Properties and distribution of glutamine synthetase in the southern armyworm, Prodenia eridania

BIOCHIMICA ET BIOPHYSICA ACTA

219

BBAI2IO 5

PROPERTIES AND DISTRIBUTION OF GLUTAMINE SYNTHETASE IN T H E S O U T H E R N A R M Y W O R M , P R O D E N I A E R I D A N I A L. L E V E N B O O K AND J E A N N E K U I t N

National Institute of A rth~itis and Metabolic Diseases, National Institutes of Health, Bethesda, Md., and Department of Biochemistm,, Jefferson Medical College, Philadelphia, Pa. ( U.S.A .) (Received March 6th, t962)

SUMMARY

Glutamine synthetase prepared from internal tissues of Prodenia eridania larvae was purified about 4-fold by isoelectric precipitation of acetone-powder extracts. The relatively unstable enzyme incubated with L-glutamate, NH4+ (or NH20H ), ATP and Mg2+ formed stoichiometric amounts of glutamine (or 7-glutamylhydroxamate), ADP and inorganic phosphate. The hydrox~/mate reaction with D-glutamate was considerably slower than with the L-isomer. 7-Glutamyl-transferase activity was about three times that of the synthetase. Characterization of the insect enzyme did not reveal any special or unique properties. Synthetase activity was highest in the larval fat-body, the gut and Malpighian tubules had a much lower titer, and little or no activity was found in the hemolymph. During adult development a more or less progressive decrease in synthetase activity occurred until just before emergence of the adult moth when the titer increased slightly. INTRODUCTION

In a companion paper 1 it has been shown that free glutamine is an important constituent of the hemolymph (blood) and tissues of the Southern Armyworm (Prodenia eridania) larva. It seemed likely that the enzyme glutamine synthetase, which mediates the reaction: g l u t a m a t e + NH~ + A T P -- glutamine + A D t ' + Pi

and is widely distributed in nature, might be concerned with glutamine synthesis in the insect. The enzyme has been purified and characterized from various sources including animal tissues, plants and bacteria ~-5, but information is almost completely lacking concerning GS of invertebrate organisms generally, and of insects in particular. ELLIOTTe detected GS activity in extracts of meal worm (Tenebrio molitor) larvae, and KILBY AND NEVILLE7 implicated GS as the enzyme responsible for glutamine synthesis by tissues of the desert locust Schistocerca gregaria in vitro, but Abbreviations: GS, g l u t a m i n e s y n t h e t a s e ; GHA, T - g l u t a m y l h y d r o x a m a t e ; PCMB, pchloromercuribenzoate.

Biochim. Biophys. Acta, 65 (I962) 210--232

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L. LEVENBOOK, J. KUHN

in neither instance was the enzyme characterized. Hence, apart from a preliminary report 8 of the present findings, the properties of the insect enzyme are now described for the first time. MATERIALS AND METHODS Larvae of the Southern Armyworm were raised first on bean seedlings and subsequently on potato slices at 25 ° . Mature, fully grown larvae were selected for the various experiments, or were transferred to dishes containing moist wood-shavings for pupation and adult development.

Acetone powders Larvae were incised longitudinally and the hemolymph and gut contents were removed b y rapid washing under a stream of cold water. After superficial drying on filter paper the combined internal organs were scraped off the cuticle into a beaker standing on ice. From the pooled organs of 2o-3 o larvae, acetone powders were prepared b y homogenization with approx. 2o volumes (w/v) of ice-cold acetone in a chilled Waring blendor. The extract was rapidly filtered on a Buchner funnel, and the insoluble material thus obtained was rehomogenized with a further io volumes of cold acetone. Following filtration the resulting cake was broken up, spread thinly on a large sheet of filter paper, and air dried. This dry powder, the starting material for preparation of the enzyme, could be stored in a desiccator at --4 °0 for several weeks without any apparent loss of enzyme activity.

