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1.
DNA-binding vesicles released from the surface of a competence-deficient mutant of Haemophilus influenzae 总被引:6,自引:2,他引:4
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Haemophilus influenzae com-51, a mutant deficient in DNA uptake, produces an extracellular DNA-binding activity. The activity was specific for Haemophilus DNA and was isolated from cell-free competence medium after incubation for 100 to 130 min. Initial steps in the purification procedure resulted in the loss of detectable binding activity, but activity was restored by the addition of a nonionic detergent. The active fractions contained vesicles derived from the outer membrane of the cells. The vesicles were produced only under conditions that normally lead to competence development. The lack of competence of com-51 cells was not due to loss of protein synthesis in M-IV competence medium or to competition of extracellular protein for exogenous DNA. Results suggest that the inability of cells to bind DNA was due in part to the loss of DNA receptors that are released into the medium in membrane fragments. 相似文献
2.
Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii. 总被引:14,自引:10,他引:4
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Azotobacter vinelandii produced three major proteins of 93,000, 85,000, and 81,000 daltons and a minor 77,000-dalton protein in the outer membrane of Fe-limited cells, and these cells were competent for transformation by DNA. The synthesis of these proteins was repressed in Fe-sufficient medium. Mo limitation of nitrogen-fixing cells resulted in the hyperproduction of a 44,000-dalton protein and the production of a minor 77,000-dalton protein in the outer membrane. Mo limitation enhanced competence in Fe-limited medium and induced competence in Fe-sufficient medium. The 44,000-dalton protein was replaced by a 45,000-dalton protein when Fe-sufficient medium also contained NH4+, but the cells were noncompetent. The synthesis of these proteins was repressed in Mo-sufficient medium and by NH4+ in Fe-limited medium. All of the culture supernatants contained a blue-white fluorescent material (absorbance maximum, 214 nm) which appeared to coordinate Fe3+, Fe2+, MoO4(2-), WO3(2-), and VO3(-). 相似文献
3.
Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication. 相似文献
4.
Haemophilus influenzae polypeptides involved in deoxyribonucleic acid uptake detected by cellular surface protein iodination. 总被引:11,自引:8,他引:3
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Polypeptides that appear to be involved in competence development and deoxyribonucleic acid (DNA) uptake by Haemophilus influenzae were detected with a surface-specific iodinating reagent 1,3,4,6,-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril. As shown on electrophoretograms, a number of polypeptides became sensitive to 125I protein labeling with the ability of these cells to bind DNA. Of these polypeptides, nine were reduced in their ability to be labeled (ral polypeptides) extensively after the incubation of competent cells with homologous, but not with heterologous, DNA. Iodination of many of these ral polypeptides was reduced in competence-deficient mutants compared with wild-type competent cells. One 125I-labeled polypeptide corresponding to a molecular weight of 29,000 was present at reduced levels in mutants reduced in the ability to bind DNA. Our results suggest that the 29,000-molecular-weight polypeptide corresponds with the ability of H. influenzae to take up DNA and that a complex of proteins is involved in DNA uptake and transformation. 相似文献
5.
Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src. 总被引:14,自引:2,他引:12
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Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src. 相似文献
6.
