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The presence of isocitrate lyase activity in the southern army worm, Prodenia eridania (Cramer)
369
SHORT COMMUNICATIONS
(5) 5-Hydroxyindole-O-methyltransferase: This enzyme, involved in the biosynthesis of melatonin 1~, was not found. A partial purification of these enzymes and a study of their properties is under investigation.
National Institute of Arthritis and Metabolic Diseases and National Institute of Mental Health, Bethesda, Md. (U.S.A.)
FRITZ M~,RKI* JuLIus AXELROD BERNHARD WITKOP
L. BRISTOL AND G. W. BARTELMEZ, Science, 27 (19o8) 455. J. J. ABEL AND D. I. MACHT, J. Pharmacot. Exptl. Therap., 3 (1911-1912) 319 . 3 Z. M. BACQ AND J. LECOMTE, Compt. rend. soc. biol., 141 (1947) 861. P. FISCHER AND J. LECOMTE, Arch. intern, pharmacodynamie, 81 (195o) 387. 5 E. CABIB, Rev. soc. arg. biol., 27 (1951) 19. 6 H. M. LEE AND K. K. CHEN, J. Pharmacol. Exptl. Therap., lO2 (1951) 286. "/ H. WEIL-MALHERBE AND A. D. BONE, Biochem. J., 51 (1952) 311. 8 j . AXELROD AND R. TOMCHICK, J. Biol. Chem., 233 (1958) 702. 9 G.L. CANTONI, in D. SHEMIN, "Biochemical Preparations", Vol. 5, J o h n Wiley & Sons, N e w York, 1957, p. 5810 A. LUND, Acta Pharmacol. Toxicol., 5 (1949) 231. 11 S. SENOH &ND B. WITKOP, J. Am. Chem. Soc., 81 (1959) 6222. x~ j . AXELROD, Federation Proc., 2o (1961) 236. i s j. AXELROD, u n p u b l i s h e d observation. 14 j. AXELROD, Science, 133 (1961) 343. xb D. D. BROWN, R. TOMCHICK AND J. AXELROD, ]. Biol. Chem., 234 (1959) 2948. 16 R. W. SCHAYER AND S. A. KARJALA, J. Biol. Chem., 221 (I956) 307. x7 j . AXELROD AND H. WEISSEACH, J. Biol. Chem., 236 (1961) 211. 18 M. GOLDENBERG, M. FABER, E. J. ALSTON AND E. C. CHARGAFF, Science, lO 9 (1949) 534z C.
Received November I6th, 1961 * Associate in t h e Visiting P r o g r a m of t h e U.S. Public H e a l t h Service, N I A M D .
Biochim. Biophys. Acta, 58 (1962) 367-369
The presence of isocitrate lyase activity in the southern army worm, Prodenia eridania (C ramer) Isocitrate lyase (Ls-isocitrate glyoxalatedyase, EC 4.1.3.1, formerly known as isocitritase) has not been found in animal tissue as reported by several investigators 1-s. LEVENBOOK~ tentatively concluded t h a t the glyoxylate cycle is inoperative in Prodenia eridania (Cramer) and isocitrate lyase apparently is absent in animal tissues. Evidence is presented here for the presence of isocitrate lyase activity in the pre-pupae and pupae of P. eridania. P. eridania larvae were grown on pole-bean plants at 26-27 °. At the appropriate stage of development 6-1o larvae or pupae (1.5-2.7 g) were blended in 20 ml of 0.2 M potassium phosphate buffer (pH 7.6) for 30 sec in a Lourdes homogenizer. All operations were carried out at o ° in pre-chilled equipment. The homogenate was centrifuged at 15 ooo × g for 20 min at o °. The f a t t y layer was removed, when present, and the supernatant decanted into another tube. Darco charcoal (5 mg/ml) was added to the supernatant and the suspension centrifuged again at 15 ooo × g for 15 min. This treatment removed pyridine nucleotides from the supernatant and thus eliminated possible interference from isocitrate dehydrogenase activity which might be Biochim. Biophys. Acta, 58 (1962) 369-371
37 °
SHORT COMMUNICATIONS
present. The final supernatant contained 9-z4 mg of protein]ml and was used in the enzyme assay with no further treatment. The methods for assay of the isocitrate lyase activity were those described b y CARPENTER AND BEEVERS4. The semi-carbazone method was used in the developmental study, and the FRmDEMANN AND HAUGEN~ method was used for the larger scale experiments to isolate the glyoxylate formed as the z,4-dinitrophenylhydrazone. Protein estimations in the enzyme preparation were carried out b y the spectrophotometric method of LAYSEe. DL-ISOcitric acid lactone, allo free, was converted to the free acid according to the method described b y CARPENTER AND BEEVERS4. A fresh solution of the isocitrate was prepared daily and used as the substrate. In all previous cases where isocitrate lyase activity has been detected, it has been confined to those tissues in which fat or acetate utilization was occurring. No isocitrate lyase activity has been detected in tissues t h a t were lacking in fat- or acetate-utilizing processes. Also, isocitrate lyase activity has not been