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1.
Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium. 相似文献
2.
Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium. 相似文献
3.
Erik J. Wiersma Gunnar Fröman Staffan Johansson Torkel Wadström ∗ 《FEMS microbiology letters》1987,44(3):365-369
Cells of 10 strains of Vibrio cholerae were grown on Trypticase Soy Broth and were tested for different surface porperties such as expression of surface haemagglutinins, cell-surface hydrophobicity and binding to 3 connective tissue proteins: fibronectin, type II collagen and fibrinogen.All strains bound fibronectin and one selected strain was shown to bind in a time-dependent and saturable manner.The binding of 125I-labelled fibronectin could be completely inhibited by unlabelled fibronectin, and also partly by some other glycoproteins. Mannose inhibited binding of fibronectin up to 60%. The data indicate that carbohydrate structures within the 40 kDa (gelatin binding) and 105 kDa (cell binding) fragments of fibronectin are recognized by lectins on V. cholerae. The binding of collagen or fibrinogen was low or negligible. 相似文献
4.
G.W. Blackburn E.H. Morgan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,497(3):728-744
59Fe- and 125I-labelled transferrin-labelled rabbit reticulocyte ghosts were incubated at 37°C for 60 min with unlabelled reticulocyte and erythrocyte stroma-free haemolysates, and the ability of these haemolysates to release 59Fe- and 125I-labelled transferrin was investigated. Reticulocyte and erythrocyte haemolysates were equally effective in releasing 59Fe from the ghosts, but only the reticulocyte haemolysate was able to release 125I-labelled transferrin. The elution profiles of the post-incubation haemolysates upon AcA 44 gel filtration were similar. The 59Fe appeared as five separate peaks and the 125I-labelled transferrin appeared as a single, unbound peak. In the post-incubation reticulocyte haemolysate, 25% of the 59Fe was bound to ferritin and transferrin, and 69% was associated with the haemoglobin fraction; 52.8% of the 59Fe was present as haem-59Fe intimately associated with haemoglobin. Another 12.5% of the 59Fe was loosely bound to proteins in the haemoglobin fraction. The haem-59Fe released to the haemoglobin fraction was derived from preformed haem in the reticulocyte ghost. 59Fe release was not impaired in experiments in which haem and protein synthesis were inhibited with isonicotinic acid hydrazide and cycloheximide. When tested alone, the haemoglobin fraction was able to release 59Fe from the ghosts to an even greater degree than reticulocyte haemolysate. It is concluded that protein in the haemoglobin fraction function as heme carriers.Less than 6% of the 59Fe released by reticulocyte haemolysate was associated with a low molecular size protein fraction. Removal of this fraction from the unlabelled haemolysate by ultrafiltration did not impair the 59Fe-releasing capacity of the haemolysate. However, both this fraction and the ferritin fraction were able to bind some 59Fe from the ghosts. Ferrous and ferric chelators, as well as defatted bovine serum albumin, were also able to bind 59Fe from the ghosts, but not to the same degree as the haemolysates.The release of 125I-labelled transferrin from the ghosts by the reticulocyte haemolysate was affected by stimulatory and inhibitory factors. The stimulatory factor(s) was present in the non-haemoglobin components of the haemoglobin fraction. The inhibitory effect was dependent on the low molecular weight fraction. 相似文献
5.
Hamid-Reza Amini Felipe Ascencio Eduardo Ruiz-Bustos Maria J. Romero Torkel Wadström 《FEMS immunology and medical microbiology》1996,16(3-4):247-255
Abstract Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori . Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with α-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a k d 2.88 × 10−6 M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or α-chymotrypsin after isolation from iron-restricted and iron-containing media. 相似文献
6.
Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding. 相似文献
7.
Following electrophoresis of ovalbumin in sodium dodecyl sulfate (SDS) this glycoprotein bound 125I-labelled concanavalin A (Con A). The reaction was specific and proportional to the amount of glycoprotein present on the gel. This technique was used to study the Con-A-binding glycoproteins of liver cell surfaces. Mouse liver plasma membranes were purified and subfractionated to yield two fractions corresponding to the bile canalicular surface and the surface between adjacent hepatocytes (Evans, W.H. (1970) Biochem. J. 116, 833-842). Both fractions bound 125I-labelled Con A, the former binding two to three times more lectin than the latter. Following SDS gel electrophoresis individual membrane glycoproteins reacted with 125I-labelled Con A. Both membrane subfractions yielded qualitatively similar Con A binding profiles, seven binding proteins being present in each. The results are consistent with a generally uniform distribution of glycoproteins over the hepatocyte surface. The reaction of lectins with glycoproteins following SDS gel electrophoresis should find general application in the study of membrane composition. 相似文献
8.
