Study On Fruit Acid Invertases

Acid invertase ( 3 -fructosidase; EC 3.2.1.26) can be categorized into two kinds, one being located in the vacuole, called soluble acid invertase and the other in the apoplasmic space, called cell wall-bound acid invertase. The enzymes play key roles in sucrose metabolism and storage in economic sink cells and the sink strength modulation in crops. So, the study on regulating mechanism of acid invertases in developing fruit should constitute an important step toward elucidating the theory of fruit quality formation. In this paper, soluble and cell wall acid invertases, respectively, were purified from developing apple fruits and the corresponding polyclonal antisera were prepared. Enzymological mechanism of acid invertase regulation by abscisic acid (ABA), a vital hormone that enhances the accumulation of carbohydrate in fruit, was then studied in developing apple fruit and grape berry, respectively. These results provide a substantial base for the further studies on the molecular mechanism and biological significance of acid invertase modulation in developing fruit.Soluble and cell wall- bound acid invertases were respectively purified to apparent electrophoretic homogeneity from apple fruit by the successive procedures including ammonium sulphate precipitation, ion-exchange chromatography, gel filtration and affinity chromatography. The fractions were both shown to have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kD composed of the identical subunits of 50 kD. The two kinds of isoenzymes had similar enzymological characterization and were distinct from the other plant acid invertases in some properties such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose and a mixed form of inhibition by fructose to their activities. The proteinous inhibitor to acid invertase was not found in apple fruit. The antibodies directed against the soluble and cell wall-bound acid invertases, respectively, were purified by sequential ammonium sulphate precipitation, DEAE-Sepharose FF and POD-Sepharose chromatography. The purified antibodies were shown highly specific for apple acid invertases by both Western blotting and immunoinhibition assays and highly sensitive to the enzymes by both enzyme linked immunosorbent assay and protein dot blot. The antibodies immuno-recognized from the crude extract of apple tissues three classes of apple acid invertases with molecular mass of, respectively, 50 kD, 68 kD and 30 kD. The last two invertases may be lost during the purification. Acid invertase in apple fruit may have three isoforms.The present experiment, using the techniques of the injection of ABA into the intact apple (Mains Domestica Borkh cv. Starkrimson) fruit attached to growing apple tree and the incubation of the excised fruit discs in the ABA-contained medium, showed that ABA strongly activated the fruit soluble acid invertase and cell wall-bound acid invertase. The ABA-induced acid invertase activation was time course, ABA dose-, medium pH-, in vivo tissue- and fruit developmental stage dependent with the maximum induced effects being attained, respectively, in the medium of pH 5.5 and at 100 lhday afterfull bloom. The ABA-induced acid invertase activation appeared after 40 min of the in vivo incubation in contained-20 umol/LABA medium. The results of enzyme linked immunosorbent assay and Western blotting study indicated that the acid invertase activation by ABA could involve a post-translational regulation mechanism but no increase in the synthesis of enzyme proteins. These results suggest that the improvement of sink strength by ABA in the economic sink organs may be attributed, at least partly, to the ABA- induced cell wall-bound acid invertase and soluble acid invertase activation.The present experiment, performed with the in vivo incubation of the grape (Vitis vinifera x K labrnsca L.) berry discs in the ABA-contained medium and the in vivo infiltration of ABA into the entire berries, demonstrated that ABA activated both the solu...