- Email: [email protected]
Degradation of adipokinetic hormones
Insect Biochem. Molec. BioL Vol. 22, No. 7, pp. 657~63, 1992 Printed in Great Britain. All fights reserved
0965-1748/92 $5.00+ 0.00 Copyright © 1992PergamonPress Ltd
D E G R A D A T I O N OF ADIPOKINETIC HORMONES KARL J. SIEGERTand WILLIAM MORDUE Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scotland (Received I0 September 1991; revised and accepted 21 May 1992)
Abstraet--Homogenates from various tissues of the desert locust, Schistocerca gregaria, produced identical patterns of breakdown products when incubated with the neuropeptide adipokinetic hormone I (AKH I). Comparison of disappearance of AKH I from the incubation medium of semi-isolated and isolated Malpighian tubules (MTs) and fat body pieces indicates that both tissues contain substantial capacities to take up and/or breakdown peptide. While the MTs may remove peptide circulating in the haemolymph, the fat body may metabolize peptide which binds to cell surface receptors. Locusta migratoria and Periplaneta americana MTs contain the same complement of degrading enzymes since they produced identical primary products. The initial proteolytic attack on AKH I took place through a postproline cleaving enzyme (PPCE, residue 6). The use of the synthetic substrate benzyloxycarbonyl-Gly-Pro7-amido-4-methyl-coumarinconfirmed the presence of PPCE. Adipokinetic hormone II from S. gregaria was cleaved between phenylalanine and serine (residues 4 and 5) indicating the action of a chymotrypsinlike endopeptidase. The synthetic substrates glutaryl-Phe-7AMC and N-succinyl-Ala-Phe-Lys-7AMC were not cleaved by MT homogenates; however, such homogenates degraded a tyrosine-containing substrate, N-benzoyl-Tyr ethyl ester. Chymotrypsin from bovine pancreas cleaved all the above substrates. Key Word Index: AKH; Malpighian tubules; enzymatic breakdown of peptides
INTRODUCTION Homogenates of the Malpighian tubules (MTs) from the desert locust, Schistocerca gregaria, have been shown to cleave the adipokinetic hormone I ( A K H I; pGiu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2); using reversed-phase high-performance liquid chromatography (RP-HPLC) three breakdown products were identified ( A K H I-1: pGlu-Leu-Asn-Phe-ThrPro, A K H 1-2: Trp and A K H I-3: Trp-Gly-ThrNH2; Siegert and Mordue, 1987). B a u m a n n and Penzlin (1987) demonstrated with an in vitro system that in the American cockroach, Periplaneta americana, only the MTs contain the ability to remove neurohormone D ( = myotropic peptide I, MI, periplanetin CC-I or hyperglycaemic hormone I; pGlu-Val-Asn-Phe-SerPro-Asn-TrpNH2) from the incubation mixture and/ or to destroy this peptide. Other tissues such as muscle, fat body, haemolymph, etc. were also tested but negative results were obtained. By contrast, Skinner et al. (1987) reported that homogenates of P. americana fat body cleaved periplanetin CC-II. Because of their physiological role in transporting water and ions and the large area exposed to the haemolymph, the MTs are indeed a very likely candidate for the removal of peptide hormones from the haemolymph. On the other hand, it is known from vertebrate studies that peptide hormones not only bind to the hormone receptors on the cell surface but are also internalized (Terris and Steiner, 1975; Suzuki and Kono, 1979). If this were also the case in insects then peripheral tissues such as the fat body must have the enzyme complement to breakdown A K H peptides. To this end, the breakdown of A K H I and A K H II-S (pGluLeu-Asn-Phe-Ser-Thr-Gly-TrpNH2) from S. gregaria by tissue homogenates was investigated.
MATERIALS
AND METHODS
Animals S. gregaria and Locusta migratoria were kept as described by Morgan and Mordue (1983). P. americana were purchased from Bioserv Ltd and kept as described by Siegert et al. (1986). Chemicals The chemicals used in the present study were from the sources listed by Siegert and Mordue (1987). Additionally, benzyloxycarbonyl- Gly - Pro - 7 - amido - 4 - methylcoumarin (7AMC) (ZGPA) was from Cambridge Research Biochemicals and tryptophan, tryptophanamide, glutaryl-Phe7AMC (GFA), N-succinyl-Ala-Phe-Lys-7AMC (SAFKA), N-benzoyl-Tyr ethyl ester (BTEE) and ~-chymotrypsin (bovine pancreas) were purchased from Sigma. Preparation and incubation of homogenates from different tissues with peptides, substrates etc. Tissue homogenates were prepared as outlined by Siegert and Mordue (1987) for MTs. All incubations were performed at 30°C. Haemolymph (50 #1) was mixed with 400/11 buffer (90 mM KH2PO4, 20 mM Na2EDTA, pH 8), vortexed and then centrifuged for 10 min at 12,000g. An aliquot of the supernatant (400 #1) was then mixed with the peptide solution (1-5/zg/400/zl) and samples withdrawn at t = 0 and 60 min. Semi-isolated M T preparations The method described by Morgan and Mordue (1984) was adopted using the same insect saline (I 15mM NaCI, 20 mM KCI, 2 mM CaClz, 2 mM MgC12, 10 mM glucose, 22 mM NaHCO3, 6 mM NaH2PO 4, pH 7.2). Generally, the preparation was incubated in 450/~1 saline plus additives and 100/zl removed at t = 0 and 60 min, 50/zl acetonitrile and 850/~1 0.1% trifluoroacetic acid (TFA) added, mixed, centrifuged (5 min, 12,000g) and injected onto the RP-HPLC.
657
658
KARL J. SIEGERTand WILLIAMMORDUE 1.5
The in vitro system The alimentary canal with the attached MTs was placed into simple insect saline (128 mM NaCI, 5 mM KCI) and then the MTs gently removed. Fat body pieces and MTs were briefly blotted on filter paper and added to the incubation tubes (Eppendorf) which contained 450/zl saline (see above) plus peptide. Aliquots (100/d) were removed at t = 0, 15 and 60 min and processed as outlined above.
Control
E c
RP-HPLC The equipment described by Siegert et al. (1985) was used. Separation of peptides was performed on an Aquapore RP-300 column (4.6 × 250 turn) using 0.1% TFA (pump A) and acetonitrile (pump B) as solvents at a flow rate of 1 ml/min. Two different gradients were employed. Gradient 1 started at 0% B and increased at 2% B/rain and allowed the analysis of AKH breakdown products (Siegert and Mordue, 1987). Gradient 2 started at 7% B with a 0.3% B/rain increase and allowed the separation of tryptophan and tryptophanamide. Absorbance was monitored with a Pye Unicam LC-u.v. detector (206 nm) and peak areas were calculated by a Shimadzu C-R1 B chromatopak integrator. Peak areas were converted to pmol peptide as described by Siegert and Mordue (1986). In some experiments the absorbance was simultaneously measured at 280 nm with a Gilson dual wavelength spectrophotometer (model 116).
1.0
8 0 z < en
0 m <
0.5
60mln
0,0
Amino acid analysis The analysis was carried out on an Applied Biosystems 420A Amino Acid Analyzer with automatic hydrolysis and derivatization. The PTC amino acids generated were identified on-line, using a 130A separation system employing a C-18 reversed-phase narrow bore cartridge. The system was calibrated using 25 pmol fl-lactoglobulin standard (A and B variants).
. 0
.
AKHI
--4_jt .
5
. 10
. . 15 20
25
RETENTION TIME [min] Fig. 1. Breakdown of AKH I by a homogenate from S. gregaria fat body, Brain, dorsal heart and midgut also cleaved AKH I producing the same breakdown products. Gradient 1 was used. Retention times: AKH 1 24.2 rain; 1, AKH I-1 23.2 min; 2, AKH I-2 15.9min.
