept aside, and the cell constituents were separated from the rest of the homogenate by differential centrifugation (7), using a Spinco ultracentrifuge. The nuclear fraction was washed twice by resuspension in about 5 ml. of 0.25 M sucrose solution and resedimentation, and the collected washings were added to the supernatant. The mitochondrial fraction received a similar treatment, and the pink-white submicroscopic particles settling above the mitochondrial layer were removed and added to the supernatant. The microsomal fraction, however, was not washed. All manipulations were carried out at 5°C.) and a blunted needle attached to a syringe was used to remove the supernatant fluid carefully after each centrifugation. All fractions, and the whole homogenate, were diluted suitably with the same sucrose solution so as to yield samples equivalent to 20 mg. of the original liver tissue/ml. Incubation experiments were carried out as described by McGugan and Laughland (l), and l-ml. samples were withdrawn at the given time intervals and were added to 10 ml. of ethanol to stop the reaction; 10 ml. of water was added to each of these samples, and they were then extracted twice with light petroleum ether in a separatory funnel. The two forms of vitamin A were separated and determined as described by Ganguly et al. (4).
RESULTS AND DISCUSSION
The results of one typical experiment are presented in Fig. 1. The activity was almost quantitatively present in the microsomal fraction, whose activity compared very well with that of the whole homogenate, while the other fractions were practically inactive. The small activity found in the nuclear fraction here was probably due to contamination, as in all other experiments this fraction was completely inactive. These results are in disagreement with those of Krause and Powell (3). The results of another report by Powell and Krause (8) are also in disagreement with an earlier communication from this California laboratory, where, by employing the technique of differential centrifugation, it was demonstrated that the vitamin A ester is localized in a centripetally migrating material (‘kream fraction”), whereas the nuclear and mito-
188
J. GANGTJLY
-VITA
0
---VITA o=w
ALCOHOL ACETATE x= M,
A=N
v= M,
60
15 3tME
(MIN.)
Fro. 1. The rate of hydrolysis N = Nuclear; Mt = Mitochondrial; tions.
of vitamin A acetate. W = Whole homogenate; MC = Microsomal; and S = Supernatant frac-
chondrial fractions have practically none of the ester (9). It was pointed out in the same communication (9) that it is very difficult to avoid contamination during decantation. During the present study, in a few preliminary experiments in which decantation was used instead of the syringe and a blunted needle, and in which the fractions were not washed carefully, considerable vitamin A esterase activity could be found in the nuclear and mitochondrial fractions. Although the effect of the vitamin A depletion of the rats should not be overlooked, it is more probable that contamination during fractionation is the cause of disagreement in the reports of the West Virginia workers. ACKNOWLEDGMENTS The author wishes to acknowledge gratefully the constant help and encouragement of Professors H. J. Deuel, Jr., and J. W. Mehl. The author also wishes to express his gratitude for a gift of crystalline vitamin A acetate from Dr. P. L. Harris of Distillation Products Industries. SUMMARY
1. Various cell fractions from livers of vitamin A-depleted rats were obtained by using the differential centrifugat,ion technique. Their relative ability to hydrolyze vitamin A acetate was also studied,
VITAMIN
A ESTERASE
ACTIVITY
189
2. The activity was quantitatively localized in the microsomal fraction, whereas the nuclear, mitochondrial, and supernatant fractions were ina&ive . REFERENCES 1. MCGUGAN, W. A., AND LAUGHLAND, D. H., Arch Biochem. and Biophys. 36, 428 (1952). 2. GANGULY, J., AND DEUEL, H. J., JR., Nature 173, 120 (1953). 3. KRAUSE, R. F., AND POWELL, L. T., Arch Biochem. and Biophys. 44,57 (1953). 4. GANGULY, J., KRINSKY, N. I., MEHL, J. W., AND DEUEL, H. J., JR., Arch. Biothem. and Biophys. 38, 275 (1952). 5. THE UNITED STATES PHARMACOPEIA, 14th ed., p. 789. black Pub. Co. EnstoIl, 1950. 6. POTTER, V. R., AND ELVEHJEM, C. A., J. Biol. Chem. 114, 495 (1936). 7. SCHNEIDER, W. C., AND HOGEBOOM, G. H., J. Biol. Chem. 183, 123 (1950). 8. POWELL, L. T., AND KRAUSE, R. F., Arch. Biochem. and Biophys. 44,102 (1953). 9. KRINSKY, X. I., AND GANGULY, J., J. Biol. Chem. 202, 227 (1953).
