Table 8.1 Characteristics of isolated chloroplasts


Chloro-
plast
type
Description
Preparation method
Appearance under phase contrast microscopy
Envelope
Rate of CO_2 fixation (CO_2/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


Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu