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1.
《生物化学与生物物理学报:疾病的分子基础》1999,1453(1):83-91
We previously reported that sodium-dependent glucose uptake is present in bovine retinal pericytes and that phlorizin normalizes its glucose consumption under high glucose conditions. To clarify the effect of phlorizin on morphological and functional change of retinal pericytes under high glucose conditions, retinal pericytes were incubated in media with 5 mM glucose, 30 mM glucose, and 30 mM glucose plus 0.2 mM phlorizin for 7 days. The diameter of cells in the concentrations of glucose more than 10 mM were significantly larger than those in 5 mM glucose and 30 mM glucose plus phlorizin. Glucose, sorbitol and fructose contents of the cells in 30 mM glucose were significantly increased compared with those in 5 mM glucose, and were normalized by phlorizin. Thymidine uptake in the concentrations of glucose more than 20 mM was significantly decreased compared with that in 5 mM glucose. Myoinositol uptake, and DNA in 30 mM glucose were significantly reduced, and were normalized with phlorizin. Myoinositol content in 30 mM glucose was the same as that in 5 mM glucose, but was significantly decreased by phlorizin. The ratios of glucose to sorbitol or fructose in 30 mM glucose were significantly decreased, compared with those in 5 mM glucose and 30 mM glucose plus phlorizin. Therefore, the cellular enlargement and decreased DNA synthesis in cultured bovine retinal pericytes with abnormal glucose metabolism under high glucose conditions are attenuated by phlorizin, independent of the cellular myoinositol content. 相似文献
2.
R D Lumsden W A Murphy 《Comparative biochemistry and physiology. A, Comparative physiology》1984,79(1):137-141
Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border. 相似文献
3.
The effect of phlorizin on the parameters of cevadine induced membrane potential oscillation and the development of the potential changes were investigated in frog (Rana esculenta) sartorius muscles. The action of phlorizin on Na transport, water and cation contents of cevadine-treated muscles were also studied. On the effect of phlorizin applied at a concentration of 1 mmol/1 the frequency of the membrane potential oscillation evoked by cevadine decreased by about half, parallel with an about four-fold increase in the duration of the resting period and the prepotential. Phlorizin, applied at a concentration of 2 mmol/l on the neural part of the muscle before cevadine treatment, delayed the development of depolarization evoked by cevadine. In the cevadine-pretreated muscles the enhanced 24Na-uptake was not reduced by 2 mmol/l phlorizin. 2 mmol/l phlorizin, applied during the radioactivity washout period, diminished reversibly the rate coefficient for 24Na loss by 49% in 120 min. The 24Na-efflux increasing effect of cevadine, which is characteristic otherwise, was prevented by phlorizin. This action was also reversible. The intracellular water, Na, and K contents of muscles were not altered significantly by 2 mmol/l phlorizin even in 3 hours. Under the effect of cevadine the characteristic gain in intracellular water, Na content and [Na]i developed despite phlorizin treatment, but the changes mentioned above evolved more slowly. In the phlorizin-pretreated muscles the K-content decreasing effect of cevadine failed to come about. In the muscles pretreated with phlorizin the [K]i was reduced by cevadine at a proportional degree to water-uptake. 相似文献
4.
High affinity phlorizin binding to the LLC-PK1 cells exhibits a sodium:phlorizin stoichiometry of 2:1 总被引:1,自引:0,他引:1
The phlorizin binding properties of luminal membrane vesicles isolated from the LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, are studied. Scatchard analysis of this binding indicates the existence of a single high affinity sodium-dependent site with KD = 0.4 microM at 266 mM sodium. The specificity properties of this site indicate that it represents the binding of phlorizin to the hexose binding site of the sodium-dependent D-glucose transporter previously identified in this cell line. Both phlorizin equilibrium binding and the rate of phlorizin binding were found to be sigmoidal functions of sodium concentration. A Hill analysis of these data was consistent with a sodium:phlorizin stoichiometry of 2:1 in good agreement with the sodium:glucose stoichiometry already established in these cells. Phlorizin dissociation was also found to be sodium-dependent. On the basis of the phlorizin binding data presented here, a number of models of the binding of phlorizin and sodium to the transporter can be excluded. An analysis of a random binding model consistent with the data is presented. The significance of the LLC-PK1 sodium-dependent D-glucose transporter as a model system for related renal and intestinal transporters is discussed. 相似文献
5.