RESULTS
1.5
Control
Breakdown o f A K H [ by homogenates from different S. gregaria tissues The ability of different locust tissues to breakdown the neuropeptide A K H I was studied using homogenates. All tissues tested (brain, heart, midgut and fat body) were found to cleave A K H I in considerable amounts comparable to the result shown for fat body (Fig. 1). H a e m o l y m p h preparations, however, failed to digest A K H I (Fig. 2). It is important to note that none of the peak areas in Fig. 2 changed during the 60 min incubation period. A control experiment with tryptophanamide as a substrate for leucine aminopeptidase showed that the haemolymph preparation contained active proteolytic enzymes (Fig. 3).
Breakdown o f A K H I by M T homogenates from L. migratoria and P. americana Locusta migratoria MTs also contain enzymes which cleave A K H I as do MTs from P. americana (Fig. 4). Three sets of MTs from P. americana had to be used to produce a substantial breakdown of A K H [. Unlike the situation in L. migratoria, peaks corresponding to breakdown products A K H I-1 and A K H I-2 were found, A K H I-3 (Trp-Gly-ThrNH2) could not be detected which either indicates a high leucine aminopeptidase activity in cockroach MTs or that A K H 1-3 had not been formed.
Semi-isolated MTs A semi-isolated system as described by Morgan and Mordue (1984) was used to test whether intact
E c,.(3
1.0
AKHII
Omin
o
0 z < r~
0 m <
0.,5
AKXI! 60 rrdn
00.
. 0
.
5
.
. I0
. . 15 20
25
RETENTION TIME [mini Fig. 2. During a 60 min incubation of a haemolymph preparation from S. gregaria with AKH I the peptide remained intact. Gradient I was employed. None of the peaks in the chromatogram changed in their peak area. AKH I 24.2 min.
Degradation of adipokinetic hormones Table I. In a semi-isolated S. gregaria MT preparation A K H ! is removed from the bathing medium. Aliquots (100 #1) were analysed on gradients I and 2
0.50 Trp-NH 2
<
E t-
8 Lid
7" ro r¢ 0 c,O
t = 0 rain t = 60 min (pmol A K H I) (pmol A K H I) n
No EGTA 5 mM EGTA No tissue
0 0.25 .<
60rnin ~P
<
0,00
t
!
0
5
i,
i
.,
i
10 15 20
RETENTION TIME [min] Fig. 3. Breakdown of tryptophanamide by homogenates of S. gregaria MTs. Gradient 2 was used to separate tryptophanamide (retention time 8.6 min) and tryptophan (12.4 min). MTs could remove or take up A K H I from the incubation medium. The alimentary canal with the attached MTs was placed in saline containing A K H I and aliquots were withdrawn for the quantification of A K H I and breakdown products. The u.v. detection system required the presence of considerably higher peptide concentrations than would normally be present in the haemolymph in vivo. A total of 1.5
L< Contml
E
C ,O O CN
1.0
LU
O 7< t'v
O
cO t~
<
659
0.5
269 ± 28 271 ± 11 277 ± 7
169 +_34 191 + 24 278 ± 17
4 4 4
270pmol A K H I was present in 450/11 bathing medium at the beginning of the experiment (Table 1). Within 60min approx, one third of the peptide disappeared. This is entirely due to the action of the biological preparation since in controls without MTs no change of the A K H I titre was found. Since the MTs secrete fluid, peptides may either be transported into the MTs with the fluid or there may be an independent transport system which does not rely on fluid transport. The inclusion of 5 mM ethyleneglycol-bis-(fl-aminoethyl ether) N,N,N',N'tetraacetic acid (EGTA) in the bathing medium efficiently stops fluid secretion by the MTs (Morgan and Mordue, 1985). Table 1 shows that this treatment does not stop the removal of A K H I from the incubation medium. In vitro incubation o f intact MTs and fat body with AKH I In the semi-isolated preparation the MTs and/or parts of the alimentary canal may be responsible for the disappearance of A K H I. The MTs were separated from the midgut of freshly dissected semi-isolated preparations and incubated under the same conditions as above. Approximately 30% of the peptide disappeared from the incubation medium within the first 15 min, during 60 min a total of ca 80% was lost (Table 2). This was more than during the above experiment with semi-isolated MTs. The difference may be due to the release of intracellular peptidases from damaged cells into the incubation medium. Thus, the total decrease may not be related solely to transport but also to enzymatic breakdown. Peaks corresponding to A K H I-1 and A K H 1-2 (see Siegert and Mordue, 1987) were found in substantial amounts after 15 and 60 min (data not shown). After 60 rain, however, one third of the initial amount had disappeared. Only relatively small amounts of A K H I-1 were found after 60 min, but substantial amounts of A K H 1-2. When fat body was incubated without A K H I no A K H I-1 and only small amounts of A K H 1-2 could be detected. Incubation of A K H I in saline alone for 60 min without MTs or fat body showed that no peptide was lost during the experiment due to adsorption or non-enzymatic degradation. The dry Table 2. Isolated S. gregaria MTs and fat body (FB) pieces remove A K H I from the bathing medium. Samples were analysed on gradients 1 and 2
. . . . . . 0 5 10 15 20 25 RETENTION TIME [min] Fig. 4. Breakdown of AKH I by homogenates of MTs from P. americana. Gradient 1 was used to separate the breakdown products. Retention times of the peptide fragments as in Fig. I. A yellow pigment (R), which was not related to the breakdown of AKH, eluted after 17.4 min.
Specific uptake by tissue t = 0 min A 60 min (Apmol A K H I/ (pmol A K H I)(Apmol A K H I) mg dry wt/h)
0.0
MTs FB MTs, no A K H I FB, no A K H I No tissue
342 _+ 31 3 2 5 ± 18 0 0 346 _+ 4
- 2 7 2 ± 12 - 1 3 4 ± 38 0 0 +2± 5
85 ± 13 6±2 0 0 0
4 4 4 4 4
660
K A R L J. SIEGERT a n d WILLIAM MORDUE
1.0
GIt-Phe- 7 A M (
1.2
1.(3 ~0.6
,o 0,8
0
o,4
7A.c I
~1/
i 0,2 -
~ Z-Gly-Pro
u.J
0 0.6 < O 0.4
2
r,n
<(
0,0
.
0
.
.
.
.
.
.
.
0.2
5 10 15 20 25 30 35 RETENTION TIME [rain]
Fig. 5. Breakdown of the synthetic substrate ZGPA by homogenates of S. gregaria MTs. Gradient 1 separated the breakdown products. Retention times: Z-GIy-Pro-7AMC 29.8 min. Amino acid analysis revealed that the first peak (20,4min) contained neither Gly nor Pro. From other experiments it can be concluded that the 7AMC component of the substrate eluted after 20.4rain. Gly and Pro were present in equimolar amounts in the second peak [22.7 min; Gly 1046 pmol (1 ×), Pro 954pmol (1 x)]. weight of the fat body pieces in this experiment was ca 20 mg while the MTs amounted to 2-3 mg suggesting that the MTs have a larger specific transport and/or breakdown capacity than the fat body or other tissues. Enzymes invoh~ed in the breakdown of A K H I by MTs The primary cleavage site in A K H I may either be after the proline (position 6) or the asparagine residue (position 7; see Siegert and Mordue, 1987). We analysed the possibility of the presence of a postproline cleaving enzyme (PPCE) using the synthetic substrate ZGPA. Indeed, this substrate was cleaved readily by MT homogenates under the conditions for the breakdown of A K H I. Two products were formed from Z G P A which eluted with retention times of 20.4 and 22.7 min, respectively, while Z G P A eluted after 29.8 min (Fig. 5). Amino acid analysis of the two peaks revealed that the former did not contain any peptide fragment, while the latter peak contained glycine and proline (see Fig. 5 caption). This result is consistent with the presence of PPCE activity in the MTs. The first product peak probably contains the 7AMC component of the substrate. Breakdown o f A K H II-S by MTs The involvement of PPCE in the breakdown of A K H I poses the question of the breakdown of A K H II-S which is also blocked at both termini but does not contain a proline residue (Siegert et al., 1985). It is likely that a different endopeptidase is involved than the one which breaks down A K H I. Two major breakdown products were identified when MT homogenate was incubated with A K H II-S: A K H II-S-1 and A K H II-S-2 (Fig. 6). Amino acid analysis of A K H II-S-1 revealed the presence of the first four residues of A K H II-S (see Fig. 6 caption for analysis data).
0,0
'
0
5
, ,
10 15 20 25 30
RETENTION TIME [r'nin]
Fig. 6. Breakdown of AKH 1I-S by homogenates of S. gregaria MTs. Gradient 1 was used to separate the products. Retention times: AKH II-S 24.drain; 1, AKH II-S-1 22.9 min: pGlu-Leu-Asn-Phe; Glx 5833 pmol (1 ×), Asx 5281 pmol (1 x ), Leu 4997 pmol (1 x ), Phe 5495 pmol (1 x); 2, AKH II-S-2; 3, Trp 15.9 min, 3 was not analysed. The synthetic chymotrypsin substrate GFA (27.9min) remained intact. The occurrence of this fragment indicated the presence of a chymotrypsin-like endopeptidase which we propose to call post-phenylalanine cleaving enzyme (PFCE). Since A K H II-S-2 absorbed at 280 nm and co-eluted with Trp, it was concluded that this peak was indeed Trp which is present in A K H II-S in position 8. The chymotrypsin substrate G F A was not cleaved by MT homogenate (Fig. 6) nor was the substrate S A F K A (not shown). One unit of bovine pancreas chymotrypsin, however, cleaved 3 # g G F A and 6 # g S A F K A within 60min at 30°C (not shown). 1.0
E t-O O
0.8
0.6
LLJ
L) Z < 0.4 CO
1
r'.,
O co CO <
0.2
0.0
~]
. 5
. . . . . 10 15 20 25 30
RETENTION TiME [rain)
Fig. 7. Breakdown of BTEE by homogenates of S. gregaria MT. Gradient 1 was used. Retention times: BTEE 27.5 rain; M I 23.4rain; M I-1 22.3rain; Trp 15.9min; R 17.4rain.
Degradation of adipokinetic hormones 1.2
interestingly were identical for A K H I and A K H II-S: 22.4 and 17.4min respectively. The third product formed from A K H II-S eluted with the retention time of tryptophan: 15.9 min. Amino acid analysis of the A K H I fragments confirmed that the 22.4 min peak contained the N-terminal fragment of A K H I (pGluLeu-Asn-Phe) while the C-terminal fragment (ThrPro-Asn-Trp-Gly-ThrNH2) eluted after 17.4 min (for amino acid analysis data see legends to Figs 8 and 9).
1.0 E t"
0.8
-O O
661
tit
O 0.6 z<
B DISCUSSION
rm
S
0.4
GO rr~
<
0.2
0,0
i
'
0
i
i
i
i
i
5 10 15 20 25 30 RETENTION TIME [rain]
Fig. 8. Breakdown of AKH I by bovine pancreas chymotrypsin. Retention times and composition of fragments: A 22.5 min (AKH I-A) pGlu-Leu-Asn-Phe; Glx 4758 pmol (Ix), Leu 4139pmol (Ix), Asx 4228pmol (Ix), Phe 4372 pmol (1 x ); B 17.4 rain (AKH I-B) Thr-Pro-Asn-TrpGIy-ThrNH2; Thr 6640 pmol (2 x ), Pro 4322 pmol (1 x ), Asx 3049pmol (1 ×), Trp was not protected, but peak absorbed at 280 nm (1 x ), Gly 4347 pmol (1 x ), Tbr (see above). By contrast the tyrosine-containing chymotrypsin substrate BTEE was readily broken down by MT homogenates (Fig. 7); 3/ag BTEE were also broken down by one unit of chymotrypsin (conditions as above, no chromatogram shown) as were A K H I and A K H II-S (5-10 nmol; Figs 8 and 9) respectively. The retention times of the two major breakdown products 1.2
8 z<
II-S
AKH
1.0
o.8
0.6
¢'v
0 0.4 < 0.2
0.0
J i
0
i
i
i
i
i
i
5 l0 15 20 25 30 RETENTION TIME [min]
Fig. 9. Digestion of AKH II-S by bovine pancreas chymotrypsin. Retention times and compositions of fragments: 1, AKH II-S-i/A 22.4 min pGlu-Leu-Asn-Phe; Glx 1363 pmol ( I x ) , Leu 1063pmol ( I x ) , Asx 1363pmol (Ix), Phe l174pmol (I x); B AKH II-S-B 17.3rain Ser-Thr-GlyTrpNH2; Ser 1979pmol (1 x), Thr 2543pmol (1 x), Gly 2091 pmol (1 ×), Trp 751 pmol (1 x); 2, 15.9 min Trp.
A wealth of information on the various biological activities of the A K H s is now available (for a review see Orchard, 1987). The termination of the hormonal signal, however, has received little attention. One aspect of this is the removal of the active peptides from the haemolymph and their breakdown. Baumann and Penzlin (1987) provided evidence for the active uptake of M I by MTs of the cockroach P. americana while other tissues were unable to remove the peptide from an in vitro bathing medium. By comparison Skinner et al. (1987) found that homogenates of the MTs as well as of the fat body broke down M II, In the present study we have confirmed this latter result and can extend it since all the S. gregaria tissues tested were able to degrade A K H I. The haemolymph, however, did not break down this petide, which is an agreement with Baumann and Penzlin (1987) and to some extent with Skinner et al. (1987). After injection of physiological doses of M II into P. americana the levels of M II declined very slowly (Skinner et aL, 1987). This led to the conclusion that the dynamics of peptide release from the corpora cardiaca [CC; the site of M II synthesis (O'Shea et al., 1984)] may be more important in the regulation of the haemolymph titre than the removal and/or breakdown. At the moment it is not at all clear which of the two cockroach AKHs is actually released from the CC. Only M I may be released and may have a different half-life in the haemolymph than M II. The injection of 0.1 pair of CC from adult cockroaches (corresponding to ca 10 pmol M I and 2 pmol M II; Siegert and Mordue, 1992a) produced a long-term hyperglycaemic effect which was still present after 24 h. Activation of fat body glycogen phosphorylase, however, only lasted between 1 and 2 h (Siegert et al., 1986). In ligated abdomens of the tobacco hornworm larva, Manduca sexta, the activity of glycogen phosphorylase seems to reflect the A K H titre in the haemolymph (Siegert et al., 1982). This suggests that within 1-2h the peptide titre in the cockroach haemolymph must have declined below the threshold for enzyme activation. The haemolymph contains considerable exopeptidase activity in the form of leucine aminopeptidase. This explains the rapid degradation of the unblocked neuropeptide proctolin on one hand and the need for a N-terminal pyroglutamic acid present in all known AKHs. This fact is reflected in the apparent in vivo half-life for M II of about 1 h, while the half-life of proctolin at physiological doses is < 2 min (Quistad et aL, 1984). The haemolymph of locusts and cockroaches either does not contain endopeptidases or endopeptidases other than PPCE and PFCE are present. In general agreement with these results,
662
KARL J, SIEGERTand WILLIAMMORDUE
Rayne and O'Shea (1992) showed that A K H I is very stable in S. gregaria haemolymph, but not in tissue cultures of MTs, fat body or flight muscles. We found that the S. gregaria fat body removed A K H I from an in vitro system. Thus, at least two general pathways for the removal from the haemolymph of circulating A K H seem to exist. Rayne and O'Shea (1992), however, proposed that AKH°degrading enzym~,s located on the surface of cells are responsible for A K H metabolism. The fat body is the major target tissue of A K H and binds these peptides through its receptors which may be followed by internalization and breakdown of the peptide. The concentrations used in our in vitro studies were high and may have driven A K H I into the fat body cells along a concentration gradient. The MTs may remove freely circulating hormone from the haemolymph. In the in vitro experiment the MTs constituted a much smaller mass, but still removed more peptide during the same period of time. The specific removal (in pmol x h - l x m g - i dry wt) was much higher for the MTs than for the fat body. Since the latter has a considerable size, its contribution to the overall removal of peptide from the haemolymph should not be underestimated. In the in vitro experiment the fat body only released small