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Evidence for a transport system for glycolate in Chlamydomonas was obtained. [14C]Glycolate was taken up rapidly, reaching an equilibrium in less than 2 s at 4 degrees C. Glycolate uptake was stimulated by valinomycin and high KCl or high KCl alone and inhibited by N-ethylmaleimide. This uptake was not dependent on temperature or pH in contrast to uptake of benzoate by diffusion which decreased by orders of magnitude with increasing external pH. Based on these data, a transporter for glycolate is proposed.

Summary Xylitol 1,5-diphosphate is a potent inhibitor of RuDP carboxylase/oxygenase activity. At an enzyme concentration of 400 μg/ml (less than 1 μM), RuDP carboxylase is inhibited 50% by 2 μM xylitol 1,5-diphosphate. The molar ratio of effector to enzyme is 2 to 1. The inhibition with respect to RuDP is mixed uncompetitive and noncompetitive. The response of enzyme to bicarbonate in the presence of xylitol 1,5-diphosphate is sigmoidal. The inhibition is time-dependent and pH-dependent, with a pKa for inhibition of about 8.6. RuDP oxygenase is inhibited at pH values below 9, but at pH 9 or above, neither RuDP oxygenase nor carboxylase is significantly inhibited. Xylitol 1,5-diphosphate does not produce a differential regulation of the RuDP carboxylase and oxygenase activities.

Ribulose-1,5-diphosphate carboxylase was purified fifteenfold from Rhodospirillum rubrum grown autotrophically under H 2 and CO 2 . There was RuDP oxygenase activity associated with the carboxylase. The oxygenase had maximal activity at pH 9.4. Although these bacterial RuDP oxygenase and carboxylase activities were cold labile, activity could not be restored by treatment at 50° in the presence of Mg ++ and a sulfhydryl reagent, in contrast to results with the enzyme from eukaryotes.

Three products formed during the paper chromatography of glycine with water-saturated phenol as a solvent have been investigaetd. These unknowns were stable condensation products whose formation was dependent upon the presence of glycine ad formaldehyde. They were ninhydrin sensitive. Alkaline hydrolysis yielded glycine. For chromatographic practices, the amount of glycine or formaldehyde which may be converted into the condensation products will depend upon the amount of the other component in the mixture. Addition of 1% formaldehyde normally complexes all of the glycine. In usual chromatographic experiments part of the glycine may be complexed by residual formaldehyde present in chromatographic phenol. Glycine condensation may be minimized by redistilling the phenol solvent with aluminum turnings and sodium bicarbonate and by washing the filter paper with 1% oxalic acid.

Xylulose 1,5-bisphosphate is a potent inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase. The enzyme was inhibited 50% at a xylulose 1,5-bisphosphate concentration of 0.56 micrometer and an enzyme concentration of 0.14 micrometer. When both ribulose 1,5-bisphosphate and xylulose 1,5-bisphosphate were added simultaneously to the enzyme, this inhibition appeared to be competitive. A preincubation of 20 to 30 min was needed for maximum inhibition. The inhibitory effect of this compound is probably exerted through its binding at the ribulose 1,5-bisphosphate active site, since both the carboxylase and oxygenase activities were inhibited similarly.

Publisher Summary This chapter discusses the determination of phosphoglycolate phosphatase. Inorganic phosphate (P i ) is determined colorimetrically after enzymatic hydrolysis of phosphoglycolate. Cacodylate buffer is only slightly inhibitory and it does not interfere with the P i determination. Maleic acid is a good alternate buffer. Phosphoglycolate has been synthesized from phosphoglycerate by controlled permanganate oxidation or by phosphorylation of glycolic acid. The tricyclohexylammonium salt of phosphoglycolate (General Biochemicals) is treated with Dowex 50 (H + ) and then the free phosphoglycolic acid solution is adjusted to pH 6.3 with base. One unit is defined as the amount of enzyme that catalyzes the formation of 1 μg of phosphorus in 10 minutes. Specific activity is units per milligram of protein. The purification procedure was developed with field-grown ‘Maryland Mammoth’ tobacco leaves. The enzyme from leaves of spinach, wheat, and alfalfa is also stable to acetone precipitation. The enzyme from tobacco leaves has been alternately purified by a sequence of ammonium sulfate precipitation, calcium phosphate gel adsorption, and elution and chromatography on diethylaminoethyl (DEAE)-cellulose.

Publisher Summary This chapter discusses the procedure for the isolation of leaf peroxisomes. Leaf peroxisomes are respiratory microbodies present in the leaves of all plants. In appearance, size, and enzymatic composition, leaf peroxisomes belong to a class of microbodies that are similar to peroxisomes from liver and kidney, and they are similar but not identical with glyoxysomes from germinating seed endosperm. The main steps in the procedure for the isolation of leaf peroxisomes are (1) grinding of the leaves; (2) differential centrifugation between 100 and 6800 g for partial separation and for enrichment of the particulate fraction; (3) an isopycnic, nonlinear sucrose density gradient centrifugation at 39,000 g; and (4) a linear sucrose density gradient centrifugation. The first nonlinear sucrose density gradient acts as a filtration process in which the chloroplasts and mitochondria are stopped by layers of sucrose between 1.3 and 1.75 M . The peroxisomes are trapped at an interface below 1.75 M sucrose generally on top of 2.3 M sucrose. In isolating leaf peroxisomes, a major difference encountered from work with liver or seed endosperm tissue is the immense bulk of chloroplasts from which the peroxisomes must be separated.

Publisher Summary Glycolate oxidase or glycolate: O 2 oxidoreductase was originally isolated by ammonium sulfate precipitation, further purified by ethanol fractionation, and crystallized from ammonium sulfate. Flavin mononucleotide (FMN) (10 -4 M) is a required cofactor. The enzyme catalyzes the oxidation of both glycolate and glyoxylate. Three methods are available: (1) manometric; (2) measurement of reduction of 2,6-dichlorophenolindophenol at 620 mμ; or (3) measurement of glyoxylate phenylhydrazone formation at 324 mμ. Most of the research on glycolate oxidase has been carried out with the manometric assay. The phenylhydrazone assay for glycolate oxidase has not been used extensively. For the manometric assay one enzyme unit is defined as the amount required for the uptake of 1 micromole of oxygen per minute during a 25-minute period of incubation. Specific activity is expressed as units per milligram of protein. For the glyoxylate phenylhydrazone assay, one unit of activity is the production of 1 micromole of glyoxylate per minute. The chapter describes the procedure that applies only for purification of the alternate form of glycolate oxidase. Etiolated leaves are used because only the alternate form of the oxidase is present. Although both forms of the enzyme are present in young green leaves of tobacco and wheat, no extensive purification of the alternate form from green leaves has been tried. Throughout the procedure the enzyme must be maintained in the presence of excess glycolate (generally, 0.01 M) or otherwise all activity even at 2° is either lost, or there is a change the original form of the enzyme which requires excess FMN.