Inhibition of trehalase activity in the hemolymph of Phormia regina Meig

ARCHIVESOF BIOCHEMISTRYAND BIOPI-IYSlCS93, 550--554 (1961)

Inhibition of Trehalase Activity in the Hemolymph of Phormia regina Meig. S. F R I E D M A N 1

From the Department o] Entomology, Purdue University, Lafayette, Indiana ~ Received January 30, 1961 The blood of Phormia regina Meig. has been shown to contain trehalose, trehalase, and a system which prevents hydrolysis of the substrate by the enzyme. Further investigation of the inhibitory system has resulted in the discovery of what appear to be at least two components: a large molecule, possibly proteinaceous in nature, and a metal ion. Consideration has been given to the role of this system in regulating trehalose breakdown in the insect. INTRODUCTION

The degradation of trehalose has long been thought to be mediated by the enzyme trehalase which is responsible for the hydrolytic cleavage of this compound to glucose, and which has been shown to be present in fungi (5) and insects (6). However, the discovery of the presence of this enzyme in the blood of P. regina Melt. in coexistence with high concentrations of trehalose (7) has provided the first indication t h a t it m a y in some manner be involved in a system responsible for the controlled breakdown of this sugar. This paper describes the properties of the compounds involved in maintaining the existing relationship between the enzyme and its nominal substrafe.

The discovery of a,a-trehalose in the hemolymph of certain insects and the subsequent realization that this disaccharide is the major carbohydrate component in the blood of a large number of insect genera (1) have awakened some interest in its role in insect metabolism. The mechanisms of synthesis and degradation of the compound are only now under investigation, and it has lately been shown (2) t h a t enzymic synthesis in locusts m a y proceed in a fashion similar to that described for yeast by Leloir and Cabib (3): UDPG + G-6-P ~,~ UDP + T-6-P s T-6-P --> T + Pi A phosphatase exhibiting marked specificity toward T - 6 - P has been isolated from the blowfly, Phormia regina Melt. (4), lending credence to the implication that this m a y be a synthetic p a t h w a y of general significance. 1This work was partially supported by Grant No. E-2440 from the National Institutes of Health, Bethesda 14, Maryland. 2Journal Paper No. 1714 of the Purdue University Agricultural Experiment Station. Abbreviations are as follows: UDPG, uridinediphosphate glucose; G-6-P, glucose 6-phosphate; UDP, uridine diphosphate; T-6-P trehalose 6phosphate; T, trehalose; Pi, inorganic phosphate; and EDTA, sodium ethylenediamine tetraacetate.

MATERIALS AND METHODS

Phormia regina Meig. larvae were reared on ground horsemeat until pupation and then transferred to cages containing 5% glucose in distilled water. From emergence until death adults not involved in maintenance of stocks used this solution as the sole source of nutritive material. Twoto 5-day-old adults taken from these cages were the experimental animals in the investigations to be described, removal of blood from the dorsal sinus being accomplished in the same fashion as has been previously noted (7). Since the quantity of blood obtained from a single insect was small (never more than 3 t~l.) compared to the amount necessary for most of the experiments, a number of insects of the same age were bled without regard to