found in tissues known to be synthesizing fat. LEVSNBOOKS reported no isocitrate lyase activity in mature 6th-instar Prodenia larvae. At this stage the larva is probably actively synthesizing fat as evidenced b y the increase in the amount of fat in the early 6th-instar larva as compared to the 5th-instar larva. However, the presence of isocitrate lyase activity could be expected in the pre-pupa and p u p a when stored fat reserves would be utilized. Isocitrate lyase activity was therefore assayed during the various stages of development of P. ¢ridania. The results, shown in Table I, indicate t h a t isocitrate lyase activity probably appears as the insect reaches the pre-pupa stage. As the insect passes into the p u p a stage, the isocitrate lyase activity declines and disappears b y the time the pupae are 5 days old. Additional confirmation for the presence of isocitrate lyase activity in the preparations was obtained b y making the 2,4-dinitrophenylhydrazone of the reaction product according to the method of FRIEDEMANN AND HAUGEN5. The absorption spectrum of the reaction product was compared to t h a t of the known glyoxylic a n d ,,-ketoglutaric acid derivatives. Fig. I shows the absorption spectra for the reaction product, glyoxylic-2,4-dinitrophenylhydrazone and ~-ketoglutaric-z,4-dinitrophenylhydrazone. I t can be seen t h a t the derivatives of glyoxylic acid and the reaction product have an absorption peak at 45o m/z, while t h a t of the derivative of ,,-ketoTABLE I I S O C I T R A T E L Y A S E A C T I V I T Y OF H O M O G E N A T E S OF P .
Stageof ~
~53
@V~,a~a
m~Jmg proteinl~
5th instax Early 6th inst~r Late 6th instax (pre-pupae) o-day pupae I - d a y pupae z-day pupae 3-day pupae
o.ooo o.ooo 0.oo6 i o.oo2 o.oo4 + o.oor o.oo7 + o.ooI o.oo4 o.oo3
4-day pupae 5-day pupae
o.oo x o.ooo
q- Standard deviation Biochira. Biophys. Acta, 58 (x96z) 369-37x
SHORT COMMUNICATIONS
371
0.5 Z < m
x~ B
~o.4 < 025
o~
o.I
o
3150
400
430
440 460 WAVELENGTH (mJJ)
480
500
Fig. I. Absorption spectrum of the 2,4-dimtrophenylhydrazone derivatives of ~-ketoacids. A, glyoxylate; B, ketoglutarate; C, product of isocitrate lyase reaction.
glutaric acid is at 420 m F, further demonstrating the absence of isocitrate dehydrogenase activity. These results indicate that isocitrate lyase activity, while very low when compared to that found in 5-day-old castor-bean endosperm 4, is definitely present in the pre-pupae and pupae of P. eridania. No attempt, however, was made to isolate the specific tissue of the insect containing the isocitrate lyase activity. Appreciation is expressed to Drs. J. EARLY and G. LUDVlK for generously supplying the P. eridania for this work.
Research Department, Agricultural Chemicals Division, Monsanto Chemical Co., St. Louis, Mo. (U.S.A.) z
W . D . CARPENTER E . G . JAWORSKI
j. A. OLSON,J. Biol. Chem., 234 (I959) 5.
N. B. MADSEN, Biochim. Biophys. Acta, 27 (x958) r99. a L. LEVEI~OOK, Arch. Biochem. Biophys., 92 (I96I) II4. 4 W. D. CARPENXER AND H. BEEVERS, Plant Physiol., 34 (I959) 403 • s T. F. FRIEDEMANN AND G. E. HAUGEN, J. Biol. Chem., 147 (953) 415 • s E. LAYr~E, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, Inc., N.Y., I957, p. 447.
Received November I r t h , 1961 Biochim. Biophys. Acta, 58 (I962) 369--37I
Adenosine deaminase in chick embryos GORDON AND RODER1 reported that the activity of adenosine deaminase (adenosine aminohydrolase, EC 3.5-4.4) in chick embryos could be increased several fold b y the injection of adenosine. SOLOMON9, who was unable to confirm this observation, studied the distribution of activity in developing embryos and concluded that there was no striking developmental change. We, also, have been unable to confirm the results of GORDONAND RODER, but have found a very striking (4o-fold) developmental increase in activity in the duodenum. Routinely, activities were measured on whole homogenates using the ammonia method. Tissue samples were homogenized in either 0.05 M, or o.2 M phosphate buffer (pH 7.0) using a pestle homogenizer with a Teflon pestle and glass vessel. Biochim. Biophys. Acta, 58 (1962) 371-373
SHORT COMMUNICATIONS
(5) 5-Hydroxyindole-O-methyltransferase: This enzyme, involved in the biosynthesis of melatonin 1~, was not found. A partial purification of these enzymes and a study of their properties is under investigation.
