NOVEL PURIFICATION AND DETAILED CHARACTERIZATION OF PERLECAN ISOLATEDFROM THE ENGELBRETH-HOLM-SWARM TUMOR FOR USE IN AN ANIMAL-MODEL OF FIBRILLAR A-BETA AMYLOID PERSISTENCE IN BRAIN

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perle can, and beta-amyloid protein (A beta) into rodent hippocampus leads t o a consistent animal model to study the effects of fibrillar A beta a myloid in brain [Snow, A.D. et al, (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolatio n. The isolation method does not require cesium chloride centrifugatio n and exploits a newly discovered aggregating property of a similar to 220 kDa PG observed during gel filtration chromatography, which allow ed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HC l, followed by anion-exchange and gel filtration chromatography, SDS-P AGE (before and after digestion with heparitinase/heparinase or nitrou s acid) followed by staining with silver demonstrated no other contami nating proteins in the perlecan preparations. Western blots using a sp ecific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicativ e of intact perlecan core protein. Absence of contamination by other b asement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies aga inst laminin, fibronectin, or type IV collagen. One week continuous co -infusion of perlecan obtained from this methodology, with A beta (1-4 0) into rodent hippocampus, led to deposition of fibrillar A beta amyl oid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of t he highest quality, and maximizes the potential effects of A beta amyl oid deposition/persistence in brain using the animal model. High quali ty perlecan obtained from this novel isolation method will also allow future studies utilizing in, vitro assays to determine the potential i nteractions of this specific HSPG with other macromolecules.