Purification and Properties of 2-Carboxy-d-Arabinitol 1-Phosphatase

Abstract

Carboxyarabinitol 1-phosphatase (2-carboxy-D-arabinitol 1-phosphate phosphohydrolase), a chloroplast enzyme that metabolizes the naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, was isolated from tobacco (Nicotiana tabacum) leaves. The enzyme was purified more than 3500-fold using a protocol that included ammonium sulfate fractionafton and gel filtration, ion-exchange, and hydrophobic interaction chromatography. Analysis of the final preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of a single polypeptide with a molecular mass of 53 kilodaltons. The enzyme exhibited an apparent Km (carboxyarabinitol 1-phosphate) of 33 micromolar and a pH optimum of 7.5. Enzyme activity did not require divalent cations and was unaffected by the metal chelators EDTA and cysteine. Carboxyarabinitol 1-phosphatase activity was inhibited by zinc, copper and molybdate and stimulated by sulfate. Chloroplast metabolites that affected activity included inorganic phosphate and ATP, which were inhibitory, and ribulose-1,5-bisphosphate, fructose-1,6-bisphosphate and NADPH which stimulated activity 2.5-fold. Activation of carboxyarabinitol 1-phosphatase activity by these positive effectors, together with the previously reported requirement for dithiothreitol, explain the light/dark modulation of carboxyarabinitol 1-phosphatase activity in vivo.phatase (I 1, 16, 17, 20) presumably by modifying the accessibility of critical sulfhydryl groups (2, 21).In the photorespiratory carbon oxidation cycle, a chloroplast phosphatase has been described that catalyzes the phosphohydrolysis ofphosphoglycolate (8, 23), one ofthe products of the ribulose-1,5-bisphosphate oxygenase reaction (4). The enzymic properties of phosphoglycolate phosphatase include inhibition of activity by ribose-5-phosphate and activation by both anions and divalent cations (13); however, aside from these factors, phosphoglycolate phosphatase does not appear to be under extensive regulatory control. This apparent lack of regulatory control may be indicative of a housekeeping function for this enzyme of rapidly catalyzing the metabolism of phosphoglycolate. The conversion of phosphoglycolate to glycolate is not of trivial importance for it prevents the accumulation of phosphoglycolate, a potent inhibitor of triose-phosphate isomerase (31), and initiates the recycling of the carbon lost in photorespiration back to the Calvin Cycle (4,8,28).In a companion study (12), a newly discovered plant phosphatase was shown to be involved in the metabolism of 2-carboxyarabinitol 1-phosphate (CA I-P2), an endogenous inhibitor of Rubisco. In the present study, we report a detailed protocol for the purification of CA l-Pase (2-carboxy-D-arabinitol 1-phosphate phosphohydrolase) from tobacco leaves and describe some of the properties of the isolated enzyme.Three phosphohydrolytic reactions occur as steps in the pathways for photosynthetic and photorespiratory carbon metabolism. In the reductive pentose phosphate cyc...

Different location in dark‐adapted leaves of Phaseolus vulgaris of ribulose‐1,5‐bisphosphate carboxylase/oxygenase and 2‐carboxyarabinitol 1‐phosphate

“…They isolated leaf chloroplastic, cytoplasmic, and vacuolar materials by the non-aqueous fractionation method to conclude that CAIP and CA1P phosphatase did occur in chloroplasts [27]. It has also been reported that CA1P phosphatase can occur in both cytoplasm and chloroplasts of tobacco leaves [15]. However, the whole leaves were ground in liquid N2 or an isotonic medium.…”

“…2-Carboxyarabinitol 1-phosphate (CA1P) may be synthesized in the night to inhibit the activated form of the enzyme in plants including beans, tobacco, and cucumber [2][3][4][5][6][7][8][9][10][11][12][13]. CAIP that accumulates and is bound to RuBisCO in the night is released from the enzyme by RuBisCO activase and degraded [12,14,15]. However, the amount of CA1P in the leaves of these plants varies widely, up to a level more than the concentration of the active sites of RuBisCO in the stroma in Phaseolus, although the amount increases least in tobacco [3,6,9].…”

Regulation of 2-Carboxyarabinitol 1-Phosphatase

“…This implies an interaction between CA 1-P metabolism and reactions on the reducing side of PSI. Additionally, in preliminary studies, it has been noted that CA 1-Pase activity is stimulated in vitro by several chloroplast metabolites, including NADPH, RuBP, and FBP (7,20). With P. vulgaris, appreciable levels of CA 1-P accumulation occur within 60 s under low-light conditions, but in other species the increase in inhibitor concentration may require more than 1 h (6, 10).…”

“…We also present information concerning the thermal stability of CA l-Pase activity, allowing a comparison to the established heat sensitivity of Rubisco activase ( 17 (20). Reactions were initiated by adding the enzyme and terminated after 10 min by the addition of 0.1 mL of0.5 N HCOOH.…”

Inhibition of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase by 2-Carboxyarabinitol-1-Phosphate

“…Feeding DCMU (18), nigericin, or methyl viologen (15) to leaf discs prevents the lightdependent removal of CAl P from rubisco, indicating the involvement of photosynthetic electron transport. Recently, a protein effective in hydrolyzing CAI P to CA was purified from tobacco chloroplasts (6,16). The protein had a high affinity for hydrolyzing CAl P but was ineffective against other phosphate esters.…”