- Email: [email protected]
Peptidase-mediated storage of amino acids in small peptides
Insect Biochem., 1976, Vol. 6, pp. 179 to 185. Pergamon Press. Printed in Great Britain.
PEPTIDASE-MEDIATED STORAGE OF AMINO ACIDS IN SMALL PEPTIDES J. I. CoLLgr'r School of Biological Sciences, University of Sussex, Brighton, England
(Received 2 August 1975) A b s t r a c t - - A d u l t Calliphora contain a large and heterogenous pool of small peptides, which it is supposed come from protein catabolism. Their composition and rapid turn-over appears to be determined in part by a group of peptidases. The activity of the peptidases is inhibited noncompetitively by free amino acids and most effectively by the essential amino acids which are also those which stimulate feeding on protein. Thus it seems that as flies feed on protein containing the essential amino acids peptide emerging from catabolized protein tends to reanain as peptide until the internal concentrations of the essential amino acids fall. This provides the fly with a physiologically tolerable reservoir of amino acids.
INTRODUCTION ADULT female blowflies, Phormia regina (Meig.) feed heavily on protein during the prolonged period of growth of their o6cytes. A d u l t male Phormia also feed heavily on protein but only for a short period in their early adult life (DET~mR, 1961). T h e work presented here suggests that during the comparably short period of intensive protein ingestion in adult males of a closely related species Calliphora erythrocephala (Meig.) a large reservoir of small peptides is created which is able to satisfy the requirements for free amino acids t h r o u g h o u t the adult life span. T h e b u i l d - u p and the release of amino acids from the peptide reservoir is probably regulated by the concentrations of the same free amino acids which stimulate the labial sensory receptors on the proboscis and evoke feeding in Phormia (GOLDRICrI, 1973). MATERIALS A N D M E T H O D S
The flies All flies used in these experiments were males reared in a stock of Calliphora erythroeephala, which has been maintained through mass matings for many years (PRICE, 1965). Except when changes in diet were explicity a part of an experiment, adult flies were maintained on a diet of dried yeast, sugar and water and kept at room temperature.
Estimation of the free amino acids in whole flies and haemolymph and of the amino acid composition of peptides Clear haemolymph was withdrawn from living adults in capillary tubes extended into the ,thorax through the neck musculature. A total of 10 to 20/tl of haemolymph from 11 to 20 adults 5 to 15 days after their adult 179
eclosion was combined in 1 ml of 10% tricMoroacetie acid (TCA) containing a standard amount (0-160 gmole) of nor-L-leucine. Whole flies were homogenized individually in 1 ml of 10% TCA, also containing nor-Lleucine, in a ground-glass homogenizer. Both preparations were then filtered through a Millipore filter, HA 0"45. After adjusting the pH of the samples to 2"0, the amino acids of each sample were then separated and the amounts measured on an amino acid analyser (Locarte Limited, London). To estimate the amino acid composition of the small peptides, part of the filtrate was hydrolysed in 6 N HC1 for 24 hr at 110°O, and then, after removal of the acid, also separated on the amino acid analyser. The difference in the amounts of each amino acid in the hydrolysed and unhydrolysed fractions, adjusted by using the amount of nor-L-leucine present in each as a means of comparing different fractions of a sample and of estimating sample loss, provided a measure of the amount of amino acid in the small peptides. The samples were kept in T C A for a sufficiently long time before separation to deaminate asparagine and glutamine to their respective amino acids. Methionine, cysteine, and tryptophan were not measured. To determine the approximate sizes of the peptides, unhydrolyzed samples of whole flies similarly prepared in T C A were eluted from a 0.9×45 cm column of Sephadex G-10 in 0"1 N sodium chloride. Dextran 2000 was used as a measure of the void volume (Ire). To study the numbers and dynamics of the peptides, C 14-giycine was injected into adult flies and subsequently monitored in peptides in the following way. A number 30 guage needle mounted on an Agla syringe was inserted beneath the scutellum into the flight muscle cavity and 10gl of a solution of glycine-C 14 in insect ringer (EPHmrSsi and B~AVLE, 1936) Was delivered into the haemolymph. After 12 and 24 hr incubation, flies were homogenized and prepared as usual for separation on the amino acid analyser. A Tracerlab Dual Ratemeter Spectrometer Scintillation Counter with a flow cell containing plastic scintillant (NE 102A supplied by Nuclear Enterprises Limited, Edinburgh) monitored the
180
J. I. COLLETT
radioactivity in each peak in the effluent of the column of the amino acid analyser.
L-lysine-p-nitroanilide hydrolysis by L-leucine and Lphenylalanine.
Electrophoretic peptidases
The relationship of diet, amount of peptide and survival
separation
and
identification
of
Haemolymph withdrawn from adults (as described in the previous section) and supematants of whole flies homogenized in 0-2 M Tris-maleate buffer, pH 7"4 were separated in the same buffer in vertical gel slabs of 10% acrylamide with a potential of 31.2 V per cm for 2~[ hr at 2°C. T h e peptidases were identified by a modification of a technique developed by LEwis and HAaam (1967) in which a series of reactions take place at the interface of the acrylamide gel containing the peptidases and a 1% agar gel overlay containing a mixture of substrate, enzymes and final reducing agent. L-leueyl-L-alanine was the substrate for the peptidases, and at the positions of hydrolysis peroxide is formed in the presence of an L-amino acid oxidase from snake venom (Sigma Chem. Co. Limited) and then 3-amino-9-ethyl-carbazole becomes coloured as it is oxidized by peroxide in the presence of horse-radish peroxidase (Sigma Chem. Co. Limited). I n vivo and in vitro assays of peptidase activity Etherized adult males of 8 to 12 days of age were injected with 10/A of insect ringer (EvHaussI and BEADLE, 1936) containing peptides in either 0.01 or 0.02 M concentrations. At various intervals within a 3 hr period after injection individual flies were homogenized in T C A as usual and separated on an amino acid analyser as previously described. T h e amounts of each peptide remaining in flies at various intervals after injection provided estimates of the rates of in vivo hydrolysis. T h e hydrolyses of the L-forms of glycylleueine, leueylglycine, alanylproline, lysylphenylalanine, and prolylleucine, as well as of glycylglycylglycine and glycylglycine were followed. Peptidase activity was also assayed in vitro using a technique (PFLI~IDEREa, 1970) in which amino acid p-nitroanilides are used as substrate and the release of p-nitroaniline is followed spectrophotometricaUy. Adult males were homogenized in 0"06 M phosphate buffer, p H 7"6 and containing MgCla to 1 raM. T h e homogenates were centrifuged at 3500 g at 0°C for 15 min. T h e solution in which enzyme activity was measured contained enzyme diluted by a factor of at least 250 times compared with the in vivo concentration and substrate concentration of 0"05 to 0"30 raM. Activity was followed at 405 n m in a Zeiss P M Q II speetrophotometer with a 6-place automatic cell changer at 30°C. Comparative rates of hydrolysis of glycine-pnitroanilide (Nutritional Biochemical Corps.), L-alaninep-nitroanilide-HC1 (Cyclo Chemicals), L-leucine-pnitroanilide (British Drug House) and L-lysine-pnitroanilide-HBrs (Nutritional Biochemical Corps,) were measured. T h e inhibition of peptidase activity by different amino acids was compared by measuring the hydrolysis of the leueine- and lysine-p-nitroanilides in the presence (73 m M ) of amino acids. T h e type of inhibition and the KI of the amino acids which inhibit these substrates most strongly were determined by the graphical method of DIxoN (1964). ' Dixon plots' were constructed of the inhibition of L-leucine-p-nitroanilide hydrolysis by L-phenylalanine, L-leucine, and L-methionine and of
