Results Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol30.Issue10.Oct2000:

956 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 reverse translated. The forward primer, PPO-FS 59- CAY CAY TKB CAY TGG CAY YTN GT-39 targets HH WY HWHLVY Copper binding domain and the reverse primer PPO-RAS 59-CKR TCR AAN GGR WAN CCC AT-39, MGYPFDR conserved carboxy domain. 2.9. Reverse transcription–polymerase chain reaction RT–PCR Independent RT reactions Superscript II Gibco BRL with 5 µ l of 15 or 110 dilutions of S. bullata total RNA eluted from the RNeasy column and primed with RT-1 59-CTC TGG GCC CAA GCT TTT TTT TTT TTT TTV-59 were set up following the manufacturer’s proto- col. All RT reactions were incubated at 42 ° C. PCR reac- tions were set up using 5 µ l of 15, 110 and 150 dilutions of the RT reaction with PPO-FS and PPO-RAS. The reactions were set up as follows: 5 µ l template, 5 µ l 10 PCR buffer Promega 2.5 mM MgCl 2 , 200 µ M each dNTP, 10–20 pM each primer, 1 unit Taq Poly- merase Promega in a total volume of 50 µ l. The reac- tions were then heated to 94 ° C for 2 min, manually adding the 1 unit of TAQ to each reaction and cycled at 94 ° C 1 min, 54 ° C 1 min, 72 ° C 1 min, 35 times. A DNA fragments was cloned into a Pgem T-easy Promega and transformed into XL-1 Blue MRF9 elec- trocompetent cells Stratagene. Colonies were allowed to grow overnight and screened by contaminating a PCR reaction containing M13 forward and reverse primers with a single colony. Replicate clones were plated and grown for 5 h at 37 ° C. Selected colonies based on PCR results were grown overnight in 10 ml of LB media, purified and sequenced on an ABI model 377 sequencer, using a dye terminator kit Perkin Elmer Applied Biosys- tems, Foster City, CA. 2.10. 3 9 race Sequence data from clones with homology to other insect PPOs were then used to design nested primers of about 250–300 bases from the 39 end of the initial frag- ment. PCR reactions were set up as described above, but pairing with a gene specific primer and RT-1, using template from the initial RT reaction primed with RT-1. 2.11. 5 9 race To obtain the 59 end of each DNA fragment, two nested anti-sense primers were designed 200–250 bp away from the 59 end of the clone. Independent RT reac- tions were primed with the outer most gene specific primer and dCTP tailed with terminal transferase sup- plied in the GIBCOBRL 59RACE kit. The PCR con- ditions are the same as above, but the cycling profile was changed as follows: The anneal temperature was decreased to 48 ° C for five cycles, then increased to 60 ° C for the 30 remaining cycles. If the PCR product was heterogeneous, another round of amplification was car- ried out with the second nested primer. The DNA frag- ments were gel purified with a Gel Purification kit QIAGEN and cloned into a T-vector and sequenced. 2.12. Amplification and sequencing of entire coding domain To ensure the integrity of each identified gene, we amplified the entire coding domain. Primers were designed to the 39 and 59 untranslated regions of each gene. PCR conditions were as previously described, but the anneal temperature was altered to accommodate each primer pair and the extension time was increased to 90 s. PCR products were purified and ligated into a T-vector. Primers were designed from the original sequence data at 500 base intervals and three independent clones sequenced. 2.13. Sequence assembly and analysis Contigs for each sequence was assembled and edited with the program Sequencher Gene Codes Corporation. Sequencing primers were designed with the program Primer3 http:www.genome.wi.mit.educgi- binprimerprimer3 Fwww.cgi. Each sequence was com- pared to other sequences in GenBank with a Blastx search. Prophenoloxidase amino acid sequences were downloaded from GenBank in fasta format and aligned with Clustal W using default parameters except for changing output format to Phylip. This file was imported to PAUP Swofford, 1999 4.0b2 and used to calculate pairwise identities.

