Table 8.1 Characteristics of isolated chloroplasts

Chloro- plast type	Description	Preparation method	Appearance under phase contrast microscopy	Envelope	Rate of CO fixation (CO/mg chlorophyll/hr)	Exogenous substrate penetration and requirements	Electron transport and photophosphorylation capacity
A	Complete chloroplasts	Rapid, in isotonic or hypertonic sugar, one centrifugation	Outer mobile jacket present. Bright and highly reflecting. Grana not obvious.	Intact	50 - 250	NADP and ferricyanide do not penetrate. Slow uptake of ATP, ADP and Pi.	Presumed to be unimpaired. (ATP/2e approaching 2.0 when assayed in hypotonic medium: good photosynthetic control)
B	Unbroken chloroplasts	In isotonic or hypertonic sugar or salt, with 2 or 3 centrifugations	Bright and highly reflecting. Smooth outline. Grana not obvious. (Class I)	Morphologi- cally but not functionally intact	<3	NADP, ferricyanide and ADP penetrate. Ferredoxin may not be necessary to add for NADP reduction.	Good ATP/2e (greater than 1.0 and often aproaching 2.0 when assayed in hypotonic medium). Good photosynthetic control.
C	Broken chloroplasts	Vigorous in isotonic sugar or salt	Not bright in appearance. 2-3 times larger than types A and B. Grana conspicuous. (Class II)	Broken and usually lost in prepara- tion (stroma also lost)	Little or none	NADP, ferricyanide and ADP penetrate. Ferredoxin needs to be added for NADP reduction	ATP/2e >1.0 or <1.0 depending on isolation and assay conditions. Some photosynthetic control
D	Free-lamellar chloroplasts	Osmotic shock of type A chloroplasts immediately followed by return to isotonic medium	----	Lost	High rates if carbon pathway intermediates and chloroplast extract added	----	Good photosynthetic control
E	Chloroplast fragments	Resuspend chloroplasts in hypotonic medium	----	Lost	None	Ferredoxin needs to be added for NADP reduction	Higher rates of electron transport and lower rates of photophosphorylation than types B and C. ATP/2e <1.0. No photosynthetic control
F	Sub- chloroplast particles	By sonication or detergent treatment or French press	----	Lost	None	Ferredoxin and plastocyanin (and some reductase needs to be added for NADP reduction)	Photophosphorylation (cyclic only) low or absent. Limited electron transport when electron donors added