PLANT PHYSIOLOGY , Vol 102, Issue 2 587-593, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Respiration of Sugars in Spinach (Spinacia oleracea), Maize (Zea mays), and Chlamydomonas reinhardtii F-60 Chloroplasts with Emphasis on the Hexose Kinases
K. K. Singh, C. Chen, D. K. Epstein and M. Gibbs
Institute for Photobiology of Cell and Organelles, Brandeis University, Waltham, Massachusetts 02254
The role of hexokinase in carbohydrate degradation in isolated, intact
chloroplasts was evaluated. This was accomplished by monitoring the
evolution of 14CO2 from darkened spinach (Spinacia oleracea), maize (Zea
mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally
supplied with 14C-labeled fructose, glucose, mannose, galactose, maltose,
and ribose. Glucose and ribose were the preferred substrates with the
Chlamydomonas and maize chloroplasts, respectively. The rate of CO2 release
from fructose was about twice that from glucose in the spinach chloroplast.
Externally supplied ATP stimulated the rate of CO2 release. The pH optimum
for CO2 release was 7.5 with ribose and fructose and 8.5 with glucose as
substrates. Probing the outer membrane polypeptides of the intact spinach
chloroplast with two proteases, trypsin and thermolysin, decreased 14CO2
release from glucose about 50% but had little effect when fructose was the
substrate. Tryptic digestion decreased CO2 release from glucose in the
Chlamydomonas chloroplast about 70%. 14CO2 evolution from
[1-14C]-glucose-6-phosphate in both chloroplasts was unaffected by
treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the
lysed spinach chloroplast indicated a hexokinase active primarily with
fructose but with some affinity for glucose. The pellet (membranal
fraction) contained a hexokinase utilizing both glucose and fructose but
with considerably less total activity than the stromal enzyme. Treatment
with trypsin and thermolysin eliminated more than 50% of the glucokinase
activity but had little effect on fructokinase activity in the spinach
chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in
a loss of about 90% of glucokinase activity.
