The Proteins of Green Leaves. VI. Centrifugal Fractionation of Tobacco Leaf Homogenates and Some Properties of Isolated Chloroplasts.

Chloroplasts of higher plants have been isolated from leaf homogenates, with greater or lesser attempts at purification from other types of particles, using differential centrifugation. Such preparations of chloroplasts, or "chloroplastic matter" (see earlier reviews, 27, 36), have in some cases been reported to contain a series of components; for instance nucleic acid (10, 15, 26), catalase (23, 24), or cytochrome oxidase (11, 12, 28, 31). Critical proof of the presence of these in the chloroplasts, however, requires the preparation of plastids freed from all other types of subeellular particles. We have accordingly investigated the process of purifying tobacco leaf chloroplasts, and have been able to obtain intact whole chloroplasts evidently free from most other leaf components. These plastid preparations had negligible amounts of the two enzymes mentioned above, and had very little nucleic acid. Our preparation of purified chloroplasts has enabled us to study the non-plastid particles of tobacco leaf homogenates. All other centrifugal fractions are contaminated with chloroplasts or chloroplast fragments, in our experience. Having purified chloroplasts, it becomes possible to estimate the contribution of the chloroplasts to each successive fraction, and the properties of the non-plastid particles are found by difference. As a result of our studies, we are able to point out a number of complications to the problem of separating subeellular organelles from leaf tissue. With full recognition of these complications, we have been able to devise methods for analyzing a leaf homogenate for four different classes of particles, as well as for purifying ehloroplasts.