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Vitamin A as a component in the mechanism of aerobic energy transport
494 VITAMIN
A AS A COMPONENT OF AEROBIC
L. ERNSTER, Department
ENERGY
R. ZETTERSTROM,
IN THE
MECHANISM
TRANSPORT and 0. LINDBERG
of Metabolic Research, The Wenner-Gren for Experimental Biology, Stockholm
Institute
Received April 15, 1950
Vn-AmN
A is, accor$ing to earlier investigations, included in the lipid phase of mitochondria (1, 3) and the theory has been stated that this vitamin has some correlation with the mechanism of oxidation. The oxygen uptake by liver in vitro, in the presence of Fe-containing porphyrines, rises with the addition of increasing amounts of vitamin A (2). When isolating mitochondria from rabbit kidney with 0.25 M sucrose solution, according to Hogeboom, Schneider & Pallade (5), these authors made this solution 0.05 M with sodium fluoride. This procedure rendered possible the partial prevention of the decomposition of the highly labile phdsphate bonds dccurring in the adenylic acid complex of the mitochondria; similarly the more effective preservation of the amount of other coenzymes was achieved by the addition of the fluoride. A yellow extract with yellowish-green fluor’escence was obtained on the extraction with 10 per cent trichloroacetic acid of mitochondria prepared in this manner. This extract contains vitamin A, as proved spectrophotometrically (absorption maximum at 325 mp) and by the Carr-Price reaction as modified by Miiller (6). Since vitamin A can thus be obtained in a trichloroacetic acid extract and since it does not leave the water phase on shaking the extract with ether, it is suggested that vitamin A is linked - directly or indirectly - to a nonproteinlike hydrophil compound included in the water ljhase of mitochondria. All substances in this water phase are connected in their function with oxidative energy transport (4, 8). The fact that vitamin A participates in this enzyme system shows that it may be a component active in hydrogen transport. The following experiment was m’ade to prove this hypothesis. Since fermenting yeast is not considered to contain vitamin A, it was of interest to investigate whether the vitamin was active in yeast maintained under aerobic conditions. 1 kg of bakers’ yeast suspended in 4 liters of water containing 50 g.glucose, 50 ml ethanol, 5 g ammonium sulphate and 950 mg set sodium phosphate was cultivated under a vigorous (2 kg) &-stream. The temperature was maintained between 25-30” C and the pH at 4.5-5.0 adjusted occasionally by addition of cocentrated ammonia. After two hours the yeast was centrifuged in the Sharpless Super Centrifuge (50,000 R. P. RI.), suspended in 700 ml of peroxide-free ether and extracted in the Waring blendor by the addition of 700 ml ethenol, a clear, pro&in-free extract was obtained after centrifuging in the cold at moderate’ speed. Two phases were obtained on
Vitamin
of Yeast
A in aerobic energy transport
Extract
----
fermenting
yeast
-
respiring
yeart
Extracts
prepared
as described
in the text
adding to this extract a further 500 ml ether, the water phase being approximately l/4 of the original volume. This water solution was washed three times with ether, The extracted lipids not strongly bound to hydrophilic compounds were thus removed. The remaining ether and a part of the ethanol were removed in vacua. The clear solution thus obtained gave a positive Carr-Price test. A quantitative estimat~o~ gave 30 pg vitamin A/ml extract. As a point of reference it can be mentioned that an equal sample contained 200 pg inorganic phosphorus after twenty minutes’ hydrolysis in N H,SO,. Fig. 1 shows the absorption spectrum of extracts of fermenting and respiring yeast. The 329 rnp band of the respiring yeast is given by vitamin A which, in this preparation, seems to be present in the form of neovitamin A, a derivate isolated by Robeson & Baxter (7) from cod liver oil. This compound, which possesses the same biological activity as common vitamin A, has an absorption maximum at 328329 rnp. The other maximum, at shorter ultraviolet wave length, is in both extracts due mainly to adenine. The extract of fermenting yeast has ouly this maximum. The synthesis of vitamin A, for which carotines may be the precursors, begins, when the yeast, loses its fermenting power, because of the high oxygen pressure. This indicates that the vitamin A is necessary for,, and involved in, the mechanism of aerobic energy transport. The occurrence of the vitamin in a water extract of mitochondria and respiring yeast shows that it is bound, in v&o, to a hydrophilic compound. Further investigations are in progress to elucidate the structure and function of this compound which is essential for tissue respiration.
496
L. Ernster, R. Zetferstriim, and 0. Lindberg REFERENCES
1. DE ROBERTIS, E. D. P., NOWINSKI, W. W., and SAEZ, F. A., General Cytology, Philadelphia, 95 (1949). 2. EULER, H. von, and AHLSTRBM, L., Z. physiol. Chem., 204, 168 (1932). 3. GOERNER, A., J. Biot. Chem., 122, 529 (1938). 4. HOGEBOOM, G. H., CLAUDE, A., and HOTCHKISS, R. D., J. Biol. Chem., 165,615 (1946). 5. HOGEBOOM. G. H., SCHNEIDER, W. C., and PALLADE, G. E., J. BioZ. Chem., 172, 619 (1948). 6. MILLER, P. B., Hetu. Chim. Aeta, 30, 1172 (1947). 7. ROBESON, C. D., and BAXTER, J. G., J. Am. Chem. Sot., 69, 136 (1947). 8. TEPLY, L. J., Federation Roe., 8, 259 (1949).