Enzyme extracts These were prepared from approx, o.5-g portions of the acetone powder largely as described by ELLIOTT6. However, somewhat higher yields were obtained by a slight modification of this procedure based on the findings of SPECK9. Thus, (a) the initial extraction was made with 0.005 M N a H C 0 3 - o . I 5 M NaC1 instead of with distilled water, and (b) the iso-electric precipitation, which effected an approximately 4-fold purification, was performed b y the addition to the extract of 0.5 volume of 0.2 M acetate buffer (pH 4.2) in the cold. No further purification was attempted.

Homogenates Larval tissue and pupal homogenates were prepared by homogenizing the weighed material in 4 volumes of cold o.I M Tris buffer (pH 7.2). The pupal extracts were centrifuged for 2 min at approx. 1oo x g to separate pieces of cuticle and a top f a t t y layer, which was removed prior to assay.

Enzyme assay GS activity of acetone powder extracts was routinely assayed at 3 °° in a medium having the following composition per millilitre : 20 #moles Tris buffer (pH 7.2), 4 °/,moles KCN, 50/,moles L-glutamate, 5 °/,moles hydroxylamine .HC1, IO/,moles MgSO4, 5/,moles ATP, o.o5-o.1 ml enzyme, and HzO to I.O ml. All acidic or basic

Biochim. Biophys. Acta, 65

(I962)

219-232

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compounds were individually neutralized to p H 7.2. The ATP was added after 5 min of temperature equilibration in order to initiate the reaction. After a further 15 min the reaction was terminated by the addition of I.O ml of a solution composed of 7 volumes 2.4% (w/v) FeC13 in 2 N HC1 and 3 volumes 20% (w/v) trichloroacetic acid 1°. Tile absorbancy of the resulting ferric-hydroxamate color was measured at 500 m#, and the readings converted to hydroxamate by comparison with a standard curve prepared from pure GHS kindly supplied by Dr. L. LEVINTOW. All the values reported in this paper have been corrected by the subtraction of suitable blank values obtained from control tubes lacking glutamate. For certain experiments described in the text, the NHzOH was replaced by NH4C1 and GS activity was then measured by the release of Pi. Measurements of GS activity of pupal, and especially of larval tissue homogenates were subject to error due to the high control blanks in the absence of NH~OH, and the relatively poor proportionality between GHS formed and enzyme concentrations a. These difficulties were minimized b y employing at least two different levels of homogenate, and aliquots not exceeding 0.05 ml per assay. To remove unsedimenting material from the deproteinized and centrifuged incubation mixtures, the supernatants were filtered into the o.5-ml capacity Beckman cuvettes through micro sintered funnels. Chemical determinations

P i was determined by a micro modification (final volume 2.o ml) of GIN G's method 12. Glutamine and glutamate in incubation mixtures were extracted with hot 80°/; (v/v) ethanol and, after concentration in a rotary evaporator, they were separated by ionophoresis on W h a t m a n 3 MM paper at IOOOV in a cold room, employing ammonium acetate buffer (pH 3.7). The amount of each amino acid eluted from the paper was estimated according to YEMM AND COCKING 13. Protein was determined by the procedure of LOWRY et al. 14 employing purified larval P. eridania protein as a standard. RESULTS

P. eridania GS appears to be a globulin-like protein, insoluble in distilled water, but

freely soluble in dilute salt solutions. By comparison with GS from other sources, the insect enzyme is relatively unstable; as shown in Table I, 5o% of the original TABLE [ S T A B I L I T Y OF /9. e r i d a n i a

Time (h)

o

24 48 72 144 240

GS

EXTRACTS

S T O R E D AT 3 - - 4 °

Residual activity

(%)

IOO

75 61.5 53 33.5 16

Biochim. Biophys. ,qcta, 65 (1962) 219-232

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L. LEVENBOOK, J. KUHN

activity is lost in about 82 h at 3-4 °. At 5 °o some 5o% and 85% of the activity is destroyed in 2.5 and 5 min, respectively.