amounts of the breakdown products A K H I-1, A K H I-2 and A K H I-3 indicating that the contribution of released/secreted proteases was limited (1) and/or that if A K H I was internalized and broken down the breakdown fragments were not released into the bathing medium (2). By contrast the MTs seemed to leak intermediary breakdown products into the medium. The inclusion of 5raM EGTA into the bathing medium stops fluid secretion by the MTs (Morgan and Mordue, 1985). This, however, did not reduce the MT's capacity to remove A K H I from the medium (present study). The presence of 10-8M EDTA in the homogenate of cockroach MT on the other hand inhibited the breakdown of M I, indicating the presence of a neutral metalloendopeptidase. This peptidase has an apparent molecular weight of 45 kDa and Baumann and Penzlin (1987) suggested that it may be comparable to the one present in mammalian kidney microvilli. It is also clear from these data that the enzyme which broke down M I is not chymotrypsin. The present study shows the presence of PPCE in MT homogenates which together with other data (Siegert and Mordue 1987, 1992b) suggests that A K H I (like M I a peptide which contains a Pro residue in position 6) is broken down by PPCE, while PFCE is also present in MTs. Skinner et al. (1987) found breakdown fragments in fat body homogenate which corroborates a post-proline breakdown. A tetrapeptide corresponding to the N-terminal part of M II was also detected in considerable amounts. The temporal occurrence of the two peptides, however, suggests that the N-terminal hexapeptide is a primary product from which the latter arose. It is not clear whether this peptide arose from carboxypeptidase or PFCE digestion. The present study did not detect a fragment which would indicate a PFCE attack on A K H I. The N-terminal hexapeptide and the tetrapeptide have almost identical retention times [AKH I-1 23.2 min, A K H II-S-1 (or A K H I - A ) 22.9 min], This difference in retention time should be enough to produce two
peaks or a shoulder and a peak in the chromatogram when both products appeared in MT homogenates. The selection of the degradative pathway for individual A K H peptides is being dealt with in a separate paper (Siegert and Mordue, 1992b). In the present study the MT homogenate was prepared in a buffer which contained 20 mM EDTA allowing the occurrence of both PPCE and PFCE activity. Lamb kidney PPCE was not inhibited by 1 mM EDTA but higher concentrations were not employed (Koida and Walter, 1976). Baumann and Penzlin (1987), however, found that M I breakdown was EDTA-sensitive (see above). Since P. americana MTs seem to break down A K H I in the same way as S. gregaria MTs, the presence of PPCE in cockroach MTs can be assumed. Baumann and Penzlin (1987) estimated the molecular weight of the peptidase as 45 kDa while the lamb kidney PPCE has an apparent size of 115 kDa, with two subunits of 57 kDa. These differences in the molecular features stress the need for the isolation of PPCE and PFCE from insect MTs to compare them with their counterparts from other sources. The two endopeptidases from S. gregaria MTs involved in the metabolism of adipokinetic peptides have the capacity to destroy in excess of 1 nmol of peptide per hour at 30°C. Since all the tested tissues broke down A K H I, the question is not where these peptides can be broken down, but which tissues can they enter or which have an uptake mechanism to internalize them. The MTs seem to be capable of taking up the AKHs. For the fat body a more sophisticated approach with radio-labelled peptides must be employed to elucidate whether or not it participates in the breakdown of AKH. Acknowledgements--This study was supported by grants
from the Deutsche Forschungsgemeinschaft (Si 1939/1-1), the SERC and the University of Aberdeen Research Committee. The authors wish to thank Ian Davidson for expert performance of the amino acid analyses and Val Johnston and Jim Levenie for caring for the animals. REFERENCES
Baumann E. and Penzlin H. (1987) Inactivation of neurohormone D by Malpighian tubules in an insect, Periplaneta americana. J. comp. Physiol. B 157, 511-517. Koida M. and Walter R. (1976) Post-proline cleaving enzyme. Purification of this endopeptidase by affinity chromatography. J. biol. Chem. 251, 7593-7599. Morgan P. J. and Mordue W. (1983) Separation and characteristics of diuretic hormone from the corpus cardiacum of Locusta. Comp. Biochem. Physiol. 75B, 75-80. Morgan P. J. and Mordue W. (1984) Diuretic hormone: another peptide with widespread distribution within the insect CNS. Physiol. Ent. 9, 197-206. Morgan P. J. and Mordue W. (1985) The role of calcium in diurectic hormone action on locust Malpighian tubules. Molec. cell Endocr. 40, 221-231. Orchard I, (1987) Adipokinetic hormones--an update. ,1. Insect Physiol. 33, 451-463. O'Shea M., Witten J. and Schaffer M. (1984) Isolation and characterization of two myoactive neuropeptides: further evidence of an invertebrate peptide family. J. Neurosci. 4, 521-529. Quistad G. B., Adams M. E., Scarborough R. M., Carney R. L. and Schooley D. A. (1984) Metabolism of proctolin,
Degradation of adipokinetic hormones a pentapeptide neurotransmitter in insects. Life Sci. 34, 569-576. Rayne R. C. and O'Shea M. (1992) Inactivation of neuropeptide hormones (AKH I and AKH II) studied in vivo and in vitro, lnsect Biochem. Molec. Biol. 22, 25-34. Siegert K. J. and Mordue W. (1986) Quantification of adipokinetic hormones I and II in the corpora cardiaca of Schistocerca gregaria and Locusta migratoria. Comp. Biochem. Physiol. 84A, 279-284. Siegert K. J. and Mordue W. (1987) Breakdown of locust adipokinetic hormone I by Malpighian tubules of Schistocerca gregaria. Insect Biochem. 17, 705-710. Siegert K. J. and Mordue W. (1992a) Content and localization of the hyperglycaemic hormones I and II in the corpus cardiacum of the American cockroach, Periplaneta americana. Comp. Biochem. Physiol. 101B, 315 319. Siegert K. J. and Mordue W. (1992b) Preliminary characterization of Malpighian tubule enzyme activities involved in the breakdown of adipokinetic hormones. Archs Insect Biochem. Physiol. 19, 147-161. Siegert K. J., Krippeit P. and Ziegler R. (1982) Regulation
663
of fat body glycogen phosphorylase during starvation in larvae of Manduca sexta (Lepidoptera: Sphingidae). Acta endocr. Suppl. 246, 99, 33-34. Siegert K. J., Morgan P. J. and Mordue W. (1985) Primary structures of locust adipokinetic hormones II. Biol, chem. Hoppe-Seyler 366, 723-727. Siegert K. J., Morgan P. J. and Mordue W. (1986) Isolation of hyperglycaemic peptides from the corpus cardiacum of the American cockroach, Periplaneta americana. Insect Biochem. 16, 365-371. Skinner W. S., Quistad G. B., Adams M. E. and Schooley D. A. (1987) Metabolic degradation of the peptide periplanetin CC-2 in the cockroach, Periplaneta americana. lnsect Biochem. 17, 433-437. Suzuki K. and Kono T. (1979) Internalization and degradation of fat-cell bound insulin. Separation and partial characterization of subcellular vesicles associated with iodoinsulin. J. biol. Chem. 254, 9876-9794. Terris S. and Steiner D. (1975) Binding and degradation of I-insulin by rat hepatocytes. J. biol. Chem. 250, 8389 8398.