To characterize further the Na+/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na+/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na+/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin). 相似文献
6.
Radiation inactivation studies of the renal brush-border membrane phlorizin-binding protein 总被引:3,自引:0,他引:3
Renal brush-border membrane vesicles were irradiated in the frozen state with a high energy electron beam. The integral membrane proteins, alkaline phosphatase and 5'-nucleotidase, each showed a single exponential loss of activity with radiation dose, indicating target sizes of 67,000 and 58,000 daltons, respectively. Inactivation of sodium-dependent phlorizin binding to the brush-border membrane D-glucose transporter was more complex. One-half of the phlorizin binding sites were lost after even the smallest doses of radiation suggestive of large functional units (greater than 4 X 10(6) daltons) for a subpopulation of phlorizin binding proteins. The remaining sites behaved as a single radiation target of 110,000 +/- 8,000 daltons and retained the kinetic characteristics commonly associated with phlorizin binding to the glucose transporter. Thus, the data are consistent with the assignment of a molecular weight of 110,000 to the phlorizin binding moiety of the brush-border membrane D-glucose transport protein. 相似文献
7.
Summary Phlorizin binding has been widely used to estimate the site density of glucose transporters on intestinal and renal brush-border vesicles. Glucose transport measurements in the intact intestinal mucosa show that changes in transport rate postulated to arise from changes in site density occur under many physiological and pathological conditions. Exploring the basis of these regulatory phenomena would be facilitated by comparing changes in transport rate and site density measured in the same preparation. Hence we developed methods for measuring phlorizin binding in everted sleeves of intact mouse intestine. Specific binding of phlorizin to glucose carriers reached an asymptotic value within 120 sec, while nonspecific binding continued to rise thereafter. Hence we used 120-sec incubations. The rate of dissociation of specifically bound phlorizin was accelerated by Na+-free solutions and even more by 50mm glucose, while the rate of dissociation of nonspecifically bound phlorizin was independent of these solution changes. Hence we chose a 20-sec rinse in Ringer+50mm mannitol, because it washes out 30–40% of the nonspecifically bound phlorizin but virtually none of the specifically bound phlorizin. Ligand-binding analysis of specific binding against phlorizin concentration suggested two classes of binding sites, of which the one with stronger affinity for phlorizin probably has the higher capacity for glucose transport in mouse jejunum. The calculated affinity and capacity of this component are independent of whether one estimates the specific component of total binding by adding glucose or by removing Na+. 相似文献
8.
We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:glucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin 总被引:1,自引:0,他引:1
D A Warden F F Fannin J O Evans D W Hanke D F Diedrich 《Biochimica et biophysica acta》1980,599(2):664-672
The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings. 相似文献
10.
B Tritar G Pérès 《Revue canadienne de biologie / éditée par l'Université de Montréal》1976,35(4):239-241
"The in vivo intestinal absorption of D(+)glucose by Eledone (Eledone moschata Lamarck), a cephalopod mollusc, is decreased by 2.10(-4)M phlorizin and increased by 2.10(-2)M phlorizin. It is noted that phlorizin is dissolved in the glucose solution before its introduction into the intestinal tube. The first result is similar to that known in vertebrates; the second differs." 相似文献
11.
T Billington P R Nayudu 《The Australian journal of experimental biology and medical science》1978,56(1):31-37
To identify glucose-binding proteins amongst the polypeptides of the mouse duodenal brush border membrane, three types of experiments are reported. The first involved the introduction of labelled glucose and its analogue phlorizin into the lumen of separate groups of ligatured duodenal segments. Several proteins were shown to have bound both labelled species in situ by liquid scintillation counting of slices from polyacrylamide gels on which solubilised membrane protein had been electrophoretically separated. The second type of experiment was designed to determine the competitive nature of the binding of both labelled and cold phlorizin to proteins which had already bound glucose. Only three bands could competitively bind phlorizin. Finally, gels on which solubilised protein from in situ glucose-binding experiments had been run were placed in solutions containing labelled phlorizin. The binding of phlorizin to proteins in the same three bands as above suggested a confirmation of the conclusion that there were three membrane protein types which appeared to be involved in phlorizin-sensitive glucose-binding. 相似文献
12.
The effects of phlorizin on the membrane potential changes induced by cevadine were compared in the presence and absence of external chloride anions in frog skeletal muscle. The action of the drug on 24Na-efflux was also studied in chloride-free medium. In accordance with previous results, it was found that phlorizin reduced the frequency of the membrane potential oscillation (1 mmol/l) or fully inhibited the rhythmic activity (2 mmol/l) in the presence of chloride anions. Replacing the total chloride content of bathing fluid with non-penetrating anions (glutamate, isethionate or sulphate) the inhibitory action of phlorizin on the membrane potential oscillation failed to appear while it reappeared rapidly if the chloride ions were partially restored in the incubating medium. The membrane potential changes evoked by changing the chloride concentration of Ringer solution at constant [K]0 were more expressed in the presence of phlorizin. The action of phlorizin on 24Na-transport proved to be a chloride-independent phenomenon. This finding indicates that the inhibitory effect of phlorizin on Na-transport processes may not be the reason of its blocking action on membrane potential oscillation. Furthermore, it suggests that failure of the drug to inhibit the membrane potential oscillation in the absence of chloride anions may not be accounted for the lack of phlorizin-binding under those circumstances. It is therefore assumed that the increase in chloride conductance may play a causal role in the inhibitory effect of phlorizin on membrane potential oscillation. 相似文献
13.
To characterize further the Na+/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na+/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na+/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin). 相似文献
14.
Cyclosporin binding to a protein component of the renal Na(+)-D-glucose cotransporter 总被引:2,自引:0,他引:2
K Ziegler M Frimmer G Fritzsch H Koepsell 《The Journal of biological chemistry》1990,265(6):3270-3277
The immunosuppressive and nephrotoxic agent cyclosporin binds to a renal polypeptide with an apparent molecular weight of 75,000 which has been identified as a component of the renal Na(+)-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H. (1987) J. Biol. Chem. 262, 10718-10729). The same Mr 75,000 polypeptide was covalently labeled with the D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside and with the cyclosporin analog N epsilon-(diazotrifluoroethyl)benzyl-D-Lys8- cyclosporin (CSDZ). CSDZ labeling was decreased when the brush-border membrane proteins were incubated with monoclonal antibodies against the Na(+)-D-glucose cotransporter. In the presence of 145 mM Na+, CSDZ labeling was decreased by D-glucose (1 microM, 1 mM, or 100 mM) and by phlorizin (100 or 500 microM). In the absence of Na+, CSDZ labeling was distinctly increased by 50 microM phlorizin and was slightly increased by 1 mM D-glucose, whereas CSDZ labeling was decreased by 50 microM phloretin and by 500 microM phlorizin. Furthermore, Na(+)-dependent high affinity phlorizin binding to the Na(+)-D-glucose cotransporter was competitively inhibited by cyclosporin A (Ki = 0.04 microM) while Na(+)-D-glucose cotransport was not influenced. The data suggest that a part of the cyclosporin binding domain on the Na(+)-D-glucose cotransporter is identical to the phloretin binding domain of the high affinity phlorizin binding site. While phloretin or the phloretin moiety of phlorizin may directly displace cyclosporin, interaction of D-glucose or of the D-glucose moiety of phlorizin with the transporter may alter the conformation of the cyclosporin binding site and this conformational change may be modulated by Na+. 相似文献
15.
The gill of the marine mussel, Mytilus, contains a high affinity, Na-dependent D-glucose transporter capable of accumulating glucose directly from sea water. We examined the ability of the beta-glucoside, phlorizin, to act as a high-affinity ligand of this process in intact gills and isolated brush border membrane vesicles (BBMV). The time course of association of nanomolar [3H]phlorizin to gills and BBMV was slow, with t50 values between 10 and 30 min, and a half-time for dissociation of approx. 30 min. 1 mM D-glucose reduced equilibrium binding of 1 nM phlorizin by 90-95%, indicating that there was little non-specific binding of this ligand to the gill. In addition, there was little, if any, hydrolysis by the gill of phlorizin to its constituents, glucose and phloretin. Phlorizin binding to gills and BBMV was significantly inhibited by the addition of 50 microM concentrations of D-glucose and alpha-methyl-D-glucose, and unaffected by the addition of L-glucose and fructose. Binding to gills and BBMV was reduced by greater than 90% when Na+ was replaced by K+. Replacement of Na+ by Li+ effectively blocked binding to the intact gill, although Li+ did support a limited amount of glucose-specific phlorizin binding in BBMV. The Kd values for glucose-specific phlorizin binding in intact gills and BBMV were 0.5 nM and 6 nM, respectively. We conclude that phlorizin binds with extremely high affinity to the Na-dependent glucose transporter of Mytilus gill, which may be useful in future efforts to isolate and purify the protein(s) involved in integumental glucose transport. 相似文献
16.