A AS A COMPONENT OF AEROBIC
L. ERNSTER, Department
ENERGY
R. ZETTERSTROM,
IN THE
MECHANISM
TRANSPORT and 0. LINDBERG
of Metabolic Research, The Wenner-Gren for Experimental Biology, Stockholm
Institute
Received April 15, 1950
Vn-AmN
A is, accor$ing to earlier investigations, included in the lipid phase of mitochondria (1, 3) and the theory has been stated that this vitamin has some correlation with the mechanism of oxidation. The oxygen uptake by liver in vitro, in the presence of Fe-containing porphyrines, rises with the addition of increasing amounts of vitamin A (2). When isolating mitochondria from rabbit kidney with 0.25 M sucrose solution, according to Hogeboom, Schneider & Pallade (5), these authors made this solution 0.05 M with sodium fluoride. This procedure rendered possible the partial prevention of the decomposition of the highly labile phdsphate bonds dccurring in the adenylic acid complex of the mitochondria; similarly the more effective preservation of the amount of other coenzymes was achieved by the addition of the fluoride. A yellow extract with yellowish-green fluor’escence was obtained on the extraction with 10 per cent trichloroacetic acid of mitochondria prepared in this manner. This extract contains vitamin A, as proved spectrophotometrically (absorption maximum at 325 mp) and by the Carr-Price reaction as modified by Miiller (6). Since vitamin A can thus be obtained in a trichloroacetic acid extract and since it does not leave the water phase on shaking the extract with ether, it is suggested that vitamin A is linked - directly or indirectly - to a nonproteinlike hydrophil compound included in the water ljhase of mitochondria. All substances in this water phase are connected in their function with oxidative energy transport (4, 8). The fact that vitamin A participates in this enzyme system shows that it may be a component active in hydrogen transport. The following experiment was m’ade to prove this hypothesis. Since fermenting yeast is not considered to contain vitamin A, it was of interest to investigate whether the vitamin was active in yeast maintained under aerobic conditions. 1 kg of bakers’ yeast suspended in 4 liters of water containing 50 g.glucose, 50 ml ethanol, 5 g ammonium sulphate and 950 mg set sodium phosphate was cultivated under a vigorous (2 kg) &-stream. The temperature was maintained between 25-30” C and the pH at 4.5-5.0 adjusted occasionally by addition of cocentrated ammonia. After two hours the yeast was centrifuged in the Sharpless Super Centrifuge (50,000 R. P. RI.), suspended in 700 ml of peroxide-free ether and extracted in the Waring blendor by the addition of 700 ml ethenol, a clear, pro&in-free extract was obtained after centrifuging in the cold at moderate’ speed. Two phases were obtained on
Vitamin
of Yeast
A in aerobic energy transport
Extract
----
fermenting
yeast
-
respiring
yeart
Extracts
prepared
as described
in the text
adding to this extract a further 500 ml ether, the water phase being approximately l/4 of the original volume. This water solution was washed three times with ether, The extracted lipids not strongly bound to hydrophilic compounds were thus removed. The remaining ether and a part of the ethanol were removed in vacua. The clear solution thus obtained gave a positive Carr-Price test. A quantitative estimat~o~ gave 30 pg vitamin A/ml extract. As a point of reference it can be mentioned that an equal sample contained 200 pg inorganic phosphorus after twenty minutes’ hydrolysis in N H,SO,. Fig. 1 shows the absorption spectrum of extracts of fermenting and respiring yeast. The 329 rnp band of the respiring yeast is given by vitamin A which, in this preparation, seems to be present in the form of neovitamin A, a derivate isolated by Robeson & Baxter (7) from cod liver oil. This compound, which possesses the same biological activity as common vitamin A, has an absorption maximum at 328329 rnp. The other maximum, at shorter ultraviolet wave length, is in both extracts due mainly to adenine. The extract of fermenting yeast has ouly this maximum. The synthesis of vitamin A, for which carotines may be the precursors, begins, when the yeast, loses its fermenting power, because of the high oxygen pressure. This indicates that the vitamin A is necessary for,, and involved in, the mechanism of aerobic energy transport. The occurrence of the vitamin in a water extract of mitochondria and respiring yeast shows that it is bound, in v&o, to a hydrophilic compound. Further investigations are in progress to elucidate the structure and function of this compound which is essential for tissue respiration.
496
L. Ernster, R. Zetferstriim, and 0. Lindberg REFERENCES
1. DE ROBERTIS, E. D. P., NOWINSKI, W. W., and SAEZ, F. A., General Cytology, Philadelphia, 95 (1949). 2. EULER, H. von, and AHLSTRBM, L., Z. physiol. Chem., 204, 168 (1932). 3. GOERNER, A., J. Biot. Chem., 122, 529 (1938). 4. HOGEBOOM, G. H., CLAUDE, A., and HOTCHKISS, R. D., J. Biol. Chem., 165,615 (1946). 5. HOGEBOOM. G. H., SCHNEIDER, W. C., and PALLADE, G. E., J. BioZ. Chem., 172, 619 (1948). 6. MILLER, P. B., Hetu. Chim. Aeta, 30, 1172 (1947). 7. ROBESON, C. D., and BAXTER, J. G., J. Am. Chem. Sot., 69, 136 (1947). 8. TEPLY, L. J., Federation Roe., 8, 259 (1949).