Calpain II-like proteinase of scallop (Patinopecten yessoensis) striated adductor muscle

Comp. Biochem. Physiol. Vol. 102B, No. 1, pp. 149-153, 1992 Printed in Great Britain

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

CALPAIN II-LIKE PROTEINASE OF SCALLOP (PATINOPECTEN YESSOENSIS) STRIATED A D D U C T O R MUSCLE OSAMU MAEDA, TAKAO OJIMA and KIYOYOSHINISH1TA Department of Fisheries Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan

(Received 29 July 1991) Abstract--1. A Ca2+-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of M, ~ 200,000, composed of two Mr 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18°C. In addition, it requires 1.7 mM Ca 2÷ for half-maximal activity and more than 10 mM Ca 2+ for maximal activity. Thus the enzyme can be classified as calpain II.

INTRODUCTION

DEAE Toyopearl and Sephacryl S-300 were purchased from Tosoh, Japan and Pharmacia, respectively. Casein (nach Hammersten) was obtained from Merck. Molecular weight marker proteins were purchased from Sigma. Other reagent-grade chemicals were obtained from Nacalai Tesque and Wako Pure Chemicals, Japan.

Calpain, a Ca2+-dependent cysteine proteinase (EC 3.4.22.17), is known to be widely distributed in various vertebrate tissues (Murachi et al., 1981; Murachi, 1983a,b). Although the real functions of calpains in vertebrate remain obscure, they appear to play important roles in protein degradation (Nelson and Traub, 1982; Malik et al., 1983), transformation of membrane receptors (Cassel and Glaser, 1982), regulation of various enzymes (Kishimoto et al., 1983; Mellgren et al., 1982), etc. This enzyme is subclassified into calpain I and calpain II depending on the Ca 2÷ requirement of micromolar and millimolar order, respectively. Vertebrate calpain II is usually composed of two subunits of M r ~ 80,000 and M , ~ 30,000 (Suzuki et al., 1981a,b) and autolyzed to calpain I. The larger subunit contains sequences for the active site and calcium binding site, whereas the smaller subunit is non-catalytic but also contains a calcium binding site (Ohno et al., 1984; Sakihama et al., 1985). However, little is known of the invertebrate calpain or calpain-like proteinase except for crustacean Ca2+-dependent proteinases (Mykles and Skinner, 1986). In this paper, we report on the isolation and characterization of calpain II-like proteinase from scallop striated adductor muscle.

Assay of caseinolytic activity Calpain-like proteinase was incubated with casein (5 mg/ml) in a medium containing 0.1 M Tris-acetate, pH 6.8, 10 mM CaCI2, and 10 mM 2-mercaptoethanol in a total volume of 2 ml at 17°C for 60 min. The reaction was stopped by adding 1 ml of 5% perchloric acid, and the acid-soluble products were determined from the absorbance at 280 nm of the supernatant after centrifuging the reaction mixture at 35,000 g for 20 min. One unit of enzyme was defined as the amount of enzyme that caused an increase in the absorbance at 280 nm of unity. Preparation of crude proteinase Scallop crude calpain-like proteinase was prepared according to the method of Ishiura et al. (1978) with slight modification. The striated adductor muscle (1500g) was homogenized with 3 vols of 20mM NaHCO3, I mM EDTA and 10 mM 2-mercaptoethanol in a Waring blendor for 5 min (20 times for 15 sec each) and centrifuged at 20,000g for 15 min. Solid ammonium sulfate was added to the supernatant. Precipitates formed between 20 and 40% saturation were collected by centrifugation at 10,000 g for 20 min, then dissolved in and dialyzed against 0.1 M NaCl, 20mM Tris-HCl, pH 7.0, 5mM EDTA, 10mM 2-mercaptoethanol (buffer A).

Striated adductor muscle was cut from living ezo-giant scallop (Patinopecten yessoensis) and used immediately.

Effect of SH reagents on the proteinase activity To the reaction mixture containing calpain-like proteinase except substrate, 1 mM of either IAA, NEM or DTNB was added. The mixture was then kept standing at 17°C for 10 min, followed by the addition of casein to measure the caseinolytic activity.

