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Plant Physiology 58:309-314 (1976)
© 1976 American Society of Plant Biologists

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Articles

Isolation of Intact Chloroplasts and Other Cell Organelles from Spinach Leaf Protoplasts 1

Mikio Nishimura, Douglas Graham2 and Takashi Akazawa

a Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.

2 Permanent address: Plant Physiology Unit, Commonwealth Scientific and Industrial Research Organization, Division of Food Research and School of Biological Sciences, Macquarie University, North Ryde, N. S. W. 2113, Australia. Recipient of Award from the Japan Society for the Promotion of Science under the Visiting Professorship Programme (1974).

1 This is Paper No. XXXV in the series "Structure and Function of Chloroplast Proteins." The research is supported in part by the grants from the Ministry of Education of Japan, the Toray Science Foundation (Tokyo), and the Naito Science Foundation (Tokyo).




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A. M. Timperio, G. M. D'Amici, C. Barta, F. Loreto, and L. Zolla
Proteomic, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves
J. Exp. Bot., October 10, 2007; (2007) erm219v1.
[Abstract] [Full Text] [PDF]


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