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Tryptophan pyrolase activity during pregnancy
198
SHORT COMMUNICATIONS
BBA 23 384
Tryptophan pyrrolase activity during pregnancy Recent studies in this laboratory z demonstrated that adrenal steroid administration to pregnant rats fed a nicotinamide-deficient diet prevents both the fetal death and the depletion of liver NAD which otherwise occur. In seeking an understanding of the mechanism b y which the adrenal steroids maintain the level of NAD in the liver, the activity of t r y p t o p h a n pyrrolase, the enzyme catalyzing the first step in the conversion of tryptophan to NAD, was determined under various conditions. In the course of this work, it was found that tryptophan pyrrolase activity is elevated in untreated pregnant animals, as compared to untreated non-pregnant ones. The higher level of tryptophan pyrrolase activity in pregnant compared to non-pregnant rats occurred whether the animals were fed a nicotinamide-deficient diet or a complete diet. The results of a typical experiment are shown in Table I. It can be seen that the t r y p t o p h a n pyrrolase activity of the pregnant animals was approximately twice that of the non-pregnant controls. TABLE I TRYPTOPHAN PYRROLASE ACTIVITY IN PREGNANT versus NON-PREGNANT RATS Virgin female r a t s (200-235 g) of the L o n g - E v a n s strain were placed in cages with males until conception occurred. Conception was a s s u m e d to h a v e occurred w h e n cornified cells and sperm a t o z o a a p p e a r e d in the vaginal smear, and this was t e r m e d day i of pregnancy. Animals were killed on day 13 of pregnancy. N o n - p r e g n a n t control animals of the same initial b o d y weight were used for comparison. Animals were fed a stock diet of P u r i n a L a b o r a t o r y Chow t h r o u g h o u t the experimental period. T r y p t o p h a n pyrrolase activity in liver was estimated from the rate of k y n u r e n i n e formation, according to the m e t h o d of FEIGELSON AND GREI~NGARD 4. Each value represents the m e a n _L s t a n d a r d error for d e t e r m i n a t i o n s on 20 animals.
Rats
Body wt. (g)
Liver wt. (~)
/,moles kynurenine formed per h -per g liver per total liver
Pregnant Non-pregnant
263 ± 3-5 243 -- 3.6
9-42 ~ o.64 7-84 i o.16
7-43 ± o.37 3 .88 7_ 0.26
69.5 : 3.4 30.0 ± 1.8
Preliminary experiments investigating a possible hormonal basis for the increased activity of the enzyme during pregnancy gave negative results. The increased tryptophan pyrrolase activity observed in pregnant animals does not appear to be the result of increased levels of ovarian hormones. Thus, although daily administration of I #g estrone p l u s 4 mg progesterone is effective in maintaining pregnancy in hypophysectomized-ovariectomized animals 2, administration of I, 3 or IO times these amounts of the ovarian hormones daily for 13 days did not cause a significant increase in the level of tryptophan pyrrolase activity when administered to non-pregnant animals. The possibility has also been considered that the increased level of tryptophan pyrrolase activity in pregnant animals might result from an increased level of circulating adrenal steroids, since adrenal steroids are known to cause an increase in the activity of this enzyme 3. However, measurements of adrenal size and of circulating plasma corticoids in pregnant and non-pregnant rats in one experiment did not support this idea. Thus, in the experiment of Table I the adrenal size and the concenBiochim. Biophys. dcta. 156 (i968) 198-199
SHORT COMMUNICATIONS
199
tration of plasma corticoids in the pregnant rats were not higher than in the nonpregnant ones. Department of Biochemistry, Geigy Research, Division of Geigy Chemical Corp., Ardsley, N . Y . (U.S.A.)
P . GREENGARD* H. J. KALINSKY
T. J. MANNING
P. GREENGARD, ]~. I3. SIGG, 1. FRATTA AND S. B. ZAK, J. Pharmacol. Exptl., Therap., 154 (1966) 624 . 2 W. R. LYONS, Proc. Soc. Exptl. Biol. Med., 54 (1943) 65. 3 W. E. KNox, in G. WEBER, Advances in Enzyme Regulation, Vol. 4, P e r g a m o n Press, New York, 1965, p. 287 . 4 P. FEIGELSON AND O. GREENGARD, J. Biol. Chem., 236 (1961) 15. I
Received November 2nd, i967 * P r e s e n t a d d r e s s : D e p a r t m e n t of N e u r o p h a r m a c o l o g y , I n s t i t u t e for Basic R e s e a r c h in M e n t a l R e t a r d a t i o n , N.Y. S t a t e D e p a r t m e n t of M e n t a l H y g i e n e , S t a t e n Island, N.Y. lO314 , U.S.A.