550

TREHALASE

INHIBITION

sex and the blood was pooled prior to use. Dialysis of small quantities of blood (down to 10 ~1.) was accomplished by efficacious use of the smallest commonly available tubing (8/32 in. diameter, Visking Co., Chicago, Ilk). One end of the dialysis tubing was simply tied in a knot and the other was slipped over a length of 3-ram. glass tubing and sealed to it using Dueo cement. The glass tubing, including the seal, was permitted to remain above the dialysis medium, providing a convenient entry and removal port for the blood which was usually dialyzed against 1000 times its volume. The method used in determining trehalose and glucose in blood samples is the same as that described elsewhere in detail (7). Briefly, it consists of coupling a highly purified trehalase preparation with a somewhat modified specific enzymic method for the determination of glucose (8). Since the trehalase splits trehalose to glucose, analysis of blood made in the presence and absence of this enzyme will permit calculation of the quantity of trehalose present. Thus, the reaction mixture consisted of 0.2 ml. of inactivated blood (usually 2 ~1. of blood heated to 100°C. for 2 rain. in the presence of 1.0 ml. of phosphate buffer, ptI 7.0, 0.05 M), 0.4 ml. of a mixture of peroxidase and odianisidine (both from Worthington Biochemical Co., Freehold, N. J., and mixed as follows: 50 rag. o-dianisidine is suspended in 1.0 ml. methanol and added with stirring to 49 ml. of a phosphate buffer, pH 7.0, 0.05M containing 25 mg. peroxidase. The mixture is centrifuged and the precipitate discarded. The supernatant can be used in determinations for 1-2 weeks if kept frozen (when not in use), 0.1 ml. of purified trehalase (the enzyme is or is not added depending upon whether analysis is being made for glucose alone or glucose and trehalose), 0.04 ml. of purified glucose oxidase [obrained from Worthington Biochemical Co. and further treated to remove contaminating trehalase (7)], and distilled water to 0.79 ml. The mixture is incubated for 20 min. at 32°C. and the reaction stopped by the addition of 0.02 ml. of 4 N HC1. The extent of reaction, thus the concentration of glucose (between 1 and 20 ~g), is then measured in the Beckman DU speetrophotometer at 401 mt~.

551

while u n d i l u t e d blood s u b j e c t e d to t h e s a m e c o n d i t i o n s m a i n t a i n e d a c o n s t a n t level of b o t h s u g a r s over t h e s a m e period. A m o r e careful s t u d y of this s a m e p h e n o m e n o n is d e p i c t e d in Fig. 1. F r o m t h e p l o t i t m a y be seen t h a t a 1:40 d i l u t i o n suffices to r e l e a s e 50% of all t h e s u g a r which will be r e l e a s e d a t m a x i m u m dilution. B l o o d w a s also t e s t e d b y d i l u t i o n w i t h p h o s p h a t e buffer ( p H 7.0, 0.05 M ) to insure t h a t t h e r e s u l t s were n o t c o m p l i c a t e d b y a d r i f t u p o n d i l u t i o n to a m o r e a c i d p H . (An i n c r e a s e in a c i d i t y w o u l d h a v e t h e effect of i n c r e a s i n g t h e a c t i v i t y of t r e h a l a s e since t h e e n z y m e is m a x i m a l l y a c t i v e a t p H 5.6.) T h e r e s u l t s in t h i s case were t h e s a m e as those o b t a i n e d w i t h distilled water. Since t h e evidence p o i n t e d to t h e possible existence of an i n h i b i t o r in t h e blood, an a t t e m p t was m a d e to release the i n h i b i t i o n b y m e a n s o t h e r t h a n dilution. T h e t e m p e r a t u r e response of t h e purified e n z y m e is k n o w n (7), so t h e h e a t l a b i l i t y of t h e i n h i b i t o r y s y s t e m was tested. P u r i f i e d t r e h a l a s e ret a i n s 50% of t h e a c t i v i t y m e a s u r e d a t its o p t i m u m t e m p e r a t u r e w h e n i t is i n c u b a t e d w i t h s u b s t r a t e a t 55°C. for 30 rain. F i g u r e 2 shows t h a t t h e i n h i b i t o r is m u c h m o r e l a b i l e t h a n t h e e n z y m e since exposure to 55°C. for o n l y 2 rain. is enough to cause over a 50% i n c r e a s e in a c t i v i t y of t h e blood u n d e r test.

I0~

RESULTS T h e presence of t r e h a l a s e in t h e blood of a d u l t Phormia w a s i n i t i a l l y d e m o n s t r a t e d in an e x p e r i m e n t i n v o l v i n g i n c u b a t i o n of d i l u t e d a n d u n d i l u t e d blood in vitro (7). I t was n o t e d a t t h a t t i m e t h a t blood d i l u t e d 100-fold w i t h d i s t i l l e d w a t e r a n d i n c u b a t e d for 30 rain. a t 32°C. showed a p r o g r e s s i v e increase in q u a n t i t y of endogenous glucose w i t h a c o n c o m i t a n t d e c r e a s e in t r e h a l o s e ,

o

_ _ . / / 40 BLOOD

60

80

,00

500

DILUTION

Fla. 1. Increase in trehalase activity as a function of blood dilution. Two microliters of freshly drawn blood was diluted to each level with distilled water and incubated for 30 min. at 32°C. Water was then added to make 1.0 ml., and the mixture was heated to 100°C. for 2 rain. Trehalose and glucose were assayed as in the Methods section.