The longevities of adult males on different diets were compared with a view to relating lifespan to the amounts of small peptide in flies with each diet. On the day of adult eclosion from a single lay of eggs, 50 males were put into each of three cages which differed only in the diet provided. In one cage sugar and water were provided. In the second cage, sugar and water were provided throughout the lifespan hut yeast was provided for only the first I0 days. In the third cage, yeast, sugar and water were provided throughout the lifespan. T h e yeast and sugar were in separate containers and the numbers feeding on each were recorded throughout the lifespan. T h e temperatures at which the cages were kept varied within the range of 12°C to 25°C and on average were higher toward the end of the lifespans. T h e amounts of peptide in flies on each feeding regime were estimated in flies kept in similar conditions. RESULTS
AND
DISCUSSION
The peptides A n acceptable criterion of identification of a c o m p o u n d as a p e p t i d e is t h a t it s h o u l d stain w i t h n i n h y d r i n a n d t h a t o n hydrolysis it s h o u l d p r o d u c e free a m i n o acids. W h e n w h o l e flies are h o m o g e n i z e d in 1 0 % T C A w h i c h precipitates protein, a n d t h e s u p e r n a t a n t is p a s s e d t h r o u g h a M i l l i p o r e filter to r e m o v e even v e r y fine precipitate, t h e s u p e r n a t a n t c o n t a i n s free a m i n o acid as well as m a t e r i a l w h i c h stains w i t h n i n h y d r i n , a n d hydrolysis p r o d u c e s m o r e free a m i n o acid. S e p a r a t i o n s of t h e u n h y d r o l y s e d T C A s u p e r n a t a n t s o n a n a m i n o acid analyser show n i n h y d r i n - s t a i n i n g peaks w h i c h d i s a p p e a r o n h y d r o lysis. W i t h hydrolysis t h e free a m i n o acid peaks increase a n d t h e larger t h e size of t h e p r o b a b l e p e p t i d e peaks in t h e u n h y d r o l y s e d s u p e r n a t a n t t h e greater t h e increase of t h e a m i n o acid peaks in t h e h y d r o l y s e d p a r t of t h e s u p e r n a t a n t . T h u s t h e h y d r o l y s a b l e m a t e r i a l in t h e s u p e r n a t a n t is almost certain to b e peptide. T h e p e p t i d e peaks in samples of flies separated o n t h e a m i n o acid analyser a p p e a r in positions w h e r e di- a n d t r i - p e p t i d e s are eluted (CoLLETT, u n p u b . ) . T h e s a m p l e peaks are m o s t likely also di- a n d tripeptides. F u r t h e r m o r e , e l u t i o n of t h e T C A s u p e r n a t a n t s of h o m o g e n a t e s of whole flies f r o m a S e p h a dex G - 1 0 c o l u m n indicates t h a t m o s t if n o t all of t h e p e p t i d e is small. F r a c t i o n s collected f r o m V0 1.2 to "1"9 c o n t a i n e d t h e same i n c r e m e n t of a m i n o acid after hydrolysis as t h e same T C A s u p e r n a t a n t w h i c h h a d not been separated on the Sephadex column. Thus t h e m a x i m u m size of t h e p e p t i d e s is unlikely to b e m o r e t h a n five residues. T h e a m i n o acid c o m p o s t i o n of t h e p e p t i d e s f r o m t h r e e different males, s h o w n in T a b l e 1, illustrates several o t h e r features of t h e peptides. (1) W i t h t h e possible e x c e p t i o n of t h o s e n o t m e a s u r e d ( m e t h i o nine, t r y p t o p h a n , a n d cysteine), all p r o t e i n a m i n o
181
Peptidase-mediated storage of amino acids in small peptides Table 1. Peptide amino acid, free amino acid and the inhibition of peptidase activity Free Amino' Acid Oom~ositlon
A.A. composition of Peptides
A.A. Inhibition of Peptidase Activity in vi t re,, prac%ional Activity:
2 days old ~Moles/fly*: aspartic acid asparagine threonine + serine glutamic acid glutamine
]0.127 O. 052 0.062 3 ] Oo 235
2to line glycine alanine valine + isoleucine # leucine + tyro sine +
O. 069 0.524 0.082 0.008 0.01~ 0.020
phenylalanine + his tidine + lymine + ar~inine + methionine Total:
i0 days oI~
Total Free :C.oncemtration in 55 A~dr~ Acid of HAemolymph of i0 " days old 20 ds~ old fly* day old flies ~Moles mMolar
]0.519
]0.288
] 0.217
5.4
99
o. 3 ~ 9.459
O. 150 0.261
O. 022 0.220
O. 6 2?2
89 97
]0.402 ]0.530 0.501 0.547
4.0 7.5
"95 10o
i. 9 4.6
99 89
]0.609 0.554 I. 355 0.690
O. 896 0.575
O. 187 0.237
hydrolysis of L-lysinenitroanilide ~HBr) (%) 2
Enzyme + 73mNolar A& enzJme hydrolysis of L~ le ucine-p-ni troanilide
90 71 79 75 I00 95 72 4O 24
0.215
0.008
0.025
0.4
57
o.15o o.2y5 >0.006
0.060" 0.095 >0.Ii
0.050 o.oss 0.016
0.5 0.5 0.4
50 5s
0.015 0o071 0.036 O. 008
O.i17
0.o11
0.2
lO
0.161 0.054 0.274
2.9 1.4 1.8
53
8 48
53
43
0.105
0.063 o.121 0.ii0 0.069
42
19
1.324
5.525
3.288
2.387
0.185
17
*Estimates from single flies weighing 52.6 +0.6 mg. Ages given in days after emergence. tEssential amino acids (DAOD, 1973). acids are present in the peptides, but in widely different amounts. Glycine and glutamic acid and/ or glutamine together account for about 50% of the peptide amino acid, while the amino acids which are required in the diet (essential) are together only a small proportion of the total peptide amino acid. (2) In contrast to the relative invariability of the free amino acid pool from fly to fly (CoLLET% in prep.), the total amount of peptide varies over a five-fold range from fly to fly. (3) But however large or small the peptide pool, the relative amounts of each constituent amino acid remain approximately the same. T h e number of kinds of peptides making up this peptide component which varies in size but not in composition is more difficult to ascertain, but several pieces of evidence suggest that their n u m b e r is considerable. Of these, the most convincing comes from in vivo incorporation of glycine-C14 into material which elutes intermittently throughout an amino acid analyser separation. Flies injected with C a4-glyeine and subsequently homogenized in T C A and separated on an amino acid analyser in which the radioactivity of the effluent is monitored, have many more peaks containing label than are visible from the less sensitive ninhydrin staining. The peaks disappear with hydrolysis and are therefore probably peptide. Some peaks appear before glyeine itself, indicating that they either contain 4 or 5 residues or that they contain acidic amino acids as well as glyeine. Many peaks appear after glycine
with the neutral and basic amino acids indicating a smaller size and the presence of neutral and basic amino acids with glycine. Some of these peaks are also broader than might be expected of a homogeneous molecular species. The number of peaks labelled with glycine-C14 alone suggests that there are in fact many species of peptide, each present in amounts which are usually too small to detect as peptides, but which together account for a considerable amount of amino acid. These peptides in Calliphora adults may be similar to those described by MITC~LL and SIMMONS (1962) in Drosophila melanogaster larvae which they estimated to number about 600. Comparison of the amount of label in the peptide peaks from flies injected with label 12 to 24 hr before sample preparation also indicates that the peptides, like the free amino acid pool, undergo rapid turnover. While some of the labelled glyeine was still present among the free amino acids and so able to generate more peptide, the label in the glycine containing peptides declined by 60% through this 12 hr period. The most likely source of these peptides is turning-over protein and for the following reasons. The peptides which n u m b e r in the hundreds contain only the protein amino acids and while the total amounts vary to sometimes as much as 10% of the total protein of the fly, the proportion of each amino acid remains constant. Therefore, the composition of the peptides is what one would expect from
182
J. I. COLLETT
turning-over protein. Furthermore, the rate of peptide accumulation early in adult life after emergence corresponds with the rate of protein synthesis (and turnover) (see below). And in any case, on energetic grounds alone, it would be remarkable if these peptides were synthesized de novo.
