Plant Physiology 94:538-544 (1990)
© 1990 American Society of Plant Biologists
Metabolism and Enzymology
Enzyme Sets of Glycolysis, Gluconeogenesis, and Oxidative Pentose Phosphate Pathway Are Not Complete in Nongreen Highly Purified Amyloplasts of Sycamore (Acer pseudoplatanus L.) Cell Suspension Cultures 1
Marco Frehner2,
Javier Pozueta-Romero and
Takashi Akazawa
Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusaku, Nagoya 464-01, Japan
Differential centrifugation and Percoll-gradient centrifugation of protoplast lysates of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) yielded pure amyloplasts. Contamination of the final amyloplast preparation by foreign compartments was assessed by measuring marker enzyme activities. The activity of alkaline pyrophosphatase was taken as a 100% plastid marker; relative to this marker, mitochondria (cytochrome c oxidase) averaged 0.34%, microbodies (catalase) 0.61%, and cytosol (alcohol dehydrogenase) 0.09%. Enzymatic activities of the glycolytic, gluconeogenic, pentose phosphate and the starch degradation pathways were found to be present in these amyloplast extracts in appreciable amounts. But the pyrophosphate-dependent phosphofructokinase and phosphoglyceromutase were judged to be essentially absent from amyloplasts because the activities of these enzymes were not enriched above the level of contaminating enzymatic activities in the amyloplast fractions. Additionally, the in vitro activities of starch phosphorylase, ATP dependent phosphofructokinase, NAD dependent glyceraldehyde-3 phosphate dehydrogenase, and glucose-6 phosphate dehydrogenase did not seem to support carbon fluxes from starch to triose phosphates as calculated from the rate of starch disappearance during carbon starvation of the cells. These results provide additional, indirect evidence for the recently emerged view that, in addition to the well known phosphate-triosephosphate translocator, another hexose phosphate and possibly also an ATP/ADP translocating system play major roles in nongreen plastids.
2 Department of Plant Science, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland.
1 M. F. gratefully acknowledges a 1 year fellowship from the Japanese Society for the Promotion of Science (JSPS) and J. P. is indebted to Mombusho for a pre-doctoral student fellowship. This is paper No. 90 in the series "Structure and Function of Chloroplast Proteins."
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