Stoichiometry of glutamine, Pt and GHA formation I t is evident from the stoichiometry of the GS reaction that for every mole of glutamine formed one mole of Pi is released. With GS from sheep brain ELLIOTT6 found the ratio of Pi : glutamine to be about I . I ; the average ratio from two experiments with the insect enzyme was 1.o 9. Further, as first shown by SPECK9, the ammonia m a y be replaced b y various other bases, e.g. when the base is hydroxylamine, GHA is synthesized instead of glutamine. The close agreement between the rates of GHA and Pi formation are indicated b y the time-course study shown in Fig. I. This demonstrates also the extent to which correction is required for Pi formation from ATP in the absence of glutamate, i.e. to contaminating ATPase activity. Fig. 2 2.0

< "1"(9 re

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0

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10

15 20 TIME (MIN)

25

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35

40

Fig. I. P r o g r e s s c u r v e s for g l u t a m i n e s y n t h e t a s e reaction. 0 - - O, f o r m a t i o n of G H A in presence of o.o 5 M N H 2 O H ; © - - ©, Pl f o r m e d in presence of o.o 5 M NH,C1 corrected for A T P a s e a c t i v i t y ; + - - - + , A T P a s e a c t i v i t y m e a s u r e d in a b s e n c e of g l u t a m a t e . 0.7 0.6 0.5 _x ~" 0.4 z < 0.3 "i(9

0.2 d O ~k0.1

0:2

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&

0'.8

110

ML ENZYME

;.2

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Fig. 2. P r o p o r t i o n a l i t y b e t w e e n g l u t a m i n e s y n t h e t a s e a c t i v i t y a n d e n z y m e c o n c e n t r a t i o n . T h e p r e p a r a t i o n e m p l o y e d h a d a specific a c t i v i t y of 8.0/~moles G H A f o r m e d p e r m g p r o t e i n / h .

Biochim. Biophys. Acta, 65 (1962) 219-232

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indicates that during the first 15 min the rate of GHA formation is directly proportional to enzyme concentration up to a maximal rate of about 2.0/~moles GHA/h. The specific activity of typical fresh P. eridania preparations varied from 8 to I I/,moles GHA/mg protein/h, and from the foregoing stoichiometry it follows that this is also the approximate rate of glutamine synthesis.

Component study The effect of omission of individual components from the incubation mixture is shown in Table II. I t is clear that with the exception of cyanide, the purpose of which is to maintain the enzyme-SH groups in the reduced form, there is an absolute requirement for each component of the incubation mixture. TABLE II COMPONENT STUDY" OF REQUIREMENTS FOR P. eridania G S "

/~moles GHA/i 5 rain Complete system No glutamate No NH2OH No enzyme No ATP N o M g 2+ No KCN

o.43 o.o2 o.o 3 0.03 0.04 0.03 o. 20

* Routine assay.

pH optimum The effect of p H on P. eridania GS was determined over the range indicated in Fig. 3 employing histidine buffers and NH2OH solutions of carefully adjusted pH. This latter precaution was necessitated by the fact that at about neutrality NH2OH is itself an efficient buffer. The optimum p H for the enzyme was close to 7.1. Over the p H range 7-8 enzyme activity was the same in either histidine or Tris buffer.

Effect of cation concentration The activity of all GS preparations is markedly dependent upon the concentration of an essential, activating, divalent cation. It has been also shown that the extent of activation is a function of p H (see ref. 4). At p H 7.2 Mg 2+ was the most effective cation for the insect enzyme, maximal activation being produced at 1.2. lO -2 M (Fig. 4). By contrast, under similar conditions Mn *+ was only about one-third as effective and concentrations higher than the optimum of approx. 4.0- lO -3 M inhibited the enzyme by almost 500/0 . Cobalt ions 15 were less effective than Mn 2+, while Ca 2+ was markedly inhibitory. In agreement with the findings for sheep brain GS (see ref. 6) the inhibitory effect of Ca 2÷ could be largely reversed by the addition of sufficient Mg 2+. Biochim. Biophys. Xcta, 65 (1962) 2 1 9 - 2 3 2

224

L. LEVENBOOK, J. KUHN

0,4

0.3 z :E £

0.2

O m 0.1 ::1, 0

~.~

6'.° 61~ 7.'0

71~ 81o 8:~

pH Fig. 3. Effect of p H on glutamine s y n t h e t a s e activity employing histidine buffers.

