caa a22 4500 397556705 CHVBK 20180308164804.0 cr unu---uuuuu 161202e199608 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021430 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021430 Novel Purification and Detailed Characterization of Perlecan Isolated from the Engelbreth-Holm-Swarm Tumor for Use in an Animal Model of Fibrillar Aβ Amyloid Persistence in Brain [Elektronische Daten] [Gerry M. Castillo, Joel A. Cummings, Catherine Ngo, Wenhua Yang, Alan D. Snow] Co-infusion of the specfic heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (Aβ) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar Aβ amyloid in brain [Snow, A.D. et al. (1994) Neuron 12,219-2341. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a ˜220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HC1, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibadies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with AB (1-40) into rodent hippocampus, led to deposition of fibrillar Aβ amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of Aβ amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence Alzheimer's disease nationallicence EHS tumor nationallicence heparan sulfate nationallicence perlecan nationallicence proteoglycans nationallicence Castillo Gerry M. Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut Cummings Joel A. Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut Ngo Catherine Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut Yang Wenhua Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut Snow Alan D. Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut The Journal of Biochemistry Oxford University Press 120/2(1996-08), 433-444 0021-924X 120:2<433 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021430 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021430 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Castillo Gerry M. Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut NATIONALLICENCE 700 1- Cummings Joel A. Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut NATIONALLICENCE 700 1- Ngo Catherine Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut NATIONALLICENCE 700 1- Yang Wenhua Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut NATIONALLICENCE 700 1- Snow Alan D. Department of Pathology, Neuropathology Laboratories, Box 356480, Seattle, Washington 98195-6480, USA aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/2(1996-08), 433-444 0021-924X 120:2<433 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford