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Artemocyanin: a proteinase zymogen rather than a proteinase inhibitor
, pp. 883-887, 1985
2YANIN: A PROTEINAS~, THAN A PROTEINASE
GEN ['OR
~R.ISSANSEN, CLIVE N. A. TROTMAN aJ
P. TATE ~land
f Biochemistry, University of Otago, Dur
(Received 11 July 1984) Abstract---l. The relationship between artemocyanins I and ]II from Arte a zymogen rather than a proteinase inhibitor. 2. The SDS-stable proteinase activity of artemocyanin II degrade different peptide fragments from those given by two other SDS-stabl~ 3. Artemocyanin I does not inhibit these two proteinase~ ~roteinases either o f ' polypeptide of artemocyanin I into the set of polypeptides polypeptide,, present in 4. The 67,000 Mr product lacks the characteristic prote 3roteinase activ present in artemocyanin II indicating that cleavage is ins1 insufficient to
INTRODUCTION /e have extensively characterised a large blue-green 'ansport protein complex (Mr 1.2 × 106) from the aemolymph of the entomostracan Artemia and have amed it artemocyanin (Krissansen et al., 1981, 1983a 983a). The pigment is a bile pigment closely related to biliverdin (Krissansen et al., 1984). Two closely related :lated forms of the protein have been isolated and purified urified (Krissansen et al., 1983b). The haemolymph form ~rm, artemocyanin I, is thought to contain three protomers each containing polypeptides of Mf~ 190,000 (Krissansen et al., 1983c). A modified form isolated from Artemia homogenates, artemtocyanin oc' II contain polypeptides of 104,000 and 7_000 generated ~enerated by a ~necific cleavage 87,000 specific ]c cleava cleavage of of the the 100.flOO 190,000 polypeptide and also a variable 'iable amount of a polypeptide of 67,000 which pepptide analysis suggests is derived from the 87,000 fra gment. This 67,000 polyoteinase activity which is peptide exhibits a latent proteinase potentiated by denaturing aagents and is retained in sodium dodecyl sulphate (SDS). The data are consistent with two possibilities for the origin of the proteolytic tic function of form II: either artemocyanin I couldi be an inactive precursor which is regulated by limited ~d proteolytic conversion to an active form, or it couldI be a proteinase inhibitor din. Artemocyanin and rather like ~2-macroglobulin. ~2-macroglobulin have man y similar physical characteristics (Dunn and Spiro, 1967). Several proteinase nonstrated in the haeinhibitors have been demonstr asaharu et al., 1982). molymph of silkworms (Masaharu In this paper we present evidence for a zymogen model for the relationship between ~etween the two forms of artemocyanin and information ttion on how the conversion from form I to form m II occurs, D METHODS MATERIALS AND Adult Artemia gathered att Lake Grassmere, Marlborough, New Zealand, were frozen i'ozen on dry ice and stored at -80°C. Adult shrimps were cultivat *BSA, bovine serum albumin.
at with form I being n albumin to yield einases. vert the 190,000 M r II. ivalent polypeptide roteolytic function.
lectetd at Lake i a 3501 aquarium equipped with four air lift ansen et al., 1983a). Ac Acrylamide, sq inhibitor (Type I-S), BSA* (fract (fraction V) ant idex A-50 were supplied by Sigm~ ,ma Chemical Co. Polyethylene glyc ,,lycol 6000 (PEG 6000) was Labora Hide Powder wastobtained from Koch-Light Laboratories. Azure was obtained from Calbiochem.
