COMPOSITION OF CHLOROPLASTS 1747 



including cytochrome oxidase, in seeds, bulbs, roots, and tubers of many- 

 plant species, indicating the general capacity of plants for the (cyanide- 

 sensitive) cytochrome-mediated respiration. Webster (1952) found cyto- 

 chrome oxidase in leaf material from 19 species and in many other plant 

 tissues. McClendon (1953) fractionated homogenized tobacco leaves and 

 separated the product into four fractions containing, respectively, whole 

 chloroplasts, chloroplast fragments of different size, and no chloroplastic 

 material at all. Cytochrome oxidase activity was found in all fractions, 

 but in approximately inverse relation to the chlorophyll content, in con- 

 tradiction to the conclusions of Rosenberg and Ducet, and of Sisakyan and 

 Filipovich. (Chlorophyll precipitated preferentially with the largest 

 particles, cytochrome oxidase with the smallest ones; as mentioned above, 

 desoxyribonucleic acid followed chlorophyll in this fractionation.) At- 

 tempts to prepare a chloroplast fraction quite free of cytochrome oxidase, 

 however, did not succeed. McClendon suggested that most, if not all, 

 of the cytochrome oxidase in leaves is associated with mitochondria, as in 

 animal tissues. No cytochrome / oxidase was found by Hill and co- 

 workers. 



Daly and Brown (1954) demonstrated cytochrome oxidase action by ob- 

 serving the reversal, in light, of CO2 inhibition of the 0(18)0(16) uptake 

 by live leaves. 



An alternative mechanism of hydrogen transfer to oxygen in respira- 

 tion involves the copper-containing polyphenoloxidase ( = tyrosinase, or 

 catecholase) instead of the cytochrome oxidase. This oxidase is widely 

 distributed in plants, although Holt (1950) found no evidence of it in 

 Phytolacca americana. This respiration mechanism is not inhibited by 

 cyanide and could conceivably account for the low cyanide sensitivity of 

 respiration in many plants, e. g. in Chlorella (cf. Chapter 12, section Al). 



Arnon (1948, 1949) reported that, in beet leaves, the polyphenol 

 oxidase is localized in the chloroplasts. Leaves were mashed in a Blendor, 

 and chloroplast fragments separated by high-speed centrifugation. The 

 oxidase was assayed in both fractions, using ascorbic acid as oxidation 

 substrate and catechol as intermediate catalyst, and found to be concen- 

 trated to more than 80% in the chloroplast fraction. 



According to Arnon, disintegration of Beta chloroplasts by grinding with 

 glass beads on a Nickle tissue-grinding machine and repeated high-speed 

 centrifugation (a procedure by which the more proteinaceous or lipopro- 

 teinaceous stroma of the chloroplasts presumably is separated from the 

 more strongly lipoidic grana) fails to separate the oxidase activity from the 

 green precipitate. 



These results do not agree with the observations of Warburg and Liitt- 

 gens (1946), who noted that at pU 6.5, the larger part of the oxidase 



