This series of papers is concerned with the isolation and physiological properties of 3-P-glycerate phosphatase. Purification and characterization of this phosphatase was done with extracts of sugarcane leaves (31). The enzymatic hydrolysis of P-glycolate and 3-P-glycerate has many parallels. The substrates are structural analogs at each end of the glycolate pathway ( Fig. 1), but the phosphatases are distinctly different. In C3-plants' glycolate metabolism, peroxisomes, and photorespiration are active, and in these plants P-glycolate phosphatase likewise is a major enzyme and relatively more active than 3-P-glycerate phosphatase. In C4-plants the peroxisomal system is mainly restricted to the bundle sheath cells (39). where most of the P-glycolate phosphatase is located (33). In an initial survey of C,-plants more 3-P-glycerate phosphatase activity was found than P-glycolate phosphatase (30), and a major part of the 3-P-glycerate phosphatase was in the cytosol of the mesophyll cells (33), which also contain the Cg-pathway of CO. fixation (16). It is suggested that the two phosphatases regulate different metabolic routes to the same products, glycine and serine. and are involved in regulating photorespiration. That 3-P-glycerate phosphatase may be involved in a carbon shuttle system in C-plants is to be considered.MATERIALS AND METHODS Plants were grown for 3 to 6 weeks in greenhouse or field as indicated in Table I, and only mature leaves were used. Etiolated sugarcane leaves were from stem nodes kept in a dark growth chamber in vermiculite and watered with Hoagland's nutrient solution for 21 days at 23 C. Chlorella pyrenoidosa (Marburg strain) and Chlamnydomnonas reinhardtii were grown as described previously (19). Tissues were extracted immediately after harvest. The leaves were washed, blotted dry, large midribs removed, and remaining tissue diced. The leaf samples were then homogenized for 2 min at 4 C in a Waring Blendor with five volumes of a grinding medium containing 20 mm sodium cacodylate buffer at pH 6.3 and 1 mm EDTA. As indicated 2% Polyclar AT and 20 'Plants which initially fix C02 mainly by the photosynthetic carbon reduction cycle are termed C3-plants, and plants which also contain the C4-dicarboxylic acid cycle of C02 fixation in the mesophyll cells, are termed C4-plants (16). 480www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

A particulate form of 3-phosphoglycerate phosphatase represents about 20% of this activity in spinach (Spinacia oleracea var. Longstanding Bloomsdale) leaves. By differential and isopyenic sucrose density gradient centrifugation, all the particulate activity was found in starch grains that pelleted through 2.5 M sucrose. This particulate phosphatase was extremely stable, had a pH optimum of 5.8, and an apparent Michaelis constant (3-phosphoglycerate) of 9 X 10' M. No cation requirement for activity could be demonstrated, and the enzyme was inhibited by 0.5 mM Zn2+ or Cu2+. The enzyme was most active in catalyzing the hydrolysis of 3-phospho-D-glycerate, but it was not substrate specific. The phosphatase from the starch grains could not be removed by washing, dialysis, homogenization, or treatment with a French pressure cell, but it was solubilized by prolonged sonication or by addition of 0.25 M MgC12, when the particles were suspended in 0.8 M sucrose. The solubilized enzyme was partially purified. The properties of the enzyme on the particles or after solubilization were similar to those previously described for the cytosol form of this phosphatase. It is conjectured that the phosphatase of the starch grain regulates glucan synthesis by controlling a 3-phosphoglycerate pool which is an effector for ADP-glucose pyrophosphorylase.In the two previous papers of this series the isolation and characterization of a soluble form of 3-P-glycerate phosphatase from sugarcane leaves were described (5), and the distribution and physiological properties of the enzyme were explored (6). The enzyme is very active in leaves of C4-plants and is located mainly in the cytosol of the mesophyll cells. In leaves of C3-plants much of it also appeared in the soluble fraction. However, a substantial portion (about 20%) of the 3-P-glycerate phosphatase activity of spinach leaves has been found firmly bound to starch-like particles. The MATERIALS AND METHODS Fresh field-grown spinach (Spinacia oleracea var. Longstanding Bloomsdale), leaves were used shortly after harvest or after storage for several days at 4 C. The procedure of Tolbert et al. (8) for peroxisome isolation from spinach leaves was followed. The leaves were ground in a Waring Blendor for 10 sec in medium containing 0.5 or 0.8 M sucrose and 20 mm glycylglycine at pH 7.5. Particles were removed by differential centrifugation at 0 to 6000g for 10 or 20 min and resuspended in the grinding medium. They were then separated by discontinuous isopycnic sucrose gradient centrifugation at 47,000 to 107,000g through 1.5 to 2.3 or 2.5 M sucrose. The pellet that sedimented through the densest sucrose was the source of the particulate 3-P-glycerate phosphatase.The assay for 3-P-glycerate phosphatase and other methods were the same as used previously (4-6). Heavy metal salts of the substrates were converted by resins to K+ or Na+ salts before use. Determination of reducing sugar was by the method of Nelson (3). Particles were stained with 12 by boiling for 5 min in 70% ethanol, w...

Extracts, mainly from mesophyll cells, were obtained by grinding cells in a Waring Blendor; then extracts of parenchyma sheath cells were obtained by exhaustive grinding of the blender residue in a roller mill or mortar with sand. The specific activities of P-glycolate phosphatase, glycolate oxidase, catalase and reduced nicotinamide adenine dinucleotide- (NADH-) hydroxypyruvate reductase were fourfold higher in extracts of the parenchyma sheath cells than in the mesophyll cells from corn, sugarcane, and Atriplex rosea. P-Glycerate phosphatase was mainly located in the mesophyll cells. The total activity of glycolate oxidase in plants without CO2-photorespiration averaged about one-third that found in other plants on a wet-weight basis. Glycolate oxidase activity in Atriplex rosea, without CO2-photorespiration, was about the same as in Atriplex patula, with CO2-photorespiration. It is concluded that enzymes for glycolate metabolism are present in all leaves in substantial amounts and are located in both cell types, although a higher specific activity is in the parenchyma sheath cells. Thus it is proposed that photorespiration occurs in all plants, but that CO2 evolution from glycolate metabolism is not manifested in plants which have high levels of activity for the C4-dicarboxylic acid cycle of CO2 fixation.

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