Light and Dark C14O2 Fixation by Spinach Leaf Systems

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Abstract
A cell-free homogenate, prepared from spinach leaves using minimal dilution in the absence of NaCl, incorporated C14O2 in both the light and dark. The distribution of the C14 in the 80% ethanol extract from this homogenate was similar to the distribution found in the intact leaf. The endogenous rate of photosynthetic C14O2 incorporation was 11% of the intact leaf. The addition of ribulose diphosphate, ADP and NADP+ increased the rate to 44% of that of the intact leaf. The effect of adding cofactors in the dark was studied in an attempt to identify the compounds which are produced by the light reaction involved in the reduction of CO2. Ribulose diphosphate and ATP were found to be most effective. The rate of incorporation and the distribution of C14 in the dark with the addition of ribulose diphosphate and ATP closely corresponded to the same conditions in the light. The addition of NADPH or NADH to the homogenate in the dark in the presence of ribulose diphosphate and ATP resulted in an inhibition of the rate of incorporation. However, NADPH and NADH acted by entirely different mechanisms; NADH blocked the incorporation of activity into carbohydrates, while NADPH resulted in a decrease in C14 incorporation. An electron microscope comparison of chloroplast utrastructure in this preparation, and the chloroplast structure of the intact leaf, as well as the chloroplasts isolated in 0.35 M NaCl was made. In NaCl the chloroplasts had little resemblance to those in the intact leaf; the grana were greatly distorted and swollen with a complete loss of the outer membrane. Chloroplasts from the above preparation had lost their outer membrane, but the structure of the grana and lamellae remained intact.

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