552

FRIEDMAN

than the disappearance of an inhibitory compound. This idea was effectively disposed of by experiments demonstrating an inhibition of added purified trehalase by o unheated, undiluted blood. Table I illustrates the results of such experiments. Exogenous trehalase shows high activity in the 10" presence of blood which has been heated to ?, 60°C., ostensibly destroying the inhibitor, and 80°C., ostensibly destroying inhibitor s" and endogenous trehalase; but no activity in the presence of blood which has not been I I I heated. I S :3 MINUTES In an effort to determine the general size and lability of the inhibitory factor, blood Fro. 2. T i m e - t e m p e r a t u r e curves relating to release of inhibition. Samples consisting of 2 ~1. of was dialyzed against 1000× its volume of freshly drawn blood were heated to the tempera- distilled water for periods up to 24 hr. at tures noted for the times noted, then cooled and 3°C. This treatment leads to a slight but incubated at 32°C. for 30 min. One milliliter of definite change in the inhibitory capacity of phosphate buffer (pH 7.0, 0.05 M) was added, and the blood. A 50% decrease in effectiveness the mixture was heated immediately to 100°C. upon dilution is reached at a level between for 2 min. Trehalose and glucose were assayed as 1:20 and i : 4 0 rather than between 1:40 in the Methods section. and 1:100. T h a t this is not a general lability is shown by control samples, which, TABLE I kept at 3°C. for 24 hr., do not lose any of INttIBITION OF EXOGENOUS TREHALASE their inhibitory capacity when compared to BY B L o o n freshly drawn blood. B l o o d was h e a t e d , as n o t e d , p r i o r t o a d d i t i o n of Dialysis for 3 hr. at 3°C. removes all of t r e h a l a s e , a n d 300 #g. t r e h a l o s e was a d d e d to all the measurable carbohydrate from the t u b e s . T h e m i x t u r e was t h e n i n c u b a t e d for 30 min. at 32°C., made up to 1.0 ml. with phosphate blood without affecting the inhibitor. Under buffer (pH 7.0, 0.05 M), and heated immediately these conditions it became possible, by addto 100°C. for 2 rain. Trehalose and glucose were ing trehalose, to judge whether or not the assayed as in Methods section. inhibitor was involved in the maintenance of an equilibrium condition between the two Enzyme source Glucose formed carbohydrates in the blood. Trehalose addition in quantities up to 500 ~g./2 ~l. blood ~g. in the complete absence o£ glucose gave rise 3 t,1. d i a l y z e d blood 4 to no glucose formation unless the blood 3 td. t r e h a l a s e 34 had been heated to 60°C. for at least 1 rain. 3 pl. d i a l y z e d blood ( h e a t e d to 60°C. for 75 Under those conditions trehalose disap3 rain.) -~- 3 pl. t r e h a l a s e peared with a corresponding increase in glu3 nl. dialyzed blood (heated to 60°C.) 51 cose. 3 ul. dialyzed blood -t- 3 ~I. trehalase 10 3 t*l. dialyzed blood (heated to 80°C. for 3 Experiments involving the addition to 3 rain.) the blood of compounds which might in3/A. dialyzed blood (heated to 80°C.) + 34 activate the inhibitor have led to the con3 ~i. trehalase clusion that a metal cofactor may be neces3 ~1. trehalase (heated to 80°C.) sary in the inhibition sequence. Sodium ethylenediamine tetraacetate ( E D T A ) , soThe inhibitor had not been isolated, so dium oxalate, and sodium citrate have all the possibility remained that activation of been found to be effective in reversing the endogenous trehalase was responsible for inhibition of trehalase activity when added the effects which had been noted rather to blood. In contrast, phosphate and acetate

TREHALASE INHIBITION

553

TABLE I I I~ELEASE OF TREHALASE INHIBITION ]~Y VARIOUS COMPOUNDS

Two microliters of freshly drawn blood was incubated for 30 rain. at 32°C. with 30 ~l. phosphate buffer (pH 7.0, 0.01 M) and 12 gl. of water or the compounds noted in the table in quantities large enough to give those final concentrations. Phosphate buffer (pYI 7.0, 0.05 M) was then added to make 1.0 ml. total, and the mixture was heated at 100°C. for 2 min. Trehalose and glucose were assayed as in Methods section. Compound

EDTA

Citrate

FinaI concn.