0.6

0.5

0.4 _z :E £ o3 i0.2 t~/

~

~ 0.1 IE a, 0 0

5

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MANGANESE A

10 1 20 plMOLES/ML OF CATION

2

Fig. 4- Effect of MgSO~ and MnSO 4 c o n c e n t r a t i o n s on glutamine s y n t h e t a s e activity at p H 7.2.

0.4

0.3

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Km=O'029

Q

~-

o~

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26o

Fig. 5. Effect of g l u t a m a t e concentration on glutamine s y n t h e t a s e activity. 0 - - O, G H A formation with 0.o 5 M NH~OH; © ... ©, P1 release with 0.o 5 M NH4C1.

Biochim. Biophys. Acta, 65 (1962) 219-232

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Effect of glutamate concentration The affinity of P. eridania GS for glutamate is considerably lower than for the other reactants, the K m being about 0.029 M (Fig. 5). Furthermore, employing either NH4CI or N H 2 0 H the enzyme could not be fully saturated with its substrate, even at glutamate concentrations as high as 0.25 M. With 0. 4 M glutamate, inhibition of the enzyme was observed, but this m a y have been due to the effect of high salt concentration rather than to glutamate per se. A similar low affinity for ~]utamate has been reported for guinea-pig kidney GS (see ref. 3).

Effect of A TP concentration The effect of valying the A T P concentration is .,hown in Fig. 6. "Ihe Km of approx, o.ooi M is considerably higher than that reported for pigeon liver 9, a staphylococcal preparation 16 or kidney 3, but is close to the value found for the l:urified pea enzyme 17. Concentrations of ATP in excess of 5 . o ' I o -~ M were progressively in-

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t

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5 10 ATP (juMOLES/ML)

i

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l

15

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,

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20

Fig. 6. Effect of A T P concentration on g l u t a m i n e s y n t h e t a s e activity.

hibitory. To examine whether this inhibition by high ATP concentrations might be due to the binding of Mg 2+ the effect was tried of increasing the Mg 2+ concentrations in the presence of inhibitory levels (I.O.lO -2 M, 2.O-lO -2 M) of ATP. No such reversal could be demonstrated, from which it is concluded that the inhibition by A]'P is not due to cation binding.

Effect of ammonia concentration The affinity of the insect enzyme for NH4+ is so high (Kin approx. 0.0004 M) that very little or even no effect of varying the NH4+ concentration could at first be demonstrated, Sufficient NH4+ was present in the enzyme preparations and/or the reagents to fully saturate the enzyme initially. The data for Fig. 7 were eventually Biochim. Biophys. ,4cta, 65 ([962) 219 232

226

L. LEVENBOOK, J. KUHN

# °

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2 3 4 5 NH4Cl (pMOLES/ML)

fS

20

Fig. 7- Effect of a m m o n i a c o n c e n t r a t i o n on glutamine s y n t h e t a s e activity.

obtained by treating all reagents with acid-washed Permutit to adsorb NH4+, and b y dialyzing the enzyme for 2 h in the cold. Clearly, the insect GS in vivo could act as a most efficient trapping agent for any traces of metabolic NH4+.

Effect of hydroxylamine The effect of varying the hydroxylamine concentration is shown in Fig. 8. The Km of o.oo13 M is probably subject to some error due to the presence in these assays of contaminating traces of NH4+. Although the enzyme affinity for N H , + is apparently higher than for NH2OH, nevertheless a degree of competition for the two substrates would ensue, with a consequent error in the determined Km for both. 0.3

S

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o.~

(9 J

o :E

:~ 0

0

lo

~5

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HYDROXYLAMtNE (/LIMOLES IML)

Fig. 8. Effect of N H , O H concentration on glutamine s y n t h e t a s e activity.

