purification, biochemical characterization and n ... - Science Direct

0 downloads 0 Views 458KB Size Report
Mar 24, 1992 - 1992 Pergamon Press Ltd. PURIFICATION, BIOCHEMICAL CHARACTERIZATION. AND N-TERMINAL SEQUENCE OF A SERINE-PROTEASE.
Comp. Biochem. PhysioLVol. 103B,No. 3, pp. 675--680, 1992 Printed in Great Britain

0305-0491/92 $5.00+ 0.00 © 1992PergamonPress Ltd

PURIFICATION, BIOCHEMICAL CHARACTERIZATION AND N-TERMINAL SEQUENCE OF A SERINE-PROTEASE WITH CHYMOTRYPSIC AND COLLAGENOLYTIC ACTIVITIES IN A TROPICAL SHRIMP, PENAEUS VANNAMEI (CRUSTACEA, DECAPODA) A. VAN WORMHOUDT,P. L~ CHEVALIERand D. SELLOS Laboratoire de Biologie Marine, Coll6ge de France, BP 225-29182, Concarneau, Cedex, France (Tel: 98-97-0659) (Received 24 March 1992; accepted 1 May 1992)

Ab~ract--1. Two chymotrypsin variants, with collagenolytic activities, were purified from the hepatopancreas of Penaeus vannamei using radioactive protein as the substrate. 2. These proteases are very close as far as amino acid composition, molecular weight, inhibitors studies and specificity against small synthetic substrates are concerned. 3. N-terminal amino acid sequences of both variants are identical and are very close to other known crustacean serine proteases.

This purified enzyme was partially characterized concerning amino acid composition, molecular weight, inhibitors studies and specificity against synthetic substrates. N-terminal comparison was carried out with other known serine proteases.

INTRODUCTION

In Crustacea, serine proteases play an important role in the control of digestive processes. Among serine proteases trypsins have been found in a large variety of crustacean species (Lee et aL, 1980; Kimoto et al., 1983, 1985; Zwilling and Neurath, 1981; Honjo et al., 1990), whereas chymotrypsin appears to be absent in crabs, crayfish and some shrimps as indicated by Tsai et al. (1986). However, the importance of the substrate should be considered to confirm the absence of chymotrypsin activity reported for many crustaceans (Brun and Worrowicz, 1976; Zwilling and Neurath, 1981; Lee et al., 1980; Maugle et ai., 1982; Galgani et al., 1984; Galgani and Nagayama, 1987). Recently Lee et al. (1984) could not measure chymotrypsin activity in Penaeus vannamei with glutamyl-L-pbenylalanine as the substrate. Neither BTEE nor ATEE was suitable for the measurement of Penae'ide chymotrypsin. However, with the use of new substrates such as Suc-L-(Ala)2-Pro-Phe-pNa, this enzyme was shown to be rather predominant in these species (Tsai et al., 1986). In these conditions the choice of substrate is very important. Chymotrypsin was firstly reported as a collagenolytic protease with regard to its activity against collagen and some specific substrates. In this case, two serine proteases exhibiting collagenolytic properties have been purified: one is related to chymotrypsin and the other to trypsin when considering substrate and inhibitor specificities (Grant et al., 1981; Welgus et al., 1982; Grant et al., 1983). Recently, the use of radioactive peptides as the substrate has been developed (Kirschner and Goldberg, 1981). In this work, to purify specific proteases, we have selected human growth hormone as the substrate (Van Wormhoudt and Sellos, 1992) following nonhydrolyzed peptide by radioimmunoassay.

MATERIALS AND METHODS

Prawns Penaeus vannamei were supplied by IFREMER (Tahiti). Chemicals TFA (Trifluoroacitique acid), SANA [Succinyl (Alanine)3-p Nitroanilide], TAME (Tosyl-Arginine methyl ester), SAPPA [Succinyl-(Ala)2 Proline-phenylalanine-p nitroanilide], DFP (Diisopropylfluorophosphate), PMSF (Phenyl methylsulphonyl fluoride), PZDR (p-phenylazobenzyloxycarbonyl-L prolyl-L Leucyl-glycyl-L prolyl vArginine), DNPR (dinitrophenyl-proline-glycine-isoleucinealanine-giycine-glutamine-D arginine), BTEE (benzoyl-tosyl-ethyl-ester) were purchased from Sigma Chemical Co. (St Louis, MO). Shrimp hepatopancreas extraction Nine grammes of Penaeus vannanei hepatopancreas were extracted in I00 ml Tris-HC1 buffer 10 raM, pH 7.5 containing 10 raM, MgCI2, NaN 2 0.1% and I mM diisopropylfluorophosphate (DFP) as a general serine proteas¢ inhibitor. The extract was then centrifuged (42,000 rpm, 30 rain). Detection of enzyme activity The method was described elsewhere (Van Wormhoudt and SeUos, 1992). Briefly, a residual protease activity not blocked by DFP was followed using a human growth hormone (GH) radioimmunoassay (CIS-Industry). In the standard conditions some proteases are able to digest radioactive hGH, decreasing the affinity for antibody and giving the appearance of the RIA displacement curve. Activity is given as GH-like equivalent with human growth hormone as the standard in radioimmunoassay. In our experimental conditions, the curves were parallel.

