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Uridine diphosphate derivatives in the tissues and hemolymph of insects
178
PRELIMINARY NOTES
Uridine diphosphate derivatives in the tissues and hemolymph of insects In the course of investigations of the acid-soluble phosphates of giant silkworm (Saturniid) pupae, our attention was attracted to a nucleotide fraction which occurs in rather high concentration. On the basis of its having an ultraviolet absorption m a x i m u m near 260 m/z and an apparent base: phosphate ratio, using the extinction coefficient of adenine, of approximately i : 3, this fraction, isolated from insect blood, was designated in an earlier publication z as ATP. This identification has proved to be incorrect. Analyses of representative samples are shown in Table I : the nucleotides have the spectral characteristics of uridine, and, when the appropriate extinction coefficient is used, the b a s e : P ratio is I:2. In paper electrophoresis (pH 3.5) the fraction gave a spot close to UDPGIuc, and, after mild acid hydrolysis, spots corresponding to UMP and UDP. In m a n y cases, the ion-exchange ctlromatograms partially resolved tile U D P derivatives into two peaks, the first and larger of which contained N-acetylaminohexose a, while the second gave the anthrone reaction for unsubstituted hexose. Since the colorimetric estimates of Gluc and GlucNAc were always less than the uridine, the sugar moiety was examined further. The products of mild acid hydrolysis of several such nucleotide fractions were subjected to paper chromatography (butanolacetic acid-water) and paper electrophoresis (sodium borate at p H io4), spots being revealed with benzidine trichloroacetate. In the latter system Gluc, Gal, mannose and their acetylamino derivatives separate cleanly. The nucleotides of insect origin always gave rise to a product migrating with GalNAc, usually as the major component. GlucNAc, Gluc, and Gal were in some cases also present. Identity of the major sugar component was further confirmed by exposing a sample of the nucleotide from hemolymph to hydrolysing and deacetylating conditions (6 N HC1) followed by reaction with ninhydrin ~. The principal sugar product migrated in borate electrophoresis identically with a lyxose standard prepared by treating a known sample of galactosamine in the same way. The product from glucosamine and from mannosamine is arabinose. We conclude that the principal component of the nucleotide fraction is UDPGalNAc. The level of U D P sugar derivatives in Cecropia tlemolymph changes characteristically with development of the insect. The larva has very little (about o.oI /,mole nucleotide/ml), the diapausing pupa 0.2-0. 9/~mole/ml, and with the initiation of adult development this rises to about 1.5/zmoles/ml. These concentrations refer to the cell-free plasma, in which the predominant species found is UDPGalNAc. In the pupal fat body, the level both in diapause and early adult development is about 0.2/~mole/g, and includes more nearly equal proportions of the several sugars mentioned above. In the wing epidermis, concentrations are especially high, ranging from 0.8/_,mole/g in diapause up to about 4 ~mole/g in early adult development. This last value is possibly the highest concentration of UDP sugar derivatives recorded for any animal tissue; hen oviduct, by comparison, contains a total level of about 0.5 tzmole/g6. Abbreviations: ATP, adenosine 5'-triphosphate; UMP and UDP, uridine 5'-monophosphate and -diphosphate; Gluc, glucose; Gal, galactose; GlucNAc, N-acetylglueosatnine; GalNAc, N-acetylgalactosamine.
Biochim. Biophys. Acta, 41 (i96o) t78-179
179
PRELIMINARY NOTES TABLE I C H A R A C T E R I Z A T I O N OF
U D P DERIVATIVES FROM I N S E C T S
Tissue e x t r a c t s w e r e p r e p a r e d w i t h cold 0. 3 N HC10~ a n d c h r o m a t o g r a p h e d on D o w e x - i f o r m a t e 2. T h e U D P d e r i v a t i v e s were e l u t e d w i t h a g r a d i e n t of H C O O N H 4 i n I N H C O O H . 2max.
Source
A 2so/A 2~o
pH x
pH x3
261
260
262 261
261 26o
pH •
z~l oles/ 2.oo moles total P
pH r ~
Labile P*
Uridine
"GlucNAc ....
0.39
0.37
0.96
1.17
0.44
0.37 0.39
0.34 0.33
1.33 0.98
0.96 0.99
0.36 --
Telea polyphemus
pupal hemolymph P l a t y s a m i a cecropia
p u p a l fat b o d y Known UDPGluc
* I N H 2 S O 4, IOO °, 15 min. ** M e t h o d of REISSlG, STROMINGER AND LELOIRa w i t h G l u c N A c s t a n d a r d . V a l u e s b a s e d on G a l N A c s t a n d a r d w o u l d be 2. 7 t i m e s higher.