0965-1748/92 $5.00+ 0.00 Copyright © 1992PergamonPress Ltd
D E G R A D A T I O N OF ADIPOKINETIC HORMONES KARL J. SIEGERTand WILLIAM MORDUE Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scotland (Received I0 September 1991; revised and accepted 21 May 1992)
Abstraet--Homogenates from various tissues of the desert locust, Schistocerca gregaria, produced identical patterns of breakdown products when incubated with the neuropeptide adipokinetic hormone I (AKH I). Comparison of disappearance of AKH I from the incubation medium of semi-isolated and isolated Malpighian tubules (MTs) and fat body pieces indicates that both tissues contain substantial capacities to take up and/or breakdown peptide. While the MTs may remove peptide circulating in the haemolymph, the fat body may metabolize peptide which binds to cell surface receptors. Locusta migratoria and Periplaneta americana MTs contain the same complement of degrading enzymes since they produced identical primary products. The initial proteolytic attack on AKH I took place through a postproline cleaving enzyme (PPCE, residue 6). The use of the synthetic substrate benzyloxycarbonyl-Gly-Pro7-amido-4-methyl-coumarinconfirmed the presence of PPCE. Adipokinetic hormone II from S. gregaria was cleaved between phenylalanine and serine (residues 4 and 5) indicating the action of a chymotrypsinlike endopeptidase. The synthetic substrates glutaryl-Phe-7AMC and N-succinyl-Ala-Phe-Lys-7AMC were not cleaved by MT homogenates; however, such homogenates degraded a tyrosine-containing substrate, N-benzoyl-Tyr ethyl ester. Chymotrypsin from bovine pancreas cleaved all the above substrates. Key Word Index: AKH; Malpighian tubules; enzymatic breakdown of peptides
INTRODUCTION Homogenates of the Malpighian tubules (MTs) from the desert locust, Schistocerca gregaria, have been shown to cleave the adipokinetic hormone I ( A K H I; pGiu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-ThrNH2); using reversed-phase high-performance liquid chromatography (RP-HPLC) three breakdown products were identified ( A K H I-1: pGlu-Leu-Asn-Phe-ThrPro, A K H 1-2: Trp and A K H I-3: Trp-Gly-ThrNH2; Siegert and Mordue, 1987). B a u m a n n and Penzlin (1987) demonstrated with an in vitro system that in the American cockroach, Periplaneta americana, only the MTs contain the ability to remove neurohormone D ( = myotropic peptide I, MI, periplanetin CC-I or hyperglycaemic hormone I; pGlu-Val-Asn-Phe-SerPro-Asn-TrpNH2) from the incubation mixture and/ or to destroy this peptide. Other tissues such as muscle, fat body, haemolymph, etc. were also tested but negative results were obtained. By contrast, Skinner et al. (1987) reported that homogenates of P. americana fat body cleaved periplanetin CC-II. Because of their physiological role in transporting water and ions and the large area exposed to the haemolymph, the MTs are indeed a very likely candidate for the removal of peptide hormones from the haemolymph. On the other hand, it is known from vertebrate studies that peptide hormones not only bind to the hormone receptors on the cell surface but are also internalized (Terris and Steiner, 1975; Suzuki and Kono, 1979). If this were also the case in insects then peripheral tissues such as the fat body must have the enzyme complement to breakdown A K H peptides. To this end, the breakdown of A K H I and A K H II-S (pGluLeu-Asn-Phe-Ser-Thr-Gly-TrpNH2) from S. gregaria by tissue homogenates was investigated.
MATERIALS
AND METHODS
Animals S. gregaria and Locusta migratoria were kept as described by Morgan and Mordue (1983). P. americana were purchased from Bioserv Ltd and kept as described by Siegert et al. (1986). Chemicals The chemicals used in the present study were from the sources listed by Siegert and Mordue (1987). Additionally, benzyloxycarbonyl- Gly - Pro - 7 - amido - 4 - methylcoumarin (7AMC) (ZGPA) was from Cambridge Research Biochemicals and tryptophan, tryptophanamide, glutaryl-Phe7AMC (GFA), N-succinyl-Ala-Phe-Lys-7AMC (SAFKA), N-benzoyl-Tyr ethyl ester (BTEE) and ~-chymotrypsin (bovine pancreas) were purchased from Sigma. Preparation and incubation of homogenates from different tissues with peptides, substrates etc. Tissue homogenates were prepared as outlined by Siegert and Mordue (1987) for MTs. All incubations were performed at 30°C. Haemolymph (50 #1) was mixed with 400/11 buffer (90 mM KH2PO4, 20 mM Na2EDTA, pH 8), vortexed and then centrifuged for 10 min at 12,000g. An aliquot of the supernatant (400 #1) was then mixed with the peptide solution (1-5/zg/400/zl) and samples withdrawn at t = 0 and 60 min. Semi-isolated M T preparations The method described by Morgan and Mordue (1984) was adopted using the same insect saline (I 15mM NaCI, 20 mM KCI, 2 mM CaClz, 2 mM MgC12, 10 mM glucose, 22 mM NaHCO3, 6 mM NaH2PO 4, pH 7.2). Generally, the preparation was incubated in 450/~1 saline plus additives and 100/zl removed at t = 0 and 60 min, 50/zl acetonitrile and 850/~1 0.1% trifluoroacetic acid (TFA) added, mixed, centrifuged (5 min, 12,000g) and injected onto the RP-HPLC.
657
658
KARL J. SIEGERTand WILLIAMMORDUE 1.5
The in vitro system The alimentary canal with the attached MTs was placed into simple insect saline (128 mM NaCI, 5 mM KCI) and then the MTs gently removed. Fat body pieces and MTs were briefly blotted on filter paper and added to the incubation tubes (Eppendorf) which contained 450/zl saline (see above) plus peptide. Aliquots (100/d) were removed at t = 0, 15 and 60 min and processed as outlined above.
Control
E c
RP-HPLC The equipment described by Siegert et al. (1985) was used. Separation of peptides was performed on an Aquapore RP-300 column (4.6 × 250 turn) using 0.1% TFA (pump A) and acetonitrile (pump B) as solvents at a flow rate of 1 ml/min. Two different gradients were employed. Gradient 1 started at 0% B and increased at 2% B/rain and allowed the analysis of AKH breakdown products (Siegert and Mordue, 1987). Gradient 2 started at 7% B with a 0.3% B/rain increase and allowed the separation of tryptophan and tryptophanamide. Absorbance was monitored with a Pye Unicam LC-u.v. detector (206 nm) and peak areas were calculated by a Shimadzu C-R1 B chromatopak integrator. Peak areas were converted to pmol peptide as described by Siegert and Mordue (1986). In some experiments the absorbance was simultaneously measured at 280 nm with a Gilson dual wavelength spectrophotometer (model 116).
1.0
8 0 z < en
0 m <
0.5
60mln
0,0
Amino acid analysis The analysis was carried out on an Applied Biosystems 420A Amino Acid Analyzer with automatic hydrolysis and derivatization. The PTC amino acids generated were identified on-line, using a 130A separation system employing a C-18 reversed-phase narrow bore cartridge. The system was calibrated using 25 pmol fl-lactoglobulin standard (A and B variants).
. 0
.
AKHI
--4_jt .
5
. 10
. . 15 20
25
RETENTION TIME [min] Fig. 1. Breakdown of AKH I by a homogenate from S. gregaria fat body, Brain, dorsal heart and midgut also cleaved AKH I producing the same breakdown products. Gradient 1 was used. Retention times: AKH 1 24.2 rain; 1, AKH I-1 23.2 min; 2, AKH I-2 15.9min.