Biochim. Biophys. Acta, 156 (1968) I 9 8 - I 9 9
BBA 23381
Effect of nitrogen source on cobalt t o x i c i t y in Neurospora crassa Cobalt toxicity has been correlated with iron deficiency in Neurospora crassa 1. H E A L Y , CHENG AND McELRoY2 have found the activities of the iron-containing
proteins, catalase (EC i . I i . i . 6 ) , peroxidase (EC I . I I . I . 7 ) , succinate dehydrogenase (EC 1.3.99.I), cytochrome c, and cytochrome c oxidase (EC 1.9.3.1 ) to be very low in cobalt toxicity just as in straight iron deficiency in N. crassa. An iron-binding compound has been isolated from cobalt-toxic as well as iron-deficient cultures of N. crassa 3. Cobalt toxicity also results in an increase in the nitrate reductase activity (EC 1.6.6.3) in N. crassa 2. However, all the earlier work on cobalt toxicity in N. crassa has been done by growing the organism in the Fries' medium which contains both ammonium ion and nitrate as the nitrogen sources. In the present communication the effect of the nitrogen source on cobalt toxicity in N. crassa is reported. A wild strain of N. crassa Em 5297 a was used in these studies. The media and the growth conditions were essentially the same as described by PADMANABANAND SARMA4 with a change only in the nitrogen source. Three types of media were used depending on the nitrogen source provided : the sole nitrate medium contained sodium nitrate, 2 g/1 and sodium tartrate, I g/l; the sole ammonium medium contained ammonium tartrate 2 g/1; and the ammonium plus nitrate medium contained ammonium nitrate 2 g/1 and ammonium tartrate I g/1. Iron was provided at a higher level of I mE/1 to the sole nitrate medium, as the iron requirement of N. crassa in the sole nitrate medium was found to be high s. Cobalt was added as CoC12.6 H20. N. crassa was grown for 72 h and the enzyme extracts were prepared from the mycelia as described by PADMANABAN AND SARMA 4. Catalase was assayed according to the method of RAMACHANDRANAND SARMA5. Biochim. Biophys. Acta, 156 (1968) 199-2o2
SHORT COMMUNICATIONS
BBA 23 384
Tryptophan pyrrolase activity during pregnancy Recent studies in this laboratory z demonstrated that adrenal steroid administration to pregnant rats fed a nicotinamide-deficient diet prevents both the fetal death and the depletion of liver NAD which otherwise occur. In seeking an understanding of the mechanism b y which the adrenal steroids maintain the level of NAD in the liver, the activity of t r y p t o p h a n pyrrolase, the enzyme catalyzing the first step in the conversion of tryptophan to NAD, was determined under various conditions. In the course of this work, it was found that tryptophan pyrrolase activity is elevated in untreated pregnant animals, as compared to untreated non-pregnant ones. The higher level of tryptophan pyrrolase activity in pregnant compared to non-pregnant rats occurred whether the animals were fed a nicotinamide-deficient diet or a complete diet. The results of a typical experiment are shown in Table I. It can be seen that the t r y p t o p h a n pyrrolase activity of the pregnant animals was approximately twice that of the non-pregnant controls. TABLE I TRYPTOPHAN PYRROLASE ACTIVITY IN PREGNANT versus NON-PREGNANT RATS Virgin female r a t s (200-235 g) of the L o n g - E v a n s strain were placed in cages with males until conception occurred. Conception was a s s u m e d to h a v e occurred w h e n cornified cells and sperm a t o z o a a p p e a r e d in the vaginal smear, and this was t e r m e d day i of pregnancy. Animals were killed on day 13 of pregnancy. N o n - p r e g n a n t control animals of the same initial b o d y weight were used for comparison. Animals were fed a stock diet of P u r i n a L a b o r a t o r y Chow t h r o u g h o u t the experimental period. T r y p t o p h a n pyrrolase activity in liver was estimated from the rate of k y n u r e n i n e formation, according to the m e t h o d of FEIGELSON AND GREI~NGARD 4. Each value represents the m e a n _L s t a n d a r d error for d e t e r m i n a t i o n s on 20 animals.