S ubstr ate specificity The insect GS has considerable specificity with respect to the N H , O H acceptor; no hydroxamate was formed in testing the following compounds: aspartic acid, asparagine, cysteine, methionine, lysine, arginine, histidine, phenylalanine, tyrosine, Biochim. Biophys. Acta, 65 (i962) 219-232

INSECT GLUTAMINE SYNTHETASE

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threonine, proline, glycine, alanine, valine, leucine, serine, ~-aminobutyric acid and pyrrolidone carboxylic acid. In fact, the only substance tested giving a positive reaction other than L-glutamate was D-glutamate. Earlier workersS, 18 reported that GS was specific for the L-isomer, but LEVlNTOW AND MEISTER19,~° showed that both enantiomorphs could be attacked at approximately equal rates by enzymes from various sources. The degree of optical specificity was subsequently demonstrated to TABLE III G H A FORMATION WITH L- AND D-GLUTAMATE Enzyme activity (%)

Substrate

5o #moles 5o #moles 50 /,moles 5° / * m o l e s

L-glutamate D-glutamate L-glutamate + 5 ° tlmoles D-glutamate L-glutamate + IOO /tmoles D-glutamate

ioo 23 85 68

be dependent upon the experimental conditionsl~, ~1. Three separate preparations of the insect enzyme reacted with D-glutamate* at 19%, 24°/0 and 26% of the corresponding rates with L-glutamate, and furthermore the D-isomer inhibited, apparently competitively, GHA formation with the natural substrate (Table III). D-Glutamate has also been shown to inhibit L-glutamine synthesis by the pea enzyme 2l. The specificity with respect to bases other than NH4+ and NH2OH was not examined in detail. However, as shown in Table IV, the enzyme did react with hydrazine and ammonia-free methylamine, although at slower rates than with NH4C1. TABLE IV THE EFFECT OF DIFFERENT BASES ON P. eridania GS ACTIVITY R o u t i n e assay as described in the t e x t except t h a t control t u b e s lacked base, and the progress of the reaction was followed of the rate b y PI liberation. 2o #moles of base per ml reaction m i x t u r e was in excess of the a m o u n t required to a t t a i n s u b s t r a t e saturation. Base (o.o2 M) NH4C1 Hydrazine Methylamine (ammonia-free)

Enzyme activity (%) IOO 68 48

Reversibility In experiments designed to demonstrate the reversibility of the GS reaction incubation mixtures similar to those described by LEVINTOW AND MEISTER~° were " Two samples of D-glutamate were employed: the first was p u r c h a s e d f r o m the Mann Chemical Co., and was f u r t h e r resolved w i t h Clostridium welchii according to CAMIEN et al.ll; the second was kindly d o n a t e d b y Dr. L. LEVlNTOW. Similar results were obtained with either preparation. Biochim. Biophys. Acta, 65 (1902) 219-232

228

L. LEVENBOOK, J. KUHN

employed. Starting with IO/~moles each of glutamine, A D P and Pi, approx. I/*mole of glutamate was formed after 4 h at 3 o°. Under similar conditions, b u t starting with IO/~moles each of L-glutamate, A T P and NH4+, approx. 9/~moles of glutamine were formed in the same period. These values cannot be utilized for an accurate estimate of the equilibrium constant due to the presence of the contaminating ATPase, and to the considerable experimental error (approx. 4- IO%) of the m e t h o d employed. The approximate equilibrium constant for the forward reaction of approx. 0. 7 • lO 3 is nevertheless surprisingly close to the values reported b y LEVlNTOW AN]) MEISTER 20 (0.73" 103) and b y VARNER AND WEBSTER23 (1.7"103).

