Effect of phloridzin on photosynthetic electron transport

Phloridzin (2',4',6',4-tetraoxyhydrochalcon-2'-glucoside) was used to study the localization of synthesis of ATP in the electrontransporting chain of photosynthesis. It was shown that phloridzin inhibits the rate of photoreduction of NADP+ by isolated pea chloroplasts by 40%, electron transport via cytochrome f by 100% and via plastocyanin--by 50%. The "crossover" experiments demonstrated that phloridzin inhibits ADP-induced photoreduction of cytochrome f, having no effect on plastocyanin under identical conditions. It is assumed that the site of ATP synthesis is localized on the reduced site of cytochrome f, while the carrier itself is located in the electron transporting chain coupled to phosphorylation. It is possible that only part of the plastocyanin molecules are located in the phosphorylating pathway of electron transport.     

Metabolism of phloridzin by Erwinia herbicola: nature of the degradation products, and the purification and properties of phloretin hydrolase

Erwinia herbicola Y46 degrades phloridzin to yield phloretin, phloroglucinol, and phloretic acid, when grown on defined medium containing phloridzin as the sole source of carbon. The identities of the intermediates isolated from culture filtrates were established by co-chromatography and by ultraviolet absorption spectra. Only 3 of 11 strains of this species, and none of the 12 species of bacterial phytopathogens tested could effect this breakdown. Some of the latter organisms possessed beta-glucosidase activity which liberated d-glucose from phloridzin. The enzyme phloretin hydrolase was purified from cells of E. herbicola Y46 grown on Yeast Beef Broth, by treatment of crude extracts with protamine sulfate, ammonium sulfate precipitation, elution from calcium phosphate gel, elution from diethylaminoethyl-cellulose, and concentration by ultrafiltration. The final preparation was free of beta-glucosidase, had a specific activity of 213 units per mg of protein, and represented a 142-fold purification over the crude extract. The enzyme had a pH optimum of 6.7 to 6.8, and produced only phloroglucinol and phloretic acid as products of phloretin breakdown, there being an equimolar relationship between the cleavage of phloretin and the formation of the products. The Michaelis constant (K(m)) for the enzyme with phloretin as substrate was 3.8 x 10(-5)m, and the enzyme was sensitive to Hg(2+) and Cu(2+) ions. Phloroglucinol, phloretic acid, p-chloromercuribenzoate and iodoacetamide were without effect on the activity. The enzyme did not react with phloridzin, naringin, or naringenin. The physiological significance of the results is discussed.     

Plasmodial surface anion channel-independent phloridzin resistance in Plasmodium falciparum

The plasmodial surface anion channel (PSAC) is an unusual ion channel induced on the human red blood cell membrane after infection with the malaria parasite, Plasmodium falciparum. Because PSAC is permeant to small metabolic precursors essential for parasite growth and is present on red blood cells infected with geographically divergent parasite isolates, it may be an ideal target for future antimalarial development. Here, we used chemically induced mutagenesis and known PSAC antagonists that inhibit in vitro parasite growth to examine whether resistance mutations in PSAC can be readily induced. Stable mutants resistant to phloridzin were generated and selected within 3 weeks after treatment with 1-methyl-3-nitro-1-nitrosoguanidine. These mutants were evaluated with osmotic lysis and electrophysiological transport assays, which indicate that PSAC inhibition by phloridzin is complex with at least two different modes of inhibition. Mutants resistant to the growth inhibitory effects of phloridzin expressed PSAC activity indistinguishable from that on sensitive parasites, indicating selection of resistance via mutations in one or more other parasite targets. Failure to induce mutations in PSAC activity is consistent with a highly constrained channel protein less susceptible to resistance mutations; whether this protein is parasite- or host-encoded remains to be determined.     

Plasma-induced dimerization of phloridzin as a new class of anti-adipogenic agents

Phloridzin is a natural phloretin glucoside found in several parts of apple trees and is an attractive target for structural modification as novel pharmaceutical agent. Nonthermal dielectric barrier discharge (DBD) plasma-induced structural changes in dihydrochalcone phloridzin (1) resulted in the isolation of three new methylene-bridged dihydrochalcone dimers, methylenebisphloridzin (2), deglucosylmethylenebisphloridzin (3), and methylenebisphloretin (4), along with phloretin (5). The chemical structures of these newly generated compounds were elucidated by interpretation of their spectroscopic data. The new phloretin dimer 4 connected by a methylene linkage exhibited significantly improved anti-adipogenic properties against pancreatic lipase as well as differentiation of 3T3-L1 preadipocytes compared to the parent compound phloridzin.     

Three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin was simulated with distance constraints estimated by the intensity of NOE peaks using the DADAS90 programs. Two inclusion possibilities were suggested-the large rim of beta-cyclodextrin as an entrance of the inclusion and the small rim of beta-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of beta-cyclodextrin.     

Substrate specificity and contribution of the glycosyltransferase UGT71A15 to phloridzin biosynthesis

The dihydrochalcone phloridzin (phloretin 2′-O-glucoside) is the most abundant phenolic compound in apple trees (Malus × domestica) and was also discussed to have an influence on the pathogen defence by shifting the dihydrochalcone profile from the glucosides to the more active aglycones. The final step in the biosynthesis of phloridzin is the glycosylation of phloretin at position 2′. Three cDNA clones from apple encoding glycosyltransferases are available which are able to catalyze the reaction in vitro. We investigated the possible role of glycosyltransferase UGT71A15 in phloridzin biosynthesis. The recombinant enzyme showed broad substrate acceptance but highest activities were observed with flavonols. Specific activities and the kinetic data indicated that phloretin is not the preferred native substrate of the UGT71A15. However, an increase of the molar ratio phloridzin:phloretin was found in transgenic lines, indicating a physiological relevance of UGT71A15 in planta, although a decrease of the total amount of dihydrochalcones in the majority of the samples was found. Unexpectedly, the increase of the phloridzin:phloretin ratio was not reflected by an increase of the total glucosyltransferase activities. In contrast, the majority of transgenic plants showed a reduced glucosylating activity with both phloretin and quercetin as a substrate, but the observed activity changes in a given sample were not similar for the two substrates. An increased susceptibility of M. robusta against the fire blight causing bacterium E. amylovora as a result of UGT71A15 overexpression could not be observed. Overexpression of UGT71A15 in transgenic apple trees also did not lead to morphological changes.     

Differences between fruit-bearing and non-bearing apple spurs in activity of an enzyme system decomposing phloridzin

The activity of an enzyme system decomposing phloridzin was investigated in fruitbearing and nonbearing spurs of apple trees, Landsberger Reinette cv., throughout vegetation. Acetone powder obtained from xylem sap of apple spurs was incubated with phloridzinsubstratum in citric buffer at pH 5.5 for 12, 18 and 24 h at 30 °C. A paper and thin-layer chromatography as well aa a spectrophotometric assay were employed for tentative identification of enzymic degradation products. Phloretic acid (PA), co-factor of IAA-oxidase, as well as phloretin (Pin), and phloroglucinol (PI) were found after the digestion of phloridzin. The chromatographed enzyme reaction products were measured densitometrically. The activity of the enzyme system was estimated by its efficiency in PA production and phloridzin disappearance. Obtained values, expressed in percentages, showed that the enzyme activity in fruitbearing spurs was much higher than in nonbearing ones; 30 and 10% of released PA in July, respectively. Because fruitbearing spurs of the apple tree are possibly additionally supplied with auxin translocated from developing seeds, an adaptive character of the enzyme system producing PA, a known auxin repressor, is suggested.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.