Purification of artemocyanins I and H un were purified as described The two forms of artemocyanin 1 Th previously (Krissansen et al., 1983b). Purification of SDS-stable proteinases St~ 1. Extraction of proteinases. Adult Artemia (100 g) Step A¢ were ground in a mortar and pestle ~estle on ice in 100 ml buffer A (5( 50mM Tris-chloride, pH 7.6, 125 mM n NaC1). The ho.mogenate ... was centrifuged for 20 min at a 12,000g. Step 2. Polyethylene glycol precipitati 9itation. The supernatant was fractionated with 20Yo(w/v) PEG 66000 and centrifuged at 16,000g to remove artemocyanin and an~ a large amount of fractionation of the glutinous orange material. Further fi supernatant at 30~o PEG gave a white p~ellet on centrifuging at 16,000g for 15min. This was resuspended resu in 5 ml of buffer A. DEAE-Sephadex Step 3. chromatography. The resuspended 20-30~o PEG pellet (3 ml) was wa applied to DEAESephadex A-50 equilibrated with 5( 50 mM Tris--chloride, pH 7.6, 150 mM NaCI. The column 0total vol 5 ml) was washed with equilibration buffer and the proteins eluted with a gradient of 0.15-1.2 M NaC1 in 120 ml of the same monitored for protein buffer. The column fractions were mo (E280 nm), total proteinase activity and a~ SDS-stable proSDS-stable proteinase activity. The fractions containing containi concentrated to 1 ml by teinase activity were pooled and conc~ ultrafiltration. Step 4. Non-denaturing gel electroph ~horesis. The DEAESephadex purified sample was applie )lied to a 15~o (w/v) acrylamide gel prepared according to Gabriel (1971) and electrophoresed at 15 mA and 4°C. The gel was sliced into 2 mm fractions and each was elutedt intc into 0.5 ml of buffer A. SDS-stable proteinase activThe fractions were assayed for SDS-stal ity. Analytical procedures tared at 595 nm with Hide presence or absence of 2~o 981).
GEOFFREY W. KRISSANSEN et
electrophoresis
DISCUSSION
RI
cording to Laemmli ylamide, 0.13% (w/v) 1M Tris, 100 mM glykrtemocyanin samples in to inactivate the ~t 98°C for a further gel loading solution v/v) SDS, 10~o (w/v) ycerol, 0.02% brodbumin obtained with , ..... " - ° ......... ~ . . . . . . . . . . . . . . . . ~S-stable proteinases BSA (9pg) was incubated at 37°C for 30min with either the two SDS-stable proteinases or with artemocyanin II the presence of SDS gel loading solution. Proteolysis was pped by heating the samples of 98°C for 3 min, followed electrophoresis into a 20~ (w/v) polyacrylamide SDS gel b.
9dification to artemocyanin I by SDS-stable proteinases Artemocyanin 1 was incubated with the SDS-stable pronases for 1.5 hr at 37°C in buffer A. Samples were heated • 3 min at 98°C to inactivate the SDS-stable proteinase, .~n heated at 98°C for a further 3 min in SDS gel loading ution prior to electrophoresis into a 10~ polyacrylamide IS gel slab. Artemocyanin I, incubated together with the SDS-stable 3teinases, was further fractionated by electrophoresis on 5% (w/v) polyacrylamide non-denaturing gel. The blue a 5~o artemoc emocyanin band was eluted and tested for an acquired ility to degrade BSA. The degradation of BSA was abilit' negligible•
(a)
al.
In order to d ing artemocyar
~tween models representa proteinase inhibitor or ) approaches. First, since a zymogen we 1 SDS-stable activity after artemocyanin I ,'mpted to isolate endodenaturation, , emia with this charactergenous proteim istic a n d to det~ er they were inhibited by artemocyanin ve have searched for a putative endog~ n Artemia extracts which could convert ." I into a r t e m o c y a n i n II, requiJ le zymogen model. a critical Crll T~ Two SDS-st~ ses have been purified to appaJ )arent h o m o ~G precipitation, D E A E 9hadex chro a n d electrophoresis on Seph; lide gels. A p a r t from arnon-denaturing non-c rely activities detected in ttemo, e m o c y a n i n the g stability in SDS. Both extra,Lcts of Art he gel profile of the P E G p~roteins rate were p pellet )ellet a n d the 1~ ]ex c o l u m n fraction (Fig. proteinases exhibiting la; ttracks A from the n o n - d e n a t u r i n g activity activi in SDS gel aaJn d re-elec! )n a similar gel (Fig. la; track C, fractk ~, fraction 2). The Mr of the p~roteins ha aated to be 36,000 (fraction :2) a n d 25, 1) by calibration o f the gel w: with markers. The D E A E - S e p~hadex ha profile in F i g 1b re revealed o t h e r proteinases which were unstable in SDS. A , c o m p a r i s o n has been m a d e of the peptide frag(BSA) was ments p ment: o b t a i n e d when a s t a n d a r d protein
(b)
Gradient
!
