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The presence of high-affinity membrane-associated receptor sites for phorbol ester tumor promoters has been demonstrated with [³H]phorbol dibutyrate ([³H]phorbol Bt2) in the mouse embryo fibroblast cell line C3H 10T1/2. Binding of [³H]phorbol Bt2 to intact cells occurs rapidly at 37 °C and is also rapidly reversed upon addition of excess unlabeled phorbol Bt2. The dissociation can be resolved into two phases at 23 °C. Scatchard analysis reveals a biphasic curve which is consistent with two classes of binding sites: a high-affinity class with a dissociation constant of 16 nM and a total concentration of binding sites of 1.9 pmol/106 cells, and a low-affinity class with a dissociation constant of 1000 nM and a total concentration of binding sites of 29.4 pmol/106 cells. Unlabeled phorbol Bt2, 12-O-tetradecanoyl-phorbol-13-acetate (Myrphorbol Ac), and the indole alkaloid tumor promoter teleocidin are potent inhibitors of [³H]phorbol Bt2 binding, producing 50% inhibition at 98 nM, 38 nM and 23 nM, respectively. On the other hand, two compounds that lack tumor-promoting activity, 4α-phorbol-12,13-didecanoate (4 α-phorbolDec2) and 4-O-methyl-MyrphorbolAc, only slightly inhibited phorbol Bt2 binding. The effects of these compounds on the physical state of the membrane of intact C3H 10T1/2 cells was also assessed using two different fluorescence lipophilic probes. Phorbol Bt2, MyrphorbolAc and teleocidin, but not 4 α-phorbol Dec2, induce significant decreases in the fluorescence anisotropy parameter obtained with both of the probes. However, the dose response curves for these effects are quite different from those obtained when the same compounds were tested for inhibition of [³H]phorbol Bt2 binding. This discrepancy suggests that although C3H 10T1/2 cells contain an abundance of high-affinity receptor sites for phorbol ester and related tumor promoters, some of the membrane effects of these compounds may be mediated by direct binding of the tumor promoter to lipid domains in cell membrane. [ABSTRACT FROM AUTHOR]

The fluorescent 12-O-tetradecanoylphorbol-13-acetate (TPA) analogue dansyl-TPA allowed the distribution of fluorescent molecules to be visualized in living mouse fibroblasts. The entry of dansyl-TPA into cells occurred within millisecond time scale and was found largely energy independent. Dansyl-TPA was shown to stain the endoplasmic reticulum, Golgi apparatus, mitochondria and nuclear membrane. In addition, dansyl-TPA fluorescence was found to parallel that obtained with rhodamine-conjugated anti-α tubulin. The intracellular location of the dansyl-TPA fluorescence can be taken into account to explain the pleiotropic action of TPA in the cells. [ABSTRACT FROM PUBLISHER]

We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ , showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-β-CD: G4- β-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6(1),6(3)- and 6(1),6(4)-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) s(-1) and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.