The Regulation of Metabolic Pathways by an Endog...

3.The Regulation of Metabolic Pathways by an Endogenous Inhibitor

（1）Discovery of inhibitor.In assay of G-6-P DH and 6-PG DH of rape bud by the usual procedure，the activities were found to be very low.But they increased markedly when the diluted extract was used.If the amount of extract used in the reaction mixture was reduced from 1 ml to 0.5 ml，the activity of G-6-P dehydrogenase was enhanced by 5 fold，and that of 6-PG dehydrogenase by 4 fold.These observations led us to the tentative conclusion that an endogenous inhibitor is present in the extract of rape buds and it somehow renders the G-6-P DH and 6-PG DH inactive.When the extract is diluted，the concentration of inhibitor is reduced，and the inhibitory effect disappears，resulting in a much higher enzyme activity.

To get a direct proof of the presence of the inhibitor，another experiment was carriedout by boiling the extract of bud acetone powder in a water bath for 5 min.After centrifuging off the denatured protein，the supernatant was added to the enzyme extract of rape bud.A profound inhibition of the activities of G-6-P DH and 6-PG DH was then noticed.A similar inhibition effect was observed by adding this supernatant to the enzyme extracts of older siliques，and the degree of inhibition increases with the amount of the added supernatant（see Figure 3）.The same results were obtained with the purified G-6-PDH of yeast，cotton roots，or soybean seeds（see Table 3）.

（2）The nature of the inhibitor.

① Relation to sulphydryl group.The inhibitory effect of the natural inhibitor on G-6-P DH and 6-PG DH could not be relieved by cysteine or glutathione（see Table 4），showing that the site of action is not of a sulphydryl nature.

②Solubility.By extracting the bud acetone powder with various solvents，evaporating off the solvents under reduced pressure，and resuspending the residues in water，the solubility properties of the inhibitor could be ascertained.It was found that the inhibitor is soluble in water，n-butanol，ethyl ether，and hot alcohol，but slightly soluble in chloroform，and insoluble in benzene（see Table 5）.

③Stability.The inhibitor is heat stable：it is not affected by heating in boiling water for 5 min（see Table 6）.On the other hand，it is quite sensitive to ultra-violet light；its action would be abolished on a 15-min exposure（see Figure 4）.

④ Molecular size.When the inhibitor solution was dialysed against phosphate-EDTA buffer for 6 hours，no significant loss of activity was found（see Table 6）.Thereforethe inhibitor is likely to be a compound of large molecular weight，but nonproteinous innature，as evidenced by its resistance to boiling and precipitating with 100%saturated a mmonium sulphate（see Table 6）.

⑤Electric charge.Electrophoresis showed that the inhibitor migrates from anode to cathode，bearing therefore a positive charge.

⑥ Fluorescence.On paper chromatogram the inhibitor appeared yellow in colour，and showed a blue fluorescence under ultra-violet light.When it was exposed to the fumes of concentrated ammonia or treated with AICl3 solution，the colour became more intense or the fluorescence changed from blue to green.

（3）Purification and identification of the inhibitor.A yellow supernatant was obtained after removing the protein by boiling the enzyme extract of rape buds.The supernatant was subjected to paper chromatography using the acetone：formic acid：water（20∶10∶65）solvent system.At the end of 24 hour，the inhibitor was located mainly near the solvent front（see Table 7）.

Both on paper chromatogram and on electrophoretogram，the areas where the inhibitor was located which were always yellow in colour.If the last section（designated as No.4 in Table 7）of the paper chromatogram was divided into yellow and colourless regions，the inhibitory activity of the coloured region was four times higher than the colourless.These experiments showed that the inhibitor is a yellow compound.

Spectral examination of the partly purified inhibitor showed that it has an absorption maximum in the region round 260 mμm.Exposure to ultra-violet light for 15 min abolishes the absorption peak and the inhibitory activity（see Figure 4）.

After being washed several times with acetone，the dry powder of rape buds was then extracted with redistilled water，boiled，and centrifuged to remove protemous impurities.The supernatant was further washed with chloroform or benzene twice to remove lipidimpurities.The concentrated extract was then purified twice by paper chromatography.A yellow-brown viscous residue was obtained after the eluate was concentrated by heating.This residue was then ground briefly with a drop of liquid paraffin，the resulting paste was transferred to an infrared window，and pressed into a thin layer with a second window.An infrared absorption spectrum was.obtained by an automatically recording double beam infrared spectrophotometer.Because of the difficulty met in preparing a uniformly thin layer sample，only two absorption peaks.1，600 cm-1 and 1，100 cm-1，corresponding to carbonyl and phenyl groups respectively，were observed.These are important functional groups of flavonoid compounds.

（4）Content of the inhibitor in the reproductive organs of rape at various developing stages.As shown in Figure 5，the inhibitor mainly occurs in the buds，flowers，and young siliques；it gradually diminishes as the siliques grow older and almost disappears in the old siliques.