M 6 X 10 -a 6 X 10 -4 6 X 10-s 6 X 10-~ 8 X 10 -2

8 X 10-a 8 X 10-4

Release

Compound

Final conch.

% 85 85

Oxalate

9 1.7 9 9

M × 10-2 X 10-2 X 10-a X 10-~

% 13 40 23 10

Acetate

2.8 X 10-~

0

10 8 18 41 17

are c o m p l e t e l y ineffective. T a b l e I I c o m p a r e s t h e efficiency w i t h w h i c h t h e s e c o m p o u n d s p e r f o r m this function. E D T A is b y f a r m o s t p o t e n t a n d is also m o s t successful, as m a y be seen b y t h e f a c t t h a t i t will o v e r come 85% of t h e i n h i b i t i o n , w h e r e a s o x a l a t e a n d c i t r a t e n e v e r show m o r e t h a n 40% activity. I t is i m p r o b a b l e t h a t specific r e a c t i v a t i o n of t h e i n h i b i t o r b y m e t a l s is d e m o n s t r a b l e in a r e a c t i o n m i x t u r e c o n t a i n i n g a n y of t h e a b o v e - n a m e d c o m p o u n d s , since t h e m e t a l s are as c a p a b l e of c o m b i n i n g w i t h the chel a t i n g a g e n t to reduce its effectiveness as t h e y a r e of c o m b i n i n g w i t h a n d i n c r e a s i n g the effectiveness of the i n h i b i t o r . A n o t h e r a p p r o a c h to m e t a l r e a c t i v a t i o n is d i a l y s i s , and, to this end, b l o o d was d i a l y z e d a g a i n s t one t h o u s a n d v o l u m e s of E D T A ( p H 7.0, 0.1 M ) for 24 hr., t h e d i a l y z a t e r e m o v e d , and dialysis continued against distilled wat e r for a n o t h e r 12 hr. A t t h e end of this t i m e the d i a l y z e d blood was t e s t e d for i n a c t i v a tion of t h e i n h i b i t o r , a n d it w a s f o u n d t h a t u n d i l u t e d b l o o d showed no i n a c t i v a t i o n . If, however, it w a s d i l u t e d , t r e h a l a s e a c t i v i t y a p p e a r e d a t v e r y low levels of d i l u e n t (complete a c t i v i t y a t 1 : 2 0 d i l u t i o n ) c o m p a r e d to u n d i a l y z e d b l o o d (50% a c t i v i t y b e t w e e n 1 : 4 0 a n d 1:100 d i l u t i o n ) . T h e results of an a t t e m p t to b r i n g b a c k i n h i b i t i o n b y a d d i tion of m e t a l ions to d i l u t e d , d i a l y z e d blood is d e p i c t e d in T a b l e I I I . As m a y be

Release

seen, M g + + a t 0.01 M is m o r e effective t h a n a n y of t h e o t h e r ions t e s t e d , c a u s i n g a 47% i n h i b i t i o n of t r e h a l o s e b r e a k d o w n a t t h i s c o n c e n t r a t i o n . H o w e v e r , t h e effectiveTABLE I I I RESTORATION OF TREHALASE INHIBITION BY INORGANIC IONS IN E D T A DIALYZED BLOOD Compound

Final c o n c n .