The presence of UDPGluc in insect tissues may be related to the biosynthesis of trehalose and glycogen, that of UDPGlucNAc to the biosynthesis of chitin. The finding of the Gal and GalNAc nucleotides was less expected since there are virtually no reports of compounds of these sugars produced in insects. Very recently, LIPKE7 has demonstrated insect hemolymph glycoproteins yielding galactosamine together with other sugars, and the present results suggest the desirability of further examination of insect tissues for mucopolysaccharides. Also interesting is the selective occurrence of these nucleotides in the hemolymph plasma. By contrast, ATP, which is abundant in the cellular tissue~ cannot be detected in the plasma. This work was supported by a grant (EIo28C) from the U.S. Public Health Service.
Department of Biochemistry, Yale University, New Haven, Connecticut (U.S.A.)
F. G. CAREY* G. R . WYATT
1 G. R. WYATT,I V I n t e r n . Congr. B i o c h e m . , V i e n n a 1958, ( S y m p o s i u m 12, B i o c h e m i s t r y of Insects), 1959, p . I 6 i . 2 R. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. t{. POTTER, J . B i o l . C h e m . , 209 (1954) 23. a j . L. REISSlG, J. L. STROMINGER AND L. F. LELOIR, J . B i o l . C h e m . , 217 (1955) 959. 4 M. J. CRIJMPTON, B i o c h e m . J . , 72 (1959) 479. 5 p. j . STOFFYN AND R. W. JEANLOZ, A r c h . B i o c h e m . B i o p h y s . , 52 (1954) 373. 6 j . L. STROMINaER, B i o c h i m . B i o p h y s . A c t a , 17 (1955) 2837 H. LIPKE, X I I n t e r n . Congr. E n t o m o l . , V i e n n a , 196o, a b s t r a c t in t h e press.
Received March 28th, I96o * P r e d o c t o r a l r e s e a r c h fellow of t h e U.S. P u b l i c H e a l t h Service. 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 B i o c h e m i s t r y , D u k e U n i v e r s i t y , D u r h a m , N o r t h Ca rol i na . B i o e h i m . B i o p h y s . A c t a , 41 (196o) 178-179
PRELIMINARY NOTES
Uridine diphosphate derivatives in the tissues and hemolymph of insects In the course of investigations of the acid-soluble phosphates of giant silkworm (Saturniid) pupae, our attention was attracted to a nucleotide fraction which occurs in rather high concentration. On the basis of its having an ultraviolet absorption m a x i m u m near 260 m/z and an apparent base: phosphate ratio, using the extinction coefficient of adenine, of approximately i : 3, this fraction, isolated from insect blood, was designated in an earlier publication z as ATP. This identification has proved to be incorrect. Analyses of representative samples are shown in Table I : the nucleotides have the spectral characteristics of uridine, and, when the appropriate extinction coefficient is used, the b a s e : P ratio is I:2. In paper electrophoresis (pH 3.5) the fraction gave a spot close to UDPGIuc, and, after mild acid hydrolysis, spots corresponding to UMP and UDP. In m a n y cases, the ion-exchange ctlromatograms partially resolved tile U D P derivatives into two peaks, the first and larger of which contained N-acetylaminohexose a, while the second gave the anthrone reaction for unsubstituted hexose. Since the colorimetric estimates of Gluc and GlucNAc were always less than the uridine, the sugar moiety was examined further. The products of mild acid hydrolysis of several such nucleotide fractions were subjected to paper chromatography (butanolacetic acid-water) and paper electrophoresis (sodium borate at p H io4), spots being revealed with benzidine trichloroacetate. In the latter system Gluc, Gal, mannose and their acetylamino derivatives separate cleanly. The nucleotides of insect origin always gave rise to a product migrating with GalNAc, usually as the major component. GlucNAc, Gluc, and Gal were in some cases also present. Identity of the major sugar component was further confirmed by exposing a sample of the nucleotide from hemolymph to hydrolysing and deacetylating conditions (6 N HC1) followed by reaction with ninhydrin ~. The principal sugar product migrated in borate electrophoresis identically with a lyxose standard prepared by treating a known sample of galactosamine in the same way. The product from glucosamine and from mannosamine is arabinose. We conclude that the principal component of the nucleotide fraction is UDPGalNAc. The level of U D P sugar derivatives in Cecropia tlemolymph changes characteristically with development of the insect. The larva has very little (about o.oI /,mole nucleotide/ml), the diapausing pupa 0.2-0. 9/~mole/ml, and with the initiation of adult development this rises to about 1.5/zmoles/ml. These concentrations refer to the cell-free plasma, in which the predominant species found is UDPGalNAc. In the pupal fat body, the level both in diapause and early adult development is about 0.2/~mole/g, and includes more nearly equal proportions of the several sugars mentioned above. In the wing epidermis, concentrations are especially high, ranging from 0.8/_,mole/g in diapause up to about 4 ~mole/g in early adult development. This last value is possibly the highest concentration of UDP sugar derivatives recorded for any animal tissue; hen oviduct, by comparison, contains a total level of about 0.5 tzmole/g6. Abbreviations: ATP, adenosine 5'-triphosphate; UMP and UDP, uridine 5'-monophosphate and -diphosphate; Gluc, glucose; Gal, galactose; GlucNAc, N-acetylglueosatnine; GalNAc, N-acetylgalactosamine.
