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Comparative studies on the acid proteinase activities in the digestive fluids of Nepenthes, Cephalotus, Dionaea, and Drosera PDF

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Preview Comparative studies on the acid proteinase activities in the digestive fluids of Nepenthes, Cephalotus, Dionaea, and Drosera

TechnicalRefereed Contribution Comparative studies on the acid proteinase activities in the DIGESTIVE FLUIDS OF NEPENTHES, CEPHALOTUS, DlONAEA, AND DROSERA Kenji Takahashi • KojiMatsumoto • Wataru Nishii • Miho Muramatsu • Keiko Kubota • Laboratory of Molecular Biochemistry, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences • 1432-1 Horinouchi, Hachioji • Tokyo 192-0392 • Japan ChiakiShibata•DepartmentofBiology,TheNipponDentalUniversity•Chiyoda-ku•Tokyo 102-8159•Japan Senareth B.P. Athauda • Department of Biochemistry • Faculty of Medicine • University of Peradeniya • Peradeniya • Sri Lanka Keywords: chemistry: Nepenthes, Cephcilotus, Dionaea, Drosera Received: 10 December 2007 Introduction There are various kinds ofcarnivorous plants innature, andmostofthemexcrete acidic digestivefluids which contain digestive enzymes, especially acidic proteinases, to digest trapped insects and other prey for nutrition (Juniperetal. 1989). Previous inhibitor studies (Lobareva et al. 1973; Takahashi etal. 1974; Tokes et al. 1974) indicated that the acid proteinase nepenthesin fromNepenthes belongs to the aspartic proteinase family. Recently, we purified two nepenthesins from the digestive fluid ofN. distillatoria, investigated their molecularandenzymaticproperties, andelucidatedtheirprimary structuresbycDNAcloningofnepenthesins fromN. gracilis (Athauda etal. 1998, 2002, 2004; Takahashi etal. 2005). The peptide bond cleavage speci- ficity of nepenthesin was investigated using N. alata pitcher fluid (Ann et al. 2002), partially purified nepenthesin from a mixture of several Nepenthes species (Amagase et al. 1969) and from N. macfarlanei (Tokes etal. 1974), andfully purifiednepenthesinfromN. distillatoria (Athauda etal. 2004; Takahashi etal. 2005). In addition, some enzymatic properties ofthe acid proteinase activities in the crude digestive fluids of Nepenthes sp. (Liittge, 1964) and Dionaea muscipula (Scala et al. 1969; Robins & Juniper 1980) and a partially purified acid proteinase from Droserapeltata (Amagase etal. 1972a, 1972b) were reported. Except for these studies, however, not many studies have been performed on the enzymatic properties ofthese acid proteinases. Therefore, further studies are necessary to understand more extensively the nature of the acid proteinases inthe digestive fluids ofcarnivorous plants. Since the digestion takes place inthe crude digestive fluid, it is thought to be important to characterize the acid proteinase activity as a whole as well as to purify and characterize the individual proteinases. Thus, we have investigated in the present study some enzymatic properties ofthe acidproteinase activities inthe crude digestive fluids oftypical carnivorous plants including two pitcherplants. Nepenthes alata and Cephalotusfollicularis, one plant with a snap-trap, Dionaea muscip- ula (Venus flytrap), and oneplantwithamucilagetrap,Drosera capensis (sundew), in acomparativemanner. The results demonstrated significant differences among them, presumably reflecting the phylogenic diversity ofthese carnivorous plants. Materials and Methods ThecrudedigestivefluidsofNepenthesalataandCephalotusfolliculariswerecollectedintheDaishoen plantation (Numazu). Dionea muscipula and Drosera capensis were obtained from the Daishoen plantation