The pep tidases Three different techniques have been used to describe the flies' peptidases and their activities. The electrophoresis of haemolymph and homogenates of whole flies provides an estimate of the number of peptidases and their location. T h e rate of disappearance of large amounts of peptides injected into the haemolymph of flies is a reliable gauge of in vivo activity and shows that the specificity of the peptidases corresponds to the amino acid composition of the peptides. In vitro assays of peptidase activity allow quantitative treatment of features of the peptidases which may affect their activity in vivo. Most significant is that the peptidase activity appears to be non-competitively inhibited by essential amino acids. origin
l
m
m
m
m
4.
Fig. 1. A diagrammatic comparison of electrophoretic separations of homogenates of whole flies (left) and of haemolymph (right). The thickness of the bands indicates the intensity of the activity hydrolysing L-leucyl-L-alanine.
Electrophoretic separation of peptides T h e activity of the peptidases which hydrolyse L-leucine-L-alanine is clearly visible in electrophoretic separations of fly material in 10% acrylamide gel. Six distinct molecular species with this specificity are consistently found in the homogenates of whole flies. As shown diagrammatically in Fig. 1, five of these peptidases are also present in haemolymph alone. T h u s clearly a substantial part of the peptidase activity is related to metabolism other than the digestion and assimilation of protein in the gut. In vivo hydrolysis of known peptides If the specificities of the peptidases available to the haemolymph are responsible in part for the composition of the peptides, then these specifieities ought to be reflected in the relative rates at which pcptides of known amino acid composition are
loo I~.~----_..~ eo
~.o
~o
gty~,oy ~oo gly glygly
80
•
gly leu
50 a
alapro
10
/I
~Apheval pheval
51
• leugly
x x
20
140
proteu
1~O
50
MINUTES
Fig. 2. The hydrolysis of natural peptides in vivo. Solutions containing peptides were injected into the haemolymph of the flight muscle cavity and the amount (nMoles) of unhydrolysed peptide in individual flies was measured at various intervals thereafter. hydrolysed in the haemolymph. Accordingly, Fig. 2 shows the relative rates of hydrolysis of eight peptides of the L-form after injection into the haemolymph of the flight muscle cavity. These rates range widely and in a way which corresponds to the amino acid composition of the fly's peptides. Of the eight peptides injected those containing amino acids which are present in the fly's peptides in relatively small amounts were hydrolysed more rapidly than those containing amino acids which are present in abundance in the fly's peptides. For example, glygly was hydrolysed more slowly than alapro and alapro was hydrolysed more slowly than pheleu. Thus however the peptides are generated, their amino acid composition appears to be in part the result of the specificities of the peptidases. In vitro hydrolysis of peptide analogues T h e hydrolyses of 4 amino acid p-nitroanilides by the peptidases in vitro showed specificity similar to the in vivo hydrolyses of natural peptides. Typical relative rates of hydrolysis of the four substrates tested are: nMoles p-nitroaniline/ml assay medium/minute at 30°C glycine-p-nitroanilide L-alanine-p-nitroanilide, HC1 L-leucine-p-nitroanilide L-lysine-p-nitroanilide,(HBr)2
1.43 1'95 2.70 5"53
This striking similarity of the relative rates of in vitro hydrolysis of peptide analogues and in vivo hydrolysis of natural peptides permits meaningful quantitative analysis of the kinetics of the peptidase activity in vitro.
183
Peptidase-mediated storage of amino acids in small peptides
The inhibition of peptidase activity in vitro by amino acids While the specificities of the active site of the peptidases explain at least in part the composition of the peptides, the inhibition of the peptidases by free amino acids which is described here seems to explain the wide variation in total amounts of peptide. Furthermore, the specificity, the type and the strength of the inhibition together suggest how these peptides, in varying so widely in amounts, may be used as a store of free amino acid. A comparison of in vitro peptidase activity in the presence of various amino acids ('fractional activities' in Table 1), shows not only that amino acids do inhibit peptidase activity but that some amino acids are very effective inhibitors. For example, in the presence of 73 m M L-phenylalanine only 8% as much leucine-p-nitroanilide was hydrolysed as when no amino acids were present. F r o m Table 1 it is clear that with both leucine- and lysine-p-nitroanilide the amino acids with appreciable effect are those which are required in the diet (essential). Moreover, the greatest effect comes from the largest hydrophobic amino acids, leucine, isoleucine, phenylalanine, and methionine. Not only are these essential amino acids present in relatively very low concentrations in the fly, b u t they are also the amino acids which stimulate feeding on protein in Phormia (GoLDmCH, 1973). A further feature which would affect the efficiency with which free amino acids regulate peptidase activity and thus the size of the peptide pool is whether the amino acid inhibition is predominantly of the competitive or the non-competitive type. With competitive inhibition the extent of inhibition depends upon the relative concentrations of inhibitor and substrate, whereas with the non-competitive type the inhibition depends only upon the concentration of the inhibitor. T h e type of inhibition of the amino acids which inhibited most strongly (lowest fractional activities) was established by the graphical method of Dixon (DIxoN and WEBn, 1964) in the in vitro assay system. T h e hydrolysis of leucine-p-nitroanilide is inhibited preponderantly, if not completely, non-competitively by L-leucine, L-phenylalanine, and L-methionine, and lysine-p-nitroanilide hydrolysis is inhibited non-competitively by L-leucine and L-phenylalanine. An example of the ' Dixon p l o t ' of the inhibition of L-leucine-p-nitroanilide hydrolysis b y L-phenylalanine is shown in Fig. 3. T h e K I ' s for each amino acid calculated by this method correspond to the fractional activities with both substrates shown in Table 1.
1
(F.A. = ~ .
if inhibition is non-competitive).
1 +-~l This type of inhibition would be a particular advantage in the control of peptide hydrolysis in vivo
where the amount of peptide substrate varies over a five-fold range. It is interesting too that noncompetitive inhibition depends not upon the catalytic site but upon a different site and hence is the result of independent adaptation.
6o
I
/v 8
!
f I I t 0 8 PHENYLALANINff (raM)
I 16
Fig. 3. A 'Dixon plot' of phenylalanine inhibition of leucine-p-nitroanilidehydrolysis in vitro. The reciprocal of the rate ( m o l e p-nitroaniline production per ml assay medium per 4 rain) is plotted against the concentration of the inhibitor at two substrate concentrations (0.1, 0-2 raM). The X-intercepts, calculated from the least squares regression line of the points for each substrate concentration, are --8.00 and --8.03. The inhibition is therefore non-competitive and has a Kl of 8"0. Assuming that the inhibition described by the fractional activities of the peptidases in the presence of each amino acid, is, like the inhibition b y leucine, phenylalanine and methionine, non-competitive, then the relative effectiveness of each amino acid in the hemolymph can be calculated from the fractional activities and the concentrations of each amino acid in the haemolymph (Table 1). (F.A. = ~
1
),
l+~-
T h e calculations indicate that the essential amino acids in the haemolymph would in total depress peptidase activity by more than 15% whereas the biosynthesizable amino acids would depress activity by only about 6%. T h u s despite the much lower concentrations of the essential amino acids in the haemolymph they would depress activity by at least three times as much as would the biosynthesizable amino acids. Furthermore, relatively small changes in the concentrations of the essential amino acids, would have proportionally greater effect in inhibition, as for instance, during the period of intensive feeding (see below).
184
J. I. COLLETT
The accumulation of peptides during the period of intensive feeding DETHmR (1961) has shown that adult male
Phormia feed intensively on protein only for a period of a few days shortly after eclosion. As indicated in Fig. 4, the feeding habit of Calliphora adult males is much the same. One of the signals required for feeding at this time must be the recognition of protein substrate, and this GOLDRICH (1973) has shown probably comes from the response of labial sensory receptors to a group of six essential amino acids which include isoleucine, leucine, methionine, and phenylalanine. Thus it would seem that as flies take in protein foods containing these amino acids, the activity of their peptidases ought to be depressed. In consequence as peptides emerge, probably from turning-over protein, they remain longer as peptides and so the amount of peptide increases. As shown in Fig. 4, such increases do consistently occur and dramatically so. I n contrast, when flies are provided with only sugar and water during the same period, the amounts of peptide decline somewhat further (Fig. 4). If the peptides do come from protein then their rate of accumulation during the period of intensive feeding ought to be a function of the rate of protein turnover and the concentrations of the inhibitory free amino acids. I n adult male insects, there is very little maturation after eclosion, and therefore the rate
3 4.0 O
< 3.0 0 z
< 2.0
"--~t
nl Q
7-
n 1.0 LU O-
Q
0
]
I
I
10
I
I
30 ADULT
AGE
I ,50
(days)
Fig. 4. The amount of peptide in flies with different diets. The measure of peptide is the sum (Ixmole) of the threonine, serine, glutamic acid/glutamine, glycine, alanine, phenylalanine and histidine in the peptides of individual flies. The blackened box marking the third to the seventh day after adult eclosion indicates the normal period of intensive feeding. The solid lines indicate a diet of yeast, sugar, and water. The dotted lines indicate diets of only sugar and water, commencing 1 and 22 days after eclosion. These flies were kept at 20°C to 25°C. Other estimates of the amount of peptide in 10 day old flies deprived of protein from eclosion but not comparable in every way amply confirm the estimates shown in the figure.
of protein turnover approximates the rate of protein synthesis. Very strikingly, estimates of incorporation of lysine and alanine into protein in adult male Phormia made by LEVENBOOKand KRISHNA (1971) correspond to the accumulation of these amino acids in peptides. At 24°C lysine and alanine were incorporated into the total protein at rates of 24 and 7"6 n M per hour respectively in males 5 to 6 days after eclosion. Assuming, conservatively, that 80% of this incorporation also represents protein turnover and that hydrolysis of the resulting peptides is inhibited by 15%, then the accumulation of lysine and alanine in peptide over a 5 day period would be 345 n M and 110 n M respectively. These estimates of possible peptide accumulation from protein turn-over are well within the range of the increases in amounts of lysine and alanine peptides from day 2 to day 10 as shown in Table 1. Thus the dynamics of protein turnover do correspond to the increases in peptide which take place through the period of intensive feeding.
Peptides and survival Once peptides have accumulated, they may play a r61e in providing a reservoir which provides for a fly's amino acid requirements for its entire life. In Fig. 4 it is shown that once peptide accumulates through the period of intensive feeding, it declines very little and only slightly more rapidly than in flies which are still able to feed on protein. Just as peptides turnover, the free amino acids also turnover. LEVENBOOK and KRISHNA (1971) estimated that in older adult male Phormia kept at 24°C nearly the entire pool of free lysine and alanine is replaced within an hour. Undoubtedly, the turnover of these free amino acids is very similar in Calliphora. Clearly, the continual hydrolysis of peptides, at least in part, supplies the pool of free amino acids. Since the amounts of peptide decline only gradually with age, even in the absence of protein food, the peptides must be recycled very efficiently. The value of the peptides may be indicated by the comparison of the amounts of peptides in flies with their respective life expectancies. Fig. 5 shows the survival of flies which never fed on protein after adult eclosion, those which were provided with protein throughout their adult life and those which were allowed protein only through the period of intensive feeding during which the amount of peptide increases very substantially. Adults without protein in their diet, and which never amass substantial amounts of peptide, die young (rJ~IBE, 1966 ; Fig. 5). But flies which are given protein during their first 10 days as adults and have amassed considerable amounts of peptide, survive for as long as flies which have had a diet of protein throughout their adult life. Thus it seems likely that the peptides established during the period of protein feeding are sufficient to supply the demand on the free amino acid pool for the entire lifespan.
Peptidase-mediated storage of amino acids in small peptides
185
but m a y also tend to stabilize the osmotic pressure of the milieu.
100
Aeknowledgements--I am grateful to the Laboratory of Physical Biology of the National Institute of Arthritis and Metabolic Diseases in Bethesda, Maryland, for its hospitality at the start of this work and to the Unit of Invertebrate Chemistry and Physiology of the Agricultural Research Council of Great Britain at the University of Sussex for a generous supply of blowflies. I am also grateful to DAVIDWATSONfor his able running of numerous samples on an amino acid analyser and particularly to Dr G. M. PRICE and Dr T. S. COLLETT for their critical reading of the manuscript.
"6 75
>__ 50 ~c ~0 26
25
50
ADULT AGE
75
100
(days)
REFERENCES Fig. 5. The survival of flies with different diets. One group (O) was fed only sugar and water from eclosion. One group (0) was fed protein, sugar and water from adult eclosion, and the third group ( • ) was fed sugar and water throughout the adult lifespan, but protein only until the 10th day after eclosion. The flies were kept at the 1974 seasonal temperatures in the laboratory which fluctuated within the range of 12 ° to 21°C. Owing to the lower temperatures these flies lived longer than those of Fig. 4.
Reservoirs of essential small molecules H o w e v e r special the regulatory feature of the peptidases m a y be in making amino acids in peptides readily available to adult blowflies, in principle the storage of essential small molecules in a rapidly t u r n i n g - o v e r m u l t i m e r is a powerful homeostatic device, and perhaps for obvious reasons. W h i l e the release of the metabolically active m o n o m e r m a y be simply controlled by the concentrations of the free m o n o m e r , the c o m b i n e d m o n o m e r may be protected f r o m the transformations it is subject to w h e n free, as well as being protected f r o m loss t h r o u g h a m e m b r a n e or t h r o u g h a selective excretory barrier. F u r t h e r m o r e , the contribution of the small molecule to the osmotic pressure of its milieu is lessened in proportion to the size of the multimer. In consequence, the availability of the small molecules m a y not only provide essential substrate w h e n needed
DADD R. H. (1973) Insect nutrition: current developments and metabolic implications .4. Rev. Ent. 18, 381-420. DwrHIER V. G. (1961) Behavioural aspects of protein ingestion by the blowfly Phormia Regina Meigen. Biol. Bull., Woods Hole 121, 456-470. Dixon M. and WEBB E. C. (1964) In Enzymes, second edition, pp. 315-331. Longmans, Green, London. Eenaussz B. and BEADLEL. C. (1936) A technique of transplantation for Drosophila. Am. Nat. 52~ 213225. GOLDRICH N. R. (1973) Behavioural responses of Phormia Regina (Meigen) to labellar stimulation with amino acids.J, gen. Physiol. 61, 74-88. Lmr~raOOK L. and KRISt-~A I. (1971) Effect of ageing on amino acid turnover and rate of protein synthesis in the blowfly Phormia Regina. J. Insect Physiol. 17, 9-12. LEwis W. H. P. and HARaIs H. (1967) Human red ceU peptidases. Nature, Lond. 215, 351-355. MITCHELL H. K. and SIMMONS J. R. (1962) In Amino acid pools (ed. HOLDEN, J. T.), pp. 136-146. Elsevier, Amsterdam. PFLEIDER~R G. (1970) In Methods in Enzymology (Ed. by CoLowicrt, S. P. and KAPLAN, N. O.) pp. 514-521, vol. 19, Academic Press, New York. PRICE G. M. (1965) Nucleic acids in the larva of the blowfly Calliphora erythrocephala (Meigen). J. Insect Physiol. 11, 869-878. Tamn M. A. (1966) The effect of diet on longevity in CaUiphora erythrocephala (Meigen). Exp. Geront. 1, 269-284.