D Roiron-Lagroux C Figarella 《Biochemical and biophysical research communications》1990,170(2):837-842
The fate of [125I] lactoferrin after binding on its specific receptor on HT29-D4 cells, a clone of HT29 cells, was studied and compared with that of [125I]transferrin. Time courses of protein binding in relation with temperature clearly show that different mechanisms of ligand-transportation for the two glycoproteins exist on these cells: whereas transferrin would enter into the cell probably by a relatively classic pathway of receptor-mediated endocytosis, lactoferrin and its specific receptor would not be internalized. Thus the mechanism by which lactoferrin promotes HT29 cell growth seems quite different from that of transferrin on the same cells. 相似文献
9.
Lactoferrin-binding sites at the surface of HT29-D4 cells. Comparison with transferrin 总被引:1,自引:0,他引:1
The binding of 125I-lactoferrin to HT29-D4 cells, a clone of HT29 cells, was studied and compared to the binding of 125I-transferrin to the same cells. The binding of the two iron-transport proteins is saturable and reversible suggesting the presence of specific receptors for each protein. Scatchard analysis suggests the existence of binding sites for lactoferrin with the relatively high equilibrium dissociation constant, Kd1 of 408 nM. Additionally, the cell is capable of binding large amounts of lactoferrin with very low affinity, probably in a non-receptor intermediate fashion. The dissociation constant of transferrin and its receptor was calculated 9.29 nM which corresponds well to values found in the literature. In contrast to lactoferrin, the cell was capable of binding only low amounts of transferrin in a non-receptor intermediate fashion. After chemical crosslinking of lactoferrin to the cell surface, the radiolabeled lactoferrin was found in a complex of molecular mass 300 kDa. Crosslinking of transferrin resulted in a complex of much higher molecular mass. These data clearly show a binding site for lactoferrin different from the transferrin receptor. Only if competition experiments were performed with a high molar excess of both ligand proteins did a small percentage of either of the two ligands crossreact with the receptor for the other, possibly due to a structural similarity of the two glycoproteins. 相似文献
10.
A ferritin receptor has been detected on isolated rat hepatocytes and has been partially purified from rat liver using affinity chromatography. Isolated hepatocytes exhibit approximately 30,000 ferritin binding sites/cell with a binding association constant (Ka) of 1 x 10(8) mol-1 liter. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support to facilitate separation of bound and free ligand. This method yielded a Ka of 3 x 10(8) mol-1 liter for the purified hepatic ferritin receptor. Binding of ferritin to the insolubilized receptor was partially inhibited by human lactoferrin but unaffected by 200-fold molar excess of bovine albumin, rat transferrin, or human asialoorosomucoid. 相似文献
11.
An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4). 总被引:2,自引:2,他引:0
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Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands. 相似文献
12.
Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.
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The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist. 相似文献
13.
We characterized binding and endocytosis of 125I-bovine lactoferrin by isolated rat hepatocytes. Iron-depleted (apo-Lf), approximately 30% saturated (Lf), and iron-saturated (holo-Lf) lactoferrin were used. At 4 degrees C, cells bound 125I-apo-Lf and 125I-holo-Lf with nearly identical apparent first order kinetics (t1/2 = approximately 42 min). Holo-Lf and apo-Lf competed with each other for binding. Hepatocytes bound lactoferrin optimally at pH greater than or equal to 7 but poorly at pH less than or equal to 6. Ca2+ (greater than or equal to 100 microM) enhanced Lf binding to cells, and holo-Lf remained monomeric with Ca2+ present as determined by gel filtration chromatography. With Ca2+, cells exhibited approximately 10(6) high affinity sites (Kd approximately 20 nM) and approximately 10(7) low affinity sites (Kd approximately 700 nM) for both apo- and holo-Lf. Without Ca2+, cells bound 125I-holo-Lf by the low affinity component only. EGTA and dextran sulfate together released greater than or equal to 90% 125I-Lf prebound at 4 degrees C, but individually removed separate populations of surface-bound 125I-Lf. Cells bound 125I-Lf in a Ca(2+)-dependent manner with dextran sulfate present. We conclude that the high affinity but not the low affinity sites require Ca2+; only the low affinity sites are dextran sulfate-sensitive. Neither transferrin nor asialo-orosomucoid blocked lactoferrin binding to hepatocytes. Some cationic proteins but not others inhibited lactoferrin binding. At 37 degrees C, hepatocytes endocytosed 125I-apo-Lf and 125I-holo-Lf similarly, and hyperosmolality (greater than 500 mmol/kg) blocked uptake by approximately 90%. These data support the proposal that hepatocytes regulate blood lactoferrin concentration by receptor-mediated endocytosis. 相似文献
14.
The binding of fibronectin an vitronectin to 207 Bacteroides strains and the binding of collagen and sialoproteins to 55 Bacteroides strains were investigated by means of latex agglutination tests. The binding of fibronectin, collagen and lactoferrin to the same 55 strains was also tested by using 125I-labelled proteins. The 207 strains, belonging to ten Bacteroides species, were isolated from different infections (51%) and from faeces of healthy subjects (49%). Most of the strains displaying fibronectin binding belonged in the species B. fragilis or B. vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. No inhibition of the binding was observed with carbohydrates. The vitronectin binding of the strains was less common, but was always observed to accompany fibronectin binding. None of the examined 55 strains exhibited any binding to fetuin or asialofetuin. The radiolabelling method indicated a low binding to 125I-fibronectin. The binding of 125I-collagen-I and 125I-lactoferrin in the Bacteroides strains tested was higher than that of 125I-fibronectin. 相似文献
15.