and grown at thebotanical garden ofTokyo University ofPharmacy and Life Sciences to obtain their digestive fluids. The digestive fluidofDionaea was collected 3 to4days aftergiving apiece (about 3-5 x 3-5 x2-3 mm) ofboiled egg white, thereby inducing closure ofthe two lobes by stimulating the triggerhairs. The digestive fluid ofDrosera was collected by soaking ten leaves successively (1 leaf for 1 min at a time) in 800 pi of M 0.1 sodiumacetatebuffer,pH4.0,inatesttubetowashoutthedigestivefluidthroughup-and-downstrokes. ThepHwas determined using aglass electrodein aHoribapH-meter. The samples werekeptfrozen at -20°C Volume 38 September 2009 75 until use. PorcinepepsinA, bovine hemoglobin, the B chain ofoxidizedbovine insulin and leucyl-4-methyl- coumaryl-7-amide (Leu-MCA) and arginyl-4-methylcoumaryl-7-amide (Arg-MCA) were obtained from Sigma andpepstatinA from Peptide Institute, Osaka. Otherreagents used were ofanalytical grade. Theproteinaseactivities ofthedigestivefluids ofNepenthes, Cephalotus, andDionaeaweredetermined withbovine hemoglobin as a substrate at pH 2.0 as described (Athauda etal. 1989), and expressed in pepsin equivalent using porcine pepsin A (Sigma) as a standard. The proteinase activity of the digestive fluid of Drosera capensis in the hemoglobin assay was markedly low as compared with those of the other three. ThereforeoxidizedinsulinB chainwas usedasasubstrateandthedigestionproductswereanalyzedbyHPLC. The assaymixture was composedof 1-40 pi ofenzyme solution, 10 piofoxidizedinsulin B chain (1 mg/ml), M 50 pi of0.1 sodium acetate buffer, pH 4.0, an appropriate volume (0-39 pi) ofwater in a total volume of M 100 pi, and the digestion was performed at 37°C for 1 h and terminated by the addition of 100 pi of0.5 H3BO3-KCI/NaOII buffer,pH9.8.A 100-plaliquotfromthereactionmixturewas analyzedbyreverse-phase HPLC using a Shimadzu LC10A system on a Tosoh ODS-120T column (4.6 x 250 mm) eluted with an % acetonitrile gradient (0 to 60%) in 0.1 trifluoroacetic acid at a flow rate of 1.0 ml/min and monitored at 215 nm. The activity was estimatedfromthe decreaseinthe amountofthe substrate, and expressed inpepsin equivalent determined using porcine pepsinA as a standard underthe same assay conditions atpH 4.0. AminopeptidaseactivitiesweredeterminedwithLeu-MCAandArg-MCAassubstrates.Theassaymixture mM contained 5 pi (Nepenthes), 200 pi (CephalotMus), or 10 pi (Dionaea) of enzyme solution, 5 pi of 10 substrate in dimethylsulfoxide, 310 pi of0.1 citric acid-Na2HP04 buffer, pH 3.0-6.0, and an appropriate volume (0-195 pi) ofwaterin a total volume of515 pi. Thedigestion was performed at 37°C for30 min and stopped by the addition of 2.5 ml of 5% trichloroacetic acid. The amount of 7-amino-4-methylcoumarin liberated was measured in aHitachi spectrofluorometer with excitation at 380 nm andemission at460 nm. Native electrophoresis followed by activity staining was performed as follows. An appropriate portion (20 pi)ofeachpitcherfluidwassubmittedtonativepolyacrylamidegelelectrophoresis using 10% acrylamide gel andTris-glycine buffer, pH 8.7, andthenproteinase activity wasexaminedby activity staining withhemo- globin as a substrate at pH 1.7 essentially as described (Furihataetal. 