3. Results

Employing the protein purification scheme outlined in Table 1, PPO from the hemolymph was purified 221- fold with a recovery of 16. The recovery of total activity was drastically reduced at the ammonium sulfate step due to the presence of proteins that interfere with quinonoid metabolism and high protein concentrations. However, during the succeeding steps, such interference is removed and the recovery of total activity units is restored. The finally purified PPO exhibited two closely moving bands on SDS–7 polyacrylamide gel Fig. 1. Both bands when cut and incubated with dopamine and CPC, exhibited PO activity. Without CPC treatment, neither of them showed any detectable phenoloxidase activity, thereby indicating that they are isozymes of PPO. 957 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 Table 1 Purification chart for prophenoloxidase Step Total volume ml Total units Total protein mg Specific activity Recovery Fold purification unitsmg Crude 25 6250 1200 5.2 100 1 NH 4 SO 4 2.8 1092 420 2.6 17.5 5 Phenyl sepharose 122 4148 152.5 27.2 66.4 5.2 DEAE-sepharose 30 2760 33 83.6 44.2 16.1 Sephacryl S-200 22 1012 0.88 1150 16.2 221 Fig. 1. SDS–PAGE of PPO from S. bullata. SDS–PAGE was perfor- med on 7.5 gel as outlined in Materials and methods. Proteins were stained with silver staining. 3.1. Molecular weight Fig. 2 shows the calibration curves used for the esti- mation of the molecular weight of PPO. On Sephacryl S-100 or Sephacryl S-200 gel filtration column, it eluted with an apparent molecular weight of 100,000 [Fig. 2a]. On HPLC, it exhibited a molecular weight of 85,000 [Fig. 2b]. Under denaturing conditions on SDS–PAGE, it migrated as a single band with a molecu- lar weight of 90,000 [Fig. 2c]. Thus the native proen- zyme seems to be made up of a single polypeptide chain with no apparent subunits. 3.2. Activation of PPO Fig. 3 inset shows the activation of PPO caused by different detergents. As is evident only the cationic detergent, CPC activated the proenzyme specifically and neither anionic detergents such as SDS, sodium capry- late, sodium lauryl sarcosidate, deoxycholate, nor non- ionic detergents such as octyl- β -glucopyranoside, Non- idet-P-40, Triton X-100, digitonin, Brij 58 and Tween 20 activated the proenzyme. Similarly, zwitterionic detergent, CHAPS also failed to activate the enzyme. The activation caused by CPC occurred well below the Fig. 2. Molecular weight estimation of sarcophagid PPO. a By gel filtration chromatography. A Sephacryl S-100 column 55 × 2 cm equi- librated with 10 mM sodium phosphate buffer pH 6.0 containing 200 mM sodium chloride was used at a flow rate of 12 mlh. Molecular weight markers used to calibrate the column are: A carbonic anhyd- rase 29 kDa; B ovalbumin 43 kDa; C Phosphorylase b 96 kDa; and D alcohol dehydrogenase 150 kDa. b By size exclusion HPLC. A Beckmann TSK 3000 column 30 cm × 7.5 mm was used to estimated the approximate molecular weight of PPO. Proteins were separated on the column using isocratic elution with 100 mM sodium phosphate buffer pH 6.0 at a flow rate of 0.7 mlmin. Molecular weight markers used are: A carbonic anhydrase 29 kDa; B bovine serum albumin 66 kDa; C alcohol dehydrogenase 150 kDa; D Myosin 200 kDa; E apoferritin 443 kDa and thyroglobulin 669 kDa. c By SDS–PAGE on 7.5 gel. Conditions are outlined in Materials and methods. Pre-stained molecular weight markers were used to calculate the approximate molecular weight of PPO. A carbonic anhydrase 29 kDa; B ovalbumin 43 kDa; C Phosphorylase b 96 kDa; and D alcohol dehydrogenase 150 kDa. 958 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 Fig. 2. continued Fig. 3. Activation of PPO by different concentrations of CPC. PPO was assayed using standard assay conditions except for variation in the concentration of CPC. Inset: activation of PPO by different detergents. Standard assay conditions were used except for varying the detergents. Detergents used are: a CPC; b SDS; octylglucopyranoside; Nonidet P 40; Brij 35; Tween 20; CHAPS; sodium lauryl sarcosidate; sodium caprylate; Triton X-100; deoxycholate or digitonin. critical micellar concentration of CPC. From the dose response curves shown in Fig. 3, the K a for the activation of PPO by CPC was estimated to be 40 µ M. The critical micellar concentration of CPC under these conditions is 0.8 mM. Therefore, CPC must be binding to the proen- zyme specifically below its critical micellar concen- tration and activating the enzyme. This is in confirmation with our previous result on the activation of Manduca PPO Hall et al., 1995. Phospholipids and fatty acids have been shown to be 959 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 potent activators of PPO Sugumaran and Nellaiappan, 1991; Sugumaran and Kanost, 1993. In order to check whether sarcophagid PPO can also activated by these reagents, the following studies were carried out. Various phospholipids and fatty acids have been incubated with PPO and the appearance of PO activity in the incubation mixture was assessed by standard PO assay. As shown in Fig. 4, fatty acids caused either mild or no activation at all. Among the different phospholipids tested, phos- phatidyl glycerol activated the PPO immediately, while phosphatidyl inositol activated the PPO after a lag. 3.3. pH optimum and thermal stability The activated PO exhibited a typical bell shaped pH curve with optimum activity observed at about pH 7 Fig. 5. Fig. 6 shows the comparative stability of pro- phenoloxidase and detergent activated phenoloxidase. The activated enzyme rapidly lost its activity even at room temperature 25 ° C within 15 min curve C, while the raising the temperature to 55 ° C resulted in total loss of its activity within one min curve D. In contrast, the proenzyme was much more stable towards heat treat- ment Curves A and B, Fig. 6. Heating the proenzyme for 10 min at 60 ° C resulted in 50 loss of its activity and heating at 70 ° C for the same time resulted in 100 loss of its activity data not shown. 3.4. Substrate specificity The substrate specificity of activated phenoloxidase is shown in Fig. 7. As is evident, N-acetyldopamine, cat- echol, 4-methyl catechol, norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, and N- β -alanyldopam- Fig. 4. Activation of PPO by different phospholipids. PPO was assayed using standard assay conditions except for using different lip- ids in the place of CPC. The lipids were dissolved in 100 µ l of ethanol and made up to 1 ml with water concentration of lipids = 5 mgml. An aliquot 6 µ l of this solution, was used to test the activation of PPO. Compounds used are A phosphatidyl glycerol; B phosphatidyl inositol; C phosphatidyl ethanolamine; D nondecanoic acid; E pal- mitic acid; F lauric acid; G stearic acid; H linoleicoleic acid and I phosphatidyl choline. Fig. 5. pH optimum of sarcophagid PPO. The activity of PO was determined at indicated pH values using the standard assay conditions. Sodium acetate buffer 50 mM was used from pH 3 to 4.5 No activity was detected from pH 3 to 4. Sodium phosphate buffer 50 mM was used from pH 5 to 9. Fig. 6. Thermal stability of PPO and PO. The thermal stability of PPO and PO were determined by incubating the PPO and CPC acti- vated PO at indicated temperature for varying amounts of time. After cooling to room temperature, the residual activity was determined using standard assay conditions. A stability of PPO at room tempera- ture; B stability of PPO at 55 ° C; C stability of PO at room tempera- ture; D stability of PO at 55 ° C. ine proved to be better substrates than dopa, which is routinely used for the assay of various phenoloxidases. In general, unlike mammalian tyrosinases, insect phenol- oxidases prefer dopamine better than dopa Barrett, 1991; Sugumaran, 1998. The marginal activity towards hydroquinone, methyl hydroquinone and gentisic acid indicates that the enzyme is a typical o-diphenoloxidase 960 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 Fig. 7. Substrate specificity of sarcophagid phenoloxidase. Standard assay conditions were employed except for variation of substrates instead of dopamine as indicated. Oxygen uptake assay was used. Sub- strate used are: A N-acetyldopamine; B 4-methylcatechol; C N- β -alanyldopamine; D dopamine; E 3,4-dihydroxyphenylacetic acid; F catechol; G norepinephrine; H 3,4-dihydroxymandelic acid; I dopa; J tyrosine methyl ester; K 3,4-dihydroxybenzoic acid; L hydroquinone; M gentisic acid; and base line = methylhydroquinone. and not a laccase type enzyme. The enzyme seems to possess monophenol monooxygenase activity as evi- denced by its ability to attack tyrosine methyl ester. 3.5. Inhibition studies Typical phenoloxidase inhibitors such as phenylthio- urea and diethyldithiocarbamate inhibited the activity of the enzyme drastically Table 2. Other copper chelators also showed marked inhibition of phenoloxidase activity. Iron chelators such as o-phenanthroline and α , α 9-dipyri- dyl failed to inhibit the enzyme. Interestingly, mimosine, which is a structural analog of dopa, and a typical inhibi- tor of tyrosinase, showed marginal inhibition only. This further emphasizes the superiority of dopamine over dopa as a substrate for insect PO. 3.6. Molecular cloning Using PCR amplification with degenerate primers, we have identified two distinct PPO genes in the hemocytes Table 2 Inhibition of detergent activated Prophenoloxidase Inhibitor Concentration Inhibition Phenylthiourea 10 µ M 100 1 µ M 93 0.3 µ M 90 0.1 µ M 74 Diethyldithiocarbamate 90 µ M 100 70 µ M 92 50 µ M 84 10 µ M 49 Sodium cyanide 0.5 mM 100 0.1 mM 64 Mimosine 5 mM 26 1 mM 20 Neocuprine 5 mM 74 1 mM 38 8-Hydroxyquinoline sulfonate 1 mM 100 0.1 mM 33 o -Phenanthroline 5 mM α , α 9-Dipyridyl 1 mM 14 Sodium fluoride 5 mM Ethylenediamine tetra acetate 5 mM 23 1 mM 19 of S. bullata larvae. The sequence of cDNA encoding sarcophagid PPO 1 and 2 are shown in Figs. 8 and 9, along with their deduced amino acid sequence. The cDNA for PPO-1 is 2268 bp while that for 2 is 2246 bp. The PPO cDNA-1 coded for a protein of 685 amino acids with a molecular weight of 79,088, while 2 codes for a protein of 691 amino acids with a molecular weight of 79,797. There is no evidence for the presence of the hydrophobic sorting signal sequence for the endoplasmic recticulum in any of the PPOs. Comparison of deduced amino acid sequence of the PPO 1 and 2 with other arthropod PPOs in GenBank Fig. 10 shows that the two copper binding sites are preserved in all these proteins underlined sequences. Both copper binding sites show extensive homology. The six histidine residues, which ligate the two copper atoms, are present at the conserved sites in all the phe- noloxidases. The proteolytic cleavage site RF, indicated by an arrow is conserved in all dipteran PPOs. A second possible proteolytic cleavage site, REE, also designated by an arrow is conserved in all dipteran PPOs with the exception of sarcophagid PPO 2 which has RAE and Drosophila PPOA1 which has RQE at this site. In addition, previously characterized putative thiol ester site CGCGWPQHML double underlined Hall et al., 1995 is preserved in SbPPO1 in its entirety, but the SbPPO2 has E instead of Q at position 7. At the C-terminal region, there is a conserved region in all the PPOs marked by asterisks. In particular, the motif MGYFPFDR is present in all known PPOs. 961 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 Fig. 8. Nucleotide and the deduced amino acid sequences of SbPPO 1 from S. bullata. GenBank accession No. AF 161260. 962 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 Fig. 9. Nucleotide and the deduced amino acid sequences of SbPPO 2 from S. bullata. GenBank accession No. AF 161261. 963 M.R. Chase et al. Insect Biochemistry and Molecular Biology 30 2000 953–967 Fig. 10. Alignment of known dipteran PPO sequences. The two copper binding domains in the amino acid sequence are underlined. Arrows indicate the two putative proteolytic cleavage sites. The putative thiolester sites are double underlined. Indicate the conserved C-terminal site. GenBank accession numbers are listed in Table 3.

4. Discussion