Ž . Comparitive Biochemistry and Physiology Part B 130 2001 199206 Proteolytic activity of cultured Pseudoperkinsus tapetis extracellular products M. Camino Ordas a , Beatriz Novoa a , Mohamed Faisal b , ´ Shawn McLaughlin c , Antonio Figueras a,  a ( ) Instituto de In estigaciones Marinas, Consejo Superior de In estigaciones Cientıficas CSIC , Eduardo Cabello, 6, 36208 Vigo, ´ Spain b Department of En ironmental Sciences, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, VA, USA c National Marine Fisheries Ser  ice, Oxford, MD, USA Received 6 November 2000; received in revised form 30 May 2001; accepted 3 June 2001 Abstract Several pathogenic protozoan release proteases are necessary for host invasion and initiation of infection. We have identified proteolytic activities in extracellular proteins secreted by the clam parasite Pseudoperkinsus tapetis Ž . Ž . Mesomycetozoa in vitro. The protein concentration of the P. tapetis extracellular products ECP increased only during the first week of culture. The appearance of new proteins of 10 and 157 kDa at the second week sample and of 12 kDa at the third week sample was shown by SDS-PAGE. The protease activity rapidly increased in the first 3 weeks of culture, and five clear bands of 23, 29, 60, 67 and 96 kDa with proteolytic activity were detected in the ECP on gelatin SDS-PAGE. Using inhibitors, the proteases were identified as members of the Ca 2 dependent, serine protease family. Their optimum pH was higher than pH 9.4. The protease activity of the P. tapetis ECP was different than that described for Perkinsus marinus, an oyster pathogen very similar morphologically to the clam parasite and member of the genus in which P. tapetis had been initially included.  2001 Elsevier Science Inc. All rights reserved. Keywords: Pseudoperkinsus tapetis; Mesomycetozoa; DRIP; Perkinsus marinus; Proteases; Extracellular products; ECP; Protozoan 1. Introduction Infection with Perkinsus spp. in bivalve mol- lusks has gained special attention due to its asso- ciation with tissue damage and mortalities. Le-  Corresponding author. Tel.: 34-86-23-19-30; fax: 34- 86-29-27-62. Ž . E-mail address: pato1@iim.csic.es A. Figueras . sions caused by Perkinsus spp. vary from local- Ž ized, encapsulated foci in gill tissues Montes et . al., 1995; McLaughlin and Faisal, 1998a to gener- alized deadly infections with protozoal cells dis- seminated to all organs of the infected mollusk Ž . Mackin, 1951 . The reason for this discrepancy in severity of infection can be attributed, at least partly, to variation in expression of virulence fac- tors among members of the genus Perkinsus. The recent success of establishing continuous cultures 1096-495901$ - see front matter  2001 Elsevier Science Inc. All rights reserved. Ž . PII: S 1 0 9 6 - 4 9 5 9 01 00423-7