Introduction Directory UMM :Data Elmu:jurnal:I:Insect Biochemistry and Molecular Biology:Vol31.Issue2.Feb2001:

Insect Biochemistry and Molecular Biology 31 2001 157–164 www.elsevier.comlocateibmb Preliminary characterisation of esterase and platelet-activating factor PAF-acetylhydrolase activities from cat flea Ctenocephalides felis salivary glands M.T. Cheeseman a, , P.A. Bates b , J.M. Crampton c a Department of Veterinary Pathology, University of Liverpool, Liverpool, UK b Liverpool School of Tropical Medicine, University of Liverpool, Liverpool, UK c School of Biological Sciences, University of Liverpool, Liverpool, UK Received 19 January 2000; received in revised form 1 June 2000; accepted 6 June 2000 Abstract Naphthyl esterase and platelet-activating factor PAF-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters α -naphthyl acetate |210 pmolmingland pair; 10.0 µ molminmg specific activity; K m |59 µ M and β -naphthyl acetate |110 pmolmingland pair; 5.2 µ molminmg specific activity; K m |132 µ M. Salivary gland extracts have PAF-acetylhydrolase activity |5 pmolmingland pair; 0.24 µ molminmg spe- cific activity but do not have detectable acetylcholinesterase activity. Native–PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native–PAGE. Renaturation of esterase activity after SDS–PAGE gave |56 kDa, |57 kDa and |58 kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of |59 kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain α - and β -naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Cat flea Ctenocephalides felis; Salivary gland; Saliva; Esterase; Glycoprotein; Platelet-activating factor PAF; PAF-acetylhydrolase

1. Introduction

The cat flea Ctenocephalides felis is the most important ectoparasite of domestic dogs and cats chiefly because flea bites cause flea allergy dermatitis FAD, the most common canine skin disease world-wide, and miliary dermatitis in cats Rust and Dryden, 1997; Gross et al., 1992. Cat flea salivary glands contain a mixture of allergens whose function in blood feeding is unknown McCall et al., 1997; Frank et al., 1998; Lee et al., 1997, Corresponding author. Current Corresponding address: University of London, The Royal Veterinary College, Dept Pathology Infec- tious Disease, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA. Tel.: + 44-1707-666208; fax: + 44-1707-661464. E-mail address: mcheesemanrvc.ac.uk M.T. Cheeseman. 0965-174801 - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 1 1 3 - 2 1999 and apyrase activity Cheeseman, 1998. In other insects and ticks, salivary apyrase has been suggested to act as an inhibitor of adenosine diphosphate ADP- mediated platelet aggregation Champagne and Valen- zuela, 1996; Ribeiro, 1995. A more detailed knowledge of the composition of cat flea saliva is needed to under- stand why saliva induces inflammatory and immune reactions in dogs and cats. Other species of blood-feed- ing arthropods have a range of salivary molecules such as vasodilators, anticoagulants and platelet-aggregation inhibitors that play a role in overcoming the host’s hae- mostatic mechanisms Champagne and Valenzuela, 1996; Ribeiro, 1995. The roles of other characterised salivary gland products, such as esterase in mosquitoes Poehling and Meyer, 1980; Kerlin and Hughes, 1992; Argentine and James, 1995, ticks Schleger and Lin- 158 M.T. Cheeseman et al. Insect Biochemistry and Molecular Biology 31 2001 157–164 coln, 1976 and blackflies Wirtz, 1990 and tick phos- pholipase A 2 Bowman et al., 1997; Zhu et al., 1998, are not so clear because naturally occurring ester and phospholipid substrates have not been identified. In this study we describe the heterogeneity and glyco- sylation of cat flea salivary gland naphthyl esterases and document salivary gland platelet-activating factor PAF-acetylhydrolase activity. The potential anti- inflammatory and anti-allergic role of salivary gland PAF-acetylhydrolase activity in fleas and other blood- feeding arthropods is discussed.

2. Methods and materials