PEPTIDASE-MEDIATED STORAGE OF AMINO ACIDS IN SMALL PEPTIDES J. I. CoLLgr'r School of Biological Sciences, University of Sussex, Brighton, England
(Received 2 August 1975) A b s t r a c t - - A d u l t Calliphora contain a large and heterogenous pool of small peptides, which it is supposed come from protein catabolism. Their composition and rapid turn-over appears to be determined in part by a group of peptidases. The activity of the peptidases is inhibited noncompetitively by free amino acids and most effectively by the essential amino acids which are also those which stimulate feeding on protein. Thus it seems that as flies feed on protein containing the essential amino acids peptide emerging from catabolized protein tends to reanain as peptide until the internal concentrations of the essential amino acids fall. This provides the fly with a physiologically tolerable reservoir of amino acids.
INTRODUCTION ADULT female blowflies, Phormia regina (Meig.) feed heavily on protein during the prolonged period of growth of their o6cytes. A d u l t male Phormia also feed heavily on protein but only for a short period in their early adult life (DET~mR, 1961). T h e work presented here suggests that during the comparably short period of intensive protein ingestion in adult males of a closely related species Calliphora erythrocephala (Meig.) a large reservoir of small peptides is created which is able to satisfy the requirements for free amino acids t h r o u g h o u t the adult life span. T h e b u i l d - u p and the release of amino acids from the peptide reservoir is probably regulated by the concentrations of the same free amino acids which stimulate the labial sensory receptors on the proboscis and evoke feeding in Phormia (GOLDRICrI, 1973). MATERIALS A N D M E T H O D S
The flies All flies used in these experiments were males reared in a stock of Calliphora erythroeephala, which has been maintained through mass matings for many years (PRICE, 1965). Except when changes in diet were explicity a part of an experiment, adult flies were maintained on a diet of dried yeast, sugar and water and kept at room temperature.
Estimation of the free amino acids in whole flies and haemolymph and of the amino acid composition of peptides Clear haemolymph was withdrawn from living adults in capillary tubes extended into the ,thorax through the neck musculature. A total of 10 to 20/tl of haemolymph from 11 to 20 adults 5 to 15 days after their adult 179
eclosion was combined in 1 ml of 10% tricMoroacetie acid (TCA) containing a standard amount (0-160 gmole) of nor-L-leucine. Whole flies were homogenized individually in 1 ml of 10% TCA, also containing nor-Lleucine, in a ground-glass homogenizer. Both preparations were then filtered through a Millipore filter, HA 0"45. After adjusting the pH of the samples to 2"0, the amino acids of each sample were then separated and the amounts measured on an amino acid analyser (Locarte Limited, London). To estimate the amino acid composition of the small peptides, part of the filtrate was hydrolysed in 6 N HC1 for 24 hr at 110°O, and then, after removal of the acid, also separated on the amino acid analyser. The difference in the amounts of each amino acid in the hydrolysed and unhydrolysed fractions, adjusted by using the amount of nor-L-leucine present in each as a means of comparing different fractions of a sample and of estimating sample loss, provided a measure of the amount of amino acid in the small peptides. The samples were kept in T C A for a sufficiently long time before separation to deaminate asparagine and glutamine to their respective amino acids. Methionine, cysteine, and tryptophan were not measured. To determine the approximate sizes of the peptides, unhydrolyzed samples of whole flies similarly prepared in T C A were eluted from a 0.9×45 cm column of Sephadex G-10 in 0"1 N sodium chloride. Dextran 2000 was used as a measure of the void volume (Ire). To study the numbers and dynamics of the peptides, C 14-giycine was injected into adult flies and subsequently monitored in peptides in the following way. A number 30 guage needle mounted on an Agla syringe was inserted beneath the scutellum into the flight muscle cavity and 10gl of a solution of glycine-C 14 in insect ringer (EPHmrSsi and B~AVLE, 1936) Was delivered into the haemolymph. After 12 and 24 hr incubation, flies were homogenized and prepared as usual for separation on the amino acid analyser. A Tracerlab Dual Ratemeter Spectrometer Scintillation Counter with a flow cell containing plastic scintillant (NE 102A supplied by Nuclear Enterprises Limited, Edinburgh) monitored the
180
J. I. COLLETT
radioactivity in each peak in the effluent of the column of the amino acid analyser.
L-lysine-p-nitroanilide hydrolysis by L-leucine and Lphenylalanine.
Electrophoretic peptidases
The relationship of diet, amount of peptide and survival
separation
and
identification
of
Haemolymph withdrawn from adults (as described in the previous section) and supematants of whole flies homogenized in 0-2 M Tris-maleate buffer, pH 7"4 were separated in the same buffer in vertical gel slabs of 10% acrylamide with a potential of 31.2 V per cm for 2~[ hr at 2°C. T h e peptidases were identified by a modification of a technique developed by LEwis and HAaam (1967) in which a series of reactions take place at the interface of the acrylamide gel containing the peptidases and a 1% agar gel overlay containing a mixture of substrate, enzymes and final reducing agent. L-leueyl-L-alanine was the substrate for the peptidases, and at the positions of hydrolysis peroxide is formed in the presence of an L-amino acid oxidase from snake venom (Sigma Chem. Co. Limited) and then 3-amino-9-ethyl-carbazole becomes coloured as it is oxidized by peroxide in the presence of horse-radish peroxidase (Sigma Chem. Co. Limited). I n vivo and in vitro assays of peptidase activity Etherized adult males of 8 to 12 days of age were injected with 10/A of insect ringer (EvHaussI and BEADLE, 1936) containing peptides in either 0.01 or 0.02 M concentrations. At various intervals within a 3 hr period after injection individual flies were homogenized in T C A as usual and separated on an amino acid analyser as previously described. T h e amounts of each peptide remaining in flies at various intervals after injection provided estimates of the rates of in vivo hydrolysis. T h e hydrolyses of the L-forms of glycylleueine, leueylglycine, alanylproline, lysylphenylalanine, and prolylleucine, as well as of glycylglycylglycine and glycylglycine were followed. Peptidase activity was also assayed in vitro using a technique (PFLI~IDEREa, 1970) in which amino acid p-nitroanilides are used as substrate and the release of p-nitroaniline is followed spectrophotometricaUy. Adult males were homogenized in 0"06 M phosphate buffer, p H 7"6 and containing MgCla to 1 raM. T h e homogenates were centrifuged at 3500 g at 0°C for 15 min. T h e solution in which enzyme activity was measured contained enzyme diluted by a factor of at least 250 times compared with the in vivo concentration and substrate concentration of 0"05 to 0"30 raM. Activity was followed at 405 n m in a Zeiss P M Q II speetrophotometer with a 6-place automatic cell changer at 30°C. Comparative rates of hydrolysis of glycine-pnitroanilide (Nutritional Biochemical Corps.), L-alaninep-nitroanilide-HC1 (Cyclo Chemicals), L-leucine-pnitroanilide (British Drug House) and L-lysine-pnitroanilide-HBrs (Nutritional Biochemical Corps,) were measured. T h e inhibition of peptidase activity by different amino acids was compared by measuring the hydrolysis of the leueine- and lysine-p-nitroanilides in the presence (73 m M ) of amino acids. T h e type of inhibition and the KI of the amino acids which inhibit these substrates most strongly were determined by the graphical method of DIxoN (1964). ' Dixon plots' were constructed of the inhibition of L-leucine-p-nitroanilide hydrolysis by L-phenylalanine, L-leucine, and L-methionine and of