Abbreviations--EDTA, ethylenediamine tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IAA, monoiodoacetic acid; NEM, N-ethylmaleimide; DTNB, 5,5'-dithiobis (2-nitrobenzoic acid); M , molecular weight.

Electrophoresis SDS-gel electrophoresis was carried out using 10% polyacrylamide slab-gel containing 0.1% SDS by the method of Porzio and Pearson (1977). Protein bands were stained with Coomassie Brilliant Blue R-250.

MATERIALS AND METHODS

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Fig. 1. DEAE-Toyopearl column chromatography of scallop muscle crude extract. The crude calpain-like proteinase preparation (3800 mg) was applied to a DEAE-Toyopearl column (2.2 x 65cm), and eluted with a 0.1-0.4M NaCI linear gradient in 20mM Tris-maleate, pH 7.0, 5mM EDTA, and 10mM 2-mercaptoethanol at a flow rate of 40ml/hr. Each fraction contains 10ml. ( ) Absorbance at 280 nm; ( - - 0 - - ' - - ( 3 - - ) caseinolytic activity; ( - - - - - - ) NaCI concentration.

Determination of protein concentration Protein concentration was determined according to Lowry et al. (1951) using bovine serum albumin as a standard.

RESULTS Purification of calpain-like proteinase The dialysate of the crude proteinase described in Materials and Methods was clarified by centrifugation at 100,000g for 120 min, and then applied to a DEAE-Toyopearl column. As shown in Fig. 1, a large and a small peak with caseinolytic activity were eluted at fractions 70-81 and 82-96, respectively, with a linear NaC1 gradient in buffer A (total volume of 1500 ml). After being concentrated by ultrafiltration through a UK-10 membrane (Advantec Toyo, Japan), each peak was chromatographed on a Sephacryl S-300 column (Fig. 2). Both peaks showed activities with similar optimum pH and Ca 2+ requirement but different elution positions (e.g. different M,) and protein compositions. Since the peak shown in Fig. 2B was revealed to contain more than six proteins by SDS-gel electrophoresis and to have further difficulty in obtaining enough purified enzyme, the peak in Fig. 2A was used for further purification. The peak was then successively rechromatographed on DEAE-Toyopearl and Sephacryl S-300 columns. As shown in Fig. 3, two similar sized but partially overlapped peaks were obtained by the Sephacryl S-300 rechromatography. The fractions 55-72 with caseinolytic activity were pooled and confirmed to contain only the M, 100,000 subunit, by SDS-PAGE. Calibration of the Sephacryl column showed that this enzyme has Mr 200,000 and is composed of two Mr 100,000 subunits (Fig. 4). Results in the purification steps are summarized in Table 1. This calpain-like proteinase was in very low yield with 333-fold purification.

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Fig. 2. Sephacryl S-300 column chromatography of scallop crude calpain-like proteinase. Crude proteinase (210mg) obtained by DEAE-Toyopearl chromatography was applied to Sephacryl S-300 column (2.7 x 95 cm) and eluted with 0.I M NaC1, 20mM Tris-maleate, pH 7.0~ 5mM EDTA and 10raM 2-mercaptoethanol at a flow rate of 35 ml/hr. Each fraction contains 5 ml. ( ) Absorbance at 280 nm; ( - - O - - ' - - O - - ) caseinolytic activity.

Enzymatic properties Figure 5 shows that the scallop calpain-like proteinase required approximately 1.7mM Ca 2+ for half-maximal activity and more than 10 mM Ca 2+ for maximal activity, but it was practically inactive at lower than 0.2 mM Ca 2÷. It was not activated by 30mM of Sr 2+ and Ba 2+ or by Mn 2+, Mg 2+, or Zn 2+. Moreover, it was hardly activated by 100-400/aM Ca 2+ along with up to 3 mM of either Sr 2÷, Mn 2+, Mg 2+, Ba 2+ or Zn 2+ (data not shown). As in Fig. 6, the scallop calpain-like proteinase showed maximum activity at pH 6.8 with three kinds of buffer. This was slightly lower than that of the vertebrate calpains of around pH 7.5. In addition, the activity was decreased to 6, 9 and 2% of the original by the addition of IAA, NEM and DTNB, respectively, indicating that this enzyme is a cysteine-proteinase (Table 2).