Inhibition %

MgC12 MgC1 ~ MgC12 MgC12 MnC12 MnC12 MnC12 CoCI.~ CoCI 2 CoC12 KC1 CaC12

O. 1 O.O1 O. 00l O. 0001 0.01 0.001 O. 0001 0.01 0. 001 0.0001 0.01 0.01

18 47 13 0 16 33 0 8 29 0 0 0

EDTA dialyzed blood (in an amount equivalent to 2 ~1. of undialyzed blood) was incubated for 30 rain. at 32°C. with 5 ~1. trehalose (10 mg./ml.), 10 t*l. of maleate buffer (pH 7.0, 0.3 M) and 10 ~1. of the compounds noted in the table in quantities large enough to give those final concentrations. Phosphate buffer (pit 7.0, 0.05 M) was then added to make 1.0 ml., and the mixture was heated at 100°C. for 2 rain. Trehalose and glucose were assayed as in Methods section.

554

FRIEDMAN

ness of Mg ++ as compared to that of the other ions may be somewhat misleading. All of these compounds, being acid salts, tend to decrease the pH of the reaction mixtures in which they are present. These pH changes can be controlled through the use of buffers when the compounds are present in low concentrations. Thus, up to 0.Ol M in the ease of Mn++ and Co++ and 0.1 M in the ease of Mg ++, the pH's of the reaction mixtures remain at 7.0 and the results provide a valid indication of the effects of the metals themselves. When the above levels are reached, however, the available nontoxic buffers do not have the capacity to prevent changes in the pH's of the reaction mixtures and there are swings toward lower pH's. Since any decrease in pH results in an increase in endogenous trehalase activity (optimum pH = 5.6), the over-all inhibitions appear to decrease as higher concentrations of these metals are added. And, since the levels at which the pH changes occur are different for Mn ++ and Co ++ than they are for Mg ++, it is possible that the effectiveness of the metal ions themselves are different at the highest concentrations than the figures in Table I I I would indicate. The ions are active in increasing inhibition only when incubated with EDTA dialyzed blood. They will not reverse the effect of heating blood at 60 ° for 3 min., nor will they reverse the activation of undialyzed blood which has been brought about by dilution. DISCUSSION The reality of a system in P h o r m i a blood which prevents the enzyme trehalase from acting on its nominal substrate, trehalose, is beyond dispute. Two questions arise from this fact: The first, what are the components of this inhibitory system and how do they function?; the second, clearly tied to the first, is this system active in the control of the metabolic utilization of trehalose? At the present time, only the first can be answered, and, at that, only on a purely descriptive level. From experiments reported in this paper, it seems that at least two factors are involved in the system, the first, a large molecule, possibly a protein

judging from its heat lability and resistance to dialysis, and the second, a metal ion, to all appearances a bivalent ion of the group embracing Mg + +. There remains the entire question of how inhibition takes place and whether the role of the metal involves both enzyme and inhibitor. The physiological conditions involved in the control of trehalose breakdown have been investigated in a rather summary fashion up to now. Since, according to one of the more attractive hypotheses, inhibition would be released in response to stress, animals have been subjected to various pressures and records made of presence or absence of inhibitor. The two most obvious conditions of challenge are flight, during which large, rapid energy bursts are required, and starvation, during which the animal utilizes its reserve carbohydrate. In neither of these situations, singly or in combination, has any change in the level of inhibitor in the blood been shown to occur, although when tested by the usual methods trehalose and glucose have both been shown to have decreased. It is entirely possible, however, that the usual methods of test are not valid here, since a mechanism could conceivably be operative which involves some localized splitting of trehalose, with the general picture remaining one of complete inhibition. This problem is at present under consideration. ACKNOWLEDGMENT The assistance of Mrs. R. Witherspoon is greatly appreciated. REFERENCES 1. WYATT, G. R., AND KALF, G. F. J. Gen. Physiol.

40, 833 (1957). 2. CA~DY,D. J., A~l) KILBY,B. A. Biochem. J. 74, 19P (1960). 3. LELOIR, L. F., AND CABIB, E. J. Am. Chem. Soc.

75, 5445 (1953). 4. FRIEDMAN, S. Arch. Bioehem. Biophys. 88, 339 (1960). 5. BOURQUELOT,E. Bull. soc. mycol. France 9, 189 (1893). 6. FRE~EJACQUE,M. Compt. rend. 213, 88 (1941). 7. FRIeDmAN,S. Arch. Biochem. Biophys. 87, 252 (1960). 8. KESTO~, A. S. Abstracts p. 31c. Am. Chem. Soc., Div. Biol. Chem. April 1956.