The longevities of adult males on different diets were compared with a view to relating lifespan to the amounts of small peptide in flies with each diet. On the day of adult eclosion from a single lay of eggs, 50 males were put into each of three cages which differed only in the diet provided. In one cage sugar and water were provided. In the second cage, sugar and water were provided throughout the lifespan hut yeast was provided for only the first I0 days. In the third cage, yeast, sugar and water were provided throughout the lifespan. T h e yeast and sugar were in separate containers and the numbers feeding on each were recorded throughout the lifespan. T h e temperatures at which the cages were kept varied within the range of 12°C to 25°C and on average were higher toward the end of the lifespans. T h e amounts of peptide in flies on each feeding regime were estimated in flies kept in similar conditions. RESULTS
AND
DISCUSSION
The peptides A n acceptable criterion of identification of a c o m p o u n d as a p e p t i d e is t h a t it s h o u l d stain w i t h n i n h y d r i n a n d t h a t o n hydrolysis it s h o u l d p r o d u c e free a m i n o acids. W h e n w h o l e flies are h o m o g e n i z e d in 1 0 % T C A w h i c h precipitates protein, a n d t h e s u p e r n a t a n t is p a s s e d t h r o u g h a M i l l i p o r e filter to r e m o v e even v e r y fine precipitate, t h e s u p e r n a t a n t c o n t a i n s free a m i n o acid as well as m a t e r i a l w h i c h stains w i t h n i n h y d r i n , a n d hydrolysis p r o d u c e s m o r e free a m i n o acid. S e p a r a t i o n s of t h e u n h y d r o l y s e d T C A s u p e r n a t a n t s o n a n a m i n o acid analyser show n i n h y d r i n - s t a i n i n g peaks w h i c h d i s a p p e a r o n h y d r o lysis. W i t h hydrolysis t h e free a m i n o acid peaks increase a n d t h e larger t h e size of t h e p r o b a b l e p e p t i d e peaks in t h e u n h y d r o l y s e d s u p e r n a t a n t t h e greater t h e increase of t h e a m i n o acid peaks in t h e h y d r o l y s e d p a r t of t h e s u p e r n a t a n t . T h u s t h e h y d r o l y s a b l e m a t e r i a l in t h e s u p e r n a t a n t is almost certain to b e peptide. T h e p e p t i d e peaks in samples of flies separated o n t h e a m i n o acid analyser a p p e a r in positions w h e r e di- a n d t r i - p e p t i d e s are eluted (CoLLETT, u n p u b . ) . T h e s a m p l e peaks are m o s t likely also di- a n d tripeptides. F u r t h e r m o r e , e l u t i o n of t h e T C A s u p e r n a t a n t s of h o m o g e n a t e s of whole flies f r o m a S e p h a dex G - 1 0 c o l u m n indicates t h a t m o s t if n o t all of t h e p e p t i d e is small. F r a c t i o n s collected f r o m V0 1.2 to "1"9 c o n t a i n e d t h e same i n c r e m e n t of a m i n o acid after hydrolysis as t h e same T C A s u p e r n a t a n t w h i c h h a d not been separated on the Sephadex column. Thus t h e m a x i m u m size of t h e p e p t i d e s is unlikely to b e m o r e t h a n five residues. T h e a m i n o acid c o m p o s t i o n of t h e p e p t i d e s f r o m t h r e e different males, s h o w n in T a b l e 1, illustrates several o t h e r features of t h e peptides. (1) W i t h t h e possible e x c e p t i o n of t h o s e n o t m e a s u r e d ( m e t h i o nine, t r y p t o p h a n , a n d cysteine), all p r o t e i n a m i n o
181
Peptidase-mediated storage of amino acids in small peptides Table 1. Peptide amino acid, free amino acid and the inhibition of peptidase activity Free Amino' Acid Oom~ositlon
A.A. composition of Peptides
A.A. Inhibition of Peptidase Activity in vi t re,, prac%ional Activity:
2 days old ~Moles/fly*: aspartic acid asparagine threonine + serine glutamic acid glutamine
]0.127 O. 052 0.062 3 ] Oo 235
2to line glycine alanine valine + isoleucine # leucine + tyro sine +
O. 069 0.524 0.082 0.008 0.01~ 0.020
phenylalanine + his tidine + lymine + ar~inine + methionine Total:
i0 days oI~
Total Free :C.oncemtration in 55 A~dr~ Acid of HAemolymph of i0 " days old 20 ds~ old fly* day old flies ~Moles mMolar
]0.519
]0.288
] 0.217
5.4
99
o. 3 ~ 9.459
O. 150 0.261
O. 022 0.220
O. 6 2?2
89 97
]0.402 ]0.530 0.501 0.547
4.0 7.5
"95 10o
i. 9 4.6
99 89
]0.609 0.554 I. 355 0.690
O. 896 0.575
O. 187 0.237
hydrolysis of L-lysinenitroanilide ~HBr) (%) 2
Enzyme + 73mNolar A& enzJme hydrolysis of L~ le ucine-p-ni troanilide
90 71 79 75 I00 95 72 4O 24
0.215
0.008
0.025
0.4
57
o.15o o.2y5 >0.006
0.060" 0.095 >0.Ii
0.050 o.oss 0.016
0.5 0.5 0.4
50 5s
0.015 0o071 0.036 O. 008
O.i17
0.o11
0.2
lO
0.161 0.054 0.274
2.9 1.4 1.8
53
8 48
53
43
0.105
0.063 o.121 0.ii0 0.069
42
19
1.324
5.525
3.288
2.387
0.185
17
*Estimates from single flies weighing 52.6 +0.6 mg. Ages given in days after emergence. tEssential amino acids (DAOD, 1973). acids are present in the peptides, but in widely different amounts. Glycine and glutamic acid and/ or glutamine together account for about 50% of the peptide amino acid, while the amino acids which are required in the diet (essential) are together only a small proportion of the total peptide amino acid. (2) In contrast to the relative invariability of the free amino acid pool from fly to fly (CoLLET% in prep.), the total amount of peptide varies over a five-fold range from fly to fly. (3) But however large or small the peptide pool, the relative amounts of each constituent amino acid remain approximately the same. T h e number of kinds of peptides making up this peptide component which varies in size but not in composition is more difficult to ascertain, but several pieces of evidence suggest that their n u m b e r is considerable. Of these, the most convincing comes from in vivo incorporation of glycine-C14 into material which elutes intermittently throughout an amino acid analyser separation. Flies injected with C a4-glyeine and subsequently homogenized in T C A and separated on an amino acid analyser in which the radioactivity of the effluent is monitored, have many more peaks containing label than are visible from the less sensitive ninhydrin staining. The peaks disappear with hydrolysis and are therefore probably peptide. Some peaks appear before glyeine itself, indicating that they either contain 4 or 5 residues or that they contain acidic amino acids as well as glyeine. Many peaks appear after glycine
with the neutral and basic amino acids indicating a smaller size and the presence of neutral and basic amino acids with glycine. Some of these peaks are also broader than might be expected of a homogeneous molecular species. The number of peaks labelled with glycine-C14 alone suggests that there are in fact many species of peptide, each present in amounts which are usually too small to detect as peptides, but which together account for a considerable amount of amino acid. These peptides in Calliphora adults may be similar to those described by MITC~LL and SIMMONS (1962) in Drosophila melanogaster larvae which they estimated to number about 600. Comparison of the amount of label in the peptide peaks from flies injected with label 12 to 24 hr before sample preparation also indicates that the peptides, like the free amino acid pool, undergo rapid turnover. While some of the labelled glyeine was still present among the free amino acids and so able to generate more peptide, the label in the glycine containing peptides declined by 60% through this 12 hr period. The most likely source of these peptides is turning-over protein and for the following reasons. The peptides which n u m b e r in the hundreds contain only the protein amino acids and while the total amounts vary to sometimes as much as 10% of the total protein of the fly, the proportion of each amino acid remains constant. Therefore, the composition of the peptides is what one would expect from
182
J. I. COLLETT
turning-over protein. Furthermore, the rate of peptide accumulation early in adult life after emergence corresponds with the rate of protein synthesis (and turnover) (see below). And in any case, on energetic grounds alone, it would be remarkable if these peptides were synthesized de novo.
