- Email: [email protected]
An iodide-complexing phospholipid
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
97
Letters An Iodide-Complexing
(1962)
to The
Editors
fresh frozen thyroid. After preliminary extraction the lipids were chromatographed in silicic acid with results shown in Fig. 1. Individual phospholipids were identified by paper chromatography on silicic acid impregnated paper with diisobutylketone-acetic acid-water (9, 10) and silicateimpregnated glass fiber paper with benzene-pyridine-water (11). The various fractions obtained were assayed for iodide biding by a dialysis system in which 20 ml. of chloroform was added to a dialysis bag which was suspended in 500 ml. of H&. Both inside and outside phases were stirred continuously. Sodium hisulfite (10-a M) was added to the aqueous phase to prevent air oxidation of iodide. Iodide (lOme M and 1OV M) labeled with 1131 was introduced into the aqueous phase and the two phases were sampled for radioactivity at various time intervals, care being taken to keep their volumes constant,. Figure 2 shows the results
Phospholipid
The mechanism of iodide concentration in t,hyroid is unknown although a type of active transport of iodide seems indicated. A role for phospholipids as a transport carrier has seemed attractive because of some evidence for this function of such substances in the transport of sodium ion in the salt gland of certain birds (1) and in erythrocytes and brain (2-4). In addition we have found that phospholipid synthesis (from P3W4) in the thyroid is influenced by digit’alis glycosides and Kf concentration in the same manner as is iodide concentration (5, 6). The present study was undertaken to investigate iodide binding by thyroidal phospholipids (7). Phospholipids were fractionated using the method of Hanahan et al. (8). To detect phospholipids more easily 0.5 g. of thyroid slices were incubated with phosphat,e32 and added to 30 g. of
I
6()()-
CHCI,:MeOH j 3:2
I:4
I I MeOt 1 I
FIG. 1. Pz2 radioactivity of lipids from 30 g. of calf column (60 g. of silicic acid + 30 g. of Hyflow-Super-Gel, ml. were collected and pooled into 9 fractions as shown. to obtain a flow-rate of 1.0 to 1.5 ml./min. Composit,ion 425
thyroid as eluted from silicic acid diameter 30 mm.). Portions of 8.4 Nitrogen under pressure was used of solvents appears at the top.
426
LETTERS
TO THE
EDITORS
Iodide in Hz0 Phase 10B6 M _ Lecithin iti CHCi3 Phase.25 mg./ml. ----_--__--------7
0 TIME FIG. 2. Concentration mg./ml. thyroidal lipid molar.
ll 3. X&M
40
80 120 I HR.
2
160MIN. 3
4
24 HR.
of iodide in organic phase at various times after addition of 0.25 fractions 1 to 9 indicated in Fig. 1. Iodide in the water phase 10WB
THYROIDAL PC FRACTION (Fr. 7.)
[II'
- lo+
TIME IN HOURS FIG. 3. Molar concentration of iodide in chloroform during the dialysis after addition of thyroidal lecithin (fraction 7). Concentration of lecithin is indicated for the different curves. form, and iodide in water ([I-]HzO)
at various times (PC) in chloro-
LETTERS
TO THE
obtained with the various phospholipid fractions in the two phase chloroform water system. It is apparent that only fractions 7 and 8 are active in concentrating iodide into the organic phase. Fraction 1 contained phosphat,idylethanolamine and phosphatidylserine. Fraction 5 with maximal Pa2 uptake was phosphatidylinositol. Fractions 7 and 8 bot,h contained lecithin although a small amount of a slower moving material was also present,. Increasing the concentration of fraction 7 to 0.5 mg./ml. of chloroform increased the iodide in the organic phase to 1OV M (with 10m6 M Iconcentration in the aqueous phase). With 10-T M I- in the water, 0.25 mg./ml. of fraction 7 increased the iodide to a ratio of 5: 1, organic: aqueous (Fig. 3). Chromatography of the organic phase under these conditions revealed only iodide. The decrease with time in iodide content of the organic phase may be due to degradation of the phospholipid but was not investigated. The iodide in the organic phase was partly precipitable with acetone, but not wit’h ethyl alcohol. The results above are suggestive evidence for the participation of t’hyroidal lecithin (or a similar phospholipid) in the concentration of iodide by thyroid. The precise mechanism whereby synthesis, metabolism and resynthesis of a carrier so that it may transport iodide against’ a concentration gradient remains to be elucidated. The results with thyroidal lecithin seem to have some physiological significance as other lecithins do not work in the above system. Commercial soy-bean lecithin binds iodide very slowly, practically not at all during the first 10 hr. (7), and the iodinated end product is stable in chromatography, running with the solvent front. Synthetic dipalmit’oyllecithin and lecithin from calf brain, although identical in chromatography with thyroidal lecithin, do not effect any transfer of iodide int,o the organic phase. Lecithin from calf liver has some activity but needs to be present in a five to ten-fold greater concentration to be equally active. I should like to express thanks to Dr. J. E. Rall, Chief, Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, for supervision and generous advice, and to all his staff for profitable discussions during this work. REFERENCES 1. HOKIN, L. E., AND HOKIN, M. R., J. Gen. Physiol. 44,61 (1960). 2. SOLOMON, A. K., LIONETTI, F., AND CURRAN, P. F., Nature 178, 582 (1956). 3. KIRSCHNER, L. B., J. Gen. Physiol. 42, 231 (1958).