7-Glutamyl-transferase activity It is now generally accepted t h a t the enzymic synthesis of glutamine and the transfer of the amide group of L-glutamine to various acceptors are catalyzed b y the same enzyme*2,24, 25. As indicated b y a typical experiment shown in Table V the TABLE V G L U T A M Y L T R A N S F E R A S E A C T I V I T Y OF P .

eridania GS

Glutamyl transferase assay: Iot, moles KH~PO 4, 5#moles MnCI,, 0.2 #mole ADP, 4° /*moles NH~OH (pH5.5). 5opmoles L-glutamate, 3o#moles acetate buffer (pH 5.5), o.i ml enzyme, H20 to I.O ml. i.o ml of the FeClatrichloroacetic acid solution was added after an incubation period of 15 rain at 3o°. Hydroxamate formed (#moles )

(i) (2) (3) (4) (5)

Complete glutamyl transferase system As above, no inorganic phosphate As (2), plus 5/~moles potassium arsenate As (3), no ADP GS, routine assay

o.61 0.28 0.77 o.17 o.2x

insect enzyme likewise manifests these dual properties, the transferase, like t h a t of other forms, being activated b y Pl, A D P and arsenate. The ratio of transferase to synthetase activity was close to three in two insect preparations; ratios of just under one were found for purified pea and crude pigeonliver enzymes 24, and as high as I0 for h u m a n H e L a cells ~.

Inhibitors A variety of GS inhibitors has been described, and the effect of some of these has been tested on the insect enzyme. PCMB was the most potent inhibitor examined (Fig. 9). The enzyme was inhibited 50% and 9 8 % b y 4 - I 0 - 5 M and I . I o - 4 M PCMB, respectively. This inhibition indicates functional thiol groups on the enzyme protein 2v, and suggested t h a t prior incubation of the enzyme with cysteine or some other - - S H c o m p o u n d might afford protection against PCMB inhibition. This was found to be the case, 3 "10-2 M cysteine giving almost I 0 0 % protection against I . i0 4 M PCMB, and about 66~o protection against I . 10 -3 M PCMB. Fluoride was also a powerful inhibitor of P. eridania GS, although somewhat

Biochim. Biophys. Acta, 65 (1962) 219 232

INSECT GLUTAMINE SYNTHETASE

229

100 9O

9O

80'

80

70

70

6O z o

5c

z

4c

~u/

3c

o

60 z

0

5O

z 4o ~

u a: lal 2C 10 5

3o

o r~ 20 hl 10 I

I

=

0

I

I

4 -LOGIo MOLAR PCMB

I

i

5

Fig. 9. I n h i b i t i o n of g l u t a m i n e s y n t h e t a s e b y PCMB. The o p e n circles show t h e degree of i n h i b i t i o n p r o d u c e d b y t h e t w o P C M B conc e n t r a t i o n s i n d i c a t e d w h e n t h e e n z y m e was first i n c u b a t e d w i t h 3 " 1°-2 M c y s t e i n e for 5 m i n prio r to a d d i t i o n of t h e i n h i b i t o r .

/

i

i

i

,4 3 -LOGIo MOLAR NctF

I

2

Fig. lO. i n h i b i t i o n of g l u t a m i n e s y n t h e t a s c b y s o d i u m fluoride. The t w o series of p o i n t s represent two separate experiments.

less so t h a n PCMB. As i n d i c a t e d in Fig. IO, the enzvme~ was 5o ~)~ a n d i o o Oj/oinhibited b y 1.5" lO .4 M a n d I • lO -2 M N a F , respectively. One of the p r o d u c t s of t h e GS reaction, n a m e l y A D P , is itself an i n h i b i t o r of the reaction (Fig. I I ) . Since the e x t e n t of t h e inhibition was p r o p o r t i o n a l not to the a b s o l u t e A D P concentration, b u t to the A D P / A T P ratio, t h e inhibition b y A D P would a p p e a r to be c o m p e t i t i v e . Similar findings h a v e been r e p o r t e d also b y other workers. BacteriaPS, 2s a n d pea GS 17 are i n h i b i t e d b y the d y e c r y s t a l violet, whereas pigeon-liver s a n d sheep-brain s p r e p a r a t i o n s are not. The insect enzyme falls into the l a t t e r category, c r y s t a l violet at a final c o n c e n t r a t i o n of 2.5" lO _3 M being w i t h o u t effect on the r a t e of the s y n t h e t a s e reaction.