The pep tidases Three different techniques have been used to describe the flies' peptidases and their activities. The electrophoresis of haemolymph and homogenates of whole flies provides an estimate of the number of peptidases and their location. T h e rate of disappearance of large amounts of peptides injected into the haemolymph of flies is a reliable gauge of in vivo activity and shows that the specificity of the peptidases corresponds to the amino acid composition of the peptides. In vitro assays of peptidase activity allow quantitative treatment of features of the peptidases which may affect their activity in vivo. Most significant is that the peptidase activity appears to be non-competitively inhibited by essential amino acids. origin
l
m
m
m
m
4.
Fig. 1. A diagrammatic comparison of electrophoretic separations of homogenates of whole flies (left) and of haemolymph (right). The thickness of the bands indicates the intensity of the activity hydrolysing L-leucyl-L-alanine.
Electrophoretic separation of peptides T h e activity of the peptidases which hydrolyse L-leucine-L-alanine is clearly visible in electrophoretic separations of fly material in 10% acrylamide gel. Six distinct molecular species with this specificity are consistently found in the homogenates of whole flies. As shown diagrammatically in Fig. 1, five of these peptidases are also present in haemolymph alone. T h u s clearly a substantial part of the peptidase activity is related to metabolism other than the digestion and assimilation of protein in the gut. In vivo hydrolysis of known peptides If the specificities of the peptidases available to the haemolymph are responsible in part for the composition of the peptides, then these specifieities ought to be reflected in the relative rates at which pcptides of known amino acid composition are
loo I~.~----_..~ eo
~.o
~o
gty~,oy ~oo gly glygly
80
•
gly leu
50 a
alapro
10
/I
~Apheval pheval
51
• leugly
x x
20
140
proteu
1~O
50
MINUTES
Fig. 2. The hydrolysis of natural peptides in vivo. Solutions containing peptides were injected into the haemolymph of the flight muscle cavity and the amount (nMoles) of unhydrolysed peptide in individual flies was measured at various intervals thereafter. hydrolysed in the haemolymph. Accordingly, Fig. 2 shows the relative rates of hydrolysis of eight peptides of the L-form after injection into the haemolymph of the flight muscle cavity. These rates range widely and in a way which corresponds to the amino acid composition of the fly's peptides. Of the eight peptides injected those containing amino acids which are present in the fly's peptides in relatively small amounts were hydrolysed more rapidly than those containing amino acids which are present in abundance in the fly's peptides. For example, glygly was hydrolysed more slowly than alapro and alapro was hydrolysed more slowly than pheleu. Thus however the peptides are generated, their amino acid composition appears to be in part the result of the specificities of the peptidases. In vitro hydrolysis of peptide analogues T h e hydrolyses of 4 amino acid p-nitroanilides by the peptidases in vitro showed specificity similar to the in vivo hydrolyses of natural peptides. Typical relative rates of hydrolysis of the four substrates tested are: nMoles p-nitroaniline/ml assay medium/minute at 30°C glycine-p-nitroanilide L-alanine-p-nitroanilide, HC1 L-leucine-p-nitroanilide L-lysine-p-nitroanilide,(HBr)2
1.43 1'95 2.70 5"53
This striking similarity of the relative rates of in vitro hydrolysis of peptide analogues and in vivo hydrolysis of natural peptides permits meaningful quantitative analysis of the kinetics of the peptidase activity in vitro.
183
Peptidase-mediated storage of amino acids in small peptides
The inhibition of peptidase activity in vitro by amino acids While the specificities of the active site of the peptidases explain at least in part the composition of the peptides, the inhibition of the peptidases by free amino acids which is described here seems to explain the wide variation in total amounts of peptide. Furthermore, the specificity, the type and the strength of the inhibition together suggest how these peptides, in varying so widely in amounts, may be used as a store of free amino acid. A comparison of in vitro peptidase activity in the presence of various amino acids ('fractional activities' in Table 1), shows not only that amino acids do inhibit peptidase activity but that some amino acids are very effective inhibitors. For example, in the presence of 73 m M L-phenylalanine only 8% as much leucine-p-nitroanilide was hydrolysed as when no amino acids were present. F r o m Table 1 it is clear that with both leucine- and lysine-p-nitroanilide the amino acids with appreciable effect are those which are required in the diet (essential). Moreover, the greatest effect comes from the largest hydrophobic amino acids, leucine, isoleucine, phenylalanine, and methionine. Not only are these essential amino acids present in relatively very low concentrations in the fly, b u t they are also the amino acids which stimulate feeding on protein in Phormia (GoLDmCH, 1973). A further feature which would affect the efficiency with which free amino acids regulate peptidase activity and thus the size of the peptide pool is whether the amino acid inhibition is predominantly of the competitive or the non-competitive type. With competitive inhibition the extent of inhibition depends upon the relative concentrations of inhibitor and substrate, whereas with the non-competitive type the inhibition depends only upon the concentration of the inhibitor. T h e type of inhibition of the amino acids which inhibited most strongly (lowest fractional activities) was established by the graphical method of Dixon (DIxoN and WEBn, 1964) in the in vitro assay system. T h e hydrolysis of leucine-p-nitroanilide is inhibited preponderantly, if not completely, non-competitively by L-leucine, L-phenylalanine, and L-methionine, and lysine-p-nitroanilide hydrolysis is inhibited non-competitively by L-leucine and L-phenylalanine. An example of the ' Dixon p l o t ' of the inhibition of L-leucine-p-nitroanilide hydrolysis b y L-phenylalanine is shown in Fig. 3. T h e K I ' s for each amino acid calculated by this method correspond to the fractional activities with both substrates shown in Table 1.
1
(F.A. = ~ .
if inhibition is non-competitive).
1 +-~l This type of inhibition would be a particular advantage in the control of peptide hydrolysis in vivo
where the amount of peptide substrate varies over a five-fold range. It is interesting too that noncompetitive inhibition depends not upon the catalytic site but upon a different site and hence is the result of independent adaptation.
6o
I
/v 8
!
f I I t 0 8 PHENYLALANINff (raM)
I 16
Fig. 3. A 'Dixon plot' of phenylalanine inhibition of leucine-p-nitroanilidehydrolysis in vitro. The reciprocal of the rate ( m o l e p-nitroaniline production per ml assay medium per 4 rain) is plotted against the concentration of the inhibitor at two substrate concentrations (0.1, 0-2 raM). The X-intercepts, calculated from the least squares regression line of the points for each substrate concentration, are --8.00 and --8.03. The inhibition is therefore non-competitive and has a Kl of 8"0. Assuming that the inhibition described by the fractional activities of the peptidases in the presence of each amino acid, is, like the inhibition b y leucine, phenylalanine and methionine, non-competitive, then the relative effectiveness of each amino acid in the hemolymph can be calculated from the fractional activities and the concentrations of each amino acid in the haemolymph (Table 1). (F.A. = ~
1
),
l+~-
T h e calculations indicate that the essential amino acids in the haemolymph would in total depress peptidase activity by more than 15% whereas the biosynthesizable amino acids would depress activity by only about 6%. T h u s despite the much lower concentrations of the essential amino acids in the haemolymph they would depress activity by at least three times as much as would the biosynthesizable amino acids. Furthermore, relatively small changes in the concentrations of the essential amino acids, would have proportionally greater effect in inhibition, as for instance, during the period of intensive feeding (see below).
184
J. I. COLLETT
The accumulation of peptides during the period of intensive feeding DETHmR (1961) has shown that adult male
Phormia feed intensively on protein only for a period of a few days shortly after eclosion. As indicated in Fig. 4, the feeding habit of Calliphora adult males is much the same. One of the signals required for feeding at this time must be the recognition of protein substrate, and this GOLDRICH (1973) has shown probably comes from the response of labial sensory receptors to a group of six essential amino acids which include isoleucine, leucine, methionine, and phenylalanine. Thus it would seem that as flies take in protein foods containing these amino acids, the activity of their peptidases ought to be depressed. In consequence as peptides emerge, probably from turning-over protein, they remain longer as peptides and so the amount of peptide increases. As shown in Fig. 4, such increases do consistently occur and dramatically so. I n contrast, when flies are provided with only sugar and water during the same period, the amounts of peptide decline somewhat further (Fig. 4). If the peptides do come from protein then their rate of accumulation during the period of intensive feeding ought to be a function of the rate of protein turnover and the concentrations of the inhibitory free amino acids. I n adult male insects, there is very little maturation after eclosion, and therefore the rate
3 4.0 O
< 3.0 0 z
< 2.0
"--~t
nl Q
7-
n 1.0 LU O-
Q
0
]
I
I
10
I
I
30 ADULT
AGE
I ,50
(days)
Fig. 4. The amount of peptide in flies with different diets. The measure of peptide is the sum (Ixmole) of the threonine, serine, glutamic acid/glutamine, glycine, alanine, phenylalanine and histidine in the peptides of individual flies. The blackened box marking the third to the seventh day after adult eclosion indicates the normal period of intensive feeding. The solid lines indicate a diet of yeast, sugar, and water. The dotted lines indicate diets of only sugar and water, commencing 1 and 22 days after eclosion. These flies were kept at 20°C to 25°C. Other estimates of the amount of peptide in 10 day old flies deprived of protein from eclosion but not comparable in every way amply confirm the estimates shown in the figure.
of protein turnover approximates the rate of protein synthesis. Very strikingly, estimates of incorporation of lysine and alanine into protein in adult male Phormia made by LEVENBOOKand KRISHNA (1971) correspond to the accumulation of these amino acids in peptides. At 24°C lysine and alanine were incorporated into the total protein at rates of 24 and 7"6 n M per hour respectively in males 5 to 6 days after eclosion. Assuming, conservatively, that 80% of this incorporation also represents protein turnover and that hydrolysis of the resulting peptides is inhibited by 15%, then the accumulation of lysine and alanine in peptide over a 5 day period would be 345 n M and 110 n M respectively. These estimates of possible peptide accumulation from protein turn-over are well within the range of the increases in amounts of lysine and alanine peptides from day 2 to day 10 as shown in Table 1. Thus the dynamics of protein turnover do correspond to the increases in peptide which take place through the period of intensive feeding.
Peptides and survival Once peptides have accumulated, they may play a r61e in providing a reservoir which provides for a fly's amino acid requirements for its entire life. In Fig. 4 it is shown that once peptide accumulates through the period of intensive feeding, it declines very little and only slightly more rapidly than in flies which are still able to feed on protein. Just as peptides turnover, the free amino acids also turnover. LEVENBOOK and KRISHNA (1971) estimated that in older adult male Phormia kept at 24°C nearly the entire pool of free lysine and alanine is replaced within an hour. Undoubtedly, the turnover of these free amino acids is very similar in Calliphora. Clearly, the continual hydrolysis of peptides, at least in part, supplies the pool of free amino acids. Since the amounts of peptide decline only gradually with age, even in the absence of protein food, the peptides must be recycled very efficiently. The value of the peptides may be indicated by the comparison of the amounts of peptides in flies with their respective life expectancies. Fig. 5 shows the survival of flies which never fed on protein after adult eclosion, those which were provided with protein throughout their adult life and those which were allowed protein only through the period of intensive feeding during which the amount of peptide increases very substantially. Adults without protein in their diet, and which never amass substantial amounts of peptide, die young (rJ~IBE, 1966 ; Fig. 5). But flies which are given protein during their first 10 days as adults and have amassed considerable amounts of peptide, survive for as long as flies which have had a diet of protein throughout their adult life. Thus it seems likely that the peptides established during the period of protein feeding are sufficient to supply the demand on the free amino acid pool for the entire lifespan.
Peptidase-mediated storage of amino acids in small peptides
185
but m a y also tend to stabilize the osmotic pressure of the milieu.
100
Aeknowledgements--I am grateful to the Laboratory of Physical Biology of the National Institute of Arthritis and Metabolic Diseases in Bethesda, Maryland, for its hospitality at the start of this work and to the Unit of Invertebrate Chemistry and Physiology of the Agricultural Research Council of Great Britain at the University of Sussex for a generous supply of blowflies. I am also grateful to DAVIDWATSONfor his able running of numerous samples on an amino acid analyser and particularly to Dr G. M. PRICE and Dr T. S. COLLETT for their critical reading of the manuscript.
"6 75
>__ 50 ~c ~0 26
25
50
ADULT AGE
75
100
(days)
REFERENCES Fig. 5. The survival of flies with different diets. One group (O) was fed only sugar and water from eclosion. One group (0) was fed protein, sugar and water from adult eclosion, and the third group ( • ) was fed sugar and water throughout the adult lifespan, but protein only until the 10th day after eclosion. The flies were kept at the 1974 seasonal temperatures in the laboratory which fluctuated within the range of 12 ° to 21°C. Owing to the lower temperatures these flies lived longer than those of Fig. 4.
Reservoirs of essential small molecules H o w e v e r special the regulatory feature of the peptidases m a y be in making amino acids in peptides readily available to adult blowflies, in principle the storage of essential small molecules in a rapidly t u r n i n g - o v e r m u l t i m e r is a powerful homeostatic device, and perhaps for obvious reasons. W h i l e the release of the metabolically active m o n o m e r m a y be simply controlled by the concentrations of the free m o n o m e r , the c o m b i n e d m o n o m e r may be protected f r o m the transformations it is subject to w h e n free, as well as being protected f r o m loss t h r o u g h a m e m b r a n e or t h r o u g h a selective excretory barrier. F u r t h e r m o r e , the contribution of the small molecule to the osmotic pressure of its milieu is lessened in proportion to the size of the multimer. In consequence, the availability of the small molecules m a y not only provide essential substrate w h e n needed
DADD R. H. (1973) Insect nutrition: current developments and metabolic implications .4. Rev. Ent. 18, 381-420. DwrHIER V. G. (1961) Behavioural aspects of protein ingestion by the blowfly Phormia Regina Meigen. Biol. Bull., Woods Hole 121, 456-470. Dixon M. and WEBB E. C. (1964) In Enzymes, second edition, pp. 315-331. Longmans, Green, London. Eenaussz B. and BEADLEL. C. (1936) A technique of transplantation for Drosophila. Am. Nat. 52~ 213225. GOLDRICH N. R. (1973) Behavioural responses of Phormia Regina (Meigen) to labellar stimulation with amino acids.J, gen. Physiol. 61, 74-88. Lmr~raOOK L. and KRISt-~A I. (1971) Effect of ageing on amino acid turnover and rate of protein synthesis in the blowfly Phormia Regina. J. Insect Physiol. 17, 9-12. LEwis W. H. P. and HARaIs H. (1967) Human red ceU peptidases. Nature, Lond. 215, 351-355. MITCHELL H. K. and SIMMONS J. R. (1962) In Amino acid pools (ed. HOLDEN, J. T.), pp. 136-146. Elsevier, Amsterdam. PFLEIDER~R G. (1970) In Methods in Enzymology (Ed. by CoLowicrt, S. P. and KAPLAN, N. O.) pp. 514-521, vol. 19, Academic Press, New York. PRICE G. M. (1965) Nucleic acids in the larva of the blowfly Calliphora erythrocephala (Meigen). J. Insect Physiol. 11, 869-878. Tamn M. A. (1966) The effect of diet on longevity in CaUiphora erythrocephala (Meigen). Exp. Geront. 1, 269-284.