!
......... 0
A
B
C
D
Fraction
t l 4O
2O
number
ring polyacrylamde gels of SDS-stable proteinases. Samples from each step of the Fig. l. (a) Non-denaturin ~d in Materials and Methods were electrophoresed on non-denaturing gels. A: step purification as described 0-30%) B: step 3--DEAE-Sephadex A-50 fraction. C: step 4--proteinlase 1 from 2--PEG precipitate (20-309 a non-denaturing gel. D: step 4~oroteinase 2 from a non-denaturing gel. (b) DEAE-Septphadex A-50 /o) was fractionated chromatography of partially pu ztivity © - - 0 0: on DEAE-Sephadex and anal S
Proteolytic acitivity of artemocyanin rifled proteinases or g conditions in SDS ~roteolytic activities y different series of is shown in track D, lase fraction 1 are .~ fraction 2 in track ~ck C. These results ity of artemocyanin e two purified SDSDS-stable activities e have been able to ~tect in Artemia. Moreover when artemocyanin I as mixed with trace amounts of the purified SDSable proteinases to see whether it could inhibit them lere was no detectable inhibition. Instead the proinases were able to degrade artemocyanin I into iscrete bands as discussed below. There was no ctensive degradation as occurs when artemocyanin itself is incubated in the presence of SDS without a inhibitor such as soybean trypsin inhibitor. This as previously been shown to be caused by the .
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polypeptide of teolytic activit) A number o
Staphylococcus
.
tilisin, thermol enterokinase dc antibody raise~ been shown to not cross-reacl proteinases. Af SDS stable pro did not degrad This indicated teins had occul arten emocyanin 1 prote ~teinase de., e2-m. ~t 2-macroglobu teina teinase when Barrett, 1980). W~ have ob~ We polyl~eptide in increeases with .
which exhibits the proet al., 1983a). ~inases, trypsin, pronase, ~roteinase, bacterial subase K and hog intestine locyanin extensively. The emocyanin II which has e 67,000 polypeptide did of the two SDS-stable n of artemocyanin I with ;-isolated artemocyanin I r denaturing conditions. ciation between the proe reasons we believe that hibitor of an SDS-stable uctural resemblance to alently attaches to a proinhibitor (Salvesen and ae amount of the 67,000 ion of artemocyanin II at - 2 0 ° C implying that
I ~,~)~
I
! 2
I
Fig. 2. Proteolytic degt fractions 1 or 2. BSA was incul: inactivation was electrophorese( proteinase 1; (B) SE
i
proteinase ~1 and after heat h, (A) SDS-stable "eated.
GEOFFREYW. KRISSANSENeta].
D
A
F
B
G
samples were artemocyanin I. Artemocyanin II sa Fig. 3. Aging of artemocyanin II and conversion from art month at - 20 C freshly prepared by a rapid isolation procedure (track A), and a~ after storage for several months (B) were electrophoresed on an SDS-polyacrylamide gel after heat inactivation. Artemocyanin I was and M~ treated with SDS-stable proteinase 1 (C) or 2 (D) as described in Materials and Methods 9rofile after prolonged incubation (19 ( hr) with electrophoresed beside an untreated sample (E). The pro SDS-stable proteinase fraction I (F) is compared with the untreated samples (G). The M, of 1he 190,000, 104,000, 87,000 and 67,000. polypeptides of artemocyanin are marked (190. ere is a susceptible cleavage point in the polythere pelptide of Mr 87,000 which generates the polypeptide of M r 67,000. A freshly prepared sample and the same g. mple stored for several months are shown in Fig. sam[ :tively, demonstrating a 3, tracks A and B respectively, significant increase in the band of M r 67,000. Repeated freezing and thawinng (50 cycles) did not increase the proportion of this band. This suggests that artemocyanin II underggoes autodegradation on storage. .~ct of artemocyanin I on In our analysis of the effect ble proteinases we were the activity of the SDS-stable roteinases aases could generate surprised to find that both protelnas~ de of Mr67,000 seen in the characteristic polypeptide ack E shows the typical artemocyanin II. Fig. 3, track file tracks C and D show profile of artemocyanin I while the polypeptide pattern afterr a short incubation with Lively. In these cases the proteinases 1 and 2 respectively. remaining intact polypeptidee of M r 190,000 has been 7,000 has appeared. On cleaved and the band of 67,000 md of 104,000 also disapprolonged incubation the band peared and the bands of M r 87,000 and 67,000 were tides remaining (track F). the only prominent polypeptides Track G shows the artemoc yanin I sample incubated hese proteinases may not without proteinase. While these be responsible for the cleava vage in vivo, the results suggest that artemocyanin I is a precursor of arte,'rated by a limited promocyanin II which is generated teolysis. The SDS-unstable proteolytic fraction (Fig. l b) was unable to convert artemocv mocyanin II. While the SDS-stable proteinases
poly[)eptide pattern of artemocy~,anin II the protll polypeptide of teoly,tic activity associated with the is not generated. This implies that the M r 67,000 6( lI having the conversion of artemocyanin I into form f cony, proteolytic function is at least a two-step process some further requiring cleavage and then :lentified to date a second modification. We have not identifie( step. The active 67,000 polypeptide is unglycosylated in contrast to the other polypepti(des and therefore activation may involve a change in the carbohydrate moiety of the molecule. A model of the relationshi!cp between artemocyanins I and I! is shown in Figg. 4. It is based on ,anm is a zymogen our conclusion that artemocyani rather than a proteinase inhibitor and that patentiation of the proteolytic function functio is a multistep process. It implies that there are at least two sus)eptide. cleavage at ceptible sites in the 190,000 polype ARTEMOCYANI N II
ARTEM OCYANIN l
190 K %.. ~10l, K. . . . . . ÷ 10~,K. . . . . . . * IO/+K " ~ 87 K ~ - ~ f l T K . . . . . . . * 87K
~"-~)'/K, "~"20K"
67K
II
~. proteose
acfivify I octivofion cleavage Fi~. 4. A proposed model for the conversion c of artehe origin of its proteinase
nposition data have been ssansen et al.. t983c).
Proteolytic acitivity of artemocyanin f 104,000 a n d 87,000 :leavage at a second ;enerates the 67,000 tide of a b o u t 20,000 )usly (Krissansen et
~,. Langston (Manager) mere for his generosity raps used in this study. ant from the Medical and CNAT thanks the [Z University Grants Committee and the Medical Distribuons Committee of the NZ Lottery Board for financial ~sistance. REFERENCES ~unn J. T. and Spiro R. G. (1967) The ct2-macroglobulin of human plasma. J. biol. Chem. 242, 5549-5555. ~abriel O. (1971) Analytical disc gel electrophoresis. Meth. Enzym. 22, 565-578. ~rissansen G. W., Trotman C. N. A. and Tate W. P. (1981) A novel protease may explain widely differing models for the structure of Artemia salina haemoglobin. Biochim. biophys. Acta 671, 104-108.
Krissansen G. (1983a) Physi artemocyanin, latent proteob Biochim. biopt Krissansen G. (1983b) Purifl protein from 1 369-378. Krissansen G. W Two forms ot brine shrimp 159-164. Kriss ansen G. W Ide Identification daJ biliprotei dant Bic Biochem. Phy~, Laen~tmli U. K dul during the as: Na Nature, Lond. Masa Masaharu E., H~ of protease il wo:rms, Bomb3 cyn'nthia ricini. Salve: Salvesen G. S. ar pro aroteinases in che. J. 187, i chem.