0,4

RATIO ADP/ATP 1.2 2.0

2.8

3.6

6O 5O

g 4o _m zz 3o zLd ~ 2o

C) ~: W 10 0 ADP OuMOLES/ML)

Fig. I I. I n h i b i t i o n of g l u t a m i n e s y n t h e t a s e b y A D P i n t h e p r e s e n c e of 5/~moles A T P / m l .

Biochim. Biophys..4cta, 65 (19(,2) 219 232

230

L. LEVENBOOK, J. KUHN DISTRIBUTION

GS activity of larval tissues The GS activity of larval fat-body, gut, and Malpighian tubules was determined separately on homogenates of these tissues, and the results are shown in Table VI. Of the organs examined, the fat-body, the largest organ in the larval body, has by far the highest enzyme activity; fat-body GS, therefore, accounts for the major part of the total activity of larval acetone powder extracts. There was very little or no GS activity in the hemolymph. T A B L E VI GLUTAMINE SYNTHETASE ACTIVITY OF LARVAL P. eridania TISSUES Figures in p a r e n t h e s e s indicate the n u m b e r of samples analyzed in duplicate. Glutamine synthetase activity (/*moles GHA formed/rag protein~h)

Fat-body Gut Malpighian t u b u l e s

2.63 (4) 0.67 (3) 0.60 (i)

GS activity during metamorphosis To determine the changes in GS activity during metamorphosis, entire Prodenia pupae were individually analyzed daily throughout the period of adult development. Wide variations in enzyme activity were observed among pupae of similar age (Fig. I2), but as the pupae were taken from a number of generations at different seasons of the year, a considerable degree of variation is perhaps to be expected. As indicated in Fig. 12, GS activity during metamorphosis shows a more or less progressive decrease from the beginning of pupation until almost the end of 4.0, Iz 0T z

3.C

bJ

Q. ~ 2.C

:

T O

•

~1 J 1.C 0 IE I 0

I

I

I

I

I 50

I

I

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I

I IO0

PER CENT PUPAL DURATION (Adult) Fig. 12. G l u t a m i n e s y n t h e t a s e activity during adult d e v e l o p m e n t of P. eridania. E a c h p o i n t rep r e s e n t s the m e a n of duplicate analyses on a whole p u p a . Q, average values of the experimental points, OBiochim. Biophys. Acta, 65 (1962) 219-232

INSECT GLUTAMINE SYNTHETASE

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adult development. As judged by the scatter of the experimental data, the abrupt drop and subsequent rise in titer at about 3o% of pupal duration appears to be genuine, even though the biological significance of this finding is still quite obscure. Just before emergence of the adult, enzyme activity increases, but the enzyme of the adult moth has not been investigated. DISCUSSION

From a comparative viewpoint the only distinguishing feature of Prodenia GS is its relative instability; in other respects such as substrate specificity, co-factor requirements, equilibrium constant or transferase activity it cannot be distinguished from the corresponding enzyme of other organisms. Nor do the quantitative values for the substrate Michaelis constants or the degree of inhibition by various inhibitors suggest that the insect enzyme has any peculiar properties. Since insect GS is not inhibited by crystal violet it can perhaps be said to resemble sheep-brain 6 or pigeonliver 9 enzyme more closely than planO v or bacterial 16,2s preparations which are inhibited by the dye. The GS titer of Prodenia larva fat-body tissue approximates that of locust 7 and blowfly larva (Phormia regina29). The specific activity of these insect tissues is thus from 2 to 2o times higher than that of developing chick tissues 31, mouse liver al, rat kidney a or pigeon liver 9. Although no direct evidence has yet been adduced, it seems most likely that the high GS activity of the larval fat-body is intimately concerned with the elevated levels of free glutamine in the larval hemolymph and tissues 1. It may be noted, however, that during metamorphosis (or adult development) the flee glutamate and glutamine concentrations first increase and subsequently decrease, whereas GS activity over the same period falls more or less progressively until just before emergence of the adult moth, when the activity increases. Thus there is no obvious correlation between the various changes in GS activity and the concentration of glutamine during adult development. During metamorphosis the larval tissues undergo extensive histolysis, and of the larval fat-body in particular, only scattered remnants remain in the newly emerged adult insect. It seems plausible to suggest that the progressive decline in GS activity during adult development is therefore associated with the disintegration of this tissue. Similarly, the increase in enzyme activity observed towards the end of metamorphosis m a y be related to the formation of new, adult fat-body. However, other interpretations of the data are also possible, particularly in terms of enzyme stability, amount versus activity, and the existence of inhibitors. ACKNOWLEDGEMENTS We would like to thank Dr. L. LEVINTOW for his interest and advice. This work was supported, in part, by the National Heart Institute, PHS, Grant No. H - I 9 I 7 (c), and by the American Philosophical Society. REFERENCES t L. LEVENBOOK, J. Insect Physiol., 8 (I962) 5.59. 2 A. MEISTER, Physiol. Revs., 36 (I956) Io3. 8 R. RICHTERICH-VAN BAERLE, L. GOLDSTEIN AND E. t-I. DEARBORN, Enzymologia, 18 (~t957) 327.

