Changes of the Enzymic Activities of the HMP and...
（1）Activities of the enzymes of the HMP pathway.As shown in Figure 1（a），the activities of G-6-P DH and 6-PG DH increase during the development of siliques，particularly between the age of 10 to 20 or more days after flowering.At this period，the activity of G-6-P DH increases more than four times.The activities of enzymes utilizing R-5-P also increase gradually from the stage of budding to 30 days after flowering.
（2）Activities of the enzymes of the EMP pathway.
① Phosphoglucoisomerase.The activity of phospfioglucoisomerase decreases from 8 days to 15 days after flowering，but increases thereafter till the age of 30 days（see Table 1 and Figure 2）.In some experiments it remains more or less at a constant level.
② Phosphofructokinase.As the silique grows the activity of phosphofructokinase decreases rapidly and approaches a very low level at the age of 30 days after flowering［see Figure 1（b）］.
③ Aldolase.The activity of aldolase shows a slight increase from the stage of budding to flowering or to young siliques.Thereafter as the siliques grow older，the activity declines rapidly，so that the activity in 30 day siliques is about one third of that of 10 days old（Figure 1（b））.
④ 3-phosphoglyceraldehyde dehydrogenase.The changes of the activity of this enzyme appears to be similar to that of phosphoglucoisomerase：a slight decline takes place from 8 days to 15 days old after flowering，then a rapid increase up to 30 days（see Figure 2）.
Figure 1 Changes of activities of some enzymes of the HMP and EMP pathways during development of rape siliques
Reaction systems：
（a）G-6-P dehydrogenase and 6-PG dehydrogenase：Glycylglycine 125 μmoles（pH 7.5），MgCl2 10 μmoles，G-6-P（or 6-GP）5）μmoles，NADP 0.27 μmole（purity 78%），enzyme 0.5 ml.Total volume 3 ml.Blank without NADP.Reaction mixture was previously incubated for 5 min in 38℃water bath and the reaction was started on addition of enzyme.One unit of enzyme activity is arbitrarily defined as the optical density increase by 0.01 at 340 mμm in the first 5 min.（Beckman DU type spectrophotometer and 1 cm light path cuvette.）Enzymes utilizing R-5-P：Glycylglycine 88 μmoles（pH 7.5），R-5-P 10 μmoles，riboflavin 1.5 μmoles，cystein 1.5 μmoles，MgCl2 10 μmoles，NADP 0.5 μmole，total volume 3 ml.Blank omitted R-5-P.Enzyme 0.5 ml，temp.38℃.Unit of enzyme activity is defined as amount of converted R-5-P in μmoles.
（b）Phosphofructokinase：Tris buffer（pH 8.6）100 μmoles，sodium bisulphite（pH 8.6）250 μmoles，F-6-P 4 μmoles，ATP 6 μmoles，MgCl2 10 μmoles，enzyme 0.5 ml.Total volume 3 ml.The reaction mixture was incubated at 38℃for 15 min and the quantity of phosphotriose produced was determined.One unit of enzyme activity is defined as amount of enzyme phosphorylating 0.1 μmoles F-6-P.
Aldolase：Barbital sodium buffer（pH 8.6）100 μmoles，sodium bisulphite（pH 8.6）250 μmoles，FDP 12.5 μmoles，enzyme 0.5 ml.Total volume 3 ml.Reacted at 38℃for 30 min.Reaction was stopped by adding equal volume of 20%trichloroacetic acid.Phosphotriose produced was determined by dinitrophenyl hydrazine reaction in alkaline medium.One unit of enzyme activity is defined as amount of enzyme degradating 1 μmole FDP.
Table 1 Change of Activity of Phosphoglucoisomerase with the Age of Rape Silique
Unit=converted 1 μmole G-6-P/15 min.
Figure 2 Changes of the activities of phosphoglucoisomerase，phosphoglyceraldehyde dehydrogenase，3-PGA kinase，and enzymes involved in converting 3-PGA to pyruvic acid during the development of rape siliques
Reaction systems：
Phosphoglyceraldehyde dehydrogenase：Tris（pH 8.5）150 μmoles，FDP 10 μmoles，NaF 10 μmoles，sodium arsenate 17 μmoles，glutathione 15 μmoles，NAD 1 μmole，enzyme 0.5 ml.Total volume 3 ml.Blank omitted NAD.Light absorption was measured spectrophotometrically at 340 mμm with Hitachi EPU-2A spectrophotometer at room temperature.
3-PGA kinase：Tris（pH 7.4）100 μmoles，3-PGA 20 μmoles，ATP 8 μmoles，MgCl2 6 μmoles，hydroxylamine 1，000 μmoles，enzyme 0.1 ml.Total volume 1.3 ml.Reacted at 38℃for 30 min.
Reaction was stopped by adding 3 ml FeCl3-HCl-trichloroacetic acid.The 1，3-diphosphoglyceric acid produced was determined（FeCl3-HCl-trichloroacetic acid：FeCl3-6H20 8.3 g，trichloroacetic acid 20 g were dissolved in 42 ml concentrated HCl and distilled water was added to 500 ml）.
Enzymes involved in conversion of 3-PGA to pyruvic acid：Tris（pH 7.4）150 μmoles，3-PGA 20 μmoles，ADP 10 μmoles，MgCl2 10 μmoles，2，3-DPGA 30 μg，KCl 25 μmoles，enzyme 2 ml.Total volume 2 ml.Incubation was proceeded at 30℃for 30 min when enzymes were killed.Pytuvic acid produced was determined with dinitrophenylhydrazine.One unit of the enzymes is defined as amount of enzyme converting 1 μmole of substrate per hour.
⑤ 3-PGA kinase and enzymes involved in the conversion of 3-PGA into pyruvic acid.As shown in Figure 2，the activities of these enzymes decline steadily as the siliques grow.In comparison with the 8-day siliques，the activities of these enzymes in the 40-day siliques drop down 6 times and 1.7 times respectively.