C. N. A. and Tate W. P. lemical characterization of glycoprotein complex with m the brine shrimp Artemia. 151-158. C. N. A. and Tate W. P. smocyanin, a haemolymph lp Artemia. Biochem. Int. 7, N. A. and Tate W. P. (1983c) Lein artemocyanin from the )chim. biophys. Acta 747, N. A. and Tate W. P. (1984) ~n chromophore of an abunmolymph of Artemia. Comp. 252. ~age of structural proteins head of bacteriophage T4. vamoto A. (1982) Properties the haemolymph of silkraea pernyi and Philosamia n. Physiol. 71B, 569-576. (1980) Covalent binding of ~¢ith ~t2-macroglobulin. Bio-
2YANIN: A PROTEINAS~, THAN A PROTEINASE
GEN ['OR
~R.ISSANSEN, CLIVE N. A. TROTMAN aJ
P. TATE ~land
f Biochemistry, University of Otago, Dur
(Received 11 July 1984) Abstract---l. The relationship between artemocyanins I and ]II from Arte a zymogen rather than a proteinase inhibitor. 2. The SDS-stable proteinase activity of artemocyanin II degrade different peptide fragments from those given by two other SDS-stabl~ 3. Artemocyanin I does not inhibit these two proteinase~ ~roteinases either o f ' polypeptide of artemocyanin I into the set of polypeptides polypeptide,, present in 4. The 67,000 Mr product lacks the characteristic prote 3roteinase activ present in artemocyanin II indicating that cleavage is ins1 insufficient to
INTRODUCTION /e have extensively characterised a large blue-green 'ansport protein complex (Mr 1.2 × 106) from the aemolymph of the entomostracan Artemia and have amed it artemocyanin (Krissansen et al., 1981, 1983a 983a). The pigment is a bile pigment closely related to biliverdin (Krissansen et al., 1984). Two closely related :lated forms of the protein have been isolated and purified urified (Krissansen et al., 1983b). The haemolymph form ~rm, artemocyanin I, is thought to contain three protomers each containing polypeptides of Mf~ 190,000 (Krissansen et al., 1983c). A modified form isolated from Artemia homogenates, artemtocyanin oc' II contain polypeptides of 104,000 and 7_000 generated ~enerated by a ~necific cleavage 87,000 specific ]c cleava cleavage of of the the 100.flOO 190,000 polypeptide and also a variable 'iable amount of a polypeptide of 67,000 which pepptide analysis suggests is derived from the 87,000 fra gment. This 67,000 polyoteinase activity which is peptide exhibits a latent proteinase potentiated by denaturing aagents and is retained in sodium dodecyl sulphate (SDS). The data are consistent with two possibilities for the origin of the proteolytic tic function of form II: either artemocyanin I couldi be an inactive precursor which is regulated by limited ~d proteolytic conversion to an active form, or it couldI be a proteinase inhibitor din. Artemocyanin and rather like ~2-macroglobulin. ~2-macroglobulin have man y similar physical characteristics (Dunn and Spiro, 1967). Several proteinase nonstrated in the haeinhibitors have been demonstr asaharu et al., 1982). molymph of silkworms (Masaharu In this paper we present evidence for a zymogen model for the relationship between ~etween the two forms of artemocyanin and information ttion on how the conversion from form I to form m II occurs, D METHODS MATERIALS AND Adult Artemia gathered att Lake Grassmere, Marlborough, New Zealand, were frozen i'ozen on dry ice and stored at -80°C. Adult shrimps were cultivat *BSA, bovine serum albumin.
at with form I being n albumin to yield einases. vert the 190,000 M r II. ivalent polypeptide roteolytic function.
lectetd at Lake i a 3501 aquarium equipped with four air lift ansen et al., 1983a). Ac Acrylamide, sq inhibitor (Type I-S), BSA* (fract (fraction V) ant idex A-50 were supplied by Sigm~ ,ma Chemical Co. Polyethylene glyc ,,lycol 6000 (PEG 6000) was Labora Hide Powder wastobtained from Koch-Light Laboratories. Azure was obtained from Calbiochem.