The effects of phlorizin (2 X 10(-3) mol X l-1) on the Na transport of frog (Rana esculenta) sartorius muscle were investigated in glucose-free medium. Phlorizin decreased the rate coefficient of 24Na efflux by about 40%. The degree of inhibition was comparable to that caused by ouabain (10(-4) mol X l-1). Phlorizin could evoke a further reduction in the 24Na efflux also in the presence of ouabain. The intracellular Na content of the phlorizin-treated muscles remained unchanged, in contrast to a 60% increase induced by ouabain. 42K uptake was not affected by phlorizin. Data indicate that the ouabain-sensitive Na-K pump was not involved in the action of phlorizin. At the same time, phlorizin failed to alter the residual 24Na efflux measured in Li-Ringer solution containing ouabain. When Na: Na exchange was restored by replacing Na into the washout solution in the presence of ouabain, the increase of 24Na efflux was significantly diminished by phlorizin. Phlorizin reduced the 24Na uptake into a compartment with a half time of 6 min by about 40% without affecting the intracellular compartment. The results suggest that phlorizin inhibits the ouabain-insensitive Na: Na exchange in a superficial Na compartment. 相似文献
17.
Carl Tannenbaum Gerhard Toggenburger Markus Kessler Aser Rothstein Giorgio Semenza 《Journal of cellular biochemistry》1977,6(4):519-533
In the presence of an NaSCN gradient phlorizin binds with a high affinity (Kd ? 4.7 μM) to vesicles derived from brush border membranes of intestinal cells of rabbits. The value for Kd corresponds closely to that of Ki determined from phlorizin inhibition of sugar transport. The apparent affinity for phlorizin is decreased if NaCl is substituted for NaSCN and decreased substantially if the gradient of NaSCN is allowed to dissipate prior to the phlorizin binding. The number of high affinity binding sites is about 11 pmol/mg protein. Additional binding to low affinity sites can amount to as much as 600 pmol/mg protein after prolonged exposure to phlorizin (5 min). The high affinity sites are related to glucose transport based on the similarity of the Kd and Ki values under a variety of conditions and on the inhibition of the binding by D-glucose but not by D-fructose. The transport system and the high affinity phlorizin binding sites can be enriched by a factor of 2–3 by treatment of vesicles with papain, which does not affect the transport system, but considerably hydrolyzes nonrelevant protein. 相似文献
18.
The disulfide bonds of the Na(+)/glucose cotransporter (SGLT1) are believed to participate in the binding of the transport inhibitor phlorizin. Here, we investigated the role of the [560-608] disulfide bond on the phlorizin-binding function of the C-terminal loop 13 of SGLT1 using 3-iodoacetamidophlorizin (3-IAP) as a probe. The reactivity of 3-IAP to the fully reduced loop 13 was competitively inhibited by phlorizin, as evident from the MALDI mass spectra. It indicates that the disulfide bond is not mandatory for phlorizin binding. CD and equilibrium unfolding studies showed that the secondary structure and conformation stability of loop 13 were not affected by removing the disulfide bond. Furthermore, we generated a series of loop 13 mutants to assess the contribution of the disulfide bond to phlorizin binding. A positive correlation between the stability and phlorizin affinity of the mutant proteins was observed, implying that the protein stability, rather than the disulfide bond, is relevant to the phlorizin-binding function of loop 13. 相似文献
19.
H Skovbjerg O Norén H Sj?str?m E M Danielsen B S Enevoldsen 《Biochimica et biophysica acta》1982,707(1):89-97
Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase. 相似文献
20.
Gerhard Toggenburger Markus Kessler Aser Rothstein Giorgio Semenza Carl Tannenbaum 《The Journal of membrane biology》1978,40(3):269-290
Both the presence of sodium and of an electrical potential difference across the membrane have been found to be necessary in order to achieve optimal D-glucose-protectable phlorizin binding to brush border membranes from rabbit small intestine. The effect of delta approximately muNa on phlorizin binding shows a close similarity to that on D-glucose transport, confirming that phlorizin is indeed bound to the D-glucose transporting protein. Possible modulations of binding by a transmembrane potential are discussed on the basis of some models. 相似文献