Rat urate oxidase produced by recombinant baculovirus expression: formation of peroxisome crystalloid core-like structures.
Urate oxidase (EC 1.7.3.3), which catalyzes the oxidation of uric acid to allantoin, is present in most mammals but absent in humans and hominoid primates. In rats and most other mammals that catabolize uric acid to allantoin, this enzyme is localized within the crystalloid cores of peroxisomes present in liver parenchymal cells. To determine whether urate oxidase forms these crystalloid cores or whether core-forming protein(s) exist in association with urate oxidase, a baculovirus expression vector system was used to overproduce the full-length rat urate oxidase in Spodoptera frugiperda cells. Urate oxidase was expressed to a level of approximately 30% of the total protein in this system. Immunoblot analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties similar to those of urate oxidase expressed in rat liver. Immunofluorescence and electron microscopic examination revealed that the overexpressed recombinant urate oxidase is present in both the cytoplasm and the nucleus of infected insect cells as numerous 1- to 3-microns discrete particles. These insoluble protein aggregates, which were positively stained for urate oxidase by protein A-gold immunocytochemical approach, did not appear to be delimited by a single membrane. They revealed a crystalloid structure reminiscent of rat peroxisomal core consisting of bundles of tubules with an inner diameter of approximately 50 A. The recombinant urate oxidase particles, isolated by a single-step procedure, were composed entirely of 35-kDa urate oxidase subunit. These studies indicate that rat urate oxidase is capable of forming insoluble crystalloid core-like structures.
Alvares
K
K
Department of Pathology, Northwestern University Medical School, Chicago, IL 60611.
Widrow
R J
RJ
Abu-Jawdeh
G M
GM
Schmidt
J V
JV
Yeldandi
A V
AV
Rao
M S
MS
Reddy
J K
JK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003460:Crystallization; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008830:Microbodies / Q000201:enzymology*; D009036:Moths; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014503:Urate Oxidase / Q000737:chemistry* / Q000235:genetics
D011994:Recombinant Proteins; D014503:Urate Oxidase