From the Department of Biochemistry and Nutrition, and the Laboratories of the Allan Hancock Foundation, University of Southern California, Los Angeles, California’ Received
January
19, 1954
INTRODUCTION McGugan and Laughland (1)) in 1952, successfully demonstrated the presence of an enzyme in the rat liver homogenates capable of hydrolyzing vitamin A acetate. Ganguly and Deuel (2) recently confirmed this observation, and stated that the activity is quantitatively localized in the microsomal fraction of the rat liver homogenate. Almost simultaneously, Krause and Powell (3) reported that the activity in relation to vitamin A acetate is not concentrated in any particular fraction, but is widely distributed in the various fractions of rat liver homogenate obtained by differential centrifugation. In view of this latter report it was felt necessary to publish this short communication. EXPERIMENTAL Crystalline vitamin A acetate dissolved in n-hexane was chromatographed through an alumina column, and was eluted with 4% acetone in light petroleum (b.p. 60-70°C.) to remove any vitamin A alcohol present (4). To suitable portions of this chromatographed material, 5 g. of G 1020 (Atlas Powder Co.)’ and 200 mg. 1 This work was supported by a grant from the Nutrition Foundation, Inc., of New York. * Present address: Senior Research Fellow of the National Institute of Science of India, Depart.ment of Biochemistry, Indian Institute of Science, Bangalore, India. 8 Contribution No. 358 of the Department of Biochemistry and Nutrition, School of Medicine, and the Allan Hancock Foundation, University of South&n California. 4 Chemically known as polyoxyethylene-6-sorbitol monolaurate, and kindly contributed by the Atlas Powder Co., Wilmington, Delaware. 186
VITAMIN
A ESTERASE ACTIVITY
187
of cu-tocopherol were added, and the solvent was evaporated off; 20 mg. of ascorbic acid was now added to the residue, which was then dispersed in 100 ml. of water in a Waring blendor. Growing male rats of the University of Southern California strain were depleted of vitamin A following the U.S.P. procedure (5), and were then maintained on the U.S.P. vitamin A-free diet with additional supplements of 15-20 I.U. of vitamin A per week until used for the experiment. They were put under light Nembutal anesthesia, and were bled to death by heart puncture. The residual blood in the liver was perfused with normal saline. The liver was chilled in crushed ice, weighed, minced with a pair of scissors, and then homogenized with the help of a PotterElvehjem homogenizer (6) in 0.25 M sucrose solution, using the ratio of 1 g. liver tissue and 2 ml. of sucrose solution. The homogenate w-as filtered through a flannel, a portion of the whole homogenate was kept aside, and the cell constituents were separated from the rest of the homogenate by differential centrifugation (7), using a Spinco ultracentrifuge. The nuclear fraction was washed twice by resuspension in about 5 ml. of 0.25 M sucrose solution and resedimentation, and the collected washings were added to the supernatant. The mitochondrial fraction received a similar treatment, and the pink-white submicroscopic particles settling above the mitochondrial layer were removed and added to the supernatant. The microsomal fraction, however, was not washed. All manipulations were carried out at 5°C.) and a blunted needle attached to a syringe was used to remove the supernatant fluid carefully after each centrifugation. All fractions, and the whole homogenate, were diluted suitably with the same sucrose solution so as to yield samples equivalent to 20 mg. of the original liver tissue/ml. Incubation experiments were carried out as described by McGugan and Laughland (l), and l-ml. samples were withdrawn at the given time intervals and were added to 10 ml. of ethanol to stop the reaction; 10 ml. of water was added to each of these samples, and they were then extracted twice with light petroleum ether in a separatory funnel. The two forms of vitamin A were separated and determined as described by Ganguly et al. (4).
RESULTS AND DISCUSSION
The results of one typical experiment are presented in Fig. 1. The activity was almost quantitatively present in the microsomal fraction, whose activity compared very well with that of the whole homogenate, while the other fractions were practically inactive. The small activity found in the nuclear fraction here was probably due to contamination, as in all other experiments this fraction was completely inactive. These results are in disagreement with those of Krause and Powell (3). The results of another report by Powell and Krause (8) are also in disagreement with an earlier communication from this California laboratory, where, by employing the technique of differential centrifugation, it was demonstrated that the vitamin A ester is localized in a centripetally migrating material (‘kream fraction”), whereas the nuclear and mito-
188
J. GANGTJLY
-VITA
0
---VITA o=w
ALCOHOL ACETATE x= M,
A=N
v= M,
60
15 3tME
(MIN.)
Fro. 1. The rate of hydrolysis N = Nuclear; Mt = Mitochondrial; tions.
of vitamin A acetate. W = Whole homogenate; MC = Microsomal; and S = Supernatant frac-
chondrial fractions have practically none of the ester (9). It was pointed out in the same communication (9) that it is very difficult to avoid contamination during decantation. During the present study, in a few preliminary experiments in which decantation was used instead of the syringe and a blunted needle, and in which the fractions were not washed carefully, considerable vitamin A esterase activity could be found in the nuclear and mitochondrial fractions. Although the effect of the vitamin A depletion of the rats should not be overlooked, it is more probable that contamination during fractionation is the cause of disagreement in the reports of the West Virginia workers. ACKNOWLEDGMENTS The author wishes to acknowledge gratefully the constant help and encouragement of Professors H. J. Deuel, Jr., and J. W. Mehl. The author also wishes to express his gratitude for a gift of crystalline vitamin A acetate from Dr. P. L. Harris of Distillation Products Industries. SUMMARY
1. Various cell fractions from livers of vitamin A-depleted rats were obtained by using the differential centrifugat,ion technique. Their relative ability to hydrolyze vitamin A acetate was also studied,
VITAMIN
A ESTERASE
ACTIVITY
189
2. The activity was quantitatively localized in the microsomal fraction, whereas the nuclear, mitochondrial, and supernatant fractions were ina&ive . REFERENCES 1. MCGUGAN, W. A., AND LAUGHLAND, D. H., Arch Biochem. and Biophys. 36, 428 (1952). 2. GANGULY, J., AND DEUEL, H. J., JR., Nature 173, 120 (1953). 3. KRAUSE, R. F., AND POWELL, L. T., Arch Biochem. and Biophys. 44,57 (1953). 4. GANGULY, J., KRINSKY, N. I., MEHL, J. W., AND DEUEL, H. J., JR., Arch. Biothem. and Biophys. 38, 275 (1952). 5. THE UNITED STATES PHARMACOPEIA, 14th ed., p. 789. black Pub. Co. EnstoIl, 1950. 6. POTTER, V. R., AND ELVEHJEM, C. A., J. Biol. Chem. 114, 495 (1936). 7. SCHNEIDER, W. C., AND HOGEBOOM, G. H., J. Biol. Chem. 183, 123 (1950). 8. POWELL, L. T., AND KRAUSE, R. F., Arch. Biochem. and Biophys. 44,102 (1953). 9. KRINSKY, X. I., AND GANGULY, J., J. Biol. Chem. 202, 227 (1953).