The Distribution of Carbonic Anhydrase and Ribulose Diphosphate Carboxylase in Maize Leaves

Abstract

(5,11,12,15). These activity differences between species may be as great as 18-fold, although in some cases there may only be a 2-or 3-fold greater activity. One exception has been described in extracts of Amaranthus edulis, which had carbonic anhydrase activity similar to that of spinach in one experiment (11). However, A. viridis and A. palmeri had considerably lower activities.In spinach leaves most of the carbonic anhydrase is found in the chloroplast (12,20), where it is located in the stromal fraction (20). But in maize, it was found to be a cytoplasmic enzyme (12), although the activity in the preparation was low.The complete extraction of enzymes from leaves such as maize is not always easily accomplished (1,3,24) Enzyme Extraction. Leaves from young plants (1-2 weeks) were picked and used immediately for extraction. Whole leaves (2 g) were placed in a chilled mortar (4 C), and 1 g of sharp sand was added. When expanding leaves of plants 6 to 8 weeks old were used, the main ribs were removed and the blades were cut into 2-cm strips crosswise prior to grinding. The following progressive grinding procedure was based on the method of Bucke and Long (3).Twenty-five ml of a medium (4 C) containing 50 mM veronal buffer, pH 8.2, 1 mm EDTA, and 10 mM 2-mercaptoethanol were added and the contents were gently ground under a stream of nitrogen gas until the solution was light green. This was filtered through six layers of cheesecloth, the residue was washed with 5.0 ml of medium, and the filtrates were combined. The residue was returned to the mortar and 10.0 ml of medium was added. Grinding under nitrogen was continued until the tissue appeared translucent. The contents were filtered through the same cheesecloth, washed, and the residue returned to the mortar. The grinding, filtering, and washing were repeated twice more for a total of four grindings. Filtrates were usually combined into one vessel under nitrogen, unless separate extracts for each grinding were collected.Enzyme extracts were assayed within 30 min of preparation. A stream of nitrogen was blown over the extracts throughout the operation.Fractionation of Leaves. As described by Bucke and Long (3) and based also on microscopic examination of the chloroplast types in each successive extract, the combined filtrates from the first and second grinding were taken together when an enzymic extract of mesophyll cells was desired. The combined solutions from the third and fourth grindings were used if an enzymic extract of the bundle-sheath cells was needed.Five fields each containing 100 to 300 chloroplasts were counted for each fraction in three separate experiments. Preparations consisting mainly of mesophyll or bundlesheath cells were also obtained by the differential grinding method of Woo et al. (23). The isolation medium consisted 336 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

“…Within the malate-pyruvate C 4 shuttle, we observed strong M expression for PPDK (TC233444 and TC286559) and MDH (TC300313). Carbonic anhydrase (TC280282) was only identified in M membranes; this is consistent with activity assays (121). As expected the NADPmalic enzyme (TC280868) was only identified in the BS.…”

Isolation and Bicarbonate Transport of Chloroplast Envelope Membranes from Species of Differing Net Photosynthetic Efficiency

“…Assays of ribulose bisphosphate carboxylase activity, located in the stroma of mesophyll chloroplasts of spinach, sunflower, and undifferentiated maize chloroplasts (2,15,28), showed less than 1% contamination by stromal material for these chloroplast envelope membranes. With envelope membranes of mesophyll maize chloroplasts, similar levels of ribulose bisphosphate carboxylase activity were observed, indicating no contamination from stromal contents of mesophyll or bundle-sheath chloroplasts, although the presence of ribulose bisphosphate carboxylase in mesophyll chloroplasts is subject to question (2,18). Assays for NAD(P)H:Cyt c oxidoreductase indicated less than 1 % contamination of any envelope membranes by membranes from mitochondria or microsomes (5 (22).…”

Photosynthetic Carbon Metabolism in Isolated Maize Bundle Sheath Strands

“…These authors propose an identical reaction sequence for C, photosynthesis, but suggest that all the enzymes for photosynthetic carbon metabolism are compartmented between the mesophyll cytoplasm or nongreen leaf cells and the mesophyll chloroplasts, relegating the bundle sheath to a mere amyloplast-like function. A third scheme of C4 photosynthesis, suggesting that photosynthetic carbon metabolism is similar in both cell types, has also been proposed (29)…”