1972). To investigate the substrate specificity, oxidized insulin B chain was used as a substrate. The reaction mixture contained 10 pi (Nepenthes), 40 pi (Cephalotus), or 3 pi (Dionaea) ofthe digestive fluid containing about 1 pmol enzyme as porcine pepsin equivalent, 10 pi of oxidized insulin B chain (1 mg/ml), 50 pi of 0.1 m sodium formate buffer, pH 3.0, and an appropriate volume (0-37pl) of water in a total volume of 100 pi.Thedigestionwas performedat 37°Cfor3 h, andtheresultingpeptideswereanalyzedusing anHPLC apparatus (1100 series,AgilentTechnology) with aTSKgel ODS-120Tcolumn (2.2 x 150 mm) connected to anLC™-DUOmass spectrometer(ThermoQuest).Amino acidsequencesofthepeptidesproducedweredeter- mined from the mass spectra ofthe original and fragmented ions by using Xcalibur Bioworks 1.0 software installed in the apparatus as described (Nishii etal. 2002). Results and Discussion The collected fluid samples ofNepenthes alata Cephalotusfollicularis, and Dionaea muscipula had a , pHof2.9, 2.9, and3.9,respectively, andwere showntocontainapproximately 10pmol, 2pmol, and40pmol, respectively, ofacidproteinase in pepsinequivalentper 100 pi as determined with hemoglobin as a substrate. Ontheotherhand, thedigestivefluidofDroseracapensishadapHofabout2.5, andthewashateoftenleaves with 800 piofthepH4.0buffercontainedapproximately 100pmolofacidproteinaseinpepsinequivalentper 100 pi as determined with oxidized insulin B chain as a substrate. Figure 1 showstheactivitystainingafternativepolyacrylamidegelelectrophoresis(PAGE)ofthecrudediges- tivefluids. Twobroadbands areseenfortheNepenthessample; themajor, slow-movingbandshouldcorrespondto nepenthesinIandtheminor,fast-movingbandtonepenthesinII.Thesebroadbandsmaycontainmorethenoneacid proteinase isozyme as shown for nepenthesins I and II fromN. gracilis andN. distillatoria (Athauda et al. 2004; Takahashietal. 2005).TheDionaeasample gaveasinglebroadbandmovingalmostinparallelwithnepenthesinI, butthebandcorrespondingtonepenthesinIIwashardlyseen.Themajorbandmaycontainmorethanone acidpro- teinase component. Indeed, it was reported previously that two majorbands were detected on native PAGE ofthe digestivefluidofDionaeamuscipula(Robins&Juniper 1980). InthecaseofCephalotus,therearetwobandscom- parable withthe nepenthesinbands,but the majorbandcorrespondingto nepenthesinIhadhighermobility toward cathode.Thus,theelectrophoreticpatterns aresimilarbutnotidenticalamongthethreesamples.Theanalysisofthe Droserasamplegavenoclearbandpresumablyduetothelowsensitivityoftheenzymetowardhemoglobin(datanot shown). 76 Carnivorous Plant Newsletter Figure 2 shows the pH profiles ofthe enzymatic a b c Figure 1: activities. Both the Nepenthes and Cephalotus Native polyacrylamide samples had the pH optimum at around 2.5, whereas the Dionaea and Drosera samples showed the pH gel electrophoresis with optimum at 3.0 and 3.5, respectively. Theresult with proteinase activity theNepenthes sample is similarto the pH profiles of staining of the digestive nepenthesins I and II from N. distillatoria having an fluids of carnivorous optimum at pH 2.6 and that of the crude fluid from plants, (a) Nepenthes N. distillatoria with an optimum at pH 2.8 (Athauda alata, (b) Dionaea mus- etal. 2004; Takahashi etal. 2005).As comparedwith cipula, (c) Cephalotus the pH profile ofthe Cephalotus sample, those ofthe follicularis. other three had broader pH profiles, which might mean that they are composed of more than one enzyme component with fairly different pH optima. As for the Dionaea digestive fluid, the pH-activity profile with casein as a substrate was reported to show two peaks at pH4.0 and 5.0 (Robins & Juniper 1980) oronesharppeakatpH 5.3 (Scalaetal. 