RESULTS
1.5
Control
Breakdown o f A K H [ by homogenates from different S. gregaria tissues The ability of different locust tissues to breakdown the neuropeptide A K H I was studied using homogenates. All tissues tested (brain, heart, midgut and fat body) were found to cleave A K H I in considerable amounts comparable to the result shown for fat body (Fig. 1). H a e m o l y m p h preparations, however, failed to digest A K H I (Fig. 2). It is important to note that none of the peak areas in Fig. 2 changed during the 60 min incubation period. A control experiment with tryptophanamide as a substrate for leucine aminopeptidase showed that the haemolymph preparation contained active proteolytic enzymes (Fig. 3).
Breakdown o f A K H I by M T homogenates from L. migratoria and P. americana Locusta migratoria MTs also contain enzymes which cleave A K H I as do MTs from P. americana (Fig. 4). Three sets of MTs from P. americana had to be used to produce a substantial breakdown of A K H [. Unlike the situation in L. migratoria, peaks corresponding to breakdown products A K H I-1 and A K H I-2 were found, A K H I-3 (Trp-Gly-ThrNH2) could not be detected which either indicates a high leucine aminopeptidase activity in cockroach MTs or that A K H 1-3 had not been formed.
Semi-isolated MTs A semi-isolated system as described by Morgan and Mordue (1984) was used to test whether intact
E c,.(3
1.0
AKHII
Omin
o
0 z < r~
0 m <
0.,5
AKXI! 60 rrdn
00.
. 0
.
5
.
. I0
. . 15 20
25
RETENTION TIME [mini Fig. 2. During a 60 min incubation of a haemolymph preparation from S. gregaria with AKH I the peptide remained intact. Gradient I was employed. None of the peaks in the chromatogram changed in their peak area. AKH I 24.2 min.
Degradation of adipokinetic hormones Table I. In a semi-isolated S. gregaria MT preparation A K H ! is removed from the bathing medium. Aliquots (100 #1) were analysed on gradients I and 2
0.50 Trp-NH 2
<
E t-
8 Lid
7" ro r¢ 0 c,O
t = 0 rain t = 60 min (pmol A K H I) (pmol A K H I) n
No EGTA 5 mM EGTA No tissue
0 0.25 .<
60rnin ~P
<
0,00
t
!
0
5
i,
i
.,
i
10 15 20
RETENTION TIME [min] Fig. 3. Breakdown of tryptophanamide by homogenates of S. gregaria MTs. Gradient 2 was used to separate tryptophanamide (retention time 8.6 min) and tryptophan (12.4 min). MTs could remove or take up A K H I from the incubation medium. The alimentary canal with the attached MTs was placed in saline containing A K H I and aliquots were withdrawn for the quantification of A K H I and breakdown products. The u.v. detection system required the presence of considerably higher peptide concentrations than would normally be present in the haemolymph in vivo. A total of 1.5
L< Contml
E
C ,O O CN
1.0
LU
O 7< t'v
O
cO t~
<
659
0.5
269 ± 28 271 ± 11 277 ± 7
169 +_34 191 + 24 278 ± 17
4 4 4
270pmol A K H I was present in 450/11 bathing medium at the beginning of the experiment (Table 1). Within 60min approx, one third of the peptide disappeared. This is entirely due to the action of the biological preparation since in controls without MTs no change of the A K H I titre was found. Since the MTs secrete fluid, peptides may either be transported into the MTs with the fluid or there may be an independent transport system which does not rely on fluid transport. The inclusion of 5 mM ethyleneglycol-bis-(fl-aminoethyl ether) N,N,N',N'tetraacetic acid (EGTA) in the bathing medium efficiently stops fluid secretion by the MTs (Morgan and Mordue, 1985). Table 1 shows that this treatment does not stop the removal of A K H I from the incubation medium. In vitro incubation o f intact MTs and fat body with AKH I In the semi-isolated preparation the MTs and/or parts of the alimentary canal may be responsible for the disappearance of A K H I. The MTs were separated from the midgut of freshly dissected semi-isolated preparations and incubated under the same conditions as above. Approximately 30% of the peptide disappeared from the incubation medium within the first 15 min, during 60 min a total of ca 80% was lost (Table 2). This was more than during the above experiment with semi-isolated MTs. The difference may be due to the release of intracellular peptidases from damaged cells into the incubation medium. Thus, the total decrease may not be related solely to transport but also to enzymatic breakdown. Peaks corresponding to A K H I-1 and A K H 1-2 (see Siegert and Mordue, 1987) were found in substantial amounts after 15 and 60 min (data not shown). After 60 rain, however, one third of the initial amount had disappeared. Only relatively small amounts of A K H I-1 were found after 60 min, but substantial amounts of A K H 1-2. When fat body was incubated without A K H I no A K H I-1 and only small amounts of A K H 1-2 could be detected. Incubation of A K H I in saline alone for 60 min without MTs or fat body showed that no peptide was lost during the experiment due to adsorption or non-enzymatic degradation. The dry Table 2. Isolated S. gregaria MTs and fat body (FB) pieces remove A K H I from the bathing medium. Samples were analysed on gradients 1 and 2
. . . . . . 0 5 10 15 20 25 RETENTION TIME [min] Fig. 4. Breakdown of AKH I by homogenates of MTs from P. americana. Gradient 1 was used to separate the breakdown products. Retention times of the peptide fragments as in Fig. I. A yellow pigment (R), which was not related to the breakdown of AKH, eluted after 17.4 min.
Specific uptake by tissue t = 0 min A 60 min (Apmol A K H I/ (pmol A K H I)(Apmol A K H I) mg dry wt/h)
0.0
MTs FB MTs, no A K H I FB, no A K H I No tissue
342 _+ 31 3 2 5 ± 18 0 0 346 _+ 4
- 2 7 2 ± 12 - 1 3 4 ± 38 0 0 +2± 5
85 ± 13 6±2 0 0 0
4 4 4 4 4
660
K A R L J. SIEGERT a n d WILLIAM MORDUE
1.0
GIt-Phe- 7 A M (
1.2
1.(3 ~0.6
,o 0,8
0
o,4
7A.c I
~1/
i 0,2 -
~ Z-Gly-Pro
u.J
0 0.6 < O 0.4
2
r,n
<(
0,0
.
0
.
.
.
.
.
.
.
0.2
5 10 15 20 25 30 35 RETENTION TIME [rain]
Fig. 5. Breakdown of the synthetic substrate ZGPA by homogenates of S. gregaria MTs. Gradient 1 separated the breakdown products. Retention times: Z-GIy-Pro-7AMC 29.8 min. Amino acid analysis revealed that the first peak (20,4min) contained neither Gly nor Pro. From other experiments it can be concluded that the 7AMC component of the substrate eluted after 20.4rain. Gly and Pro were present in equimolar amounts in the second peak [22.7 min; Gly 1046 pmol (1 ×), Pro 954pmol (1 x)]. weight of the fat body pieces in this experiment was ca 20 mg while the MTs amounted to 2-3 mg suggesting that the MTs have a larger specific transport and/or breakdown capacity than the fat body or other tissues. Enzymes invoh~ed in the breakdown of A K H I by MTs The primary cleavage site in A K H I may either be after the proline (position 6) or the asparagine residue (position 7; see Siegert and Mordue, 1987). We analysed the possibility of the presence of a postproline cleaving enzyme (PPCE) using the synthetic substrate ZGPA. Indeed, this substrate was cleaved readily by MT homogenates under the conditions for the breakdown of A K H I. Two products were formed from Z G P A which eluted with retention times of 20.4 and 22.7 min, respectively, while Z G P A eluted after 29.8 min (Fig. 5). Amino acid analysis of the two peaks revealed that the former did not contain any peptide fragment, while the latter peak contained glycine and proline (see Fig. 5 caption). This result is consistent with the presence of PPCE activity in the MTs. The first product peak probably contains the 7AMC component of the substrate. Breakdown o f A K H II-S by MTs The involvement of PPCE in the breakdown of A K H I poses the question of the breakdown of A K H II-S which is also blocked at both termini but does not contain a proline residue (Siegert et al., 1985). It is likely that a different endopeptidase is involved than the one which breaks down A K H I. Two major breakdown products were identified when MT homogenate was incubated with A K H II-S: A K H II-S-1 and A K H II-S-2 (Fig. 6). Amino acid analysis of A K H II-S-1 revealed the presence of the first four residues of A K H II-S (see Fig. 6 caption for analysis data).