National Institute of Arthritis and Metabolic Diseases and National Institute of Mental Health, Bethesda, Md. (U.S.A.)
FRITZ M~,RKI* JuLIus AXELROD BERNHARD WITKOP
L. BRISTOL AND G. W. BARTELMEZ, Science, 27 (19o8) 455. J. J. ABEL AND D. I. MACHT, J. Pharmacot. Exptl. Therap., 3 (1911-1912) 319 . 3 Z. M. BACQ AND J. LECOMTE, Compt. rend. soc. biol., 141 (1947) 861. P. FISCHER AND J. LECOMTE, Arch. intern, pharmacodynamie, 81 (195o) 387. 5 E. CABIB, Rev. soc. arg. biol., 27 (1951) 19. 6 H. M. LEE AND K. K. CHEN, J. Pharmacol. Exptl. Therap., lO2 (1951) 286. "/ H. WEIL-MALHERBE AND A. D. BONE, Biochem. J., 51 (1952) 311. 8 j . AXELROD AND R. TOMCHICK, J. Biol. Chem., 233 (1958) 702. 9 G.L. CANTONI, in D. SHEMIN, "Biochemical Preparations", Vol. 5, J o h n Wiley & Sons, N e w York, 1957, p. 5810 A. LUND, Acta Pharmacol. Toxicol., 5 (1949) 231. 11 S. SENOH &ND B. WITKOP, J. Am. Chem. Soc., 81 (1959) 6222. x~ j . AXELROD, Federation Proc., 2o (1961) 236. i s j. AXELROD, u n p u b l i s h e d observation. 14 j. AXELROD, Science, 133 (1961) 343. xb D. D. BROWN, R. TOMCHICK AND J. AXELROD, ]. Biol. Chem., 234 (1959) 2948. 16 R. W. SCHAYER AND S. A. KARJALA, J. Biol. Chem., 221 (I956) 307. x7 j . AXELROD AND H. WEISSEACH, J. Biol. Chem., 236 (1961) 211. 18 M. GOLDENBERG, M. FABER, E. J. ALSTON AND E. C. CHARGAFF, Science, lO 9 (1949) 534z C.
Received November I6th, 1961 * Associate in t h e Visiting P r o g r a m of t h e U.S. Public H e a l t h Service, N I A M D .
Biochim. Biophys. Acta, 58 (1962) 367-369
The presence of isocitrate lyase activity in the southern army worm, Prodenia eridania (C ramer) Isocitrate lyase (Ls-isocitrate glyoxalatedyase, EC 4.1.3.1, formerly known as isocitritase) has not been found in animal tissue as reported by several investigators 1-s. LEVENBOOK~ tentatively concluded t h a t the glyoxylate cycle is inoperative in Prodenia eridania (Cramer) and isocitrate lyase apparently is absent in animal tissues. Evidence is presented here for the presence of isocitrate lyase activity in the pre-pupae and pupae of P. eridania. P. eridania larvae were grown on pole-bean plants at 26-27 °. At the appropriate stage of development 6-1o larvae or pupae (1.5-2.7 g) were blended in 20 ml of 0.2 M potassium phosphate buffer (pH 7.6) for 30 sec in a Lourdes homogenizer. All operations were carried out at o ° in pre-chilled equipment. The homogenate was centrifuged at 15 ooo × g for 20 min at o °. The f a t t y layer was removed, when present, and the supernatant decanted into another tube. Darco charcoal (5 mg/ml) was added to the supernatant and the suspension centrifuged again at 15 ooo × g for 15 min. This treatment removed pyridine nucleotides from the supernatant and thus eliminated possible interference from isocitrate dehydrogenase activity which might be Biochim. Biophys. Acta, 58 (1962) 369-371
37 °
SHORT COMMUNICATIONS
present. The final supernatant contained 9-z4 mg of protein]ml and was used in the enzyme assay with no further treatment. The methods for assay of the isocitrate lyase activity were those described b y CARPENTER AND BEEVERS4. The semi-carbazone method was used in the developmental study, and the FRmDEMANN AND HAUGEN~ method was used for the larger scale experiments to isolate the glyoxylate formed as the z,4-dinitrophenylhydrazone. Protein estimations in the enzyme preparation were carried out b y the spectrophotometric method of LAYSEe. DL-ISOcitric acid lactone, allo free, was converted to the free acid according to the method described b y CARPENTER AND BEEVERS4. A fresh solution of the isocitrate was prepared daily and used as the substrate. In all previous cases where isocitrate lyase activity has been detected, it has been confined to those tissues in which fat or acetate utilization was occurring. No isocitrate lyase activity has been detected in tissues t h a t were lacking in fat- or acetate-utilizing processes. Also, isocitrate lyase activity has not been found in tissues known to be synthesizing fat. LEVSNBOOKS reported no isocitrate