1969). Several pH optima (Chandler & Anderson 1976) or an irregularpH profile (Clancy & Coffey 1977) with casein as a substrate were reported for the digestive fluids ofDrosera species. The reason for the differ- ences between the present results and those reported previouslyisnotclearatpresent, butmightbe atleast partly due to the difference in the assay conditions, especially in the substrate used. The temperature dependence ofactivity ofeach sampleis shownin Figure 3a. TheNepenthes sample showed a temperature-activity profile with a maximum at about 57°C. This is similar to that of nepenthesin I from N. distillatoria which has a maximum at 55°C, but the maximum temperature was higherthan thatofthe crudefluidfromN. distil- latoria with a maximum at 50°C (Athauda et al. 2004; Takahashi etal. 2005). Theresults areroughly similar to that reported for the digestive fluid of N. khasiana (Liittge 1964). On the other hand, the Cephalotus Dionaea andDrosera samples showeda , , temperature-activityprofilewithamaximumat47°C, Figure 2: 47°C, and 42°C, respectively, which are apparently pH dependence of the proteinase activities of similar to that ofnepenthesin II from N. distillatoria the digestive fluids of carnivorous plants. (Athaudaetal. 2004;Takahashietal. 2005).Thepro- file ofthe Dionaea sample has a shoulder at around Closed circle, Nepenthes alata; open circle, 60°C,whichmightindicatethepresenceofmorethan Cephalotus follicularis; closed triangle, one component with fairly different temperature Dionaea muscipula; open triangle, Drosera dependence ofactivity. capensis. The same symbols are used in Theresultsoftemperature-stabilityexperiments Figures 3-5. Buffers used were 0.1 M HCI/KCI are shown in Figure 3b, in which each crude sample buffers (pH 1.1 to 2.0) and 0.1 M citric was incubated at different temperatures for 1 h, then acid/Na2HP04 buffers (pH 2.5 to 6.5) except the remaining activity was determined. As for the M for Drosera for which 0.1 HCI/KCI buffers Nepenthes sample, the activity was stable up to M around 53°C, then started to decrease and was lost (pH 0.7 to 1.4), 0.M1 glycine/HCI buffers completely ataround 80°C. This result was similarto (pH 2.6 to 3.5), 0.1 potassium acetate/HCI those obtained withnepenthesin I andthe crude fluid buffers (pH 4.0 to 5.4) and 0.1 M fromN. distillatoria (Athauda etal. 2004; Takahashi KH2P04/Na0H buffers (pH 6.1 to 6.6) were et al. 2005). A similar result was obtained with the used. Volume 38 September 2009 77 ) Droserasample. Ontheotherhand,theactivityofthe Cephalotussample wasless stable. Itwas stableupto 42°C, where it started to decrease to zero at around 70°C. Interestingly, the Dionaea sample was appar- ently morestablethantheNepenthes sample at above 55°C. Thus,theTmvalues wereapproximately 63°C, 59°C, 58°C, and47°C, respectively, fortheDionaea , Drosera Nepenthes, and Cephalotus samples. The , temperature-stability profile of the Dionaea sample was different from those of the other three. The activity was stable at around 50°C, but above this temperature the inactivation appeared to proceed in TEMPERATURE(°C) two steps, suggesting the presence of at least two enzyme components different in heat stability. These Figure 3: results are consistent with the temperature depen- Effects of temperature on the proteinase dences ofthe activities shown in Figure 3a. activities and stabilities of the digestive flu- Figure 4 shows the changes of the acid pro- ids of carnivorous plants, (a) The activity teinaseactivitiesuponlongerincubationat37°C.The was measured at various temperatures at activity oftheNepenthes sampledecreased only very pH 2.0 except for the Drosera sample slowly on longer incubation; about 60% and 30% of which was assayed at pH 4.0. (b) Each the original activity were still retained after 30 days sample was incubated at various tempera- and 65 days of incubation, respectively. Thus, the tures and pH 2.9 Nepenthes and activityofthecrudedigestivefluidofN. alata is con- ( siderably stable under the conditions used. However, Cephalotus), 3.9 Dionaea or 4.0 ( ), under similar conditions, that of N. distillatoria (Drosera for 1 h at an enzyme concentra- retained nearly full activity after 30 days of incuba- tion of approximately 5-10 pmol pepsin tion(Athaudaetal. 