Biochim. Biophys. Acta, 41 (i96o) t78-179
179
PRELIMINARY NOTES TABLE I C H A R A C T E R I Z A T I O N OF
U D P DERIVATIVES FROM I N S E C T S
Tissue e x t r a c t s w e r e p r e p a r e d w i t h cold 0. 3 N HC10~ a n d c h r o m a t o g r a p h e d on D o w e x - i f o r m a t e 2. T h e U D P d e r i v a t i v e s were e l u t e d w i t h a g r a d i e n t of H C O O N H 4 i n I N H C O O H . 2max.
Source
A 2so/A 2~o
pH x
pH x3
261
260
262 261
261 26o
pH •
z~l oles/ 2.oo moles total P
pH r ~
Labile P*
Uridine
"GlucNAc ....
0.39
0.37
0.96
1.17
0.44
0.37 0.39
0.34 0.33
1.33 0.98
0.96 0.99
0.36 --
Telea polyphemus
pupal hemolymph P l a t y s a m i a cecropia
p u p a l fat b o d y Known UDPGluc
* I N H 2 S O 4, IOO °, 15 min. ** M e t h o d of REISSlG, STROMINGER AND LELOIRa w i t h G l u c N A c s t a n d a r d . V a l u e s b a s e d on G a l N A c s t a n d a r d w o u l d be 2. 7 t i m e s higher.
The presence of UDPGluc in insect tissues may be related to the biosynthesis of trehalose and glycogen, that of UDPGlucNAc to the biosynthesis of chitin. The finding of the Gal and GalNAc nucleotides was less expected since there are virtually no reports of compounds of these sugars produced in insects. Very recently, LIPKE7 has demonstrated insect hemolymph glycoproteins yielding galactosamine together with other sugars, and the present results suggest the desirability of further examination of insect tissues for mucopolysaccharides. Also interesting is the selective occurrence of these nucleotides in the hemolymph plasma. By contrast, ATP, which is abundant in the cellular tissue~ cannot be detected in the plasma. This work was supported by a grant (EIo28C) from the U.S. Public Health Service.
Department of Biochemistry, Yale University, New Haven, Connecticut (U.S.A.)
F. G. CAREY* G. R . WYATT
1 G. R. WYATT,I V I n t e r n . Congr. B i o c h e m . , V i e n n a 1958, ( S y m p o s i u m 12, B i o c h e m i s t r y of Insects), 1959, p . I 6 i . 2 R. B. HURLBERT, H. SCHMITZ, A. F. BRUMM AND V. t{. POTTER, J . B i o l . C h e m . , 209 (1954) 23. a j . L. REISSlG, J. L. STROMINGER AND L. F. LELOIR, J . B i o l . C h e m . , 217 (1955) 959. 4 M. J. CRIJMPTON, B i o c h e m . J . , 72 (1959) 479. 5 p. j . STOFFYN AND R. W. JEANLOZ, A r c h . B i o c h e m . B i o p h y s . , 52 (1954) 373. 6 j . L. STROMINaER, B i o c h i m . B i o p h y s . A c t a , 17 (1955) 2837 H. LIPKE, X I I n t e r n . Congr. E n t o m o l . , V i e n n a , 196o, a b s t r a c t in t h e press.
Received March 28th, I96o * P r e d o c t o r a l r e s e a r c h fellow of t h e U.S. P u b l i c H e a l t h Service. 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 B i o c h e m i s t r y , D u k e U n i v e r s i t y , D u r h a m , N o r t h Ca rol i na . B i o e h i m . B i o p h y s . A c t a , 41 (196o) 178-179