Rats
Body wt. (g)
Liver wt. (~)
/,moles kynurenine formed per h -per g liver per total liver
Pregnant Non-pregnant
263 ± 3-5 243 -- 3.6
9-42 ~ o.64 7-84 i o.16
7-43 ± o.37 3 .88 7_ 0.26
69.5 : 3.4 30.0 ± 1.8
Preliminary experiments investigating a possible hormonal basis for the increased activity of the enzyme during pregnancy gave negative results. The increased tryptophan pyrrolase activity observed in pregnant animals does not appear to be the result of increased levels of ovarian hormones. Thus, although daily administration of I #g estrone p l u s 4 mg progesterone is effective in maintaining pregnancy in hypophysectomized-ovariectomized animals 2, administration of I, 3 or IO times these amounts of the ovarian hormones daily for 13 days did not cause a significant increase in the level of tryptophan pyrrolase activity when administered to non-pregnant animals. The possibility has also been considered that the increased level of tryptophan pyrrolase activity in pregnant animals might result from an increased level of circulating adrenal steroids, since adrenal steroids are known to cause an increase in the activity of this enzyme 3. However, measurements of adrenal size and of circulating plasma corticoids in pregnant and non-pregnant rats in one experiment did not support this idea. Thus, in the experiment of Table I the adrenal size and the concenBiochim. Biophys. dcta. 156 (i968) 198-199
SHORT COMMUNICATIONS
199
tration of plasma corticoids in the pregnant rats were not higher than in the nonpregnant ones. Department of Biochemistry, Geigy Research, Division of Geigy Chemical Corp., Ardsley, N . Y . (U.S.A.)
P . GREENGARD* H. J. KALINSKY
T. J. MANNING
P. GREENGARD, ]~. I3. SIGG, 1. FRATTA AND S. B. ZAK, J. Pharmacol. Exptl., Therap., 154 (1966) 624 . 2 W. R. LYONS, Proc. Soc. Exptl. Biol. Med., 54 (1943) 65. 3 W. E. KNox, in G. WEBER, Advances in Enzyme Regulation, Vol. 4, P e r g a m o n Press, New York, 1965, p. 287 . 4 P. FEIGELSON AND O. GREENGARD, J. Biol. Chem., 236 (1961) 15. I
Received November 2nd, i967 * P r e s e n t a d d r e s s : D e p a r t m e n t of N e u r o p h a r m a c o l o g y , I n s t i t u t e for Basic R e s e a r c h in M e n t a l R e t a r d a t i o n , N.Y. S t a t e D e p a r t m e n t of M e n t a l H y g i e n e , S t a t e n Island, N.Y. lO314 , U.S.A.
Biochim. Biophys. Acta, 156 (1968) I 9 8 - I 9 9
BBA 23381
Effect of nitrogen source on cobalt t o x i c i t y in Neurospora crassa Cobalt toxicity has been correlated with iron deficiency in Neurospora crassa 1. H E A L Y , CHENG AND McELRoY2 have found the activities of the iron-containing
proteins, catalase (EC i . I i . i . 6 ) , peroxidase (EC I . I I . I . 7 ) , succinate dehydrogenase (EC 1.3.99.I), cytochrome c, and cytochrome c oxidase (EC 1.9.3.1 ) to be very low in cobalt toxicity just as in straight iron deficiency in N. crassa. An iron-binding compound has been isolated from cobalt-toxic as well as iron-deficient cultures of N. crassa 3. Cobalt toxicity also results in an increase in the nitrate reductase activity (EC 1.6.6.3) in N. crassa 2. However, all the earlier work on cobalt toxicity in N. crassa has been done by growing the organism in the Fries' medium which contains both ammonium ion and nitrate as the nitrogen sources. In the present communication the effect of the nitrogen source on cobalt toxicity in N. crassa is reported. A wild strain of N. crassa Em 5297 a was used in these studies. The media and the growth conditions were essentially the same as described by PADMANABANAND SARMA4 with a change only in the nitrogen source. Three types of media were used depending on the nitrogen source provided : the sole nitrate medium contained sodium nitrate, 2 g/1 and sodium tartrate, I g/l; the sole ammonium medium contained ammonium tartrate 2 g/1; and the ammonium plus nitrate medium contained ammonium nitrate 2 g/1 and ammonium tartrate I g/1. Iron was provided at a higher level of I mE/1 to the sole nitrate medium, as the iron requirement of N. crassa in the sole nitrate medium was found to be high s. Cobalt was added as CoC12.6 H20. N. crassa was grown for 72 h and the enzyme extracts were prepared from the mycelia as described by PADMANABAN AND SARMA 4. Catalase was assayed according to the method of RAMACHANDRANAND SARMA5. Biochim. Biophys. Acta, 156 (1968) 199-2o2