R T Parmley F Ostroy R A Gams L DeLucas 《The journal of histochemistry and cytochemistry》1979,27(2):681-685
To evaluate the ultrastructural distribution of transferrin on the surface of L1210 ascites tumor cells, we used ferrocyanide to stain ferric iron (Prussian blue reaction) in transferrin, as well as in ferritin conjugated to antibody that was immunologically attached to the transferrin. Small deposits averaging 5 nm in diameter identified transferrin iron, whereas large cuboidal deposits averaging 50 nm in diameter stained ferritin conjugated-antibody that was bound to both transferrin and apotransferrin on the cell surface. The ability of transferrin to deliver iron to ascites tumor cells was confirmed by kinetic studies of transferrin labeled with 59Fe and 125I. These preliminary results are consistent with release of transferrin iron at the cell surface and demonstrate additional uses for ferrocyanide in ultrastructural cytochemical techniques. 相似文献
16.
T. Toba R. Virkola B. Westerlund Y. Bjorkman J. Sillanpaa T. Vartio N. Kalkkinen T. K. Korhonen 《Applied microbiology》1995,61(7):2467-2471
Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer. 相似文献
17.
Premysl Ponka Jitka Borova Jan Neuwirt 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,632(4):527-535
A high level of non-heme iron (either labelled or unlabelled) in mitochondria, ferritin and low-molecular-weight pool of reticulocytes was induced by preincubation with isonicotinic acid hydrazide or penicillamine together with either 59Fe- or 56Fe-labelled transferrin. Addition of apotransferrin during reincubation of 59Fe-labelled reticulocytes was accompanied by the transfer of 59Fe from low-molecular-weight pool to transferrin, which was found in the reticulocyte cytosol both free and bound to a carrier. Similarly, when cells were reincubated with 125I-labelled transferrin, more 125I-labelled radioactivity was found, in both free and carrier-bound transferrin peaks, in reticulocytes with a high level of low-molecular-weight cold iron than in control ones. These results suggest that transferrin enters reticulocytes takes up iron from low-molecular-weight pool. 相似文献
18.
The uptake of 125I-labelled LH by equal numbers of granulosa cells from small, medium or large follicles was greater by cells from large follicles. In contrast, granulosa cells obtained from small follicles bound much more 125I-labelled FSH per cell than did cells obtained from medium and large follicles. Competition studies with unlabelled hormones indicated that porcine granulosa cells have specific receptors for LH and FSH. The addition of diethylstilboestrol enhanced the binding of 125I-labelled LH and inhibited the binding of 125I-labelled FSH to granulosa cells harvested from small and medium-sized follicles, but had no effect on those from large follicles. 相似文献
19.
Role of transferrin in iron transport between maternal and fetal circulations of a perfused lobule of human placenta 总被引:1,自引:0,他引:1
The transfer of iron between the maternal and fetal circulations of an isolated perfused lobule of term human placenta was investigated using 125I-labelled or 59Fe-labelled diferric transferrin. There was negligible transplacental transfer of intact transferrin whereas nearly 4 per cent of the added 59Fe was transferred into the fetal circulation after 2 h, where it became associated with fetal transferrin. Over 20 per cent of the added 59Fe radioactivity was sequestered within the placental tissue during this period, associated with transferrin, ferritin and other uncharacterized molecules. This suggests an important role for an intracellular pool in regulating transfer. The presence of 10 mM chloroquine in the maternal circulation substantially reduced tissue accumulation of 59Fe and totally inhibited transfer to the fetus. It is concluded that the initial stages of iron transfer to the fetus involve the internalization of maternal iron-saturated transferrin bound to membrane receptors by receptor-mediated endocytosis, which can be inhibited by the drug chloroquine. Subsequently, the transplacental transfer of iron to the fetus does not involve the concomitant movement of transferrin. 相似文献
20.
Identification of lactoferrin-binding proteins in bovine mastitis-causing Streptococcus uberis 总被引:4,自引:0,他引:4
All strains of Streptococcus uberis evaluated bound to lactoferrin (Lf) in milk as detected by polyacrylamide gel electrophoresis and Western blotting. A biotin-avidin-based microplate binding assay and ELISA also revealed that these bacterial strains bound to purified Lf. Binding of bacteria of Lf was not inhibited by mannose and galactose, indicating that glycosidic domains of the Lf molecule were not involved in binding. Lf binding was also unaffected by bovine transferrin. Western blot analysis demonstrated that there were at least two bacterial proteins involved in Lf-binding. Lf binding by S. uberis could enable this bacterium to acquire iron necessary for its growth. 相似文献