lyase activity in mature 6th-instar Prodenia larvae. At this stage the larva is probably actively synthesizing fat as evidenced b y the increase in the amount of fat in the early 6th-instar larva as compared to the 5th-instar larva. However, the presence of isocitrate lyase activity could be expected in the pre-pupa and p u p a when stored fat reserves would be utilized. Isocitrate lyase activity was therefore assayed during the various stages of development of P. ¢ridania. The results, shown in Table I, indicate t h a t isocitrate lyase activity probably appears as the insect reaches the pre-pupa stage. As the insect passes into the p u p a stage, the isocitrate lyase activity declines and disappears b y the time the pupae are 5 days old. Additional confirmation for the presence of isocitrate lyase activity in the preparations was obtained b y making the 2,4-dinitrophenylhydrazone of the reaction product according to the method of FRIEDEMANN AND HAUGEN5. The absorption spectrum of the reaction product was compared to t h a t of the known glyoxylic a n d ,,-ketoglutaric acid derivatives. Fig. I shows the absorption spectra for the reaction product, glyoxylic-2,4-dinitrophenylhydrazone and ~-ketoglutaric-z,4-dinitrophenylhydrazone. I t can be seen t h a t the derivatives of glyoxylic acid and the reaction product have an absorption peak at 45o m/z, while t h a t of the derivative of ,,-ketoTABLE I I S O C I T R A T E L Y A S E A C T I V I T Y OF H O M O G E N A T E S OF P .
Stageof ~
~53
@V~,a~a
m~Jmg proteinl~
5th instax Early 6th inst~r Late 6th instax (pre-pupae) o-day pupae I - d a y pupae z-day pupae 3-day pupae
o.ooo o.ooo 0.oo6 i o.oo2 o.oo4 + o.oor o.oo7 + o.ooI o.oo4 o.oo3
4-day pupae 5-day pupae
o.oo x o.ooo
q- Standard deviation Biochira. Biophys. Acta, 58 (x96z) 369-37x
SHORT COMMUNICATIONS
371
0.5 Z < m
x~ B
~o.4 < 025
o~
o.I
o
3150
400
430
440 460 WAVELENGTH (mJJ)
480
500
Fig. I. Absorption spectrum of the 2,4-dimtrophenylhydrazone derivatives of ~-ketoacids. A, glyoxylate; B, ketoglutarate; C, product of isocitrate lyase reaction.
glutaric acid is at 420 m F, further demonstrating the absence of isocitrate dehydrogenase activity. These results indicate that isocitrate lyase activity, while very low when compared to that found in 5-day-old castor-bean endosperm 4, is definitely present in the pre-pupae and pupae of P. eridania. No attempt, however, was made to isolate the specific tissue of the insect containing the isocitrate lyase activity. Appreciation is expressed to Drs. J. EARLY and G. LUDVlK for generously supplying the P. eridania for this work.
Research Department, Agricultural Chemicals Division, Monsanto Chemical Co., St. Louis, Mo. (U.S.A.) z
W . D . CARPENTER E . G . JAWORSKI
j. A. OLSON,J. Biol. Chem., 234 (I959) 5.
N. B. MADSEN, Biochim. Biophys. Acta, 27 (x958) r99. a L. LEVEI~OOK, Arch. Biochem. Biophys., 92 (I96I) II4. 4 W. D. CARPENXER AND H. BEEVERS, Plant Physiol., 34 (I959) 403 • s T. F. FRIEDEMANN AND G. E. HAUGEN, J. Biol. Chem., 147 (953) 415 • s E. LAYr~E, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, Inc., N.Y., I957, p. 447.
Received November I r t h , 1961 Biochim. Biophys. Acta, 58 (I962) 369--37I
Adenosine deaminase in chick embryos GORDON AND RODER1 reported that the activity of adenosine deaminase (adenosine aminohydrolase, EC 3.5-4.4) in chick embryos could be increased several fold b y the injection of adenosine. SOLOMON9, who was unable to confirm this observation, studied the distribution of activity in developing embryos and concluded that there was no striking developmental change. We, also, have been unable to confirm the results of GORDONAND RODER, but have found a very striking (4o-fold) developmental increase in activity in the duodenum. Routinely, activities were measured on whole homogenates using the ammonia method. Tissue samples were homogenized in either 0.05 M, or o.2 M phosphate buffer (pH 7.0) using a pestle homogenizer with a Teflon pestle and glass vessel. Biochim. Biophys. Acta, 58 (1962) 371-373