0,0
'
0
5
, ,
10 15 20 25 30
RETENTION TIME [r'nin]
Fig. 6. Breakdown of AKH 1I-S by homogenates of S. gregaria MTs. Gradient 1 was used to separate the products. Retention times: AKH II-S 24.drain; 1, AKH II-S-1 22.9 min: pGlu-Leu-Asn-Phe; Glx 5833 pmol (1 ×), Asx 5281 pmol (1 x ), Leu 4997 pmol (1 x ), Phe 5495 pmol (1 x); 2, AKH II-S-2; 3, Trp 15.9 min, 3 was not analysed. The synthetic chymotrypsin substrate GFA (27.9min) remained intact. The occurrence of this fragment indicated the presence of a chymotrypsin-like endopeptidase which we propose to call post-phenylalanine cleaving enzyme (PFCE). Since A K H II-S-2 absorbed at 280 nm and co-eluted with Trp, it was concluded that this peak was indeed Trp which is present in A K H II-S in position 8. The chymotrypsin substrate G F A was not cleaved by MT homogenate (Fig. 6) nor was the substrate S A F K A (not shown). One unit of bovine pancreas chymotrypsin, however, cleaved 3 # g G F A and 6 # g S A F K A within 60min at 30°C (not shown). 1.0
E t-O O
0.8
0.6
LLJ
L) Z < 0.4 CO
1
r'.,
O co CO <
0.2
0.0
~]
. 5
. . . . . 10 15 20 25 30
RETENTION TiME [rain)
Fig. 7. Breakdown of BTEE by homogenates of S. gregaria MT. Gradient 1 was used. Retention times: BTEE 27.5 rain; M I 23.4rain; M I-1 22.3rain; Trp 15.9min; R 17.4rain.
Degradation of adipokinetic hormones 1.2
interestingly were identical for A K H I and A K H II-S: 22.4 and 17.4min respectively. The third product formed from A K H II-S eluted with the retention time of tryptophan: 15.9 min. Amino acid analysis of the A K H I fragments confirmed that the 22.4 min peak contained the N-terminal fragment of A K H I (pGluLeu-Asn-Phe) while the C-terminal fragment (ThrPro-Asn-Trp-Gly-ThrNH2) eluted after 17.4 min (for amino acid analysis data see legends to Figs 8 and 9).
1.0 E t"
0.8
-O O
661
tit
O 0.6 z<
B DISCUSSION
rm
S
0.4
GO rr~
<
0.2
0,0
i
'
0
i
i
i
i
i
5 10 15 20 25 30 RETENTION TIME [rain]
Fig. 8. Breakdown of AKH I by bovine pancreas chymotrypsin. Retention times and composition of fragments: A 22.5 min (AKH I-A) pGlu-Leu-Asn-Phe; Glx 4758 pmol (Ix), Leu 4139pmol (Ix), Asx 4228pmol (Ix), Phe 4372 pmol (1 x ); B 17.4 rain (AKH I-B) Thr-Pro-Asn-TrpGIy-ThrNH2; Thr 6640 pmol (2 x ), Pro 4322 pmol (1 x ), Asx 3049pmol (1 ×), Trp was not protected, but peak absorbed at 280 nm (1 x ), Gly 4347 pmol (1 x ), Tbr (see above). By contrast the tyrosine-containing chymotrypsin substrate BTEE was readily broken down by MT homogenates (Fig. 7); 3/ag BTEE were also broken down by one unit of chymotrypsin (conditions as above, no chromatogram shown) as were A K H I and A K H II-S (5-10 nmol; Figs 8 and 9) respectively. The retention times of the two major breakdown products 1.2
8 z<
II-S
AKH
1.0
o.8
0.6
¢'v
0 0.4 < 0.2
0.0
J i
0
i
i
i
i
i
i
5 l0 15 20 25 30 RETENTION TIME [min]
Fig. 9. Digestion of AKH II-S by bovine pancreas chymotrypsin. Retention times and compositions of fragments: 1, AKH II-S-i/A 22.4 min pGlu-Leu-Asn-Phe; Glx 1363 pmol ( I x ) , Leu 1063pmol ( I x ) , Asx 1363pmol (Ix), Phe l174pmol (I x); B AKH II-S-B 17.3rain Ser-Thr-GlyTrpNH2; Ser 1979pmol (1 x), Thr 2543pmol (1 x), Gly 2091 pmol (1 ×), Trp 751 pmol (1 x); 2, 15.9 min Trp.
A wealth of information on the various biological activities of the A K H s is now available (for a review see Orchard, 1987). The termination of the hormonal signal, however, has received little attention. One aspect of this is the removal of the active peptides from the haemolymph and their breakdown. Baumann and Penzlin (1987) provided evidence for the active uptake of M I by MTs of the cockroach P. americana while other tissues were unable to remove the peptide from an in vitro bathing medium. By comparison Skinner et al. (1987) found that homogenates of the MTs as well as of the fat body broke down M II, In the present study we have confirmed this latter result and can extend it since all the S. gregaria tissues tested were able to degrade A K H I. The haemolymph, however, did not break down this petide, which is an agreement with Baumann and Penzlin (1987) and to some extent with Skinner et al. (1987). After injection of physiological doses of M II into P. americana the levels of M II declined very slowly (Skinner et aL, 1987). This led to the conclusion that the dynamics of peptide release from the corpora cardiaca [CC; the site of M II synthesis (O'Shea et al., 1984)] may be more important in the regulation of the haemolymph titre than the removal and/or breakdown. At the moment it is not at all clear which of the two cockroach AKHs is actually released from the CC. Only M I may be released and may have a different half-life in the haemolymph than M II. The injection of 0.1 pair of CC from adult cockroaches (corresponding to ca 10 pmol M I and 2 pmol M II; Siegert and Mordue, 1992a) produced a long-term hyperglycaemic effect which was still present after 24 h. Activation of fat body glycogen phosphorylase, however, only lasted between 1 and 2 h (Siegert et al., 1986). In ligated abdomens of the tobacco hornworm larva, Manduca sexta, the activity of glycogen phosphorylase seems to reflect the A K H titre in the haemolymph (Siegert et al., 1982). This suggests that within 1-2h the peptide titre in the cockroach haemolymph must have declined below the threshold for enzyme activation. The haemolymph contains considerable exopeptidase activity in the form of leucine aminopeptidase. This explains the rapid degradation of the unblocked neuropeptide proctolin on one hand and the need for a N-terminal pyroglutamic acid present in all known AKHs. This fact is reflected in the apparent in vivo half-life for M II of about 1 h, while the half-life of proctolin at physiological doses is < 2 min (Quistad et aL, 1984). The haemolymph of locusts and cockroaches either does not contain endopeptidases or endopeptidases other than PPCE and PFCE are present. In general agreement with these results,
662
KARL J, SIEGERTand WILLIAMMORDUE
Rayne and O'Shea (1992) showed that A K H I is very stable in S. gregaria haemolymph, but not in tissue cultures of MTs, fat body or flight muscles. We found that the S. gregaria fat body removed A K H I from an in vitro system. Thus, at least two general pathways for the removal from the haemolymph of circulating A K H seem to exist. Rayne and O'Shea (1992), however, proposed that AKH°degrading enzym~,s located on the surface of cells are responsible for A K H metabolism. The fat body is the major target tissue of A K H and binds these peptides through its receptors which may be followed by internalization and breakdown of the peptide. The concentrations used in our in vitro studies were high and may have driven A K H I into the fat body cells along a concentration gradient. The MTs may remove freely circulating hormone from the haemolymph. In the in vitro experiment the MTs constituted a much smaller mass, but still removed more peptide during the same period of time. The specific removal (in pmol x h - l x m g - i dry wt) was much higher for the MTs than for the fat body. Since the latter has a considerable size, its contribution to the overall removal of peptide from the haemolymph should not be underestimated. In the in vitro experiment the fat body only released small