675

676

A. VANWORMHOUDTet al.

Gel filtration The extract was filtered over an ACA 54 (IBF) column (140 × 2.7 cm) and then eluted with an ammonium acetate buffer 10mM, pH8.5 at a flow rate of 30ml/hr. Ten millilitre fractions were collected: aliquots of 100 ml were used for the estimation of protease activity. Ion-exchange chromatography Mono-QHR (Pharmacia, Uppsala, Sweden) was equilibrated in Tris-HCl buffer, pH 7.5. Aliquots of 200 ml were injected on the column and proteins were eluted using a NaCI gradient (0-0.5 M) and measured at 226 nM. Reverse-phase HPLC Two columns were used to achieve the purification. Firstly, a nucleosil C18 column (CIL-Cluzeau, France) 300 A 25 × 4.6 cm, was equilibrated in phosphate buffer 10 mM, pH 6.2. Peptides and proteins were ehited using an acetonitrile gradient as indicated in Fig. 1. Secondly, a Vydac column (218 TP 54) equilibrated in 40% acetonitrile/TFA 0.1% was eluted in acetonitrile/TFA 0.1% gradient (40-44% in 20 mn). Absorbance was monitored at 226 nm (Pharmacia 2158 Uvicord). To preserve protease activity at this step, fractions were immediately collected in 500 #1 NH 40AC 100 raM, pH 8.5 until analysis. Protein measurement and enzymatic specificity Protein was estimated by the Folin-Cioccalteu method (Lowry et al., 1951). Trypsin was measured using TAME as the substrate according to Hummel (1959), chymotrypsin using SAPPA according to Delmar et al. (1979) or BTEE (Hummel, 1959), collagenase using PZDR according to Wunsch and Heidrich (1963) or DNPR according to Grant and Eisen (1980) and elastase using SANA according to Yoshimaka et al. (1982). RESULTS Purification o f proteinases Fractionation of crude extract of Penaeus vannamei on the AC54 column gives one fraction (comprised between 400 and 550 ml) presenting protease activity (data not shown). The elution pattern of this concentrated fraction on an ion-exchange chromatography shows two

isoforms of protease activity (Fig. 1) eluted between 0.3 and 0.4 M NaCi. Tubes corresponding to these two fractions (BI and BII) were pooled: the fractions were lyophilized and then purified by HPLC on a nucleosil column (Fig. 2) and finally on the Vydac column (Fig. 3) as indicated in Materials and Methods. BII is a little more hydrophobic than BI. After the Vydac column about 500/~g BI and 250/tg BII proteases were purified and they corresponded to 0.05% of the total amount of proteins in crude extracts. This level is low compared to trypsin activities (about 5-10%) but it is probably underestimated due to loss in purification steps. Molecular weight determination Electrophoretic analysis of the enzyme in denaturating conditions showed that the molecular weights of the two proteins are found to be around 25 kDa. BII was seen to be a little larger than BI (Fig. 4). Amino acid composition and N-terminal sequences Amino acid analysis shows a relatively low content of basic residues (a little higher than for crab collagenolytic protease) while the level of acidic residues is quite similar (Table 1). The amino acid composition of P. vannamei proteinases presents some significant differences concerning the number of serine. Other differences were weak and should be confirmed by amino acid sequencing. The N-terminal sequence of the two enzymes is identical (Table 2) and it was compared to other crustacean chymotrypsins, vertebrate chymotrypsins and crayfish trypsin. Ninety per cent homology was found with P. monodon chymotrypsin or crab collagenase, 60% homology with mamalian chymotrypsin A and only 35% with crayfish trypsin. Tryptic mapping Ten milligrammes of each purified proteinase were hydrolysed by 5 mg of pancreatic trypsin during 5hr at 37°C in phosphate buffer 10mM, p H 7 and then separated on a HPLC column as indicated (Fig. 5).

2F

/

200 Gaog/tub

IIII

BI

e~

r/

Bll

8

J

10

rn

ql

/',

/

/All /

lll',

~

~

I

~-~

i

20

30 40 50 60 70 Elution Vol. (ml) Fig. 1. Ion-exchange chromatography of the ACA 54 extract (concentrated fraction 400-550 ml) on a

Mono-QHR Pharmacia column. Two fractions, BI and BII, with protease activity were eluted between 0.3 and 0.4 M NaC1. Activity was expressed as GH-like equivalent in the radioimmunoassay.

Penaeus

chyrnotrypsins

677 60

BI

46EO GH (ng/tube)

200

.// 30

0.5

E e. ¢o 04 ¢N

'

O

I

[

BII

i

i

,

i

r--i

20o

I

100

I i i ,,,~

Z t.3 60