THE INTRACELLULAR LOCALIZATION OF ENZYMES IN TOBACCO LEAVES. I. IDENTIFICATION OF COMPONENTS OF THE HOMOGENATE

Abstract

MANY INTERESTING and significant results concerning the relationship of subcellular structures to biochemical functions have come from studying isolated mitochondria, nuclei, etc. of mammalian tissues using ordinary biochemical procedures (see reviews of Bradfield, 1950; Schneider and Hogeboom, 1951). Plant tissues have not received as much attention, but Chibnall (1939), Granick (1938a,b) and Menke (1937, 1938) made pioneering studies of the composition and properties of chloroplasts and cytoplasm from homogenized leaves. Other studies of isolated chloroplasts and soluble proteins have followed, some indebted to Granick for the method of isolation in M/2 sugars (see reviews by Rabinowitch, 1945; Granick, 1949; Bonner, 1950). Cellular components other than chloroplasts and soluble proteins have been more or less ignored in such studies. Nuclei have usually been found to disappear upon homogenization (but see Menke, 1937, 1938, and duBuy and Woods, 1943). No one has isolated samples of them from leaves. The only mention of isolated leaf mitochondria is the brief paper of duBuy et al. (1950). While some authors have attempted to isolate pure samples of chloroplasts, others have lumped as chloroplasts all particulate material sedimenting at up to 20,000 X g. (McClendon, 1951). One purpose of this paper is to show that this procedure is unjustified. Hence, although preliminary in some respects (especially with regard to the nucleic acid data), this is an attempt to identify and to some extent characterize quantitatively the particulate material in a tobacco leaf homogenate. METHODS.-Preparation of material.-Nicotiana tabacum L. (var. Connecticut 'Seed Leaf) was grown in the greenhouse or in the adjoining garden. Mature leaves from large plants were cut immediately before use, washed, the midribs removed, and cut into pieces. All subsequent steps in preparation of the material were done in a cold room maintained at about 4?C. About 60 g. of leaf were put into a Waring, Blen-