Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol149.Issue1.2000:

Plant Science 149 1999 33 – 41 Purification and characterization of the 26S proteasome from cultured rice Oryza sati6a cells Yuki Yanagawa a,b , Akiko Ohhashi a , Yasuko Murakami c , Yasushi Saeki d , Hideyoshi Yokosawa d , Keiji Tanaka e , Junji Hashimoto b,f , Takahide Sato a , Hiroki Nakagawa a, a Department of Bioproduction Science, Faculty of Horticulture, Chiba Uni6ersity, Matsudo, Chiba 271 - 8510 , Japan b CREST Core Research for E6olutional Science and Technology of Japan Science and Technology Corporation JST , Chiyoda-ku, Tokyo 101 - 0062 , Japan c Department of Biochemistry 2 , The Jikei Uni6ersity School of Medicine, Nishishinbashi, Minato-ku, Tokyo 105 - 8461 , Japan d Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido Uni6ersity, Kita-ku, Sapporo 060 - 0812 , Japan e The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113 - 8613 , Japan f National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki 305 - 8602 , Japan Received 30 April 1999; received in revised form 12 July 1999; accepted 13 July 1999 Abstract The 26S proteasome was purified from cultured rice cells to near homogeneity by ultracentrifugation for 5 h at 85,000 × g, chromatography on Biogel A-1.5m, and glycerol density-gradient centrifugation analysis. The purified enzyme had two distinct forms, termed 26Sa- and 26Sb-type proteasomes, with different electrophoretic mobilities by nondenaturing polyacrylamide gel electrophoresis. It consisted of multiple polypeptides with molecular masses of 25 – 35 and 42 – 120 kDa, which presumably corresponded to those of the 20S proteasome and an associated PA700 regulatory complex, respectively. The rice 26S proteasome resembled, but was not identical to, one from other sources in their subunit composition and immunochemical reactivity. Intriguingly, both rice and spinach 26S proteasomes could not degrade rat ornithine decarboxylase in the presence of antizyme and ATP, unlike the rat 26S proteasome, implying the existence of functional differences between mammalian and plant 26S proteasomes. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords : 26S Proteasome; 20S Proteasome; Rice Oryza sati6a L. www.elsevier.comlocateplantsci

1. Introduction

The 26S proteasome multicatalytic endopepti- dase complex EC 3.4.99.46 is a large multisubunit complex composed of a core proteinase, known as the 20S proteasome, and a pair of symmetrically disposed PA700 also named 19S complex regula- tory particles [1 – 3]. It is known to catalyze the ATP-dependent degradation of ubiquitinated proteins and is involved in a variety of important cellular processes [4,5]. Proteasomes in higher plants have been characterized both structurally and immunologically [6 – 10]. The 20S proteasomes have been purified from dicotyledonous plants such as spinach leaves [7] and dry pea seeds [8], and monocotyledonous plants such as wheat leaves [9]. The biochemical characteristics of proteasomes isolated from these three plant sources are remarkably similar to those of the proteasomes found in other eukaryotes. The 26S proteasome was recently isolated from spinach leaves [10]. By electron microscopy, this 26S proteasome from spinach was found to be struc- turally similar to those of Xenopus [11], rat [12] and yeast [13]. However, 26S proteasome from only spinach was purified in higher plants, al- though the genes that encode many of the 26S Corresponding author. Tel.fax: + 81-47-308-8862. E-mail address : nakagawamidori.h.chiba-u.ac.jp H. Nakagawa 0168-945299 - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 4 0 - 5 proteasome subunits, including all of the 20S proteasome, have been reported [14 – 20]. The properties of some enzymes are distinct between monocotyledonous and dicotyledonous plants; for example, acetyl-CoA carboxylase is particularly distinct in gramineae such as rice [21]. Although there have been a considerable number of genetic studies on rice [14,17,19,22], little is known of the purification and properties of the rice 26S proteasome. The relationship between the properties of rice and spinach 26S proteasomes has never been clarified. In this paper, we report the purification and some properties of the 26S proteasome from cultured rice cells, compare the properties of rice and spinach 26S proteasomes, and discuss the functional differences between higher plant and mammalian 26S proteasomes.

2. Materials and methods