Purification of artemocyanins I and H un were purified as described The two forms of artemocyanin 1 Th previously (Krissansen et al., 1983b). Purification of SDS-stable proteinases St~ 1. Extraction of proteinases. Adult Artemia (100 g) Step A¢ were ground in a mortar and pestle ~estle on ice in 100 ml buffer A (5( 50mM Tris-chloride, pH 7.6, 125 mM n NaC1). The ho.mogenate ... was centrifuged for 20 min at a 12,000g. Step 2. Polyethylene glycol precipitati 9itation. The supernatant was fractionated with 20Yo(w/v) PEG 66000 and centrifuged at 16,000g to remove artemocyanin and an~ a large amount of fractionation of the glutinous orange material. Further fi supernatant at 30~o PEG gave a white p~ellet on centrifuging at 16,000g for 15min. This was resuspended resu in 5 ml of buffer A. DEAE-Sephadex Step 3. chromatography. The resuspended 20-30~o PEG pellet (3 ml) was wa applied to DEAESephadex A-50 equilibrated with 5( 50 mM Tris--chloride, pH 7.6, 150 mM NaCI. The column 0total vol 5 ml) was washed with equilibration buffer and the proteins eluted with a gradient of 0.15-1.2 M NaC1 in 120 ml of the same monitored for protein buffer. The column fractions were mo (E280 nm), total proteinase activity and a~ SDS-stable proSDS-stable proteinase activity. The fractions containing containi concentrated to 1 ml by teinase activity were pooled and conc~ ultrafiltration. Step 4. Non-denaturing gel electroph ~horesis. The DEAESephadex purified sample was applie )lied to a 15~o (w/v) acrylamide gel prepared according to Gabriel (1971) and electrophoresed at 15 mA and 4°C. The gel was sliced into 2 mm fractions and each was elutedt intc into 0.5 ml of buffer A. SDS-stable proteinase activThe fractions were assayed for SDS-stal ity. Analytical procedures tared at 595 nm with Hide presence or absence of 2~o 981).
GEOFFREY W. KRISSANSEN et
electrophoresis
DISCUSSION
RI
cording to Laemmli ylamide, 0.13% (w/v) 1M Tris, 100 mM glykrtemocyanin samples in to inactivate the ~t 98°C for a further gel loading solution v/v) SDS, 10~o (w/v) ycerol, 0.02% brodbumin obtained with , ..... " - ° ......... ~ . . . . . . . . . . . . . . . . ~S-stable proteinases BSA (9pg) was incubated at 37°C for 30min with either the two SDS-stable proteinases or with artemocyanin II the presence of SDS gel loading solution. Proteolysis was pped by heating the samples of 98°C for 3 min, followed electrophoresis into a 20~ (w/v) polyacrylamide SDS gel b.
9dification to artemocyanin I by SDS-stable proteinases Artemocyanin 1 was incubated with the SDS-stable pronases for 1.5 hr at 37°C in buffer A. Samples were heated • 3 min at 98°C to inactivate the SDS-stable proteinase, .~n heated at 98°C for a further 3 min in SDS gel loading ution prior to electrophoresis into a 10~ polyacrylamide IS gel slab. Artemocyanin I, incubated together with the SDS-stable 3teinases, was further fractionated by electrophoresis on 5% (w/v) polyacrylamide non-denaturing gel. The blue a 5~o artemoc emocyanin band was eluted and tested for an acquired ility to degrade BSA. The degradation of BSA was abilit' negligible•
(a)
al.
In order to d ing artemocyar
~tween models representa proteinase inhibitor or ) approaches. First, since a zymogen we 1 SDS-stable activity after artemocyanin I ,'mpted to isolate endodenaturation, , emia with this charactergenous proteim istic a n d to det~ er they were inhibited by artemocyanin ve have searched for a putative endog~ n Artemia extracts which could convert ." I into a r t e m o c y a n i n II, requiJ le zymogen model. a critical Crll T~ Two SDS-st~ ses have been purified to appaJ )arent h o m o ~G precipitation, D E A E 9hadex chro a n d electrophoresis on Seph; lide gels. A p a r t from arnon-denaturing non-c rely activities detected in ttemo, e m o c y a n i n the g stability in SDS. Both extra,Lcts of Art he gel profile of the P E G p~roteins rate were p pellet )ellet a n d the 1~ ]ex c o l u m n fraction (Fig. proteinases exhibiting la; ttracks A from the n o n - d e n a t u r i n g activity activi in SDS gel aaJn d re-elec! )n a similar gel (Fig. la; track C, fractk ~, fraction 2). The Mr of the p~roteins ha aated to be 36,000 (fraction :2) a n d 25, 1) by calibration o f the gel w: with markers. The D E A E - S e p~hadex ha profile in F i g 1b re revealed o t h e r proteinases which were unstable in SDS. A , c o m p a r i s o n has been m a d e of the peptide frag(BSA) was ments p ment: o b t a i n e d when a s t a n d a r d protein
(b)
Gradient
!
!
......... 0
A
B
C
D
Fraction
t l 4O
2O
number
ring polyacrylamde gels of SDS-stable proteinases. Samples from each step of the Fig. l. (a) Non-denaturin ~d in Materials and Methods were electrophoresed on non-denaturing gels. A: step purification as described 0-30%) B: step 3--DEAE-Sephadex A-50 fraction. C: step 4--proteinlase 1 from 2--PEG precipitate (20-309 a non-denaturing gel. D: step 4~oroteinase 2 from a non-denaturing gel. (b) DEAE-Septphadex A-50 /o) was fractionated chromatography of partially pu ztivity © - - 0 0: on DEAE-Sephadex and anal S
Proteolytic acitivity of artemocyanin rifled proteinases or g conditions in SDS ~roteolytic activities y different series of is shown in track D, lase fraction 1 are .~ fraction 2 in track ~ck C. These results ity of artemocyanin e two purified SDSDS-stable activities e have been able to ~tect in Artemia. Moreover when artemocyanin I as mixed with trace amounts of the purified SDSable proteinases to see whether it could inhibit them lere was no detectable inhibition. Instead the proinases were able to degrade artemocyanin I into iscrete bands as discussed below. There was no ctensive degradation as occurs when artemocyanin itself is incubated in the presence of SDS without a inhibitor such as soybean trypsin inhibitor. This as previously been shown to be caused by the .
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polypeptide of teolytic activit) A number o
Staphylococcus
.
tilisin, thermol enterokinase dc antibody raise~ been shown to not cross-reacl proteinases. Af SDS stable pro did not degrad This indicated teins had occul arten emocyanin 1 prote ~teinase de., e2-m. ~t 2-macroglobu teina teinase when Barrett, 1980). W~ have ob~ We polyl~eptide in increeases with .
which exhibits the proet al., 1983a). ~inases, trypsin, pronase, ~roteinase, bacterial subase K and hog intestine locyanin extensively. The emocyanin II which has e 67,000 polypeptide did of the two SDS-stable n of artemocyanin I with ;-isolated artemocyanin I r denaturing conditions. ciation between the proe reasons we believe that hibitor of an SDS-stable uctural resemblance to alently attaches to a proinhibitor (Salvesen and ae amount of the 67,000 ion of artemocyanin II at - 2 0 ° C implying that
I ~,~)~
I
! 2
I
Fig. 2. Proteolytic degt fractions 1 or 2. BSA was incul: inactivation was electrophorese( proteinase 1; (B) SE
i
proteinase ~1 and after heat h, (A) SDS-stable "eated.
GEOFFREYW. KRISSANSENeta].
D
A
F
B
G
samples were artemocyanin I. Artemocyanin II sa Fig. 3. Aging of artemocyanin II and conversion from art month at - 20 C freshly prepared by a rapid isolation procedure (track A), and a~ after storage for several months (B) were electrophoresed on an SDS-polyacrylamide gel after heat inactivation. Artemocyanin I was and M~ treated with SDS-stable proteinase 1 (C) or 2 (D) as described in Materials and Methods 9rofile after prolonged incubation (19 ( hr) with electrophoresed beside an untreated sample (E). The pro SDS-stable proteinase fraction I (F) is compared with the untreated samples (G). The M, of 1he 190,000, 104,000, 87,000 and 67,000. polypeptides of artemocyanin are marked (190. ere is a susceptible cleavage point in the polythere pelptide of Mr 87,000 which generates the polypeptide of M r 67,000. A freshly prepared sample and the same g. mple stored for several months are shown in Fig. sam[ :tively, demonstrating a 3, tracks A and B respectively, significant increase in the band of M r 67,000. Repeated freezing and thawinng (50 cycles) did not increase the proportion of this band. This suggests that artemocyanin II underggoes autodegradation on storage. .