427
EDITORS
4. WOGT, W., Nature 179, 300 (1957). 5. WOLFF, J., Biochem. et Biophys. Acta 38, 316 (1960). 6. VILKKI, P., “Advances in Thyroid Research, p. 231. Pergamon Press, Oxford, 1961. 7. VILKKI, P., Suomen Kemistilehti B33, 209 (1960). 8. HANAHAN, D. J., DITTMER, J. C., AND WARASHINA, E., J. Biol. Chem. 228, 685 (1957). 9. MARINETTI, G. W., AND STOTZ, E., Biochem. et Biophys. Acta 18,102 (1955). 10. HOKIN, L. E., AND HOKIN, M. R., J. Biol. Chem. 233, 805 (1958). 11. MULDREY, J. E., MILLER, 0. N., AND HAYILTON, J. G. J. Lipid Research 1,48 (1959). PANU VILKKI~ Clinical Endocrinology Branch National Institute of Arthritis and Metabolic Diseases National Institute of Health Bethesda, Maryland Received January 25, 1961 1 Present address : Department of Chemistry, Universit,y of Turku, Turku,
Effect Coenzyme
of Cobalt Content
Deficiency
Medical Finland.
on the B12
of Rhizobium
meliloti’
Recently it has been demonstrated that the growth of several species of rhizobia was increased several-fold by traces of cobalt (1). In the experiments conducted with Rhizobium meliloti, cobalt was shown to be an indispensable component, of the culture medium. Investigations of the cobalt requirements of symbiotically grown soybean plants (3) provided evidence that the vitamin B,? content of root nodules from plants grown in purified solution lacking combined nit,rogen was positively correlated with the cobalt supply in the nutrient solution. Since Barker et al. (4, 5) have isolated different types of cobamide coenzymes which have been shown to be active in several enzyme systems, it was considered desirable to determine the influence of cobalt levels in culture media on the content of the Btz coenzymes in R. meliloti cells. The methods used for cleaning glassware and for the purification and preparation of the nutrient stock solutions and water have been described earlier (3). The basal nutrient medium contained 1 This investigation a grant (G 18556) Foundation.
was supported in part by from the Piational Science
OF
BIOCHEMISTRY
AND
BIOPHYSICS
97
Letters An Iodide-Complexing
(1962)
to The
Editors
fresh frozen thyroid. After preliminary extraction the lipids were chromatographed in silicic acid with results shown in Fig. 1. Individual phospholipids were identified by paper chromatography on silicic acid impregnated paper with diisobutylketone-acetic acid-water (9, 10) and silicateimpregnated glass fiber paper with benzene-pyridine-water (11). The various fractions obtained were assayed for iodide biding by a dialysis system in which 20 ml. of chloroform was added to a dialysis bag which was suspended in 500 ml. of H&. Both inside and outside phases were stirred continuously. Sodium hisulfite (10-a M) was added to the aqueous phase to prevent air oxidation of iodide. Iodide (lOme M and 1OV M) labeled with 1131 was introduced into the aqueous phase and the two phases were sampled for radioactivity at various time intervals, care being taken to keep their volumes constant,. Figure 2 shows the results
Phospholipid
The mechanism of iodide concentration in t,hyroid is unknown although a type of active transport of iodide seems indicated. A role for phospholipids as a transport carrier has seemed attractive because of some evidence for this function of such substances in the transport of sodium ion in the salt gland of certain birds (1) and in erythrocytes and brain (2-4). In addition we have found that phospholipid synthesis (from P3W4) in the thyroid is influenced by digit’alis glycosides and Kf concentration in the same manner as is iodide concentration (5, 6). The present study was undertaken to investigate iodide binding by thyroidal phospholipids (7). Phospholipids were fractionated using the method of Hanahan et al. (8). To detect phospholipids more easily 0.5 g. of thyroid slices were incubated with phosphat,e32 and added to 30 g. of
I
6()()-
CHCI,:MeOH j 3:2
I:4
I I MeOt 1 I
FIG. 1. Pz2 radioactivity of lipids from 30 g. of calf column (60 g. of silicic acid + 30 g. of Hyflow-Super-Gel, ml. were collected and pooled into 9 fractions as shown. to obtain a flow-rate of 1.0 to 1.5 ml./min. Composit,ion 425
thyroid as eluted from silicic acid diameter 30 mm.). Portions of 8.4 Nitrogen under pressure was used of solvents appears at the top.
426
LETTERS
TO THE
EDITORS
Iodide in Hz0 Phase 10B6 M _ Lecithin iti CHCi3 Phase.25 mg./ml. ----_--__--------7
0 TIME FIG. 2. Concentration mg./ml. thyroidal lipid molar.
ll 3. X&M
40
80 120 I HR.