amounts of the breakdown products A K H I-1, A K H I-2 and A K H I-3 indicating that the contribution of released/secreted proteases was limited (1) and/or that if A K H I was internalized and broken down the breakdown fragments were not released into the bathing medium (2). By contrast the MTs seemed to leak intermediary breakdown products into the medium. The inclusion of 5raM EGTA into the bathing medium stops fluid secretion by the MTs (Morgan and Mordue, 1985). This, however, did not reduce the MT's capacity to remove A K H I from the medium (present study). The presence of 10-8M EDTA in the homogenate of cockroach MT on the other hand inhibited the breakdown of M I, indicating the presence of a neutral metalloendopeptidase. This peptidase has an apparent molecular weight of 45 kDa and Baumann and Penzlin (1987) suggested that it may be comparable to the one present in mammalian kidney microvilli. It is also clear from these data that the enzyme which broke down M I is not chymotrypsin. The present study shows the presence of PPCE in MT homogenates which together with other data (Siegert and Mordue 1987, 1992b) suggests that A K H I (like M I a peptide which contains a Pro residue in position 6) is broken down by PPCE, while PFCE is also present in MTs. Skinner et al. (1987) found breakdown fragments in fat body homogenate which corroborates a post-proline breakdown. A tetrapeptide corresponding to the N-terminal part of M II was also detected in considerable amounts. The temporal occurrence of the two peptides, however, suggests that the N-terminal hexapeptide is a primary product from which the latter arose. It is not clear whether this peptide arose from carboxypeptidase or PFCE digestion. The present study did not detect a fragment which would indicate a PFCE attack on A K H I. The N-terminal hexapeptide and the tetrapeptide have almost identical retention times [AKH I-1 23.2 min, A K H II-S-1 (or A K H I - A ) 22.9 min], This difference in retention time should be enough to produce two
peaks or a shoulder and a peak in the chromatogram when both products appeared in MT homogenates. The selection of the degradative pathway for individual A K H peptides is being dealt with in a separate paper (Siegert and Mordue, 1992b). In the present study the MT homogenate was prepared in a buffer which contained 20 mM EDTA allowing the occurrence of both PPCE and PFCE activity. Lamb kidney PPCE was not inhibited by 1 mM EDTA but higher concentrations were not employed (Koida and Walter, 1976). Baumann and Penzlin (1987), however, found that M I breakdown was EDTA-sensitive (see above). Since P. americana MTs seem to break down A K H I in the same way as S. gregaria MTs, the presence of PPCE in cockroach MTs can be assumed. Baumann and Penzlin (1987) estimated the molecular weight of the peptidase as 45 kDa while the lamb kidney PPCE has an apparent size of 115 kDa, with two subunits of 57 kDa. These differences in the molecular features stress the need for the isolation of PPCE and PFCE from insect MTs to compare them with their counterparts from other sources. The two endopeptidases from S. gregaria MTs involved in the metabolism of adipokinetic peptides have the capacity to destroy in excess of 1 nmol of peptide per hour at 30°C. Since all the tested tissues broke down A K H I, the question is not where these peptides can be broken down, but which tissues can they enter or which have an uptake mechanism to internalize them. The MTs seem to be capable of taking up the AKHs. For the fat body a more sophisticated approach with radio-labelled peptides must be employed to elucidate whether or not it participates in the breakdown of AKH. Acknowledgements--This study was supported by grants
from the Deutsche Forschungsgemeinschaft (Si 1939/1-1), the SERC and the University of Aberdeen Research Committee. The authors wish to thank Ian Davidson for expert performance of the amino acid analyses and Val Johnston and Jim Levenie for caring for the animals. REFERENCES
Baumann E. and Penzlin H. (1987) Inactivation of neurohormone D by Malpighian tubules in an insect, Periplaneta americana. J. comp. Physiol. B 157, 511-517. Koida M. and Walter R. (1976) Post-proline cleaving enzyme. Purification of this endopeptidase by affinity chromatography. J. biol. Chem. 251, 7593-7599. Morgan P. J. and Mordue W. (1983) Separation and characteristics of diuretic hormone from the corpus cardiacum of Locusta. Comp. Biochem. Physiol. 75B, 75-80. Morgan P. J. and Mordue W. (1984) Diuretic hormone: another peptide with widespread distribution within the insect CNS. Physiol. Ent. 9, 197-206. Morgan P. J. and Mordue W. (1985) The role of calcium in diurectic hormone action on locust Malpighian tubules. Molec. cell Endocr. 40, 221-231. Orchard I, (1987) Adipokinetic hormones--an update. ,1. Insect Physiol. 33, 451-463. O'Shea M., Witten J. and Schaffer M. (1984) Isolation and characterization of two myoactive neuropeptides: further evidence of an invertebrate peptide family. J. Neurosci. 4, 521-529. Quistad G. B., Adams M. E., Scarborough R. M., Carney R. L. and Schooley D. A. (1984) Metabolism of proctolin,
Degradation of adipokinetic hormones a pentapeptide neurotransmitter in insects. Life Sci. 34, 569-576. Rayne R. C. and O'Shea M. (1992) Inactivation of neuropeptide hormones (AKH I and AKH II) studied in vivo and in vitro, lnsect Biochem. Molec. Biol. 22, 25-34. Siegert K. J. and Mordue W. (1986) Quantification of adipokinetic hormones I and II in the corpora cardiaca of Schistocerca gregaria and Locusta migratoria. Comp. Biochem. Physiol. 84A, 279-284. Siegert K. J. and Mordue W. (1987) Breakdown of locust adipokinetic hormone I by Malpighian tubules of Schistocerca gregaria. Insect Biochem. 17, 705-710. Siegert K. J. and Mordue W. (1992a) Content and localization of the hyperglycaemic hormones I and II in the corpus cardiacum of the American cockroach, Periplaneta americana. Comp. Biochem. Physiol. 101B, 315 319. Siegert K. J. and Mordue W. (1992b) Preliminary characterization of Malpighian tubule enzyme activities involved in the breakdown of adipokinetic hormones. Archs Insect Biochem. Physiol. 19, 147-161. Siegert K. J., Krippeit P. and Ziegler R. (1982) Regulation
663
of fat body glycogen phosphorylase during starvation in larvae of Manduca sexta (Lepidoptera: Sphingidae). Acta endocr. Suppl. 246, 99, 33-34. Siegert K. J., Morgan P. J. and Mordue W. (1985) Primary structures of locust adipokinetic hormones II. Biol, chem. Hoppe-Seyler 366, 723-727. Siegert K. J., Morgan P. J. and Mordue W. (1986) Isolation of hyperglycaemic peptides from the corpus cardiacum of the American cockroach, Periplaneta americana. Insect Biochem. 16, 365-371. Skinner W. S., Quistad G. B., Adams M. E. and Schooley D. A. (1987) Metabolic degradation of the peptide periplanetin CC-2 in the cockroach, Periplaneta americana. lnsect Biochem. 17, 433-437. Suzuki K. and Kono T. (1979) Internalization and degradation of fat-cell bound insulin. Separation and partial characterization of subcellular vesicles associated with iodoinsulin. J. biol. Chem. 254, 9876-9794. Terris S. and Steiner D. (1975) Binding and degradation of I-insulin by rat hepatocytes. J. biol. Chem. 250, 8389 8398.