where they are well separated from early biosynthetic compartments. Using Western blotting, we show that these lysosomes contain APP co-localized with and the gamma secretase proteins nicastrin, presenilin-1, mAph1 and Pen2 and gamma secretase activity. Conclusions: This work demonstrates the usefulness of Free Flow Electrophoresis as a tool to study gamma- secretase proteins and activity and reinforces the importance of the endo- somal/ lysosomal system in the generation of beta amyloid. P3-236 ENZYMES IN NEURODEGENERATION. PEPTIDYL ARGININE DEIMINASE II IN ETIOLOGY AND PATHOGENESIS OF ALZHEIMER’S DISEASE Chris Whiteley, Cassandra Louw, Graeme Bradley, Rhodes University, Grahamstown, South Africa. Contact e-mail: c.whiteley@ru.ac.za Background: Alzheimer’s disease (AD) is a fatal form of dementia that results in progressive neurodegeneration. At present, there is no definitive pre-mortem diagnostic tool for AD, which relates to a poor understanding of disease etiology. Subsequently, development of effective diagnostic tools and treat- ment regimens requires an enhanced understanding of the etiology and patho- genesis of Alzheimer’s disease. Brains of AD patients contain large amounts of the plaque-forming -Amyloid 1-42 fragment in addition to elevated concen- trations of L-arginine. Objectives: The interaction of -amyloid with arginine deiminase and/or PADII as well as levels of arginine in the astrocytes sur- rounding amyloid plaques can serve as therapeutic tools in neurodegenerative disorders such as Alzheimer’s disease. Methods: Peptidylarginine deiminase II [PADII] was purified from bovine brain by the method of Lammensa and Moscarello (1993) and assayed by measuring the rate of conversion of arginine to citrulline according to Boyde and Rahmatullah. Arginine deiminase from Pseudomonas and PADII from bovine brain are inhibited by amyloid frag- ments [2 - 11 nM] such as the arginine containing toxic plaque-forming amyloid 1-42 as well as the arginine-free amyloid fragments like amyloid 12-28 and amyloid 22-35. The peptidyl enzyme from bovine brain also exhibited an enhanced activity [1.8 fold] in the presence of free L-arginine [38 M]. Total RNA was isolated from bovine and rat brain, primers identified and the coding regions of PADII amplified using RT-PCR. Unfortunately this was unsuccess- ful and no product corresponding in size to the PAD II coding region was obtained. Conclusions: It is suggested that PADII is transcribed only in response to certain stimuli in the brain. Furthermore since it was inhibited by several amyloid fragments it could be identified as a pathogenic marker and play a role in the regulation of formation of amyloid aggregates. The signifi- cance of these findings in the etiology and pathogenesis of Alzheimer’s disease will be discussed. References Lamensa JWE, Moscarello MA, 1993. Deimination of human myelin basic protein by a peptidylarginine deiminase from bovine brain. J Neurochem 61:987-996. Boyde TRC, Rahmatullah M. 1980. Optimization of conditions for the colorimetric determination of citrulline using monoxime. Anal Biochem 107:424-431. P3-237 REGULATION OF THE ASPARTYL PROTEASE BACE ACTIVITY Virginie Buggia 1 , Vincent Lisowski 2 , Jean-Franc ¸ois Hernandez 2 , Steffen Rossner 3 , Fre ´de ´ric Checler 1 , 1 CNRS, Valbonne, France; 2 CNRS UMR 5810, Montpellier, France; 3 Universita ¨t Leipzig, Leipzig, Germany. Contact e-mail: buggia@ipmc.cnrs.fr The aspartyl protease BACE1 has been demonstrated to be the major -secretase-like activity involved in amyloid precursor (APP) processing. This cleavage is the first step of A production, the major component of senile plaques in Alzheimer disease. We have developed a novel BACE assay based on two quenched fluorometric substrates mimicking the wild type APP sequence JMV2235 and swedish mutated APP sequence JMV2236 which increase BACE activity 1 . With this selective assay we identified a series of statine-derived BACE inhibitors and in particular JMV1195 which can inhibit A production in vitro. Because JMV1195 was unable to inhibit BACE and A production in vivo, we developed a new permeant inhibitor, JMV2764 which corresponds to the derivative JMV1195 in which a penetratin sequence has been inserted 2 . This assay was proved useful to examine the cellular regulation of BACE activity. We will discuss here novel data concerning both transcriptional and post- transcriptional regulation of BACE1 in various cell systems. 1 Andrau D. et al., J Biol Chem. 2003 Jul 11;278(28):25859-66. 2 Lefranc-Jullien S. et al., Br J Pharmacol. 2005 May;145(2):228-35. P3-238 DESIGN OF NEW FLUORIMETRIC SUBSTRATES AND NEW INHIBITORS OF GAMMA-SECRETASE AND PRESENILINASE ACTIVITIES Jean Se ´valle 1 , Erwan Eyral 2 , Jean-Franc ¸ois Hernandez 2 , Jean Martinez 2 , Fre ´de ´ric Checler 1 , 1 IPMC CNRS, Valbonne, France; 2 LAPP CNRS, Montpellier, France. Contact e-mail: sevalle@ipmc.cnrs.fr The senile plaques are one of the major histological hallmarks in Alzhei- mer’s disease-affected brains. The amyloid b peptide (Ab), the main constituent of the plaques, is generated from the b-amyloid precursor protein (bAPP) by subsequent attacks by b- and g-secretases, which liber- ate the N- and C-terminal moieties respectively. An additional -cleavage takes place downstream of the g-site and liberates the C-terminal fragment AICD. The nature of the g-secretase still remains unclear and discussed but some lines of evidence suggest that there likely exists both PS-dependent and PS-independent g-secretase activities. Furthermore, whether g- and -secretases are generated by a unique or distinct activities remains to be established. We previously set up a fluorimetric assay to characterize the b-secretase activities in-vitro (1). Following the same approach, we have designed new fluorimetric assays, in order to further characterize the biochemical and pharmacological properties of the g- and -secretase activities. We described data suggesting that g- and -secretases could be a generic name for multiple presenilin-dependent or/and presenilin-inde- pendent activities (2). The presenilins (PS) undergo proteolytic processing by a protease named presenilinase, leading to N-and C-terminal products that associate in a 1:1 stochiometric manner thought to bear the biological activity within the PS-dependent g-secretase complex. In this work, we describe new inhibitors designed to target this “presenilinase” activity and evaluate their potential as Ab production blockers. (1) Andrau D. et al. J Biol Chem. 2003 Jul 11;278(28):25859-66. (2) Armogida et al. Nature Cell Biology 2001, 3, 1030-1033. P3-239 HEPARIN ACTIVATES ETA- SECRETASE(BACE1) OF ALZHEIMER’S DISEASE AND INCREASES AUTOCATALYSIS OF THE ENZYME David H. Small 1 , R. Damian Holsinger 2 , Marie Beckman 1 , 1 Monash University, Clayton, Australia; 2 University of Melbourne, Parkville, Australia. Contact e-mail: david.small@med.monash.edu.au Background: The beta-site APP cleaving enzyme 1 (BACE1) is an aspar- tic protease that generates the N-terminus of the beta-amyloid protein from the beta-amyloid precursor protein (APP). BACE1 is a key target for Alzheimer drug development. However, little is known about the physio- logical regulation of the enzyme. Heparin can promote beta-secretase cleavage of endogenous APP in neuroblastoma cells. However, heparin has also been reported to directly inhibit BACE1 activity in vitro. Objective: To clarify the role of heparin in regulating BACE1. Methods: We exam- ined the effect of heparin on the activity of recombinant human BACE1 (rBACE1) in vitro. Conclusions: Low concentrations (1 microg/mL) of heparin were found to stimulate rBACE1, whereas higher concentrations (10 or 100 microg/mL) were inhibitory. Heparin affinity chromatography S445 Poster P3: Tuesday Posters