2
160MIN. 3
4
24 HR.
of iodide in organic phase at various times after addition of 0.25 fractions 1 to 9 indicated in Fig. 1. Iodide in the water phase 10WB
THYROIDAL PC FRACTION (Fr. 7.)
[II'
- lo+
TIME IN HOURS FIG. 3. Molar concentration of iodide in chloroform during the dialysis after addition of thyroidal lecithin (fraction 7). Concentration of lecithin is indicated for the different curves. form, and iodide in water ([I-]HzO)
at various times (PC) in chloro-
LETTERS
TO THE
obtained with the various phospholipid fractions in the two phase chloroform water system. It is apparent that only fractions 7 and 8 are active in concentrating iodide into the organic phase. Fraction 1 contained phosphat,idylethanolamine and phosphatidylserine. Fraction 5 with maximal Pa2 uptake was phosphatidylinositol. Fractions 7 and 8 bot,h contained lecithin although a small amount of a slower moving material was also present,. Increasing the concentration of fraction 7 to 0.5 mg./ml. of chloroform increased the iodide in the organic phase to 1OV M (with 10m6 M Iconcentration in the aqueous phase). With 10-T M I- in the water, 0.25 mg./ml. of fraction 7 increased the iodide to a ratio of 5: 1, organic: aqueous (Fig. 3). Chromatography of the organic phase under these conditions revealed only iodide. The decrease with time in iodide content of the organic phase may be due to degradation of the phospholipid but was not investigated. The iodide in the organic phase was partly precipitable with acetone, but not wit’h ethyl alcohol. The results above are suggestive evidence for the participation of t’hyroidal lecithin (or a similar phospholipid) in the concentration of iodide by thyroid. The precise mechanism whereby synthesis, metabolism and resynthesis of a carrier so that it may transport iodide against’ a concentration gradient remains to be elucidated. The results with thyroidal lecithin seem to have some physiological significance as other lecithins do not work in the above system. Commercial soy-bean lecithin binds iodide very slowly, practically not at all during the first 10 hr. (7), and the iodinated end product is stable in chromatography, running with the solvent front. Synthetic dipalmit’oyllecithin and lecithin from calf brain, although identical in chromatography with thyroidal lecithin, do not effect any transfer of iodide int,o the organic phase. Lecithin from calf liver has some activity but needs to be present in a five to ten-fold greater concentration to be equally active. I should like to express thanks to Dr. J. E. Rall, Chief, Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, for supervision and generous advice, and to all his staff for profitable discussions during this work. REFERENCES 1. HOKIN, L. E., AND HOKIN, M. R., J. Gen. Physiol. 44,61 (1960). 2. SOLOMON, A. K., LIONETTI, F., AND CURRAN, P. F., Nature 178, 582 (1956). 3. KIRSCHNER, L. B., J. Gen. Physiol. 42, 231 (1958).
427
EDITORS
4. WOGT, W., Nature 179, 300 (1957). 5. WOLFF, J., Biochem. et Biophys. Acta 38, 316 (1960). 6. VILKKI, P., “Advances in Thyroid Research, p. 231. Pergamon Press, Oxford, 1961. 7. VILKKI, P., Suomen Kemistilehti B33, 209 (1960). 8. HANAHAN, D. J., DITTMER, J. C., AND WARASHINA, E., J. Biol. Chem. 228, 685 (1957). 9. MARINETTI, G. W., AND STOTZ, E., Biochem. et Biophys. Acta 18,102 (1955). 10. HOKIN, L. E., AND HOKIN, M. R., J. Biol. Chem. 233, 805 (1958). 11. MULDREY, J. E., MILLER, 0. N., AND HAYILTON, J. G. J. Lipid Research 1,48 (1959). PANU VILKKI~ Clinical Endocrinology Branch National Institute of Arthritis and Metabolic Diseases National Institute of Health Bethesda, Maryland Received January 25, 1961 1 Present address : Department of Chemistry, Universit,y of Turku, Turku,
Effect Coenzyme
of Cobalt Content
Deficiency
Medical Finland.
on the B12
of Rhizobium
meliloti’
Recently it has been demonstrated that the growth of several species of rhizobia was increased several-fold by traces of cobalt (1). In the experiments conducted with Rhizobium meliloti, cobalt was shown to be an indispensable component, of the culture medium. Investigations of the cobalt requirements of symbiotically grown soybean plants (3) provided evidence that the vitamin B,? content of root nodules from plants grown in purified solution lacking combined nit,rogen was positively correlated with the cobalt supply in the nutrient solution. Since Barker et al. (4, 5) have isolated different types of cobamide coenzymes which have been shown to be active in several enzyme systems, it was considered desirable to determine the influence of cobalt levels in culture media on the content of the Btz coenzymes in R. meliloti cells. The methods used for cleaning glassware and for the purification and preparation of the nutrient stock solutions and water have been described earlier (3). The basal nutrient medium contained 1 This investigation a grant (G 18556) Foundation.
was supported in part by from the Piational Science