RESPIRATION OF SUGARS IN SPINACH (SPINACIA-OLERACEA), MAIZE (ZEA-MAYS), AND CHLAMYDOMONAS-REINHARDTII F-60 CHLOROPLASTS WITH EMPHASIS ON THE HEXOSE KINASES

The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the e volution of (CO2)-C-14 from darkened spinach (Spinacia oleracea), maiz e (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts ext ernally supplied with C-14-labeled fructose, glucose, mannose, galacto se, maltose, and ribose. Glucose and ribose were the preferred substra tes with the Chlamydomonas and maize chloroplasts, respectively. The r ate of CO2 release from fructose was about twice that from glucose in the spinach chloroplast. Externally supplied ATP stimulated the rate o f 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 membran e polypeptides of the intact spinach chloroplast with two proteases, t rypsin and thermolysin, decreased (CO2)-C-14 release from glucose abou t 50% but had little effect when fructose was the substrate. Tryptic d igestion decreased CO2 release from glucose in the Chlamydomonas chlor oplast about 70%. (CO2)-C-14 evolution from [1-C-14]-glucose-6-phospha te in both chloroplasts was unaffected by treatment with trypsin. Enzy mic analysis of the supernatant (stroma) of the lysed spinach chloropl ast indicated a hexokinase active primarily with fructose but with som e affinity for glucose. The pellet (membranal fraction) contained a he xokinase utilizing both glucose and fructose but with considerably les s total activity than the stromal enzyme. Treatment with trypsin and t hermolysin eliminated more than 50% of the glucokinase activity but ha d little effect on fructokinase activity in the spinach chloroplast. T ryptic digestion of the Chlamydomonas chloroplast resulted in a loss o f about 90% of glucokinase activity.