Purification, properties and in situ localization of the amphibolic enzymes D-ribulose 5-phosphate 3-epimerase and transketolase from spinach chloroplasts.
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 NH2-terminal amino acid sequences of mature epimerase (NH2-TSRVDKFSKSDIIVSP) and transketolase (NH2-AAVEALESTDTDQLVEG). The enzymic properties of both enzymes such as Km 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+-ATP synthase portion CF1 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.
Teige
M
M
Abteilung Molekulare Zellbiologie, Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany. Markus.Teige@Univie.ac.at
Melzer
M
M
Süss
K H
KH
D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis; D017421:Sequence Analysis; D018724:Spinacia oleracea / Q000201:enzymology* / Q000648:ultrastructure; D014174:Transketolase / Q000737:chemistry*
D010940:Plant Proteins; D014174:Transketolase; D012273:Ribulose-Bisphosphate Carboxylase; D002238:Carbohydrate Epimerases; C022553:ribulosephosphate 3-epimerase