~ct of artemocyanin I on In our analysis of the effect ble proteinases we were the activity of the SDS-stable roteinases aases could generate surprised to find that both protelnas~ de of Mr67,000 seen in the characteristic polypeptide ack E shows the typical artemocyanin II. Fig. 3, track file tracks C and D show profile of artemocyanin I while the polypeptide pattern afterr a short incubation with Lively. In these cases the proteinases 1 and 2 respectively. remaining intact polypeptidee of M r 190,000 has been 7,000 has appeared. On cleaved and the band of 67,000 md of 104,000 also disapprolonged incubation the band peared and the bands of M r 87,000 and 67,000 were tides remaining (track F). the only prominent polypeptides Track G shows the artemoc yanin I sample incubated hese proteinases may not without proteinase. While these be responsible for the cleava vage in vivo, the results suggest that artemocyanin I is a precursor of arte,'rated by a limited promocyanin II which is generated teolysis. The SDS-unstable proteolytic fraction (Fig. l b) was unable to convert artemocv mocyanin II. While the SDS-stable proteinases
poly[)eptide pattern of artemocy~,anin II the protll polypeptide of teoly,tic activity associated with the is not generated. This implies that the M r 67,000 6( lI having the conversion of artemocyanin I into form f cony, proteolytic function is at least a two-step process some further requiring cleavage and then :lentified to date a second modification. We have not identifie( step. The active 67,000 polypeptide is unglycosylated in contrast to the other polypepti(des and therefore activation may involve a change in the carbohydrate moiety of the molecule. A model of the relationshi!cp between artemocyanins I and I! is shown in Figg. 4. It is based on ,anm is a zymogen our conclusion that artemocyani rather than a proteinase inhibitor and that patentiation of the proteolytic function functio is a multistep process. It implies that there are at least two sus)eptide. cleavage at ceptible sites in the 190,000 polype ARTEMOCYANI N II
ARTEM OCYANIN l
190 K %.. ~10l, K. . . . . . ÷ 10~,K. . . . . . . * IO/+K " ~ 87 K ~ - ~ f l T K . . . . . . . * 87K
~"-~)'/K, "~"20K"
67K
II
~. proteose
acfivify I octivofion cleavage Fi~. 4. A proposed model for the conversion c of artehe origin of its proteinase
nposition data have been ssansen et al.. t983c).
Proteolytic acitivity of artemocyanin f 104,000 a n d 87,000 :leavage at a second ;enerates the 67,000 tide of a b o u t 20,000 )usly (Krissansen et
~,. Langston (Manager) mere for his generosity raps used in this study. ant from the Medical and CNAT thanks the [Z University Grants Committee and the Medical Distribuons Committee of the NZ Lottery Board for financial ~sistance. REFERENCES ~unn J. T. and Spiro R. G. (1967) The ct2-macroglobulin of human plasma. J. biol. Chem. 242, 5549-5555. ~abriel O. (1971) Analytical disc gel electrophoresis. Meth. Enzym. 22, 565-578. ~rissansen G. W., Trotman C. N. A. and Tate W. P. (1981) A novel protease may explain widely differing models for the structure of Artemia salina haemoglobin. Biochim. biophys. Acta 671, 104-108.
Krissansen G. (1983a) Physi artemocyanin, latent proteob Biochim. biopt Krissansen G. (1983b) Purifl protein from 1 369-378. Krissansen G. W Two forms ot brine shrimp 159-164. Kriss ansen G. W Ide Identification daJ biliprotei dant Bic Biochem. Phy~, Laen~tmli U. K dul during the as: Na Nature, Lond. Masa Masaharu E., H~ of protease il wo:rms, Bomb3 cyn'nthia ricini. Salve: Salvesen G. S. ar pro aroteinases in che. J. 187, i chem.
C. N. A. and Tate W. P. lemical characterization of glycoprotein complex with m the brine shrimp Artemia. 151-158. C. N. A. and Tate W. P. smocyanin, a haemolymph lp Artemia. Biochem. Int. 7, N. A. and Tate W. P. (1983c) Lein artemocyanin from the )chim. biophys. Acta 747, N. A. and Tate W. P. (1984) ~n chromophore of an abunmolymph of Artemia. Comp. 252. ~age of structural proteins head of bacteriophage T4. vamoto A. (1982) Properties the haemolymph of silkraea pernyi and Philosamia n. Physiol. 71B, 569-576. (1980) Covalent binding of ~¢ith ~t2-macroglobulin. Bio-