Biochim. Biophys. Acta, 65 (I062) 210-232

232

L. L E V E N B O O K , J. KUHN

4 j . GREENBERG AND N. LICHTENSTEIN, J. Biol. Chem., 234 (1959) 2337. 5 it,. D. BOYER, R. C. MILLS AND H. J. FROMM, Arch. Biochem. Biophys., 81 (1959) 249. 6 W . H. ELLIOTT, Biochem. J., 49 (1951) lO6. 7 B. A. I{ILBY AND E . NEVILLE, J . Exptl. Biol., 34 (1957) 276. 8 L. LEVENBOOK AND J. KUHN, Federation Proc., 17 (1958) 374. 9 j . F. SPECK, J. Biol. Chem., 179 (1949) 14o5. 10 F. LIPMANN AND L. C. TUTTLE, J. Biol. Chem., 159 (1945) 21. 11 M. N. CAMIEN, L. E . McCLURE AND M. S. DUNN, Arch. Biochem., 28 (195 o) 220. 12 N. S. GING, Anal. Chem., 28 (1956) 133o. 13 ]~. W . YEMM AND E . C. COCKING, The Analyst, 80 (1955) 209. 1, O. H . LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265 . 15 (3. DANES, Biochim. Biophys. Acta, 15 (1954) 296. 16 13. A. FRY, Biochem. J., 59 (1955) 57917 W . H. ELLIOTT, J. Biol. Chem., 2Ol (1953) 661. 18 H . A. KREBS, Biochem. J., 29 (1935) 1951. 19 L. LEVINTOW AND A. MEISTER, J. Am. Chem. Soc., 75 (1953) 3039. 2o L. LEVINTOW AND A. MEISTER, J. Biol. Chem., 2o9 (1954) 265. 21 j . E . VARNER, Arch. Biochem. Biophys., 90 (196o) 7. 22 •. LAJTHA, P. MELA AND H . WAELSCH, J. Biol. Chem., 205, (1953) 55323 j . E . VARNER AND G. C. WEBSTER, Plant Physiol., 3 ° (1951) 126. 24 L. LEVINTOW, A. MEISTER, G. H . HOGEBOOM AND E . L. I~UFF, J. An,. Chem. Soc., 77 (1955) 5304 . 25 p . K. STUMPF, W . D. LOOMIS AND C. MICBELSON, Arch. Biochem., 30 (1951) 126. 26 R. DBMARS, Biochim. Biophys. Acta, 27 (1958) 43537 L. HELLERMAN, F. lz). CHINARD AND V. R. DEITZ, J. Biol. Chem., 147 (1943) 443. 2s W . H . ELLIOTT AND E . F. GALE, Nature, 161 (1948) 129. 29 L. LEVENBOOK, u n p u b l i s h e d o b s e r v a t i o n s . 3o D. RUDNICK, I~. MELA AND H. WAELSCH, .[. Cellular Comp. Physiol., 126 (1954) 297. st L. LEVlNTOW, J. Natl. Cancer Inst., 15 (1954) 347.

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