DISCUSSION Isolation and characterization were described on calpain II-like proteinase of scallop striated adductor muscle. Two species of neutral proteinase of M r ~ 850,000 and Mr ~ 200,000 were found, both of which required millimolar order of Ca 2~ for activity. The scallop proteinase of Mr 200,000 was further investigated. Though this enzyme is unique to be composed of two Mr 100,000 subunits, unlike the vertebrate calpains, it can be classified as calpain II by taking the definition of calpain into consideration. It may be reasonable to consider that the enzymes

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Fraction Number Fig. 3. Rechromatography of scallop calpain-like proteinase on Sephacryl S-300 column. Inset are SDS-PAGE patterns; numbers at the bottom of the figure represent the fraction numbers; P, before rechromatography; 100 and 120 K, mol. wts of 100,000 and 120,000. with the same function but different molecular size are distributed a m o n g various animals. We have found that one of the scallop troponin subunits, " t r o p o n i n I" possesses an Mr more than twice as large as the vertebrate counterpart (Ojima and Nishita, 1986, 1988). A b o u t 4 mg of this scallop calpain-like proteinase was yielded from 1500g muscle and purified 333fold to a spec. act. of 10.0 U/mg. Since the activity was detected in the crude extract of the scallop muscle, though very weak as in porcine tissues (Kitahara et al., 1984), it is important to investigate the existence and properties of a specific inhibitor.

Though we could not find analogous proteinase to calpain I in scallop muscle, its existence may not be excluded, since calpain I was found recently in carp muscle (Toyohara and Makinodan, 1989) and chicken breast muscle (Wolfe et al., 1989) in which only calpain II had been found. Table I. Purification of scallop calpain-likeproteinase from striated adductor muscle (1500 g) Total protein Total activity Spec. act. Step (mg) (U) (U/rag) Crude extract 50,250 1505 0.03 (NH4)2SO4 fractionation 2130 810 0.38 First DEAE-Toyopearl 123.5 310 2.4 First Sephacryl S-300 34.3 194 5.6 Second DEAE-Toyopearl 6.6 56.4 8.5 Second Sephacryl S-300 4.3 43.0 10.0

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J. biol. Chem. 257, 9845-9848.

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The purified scallop calpain II-like proteinase showed half-maximal activity at around 1 . 7 m M Ca 2÷, and maximal activity at more than 1 0 m M Ca 2÷, which is comparable with 1.5 and 5 m M , respectively, for carp calpain II (Toyohara and Makinodan, 1989) and maximal activity at 5-10 m M Ca 2+ for American lobster Ca 2÷-dependent proteinases (Mykles and Skinner, 1986), in contrast to 0.75 and 1.5mM, respectively, for rabbit calpain II (Inomata et al., 1984). Moreover, the scallop calpain II-like proteinase was not activated by either Sr 2 + or Ba 2 ÷ (Fig. 4), in contrast to the rabbit calpain II which was activated by Sr 2+ (Inomata et aL, 1984), and carp calpain II by both Sr 2÷ and Ba 2÷. However, Mn 2 ÷ and Zn 2÷ were also ineffective for the scallop proteinase (data not shown) as well as for rabbit and carp calpains. These results indicate that the scallop proteinase is highly specific for Ca: ÷ and further suggest that the effect of these metal ions depends on the affinity of the metal ions for the proteinases. The scallop calpain II-like proteinase showed the optimal activity at pH 6.8 similarly to American lobster Ca2+-dependent proteinases (Mykles and Skinner, 1986), while vertebrate calpains showed it at pH 7.5 (Ishiura et al., 1978; Dayton et al., 1976; Taneda et al., 1983; Toyohara et al., 1985). The optimum temperature of scallop calpain II (18°C) was considerably lower than that of rabbit calpain (30°C). REFERENCES

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