Purification, properties and in situ localization of the amphibolic enzymes D‐ribulose 5‐phosphate 3‐epimerase and transketolase from spinach chloroplasts

Abstract

The amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase have been purified from stroma extracts of spinach chloroplasts using ammonium sulfate fractionation and FPLC. For the native enzymes, a molecular mass of 180 kDa for epimerase and 160 kDa for transketolase was found and the molecular masses of the subunits was determined to be 23 kDa for epimerase and 74 kDa for transketolase. Protein sequencing of the purified chloroplast enzymes revealed the NH 2 -terminal amino acid sequences of mature epimerase (NH 2 -TSRVDKFSKSDIIVSP) and transketolase (NH 2 -AAVEALES-TDTDQLVEG). The enzymic properties of both enzymes such as K m values or pH optima, were found to be very similar to those for epimerases and transketolases from other sources, including yeast and animal cells. In contrast to the light-activated enzymes of the Calvin cycle, the activity of these amphibolic enzymes was not redox-dependent. Immunogold electron microscopy on spinach leaf thin sections revealed that about 90% of the total epimerase and transketolase, and 96% of the total chloroplast H ϩ -ATP synthase portion CF 1 are associated with thylakoid membranes in situ. Ribulose-1,5-bisphosphate carboxylase/oxygenase, in contrast, was evenly distributed throughout chloroplasts. These and other results indicate that minor chloroplast enzymes are arranged in a thin layer on thylakoid membrane surfaces in vivo.Keywords : D-ribulose 5-phosphate 3-epimerase ; transketolase ; chloroplast enzymes ; reductive pentose phosphate pathway ; in situ localization.Although most of the Calvin cycle enzymes have been puriIn this study, a method is described for the simultaneous purification of epimerase and transketolase from spinach chlorofied and characterized previously from higher plants, little is known about the structural and enzymic properties of transke-plasts. The purified enzymes were used to determine their subunit compositions, enzymic properties, NH 2 -terminal amino acid tolase and epimerase, two enzymes involved consecutively in the cyclic regeneration of the CO 2 acceptor molecule ribulose-sequences, and to raise monospecific antibodies against them in rabbits. Recently, these antibodies were used for immunoscreen-1,5-bisphosphate [1]. Transketolase catalyzes the reversible transfer of a C2 ketol moiety and displays a broad substrate ing of a potato leaf cDNA library to identify, clone and sequence chloroplast transketolase and epimerase [6, 7]. Moreover, immuspecificity [2]. Epimerase converts xylulose 5-phosphate to ribulose 5-phosphate via epimerisation at position C3 [1]. Both en-nogold electron microscopy on ultrathin spinach leaf sections was employed to investigate the cellular distribution and localzymes catalyze essentially the same reactions the reductive and oxidative pentose phosphate pathway in plants [3]. Differing re-ization of these enzymes in situ. sults have been reported for the molecular and catalytic properties of plant transketolases in previous reports [2, 4, 5], and none MATERIALS AND METHODS of these studi...

Functional Replacement of Ferredoxin by a Cyanobacterial Flavodoxin in Tobacco Confers Broad-Range Stress Tolerance

“…Samples were transferred into gelatin capsules, incubated for 3 h in fresh resin, and polymerized at 358C for 3 d under indirect UV light. Thin sections, 70 nm thick, were cut with a diamond knife and used for immunogold labeling with anti-Fld as the primary antibody and 10 nM protein A-gold as described by Teige et al (1998).…”

Identification of a Novel Adenine Nucleotide Transporter in the Endoplasmic Reticulum of Arabidopsis

“…The samples were infiltrated subsequently with Lowycryl HM20 resin (Plano) as follows: 33% (v/v) HM20 resin in ethanol overnight, 50% (v/v) and 66% (v/v) for 6 h each, and then 100% (v/v) resin overnight. Samples were polymerized in gelatin capsules with fresh HM20 and accelerator at 608C for 24 h. Sections with a thickness of 70 nm were cut with a diamond knife and used for electron microscopic examination and immunogold labeling with an ER-ANT1-specific antiserum as described previously (Teige et al, 1998).…”

Identification, Cloning, and Properties of Cytosolic d-Ribulose-5-phosphate 3-Epimerase from Higher Plants

“…Molecular Mass Determination-The molecular mass of the purified RPEases was determined by gel permeation chromatography on a fast protein liquid chromatography Superdex-S200 (HP, 16/60) column that had been pre-equilibrated with the buffer used for anion-exchange chromatography and calibrated with standard proteins as described previously (13).…”

“…On the other hand, chloroplasts and chromoplasts contain the components for a complete OPPP (9, 10). Recently, nuclear-encoded genes for chloroplast enzymes with dual (amphibolic) function in the OPPP and reductive pentose phosphate cycle (Calvin cycle) have been cloned and characterized including transketolase (11-13), ribose-5-phosphate isomerase (14), and RPEase (13,(15)(16)(17).Relatively little is known about the occurrence, structure, and biochemistry of plant cytosolic (cyt) OPPP enzymes. Although cytosolic glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been isolated from green and non-green plant tissue (6, 18), cytosolic isoforms of the non-oxidative branch OPPP enzymes transketolase, ribose-5-phosphate isomerase, and RPEase were undetectable in plant cell extracts by combined cell fractionation and enzyme chromatography experiments (7,8).…”

“…On the other hand, chloroplasts and chromoplasts contain the components for a complete OPPP (9, 10). Recently, nuclear-encoded genes for chloroplast enzymes with dual (amphibolic) function in the OPPP and reductive pentose phosphate cycle (Calvin cycle) have been cloned and characterized including transketolase (11-13), ribose-5-phosphate isomerase (14), and RPEase (13,(15)(16)(17).…”