2004;Takahashi etal. 2005).This equivalents per 200 (tl and then assayed difference might be due to the difference in the as above. Nepenthes species used and/or the difference in the experimental conditions. Incontrastto theNepenthes sample, the activity of the Cephalotus sample was rather unstable; nearly 50% and 70% of the original activitywerelostin 1 hand 15 h,respectively. Onthe other hand, the Dionaea sample appeared to be con- siderably more stable than the Cephalotus sample, but somewhat less stable than the Nepenthes sample in the early phase ofincubation; about40% and 25% of the original activity were lost in 3 h and 65 h, respectively. As can be seen from Figure 4, the profilesoftheactivity changeofboth Cephalotus and Dionaea samples appeared to be biphasic. This suggests that each of these samples contains a rela- tively unstable component and a more stable compo- nent. The latter component inDionaea appears to be TIME OFINCUBATION (days) comparable in stability with the Nepenthes sample. Figure 4: The experiment with the Drosera sample has not yet beenperformed. Stabilities of the proteinase activities of the TheeffectsofpepstatinAontheactivityofeach digestive fluids of carnivorous plants on long sample are shown in Figure 5. Under the conditions incubation. The activity was measured at used,theactivitiesofallsamples appearedtobenear- 37°C after incubation of each sample for ly halflost in the initial phase at a low pepstatin con- various periods at 37°C and pH 2.9 centration(uptoabout 1 iiM).Athigherconcentration (Nepenthesanti Cephalotus) or3.9 Dionaea of pepstatin, the Nepenthes and Cephalotus samples ( ) at an enzyme concentration of approximately were inhibited further, but more weakly, and nearly 5-10 pmol pepsin equivalents/200 |xl. The complete inhibition occurred with 120 uM of the experiment with Drosera was not performed. inhibitor. On the other hand, the activity of the 78 Carnivorous Plant Newsletter Dionaea samplewasnotinhibitedcompletely; theactivity was halfinhibited atalow concentrationofpepstatin A, but the remaining about 50% ofthe activity was almost insensitive to pepstatinA and remained uninhibited M inthepresenceof100 11 inhibitor. Incontrast,theDroserasamplewasinhibitedcompletelyatapepstatincon- centrationof5 uM. Theseresultsindicate thefollowing. First, theNepenthes and Cephalotussamples eachmaycontaintwo kinds ofenzymes with ahigher and a lower affinity to pepstatin. Indeed, nepenthesins I and II ofN. distilla- toria have been shown to have different affinity to pepstatin (Athauda et al. 2004; Takahashi et al. 2005). Second, the Dionaea sample may contain a pepstatin-insensitive acid proteinase in addition to the nepenthesin-like pepstatin-sensitive acid proteinase. This pepstatin-insensitive enzyme has not yet been identified, but might be similar to glutamic peptidases such as aspergilloglutamic peptidase (Huang et al. 2000;Yabuki etal. 2004; Sasaki etal. 2004) and scytallidoglutamic peptidase (Fujinaga etal. 2004; Kataoka etal. 2005) orserine-carboxylpeptidases such asphysarolisin(Nishii etal. 2003a, 2003b) andpseudomonal- isin (Wlodawer et al. 2001), each of which is known to be insensitive to pepstatin but has an acidic pH optimum. To our knowledge, such an enzyme as a glutamic peptidase or serine-carboxyl peptidase has not beenfoundsofarinplants.Theoccurrenceofatleasttwodifferenttypesofacidproteinasesisconsistentwith the biphasic character ofthe Dionaea proteinase activity observed already. The pepstatin-insensitive activity in the Dionaea sample appeared to be less stable than the pepstatin-sensitive activity when the enzyme sample was storedat4°C. Incontrast to the present results, the acidproteinase activity in theDionaeadigestive fluid was previously reported to be insensitive to pepstatin (Robins & Juniper 1980). The reason for this difference is not certain at present, but might partly be due to the occurrence of two different types of acid proteinases.Third, theDroserasamplemaycontainonlyacidproteinaseswith arelativelyhigherpepstatinaffinity. The peptide bond cleavage specificities of the acid proteinases in the Nepenthes Cephalotus, and , Dionaea samples as examined withoxidizedinsulin B chain as a substrate are shown inFigures 6 through 8. IntheNepenthes sample, themajorcleavages occurredatLeu15-Tyr16 and Phe24-Phe25 andmoderatecleavages , at Glu13-Ala14,Alal4-Leu15, Tyrl6-Leu17, andTyr26-Thr27. These results are roughly similarto those reportedpre- viously for the crude digestive fluid ofN. alata (Ann etal. 2002) and the purified nepenthesin I fromN. dis- tillatoria (Athaudaetal. 2004; Takahashi etal. 2005). Inthe lattercase,Leu6-Cys(ox)7bond was cleaved to a significantextent, whichmightbedue to species orisozymic difference. In the Cephalotus sample, themajor cleavagesoccurredatLeul5-Tyr16 Phe24-Phe25 andLys29-Ala30 andmoderatecleavages atGlu'3-Ala14 andAla14- , , , Leu15. In the case ofthe Dionaea sample, the major cleavages occurred at Glul3-Alal4, Leul5-Tyrl6, and Phe24-Phe25, and moderate cleavages at Ala14-LeuL\ Tyr16-Leu17, Gly23-Phe24, and Lys29-Ala30. Thus, the major cleavages at Leu15-Tyr16 and Phe24-Phe25 were com- mon, but the other cleavage sites varied significantly among the three samples. Especially, the Cephalotus andDionaea samples differfrom theNepenthes sam- pleinthatLys29-Ala30andGlu13-Ala14 respectively,are , also the major cleavage sites. These differences shouldreflectthedifferenceinthe cleavage specifici- ty ofthe enzyme components ineach sample. Aminopeptidase activities in the acidic pH range were determined using Leu-MCA and Arg- MCA as substrates. Neither activity was detected with Nepenthes and Cephalotus in the pH range of 3-6 and 3-5, respectively. As for Dionaea, a trace activity towardLeu-MCA was detectedbutno activity towardArg-MCA in the pH range of3-6. Therefore, practically noaminopeptidaseactionis assumedtobe PEPSTATIN (p,M) involved in the cleavages ofoxidized insulin B chain Figure 5: observed in the present study. On the other hand, the activities of carboxypeptidases were not analyzed in Effects of pepstatin A on the proteinase the present study; the possibility for the action of a activities of the digestive fluids of carnivo- carboxypeptidase(s) cannot be completely excluded, rous plants. The enzyme concentrations however, considering the reports ofthe occurrence of used were approximately 10-20 nM pepsin carboxypeptidase in the digestive fluids of N. alata equivalents except for Drosera (400 nM), (Ann et al. 2002) and Dionaea (Robins & Juniper and the concentration of pepstatin was var- 1980). ied from 0 to 120 (tM. Volume 38 September 2009 79 | I In the present study, the hemoglobin-digestion method was not so useful for the assay ofthe Drosera capensis acid proteinase activity; instead, the oxidized insulin B chain was useful as a substrate forthe assay as coupled with HPLC. Similar results were also obtained with D.filiformis. This may be due mainly to the difference in the substrate specificity ofDrosera sp. from that ofother carnivorous plants and remains to be clarifiedinafuture study. During thecourseofthepresent studies, we have alsoexaminedthe acidproteinase activities ofthedigestive fluids ofsomeothercarnivorousplants. So far, thedigestive fluids ofDrosophyllum Figure 6: Cleavage specificity of the acid proteinase activity of the digestive fluid of Nepenthes alata on oxidized insulin B chain, (a) The chromatogram shows the HPLC pattern of the hydrolysis products, (b) The amino acid sequence of oxidized insulin B chain is given in the one-letter notation. C*, cysteic acid. Large, medium, and small closed arrowheads indicate the major, medium, and minor cleavages, respectively, and small open arrowhead, trace cleavage. The number for each pep- tide stands forthe peak number, and that in parenthesis an approximate value for the FVNQHLCGSHLVEALYLVCGERGFFYTPKA relative yield of each peptide. The relative A A A A A AA A A * 1 1 1 64 6711 1 96 4 yield was calculated by dividing the peak achelsiesgauhvmtaigbneygstihtteehnetoumebbxeteer1n0to0f%ta.hte tpheeptimdeaxbionmdusm, |III | -458(6(9()45()81)1) 10(1)1 1 1|I 1| -72(16(139))(-4) 1 11| 11(96) 1 (b) F1 VNQHLCGS1H0LVEALYLVCG10ERGFFYTPKA90 (b) F1 VNQHLCGS1H0LVEALYLVCGnERGFFYTPKA30 A A A A A A* A a A * A A AaAA a a A 2 8 8 2513403 972 90 82 18 71 261307 3070 — — — II|| 39((818)10)1((2114))—U—11(1)1—1I II II 128(1(3528))(2)1-3(1)-j1|| 1 5(68(98)) 1 1 |I|i 6(28()2)8(1198()I5I9) 1(12)1 11 I || | | 41(11307(0((23))331(0)7)) 11111| 5(57) — Figure 7: Cleavage specificity of the acid Figure 8: Cleavage specificity of the acid proteinase activity of the digestive fluid of proteinase activity of the digestive fluid of Cephalotus follieuIartson oxidized insulin B Dionaea muscipula on oxidized insulin B chain. The conditions are the same as chain. The conditions are the same as those described in the legend to Figure 6. those described in the legend to Figure 6. 80 Carnivorous Plant Newsletter . lusitanicum and Byblis Uniflora showed high activity toward both hemoglobin and oxidized insulin B chain, whereas thatofSarraceniapurpurea failedto give any activity toward hemoglobin. Furtherextensive studies along this line, as well as purification and characterization of individual enzymes, including many more carnivorous plants, are necessary for deeper understanding of the biochemistry and physiology of the acid proteinases in the digestive fluids ofcarnivorous plants. Acknowledgments: This study was supported in part by grants-in-aid for scientific research from the Japan Society forthe Promotion ofScience. References Amagase, S., Mori, M., and Nakayama, S. 1972a. Digestive enzymes in insectivorous plants. IV. 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Proc. 4thInterntl. Carnivor. PlantConf. Tokyo, 117-124. Athauda, S.B.P, Matsumoto, K., Rajapakshe, S., Kuribayashi, M., Kojima, M., Kubomura-Yoshida, N., Iwamatsu, A., Shibata, C., Inoue, H., and Takahashi, K. 2004. Enzymic and structural characterization ofnepenthesin, auniquememberofanovelsubfamilyofasparticproteinases. Biochem.J. 380: 295-306. Athauda, S.B.P, Tanji, M., Kageyama, T., andTakahashi, K. 1989.Acomparative study on theNF^-terminal amino acid sequences and some other properties of six isozymic forms of human pepsinogens and pepsins. J. Biochem. 106: 920-927. Chandler, G.E., andAnderson, J.W. 1976. Studies on theoriginofsomehydrolytic enzymes associated withthe leaves andtentacles ofDrosera species andtheirrole inheterotrophic nutrition. New Phytol. 77: 51-62. Clancy, F.G.A., and Coffey, M.D. 1977. Acid phosphatase and protease release by the insectivorous plant Drosera rotundifolia. Can. J. Bot. 56: 480-488. 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