The binding and uptake of Gd3+ ions by human erythrocytes in vitro were studied by determining the Gd contents in membrane and in cytosol by means of particle-induced X-ray emission (PIXE) spectrometry. Results obtained from varied incubation time revealed that the Gd3+ ions bind to the membrane proteins and lipids at first. Gd3+ binding to the membrane lipids and proteins lasts 0 approximately 20 and 20 approximately 100 ms respectively, as shown by the stopped-flow studies. Then a fraction of Gd3+ ions diffuses through the membrane. The kinetics of Gd3+ binding indicates that the binding to phospholipids is prior to that to the membrane proteins, but a portion of the lipid-bound Gd3+ redistributed later to the proteins. PIXE studies showed that the entry of Gd3+ increased the influx of Ca2+ and Cl-. By monitoring the changes in fluorescence of proteins and that of the Ln3+, the uptake of La3+, Eu3+, Gd3+ and Tb3+ was shown to be a process comprising a series of events. Binding to the membrane molecules induces the phase transition of lipid bilayer and conformational changes and aggregation of membrane proteins. Conformational changes of the proteins were characterized by Fourier transform IR spectroscopy (FT-IR) deconvolved spectra, i.e. alpha-helix content decreases while beta-sheet increases. ESR spectra of MSL-labeled proteins reflect the aggregation state related with the conformational change. [31P]NMR spectra of membrane lipid bilayer revealed the Ln3+ ions induced hexagonal (H(II)) phase formation. Phase transition and aggregation of membrane proteins cause the formation of domain structure and perforation in the membrane. These alterations in membrane structure are responsible for the Ln3+ enhanced membrane permeability. Thus the previous Ln3+ binding will facilitate the across-membrane transport of other Ln3+ ions through the membrane. 相似文献
7.
All Seven comG Open Reading Frames Are Required for DNA Binding during Transformation of Competent Bacillus subtilis 总被引:1,自引:0,他引:1
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The seven proteins encoded by the comG operon of Bacillus subtilis exhibit similarity to gene products required for the assembly of type 4 pili and for the secretion of certain proteins in gram-negative bacteria. Although polar transposon insertions in comG result in the loss of transformability and in the failure of cells grown through the competence regimen to bind DNA, it was not known whether the ComG proteins are all required for competence. We have constructed strains missing each of these proteins individually and found that they are all nontransformable and fail to bind transforming DNA to the cell surface. The implications of these findings are discussed. 相似文献
8.
Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells 总被引:7,自引:5,他引:2
Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium. 相似文献
9.
The adsorption properties of the tailed bacteriophage L3 to Acholeplasma laidlawii cells were studied. Adsorption followed a biphasic curve. Reversibility and virus heterogeneity were not sufficient to explain the break in the adsorption curve. Binding studies showed that each colony-forming unit could bind about 350 virions. The electrostatic nature of L3 adsorption was indicated by the effect of cations, pH, and temperature on the adsorption rate constant. L3 adsorption appeared to have a requirement for Ca2+, which could not be replaced by the mono- and divalent cations examined. Ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid inhibition of adsorption was totally reversed by added Ca2+. The effects of EDTA, proteases, and lectins on absorption indicated that membrane proteins are the L3 receptors. The model for L3 adsorption is a multivalent one involving lateral diffusion of adsorbed virions and receptor proteins. 相似文献
10.
Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites. 相似文献
11.
Heparin-binding properties of lactoferrin and lysozyme. 总被引:3,自引:0,他引:3
S Zou C E Magura W L Hurley 《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,103(4):889-895
1. Binding of biotin-heparin to immobilized lactoferrin and lysozyme was optimum at pH 6.0, 100 mM NaCl. Complex interactions between NaCl and CaCl2 concentrations were observed for heparin binding to both proteins. 2. The metal ions Cu2+, Zn2+, Fe2+ and Fe3+ inhibited heparin binding, with half-maximal inhibition of binding to lactoferrin occurring between 600 microM and 1 mM and for lysozyme between 500 and 800 microM. 3. Binding of biotin-heparin to both proteins was inhibited to varying degrees by heparin, heparan sulfate, chondroitin sulfate A, dextran sulfate and DNA. 相似文献
12.
Interactions of annexins with membrane phospholipids. 总被引:2,自引:0,他引:2
The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions. 相似文献
13.
Inhibition of anion permeability of sarcoplasmic reticulum vesicles by 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate 总被引:2,自引:0,他引:2
The permeabilities of sarcoplasmic reticulum vesicle membrane for various ions and neutral molecules were measured by following the change in light scattering intensity due to the osmotic volume change of the vesicles. 4-Acetoamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS), which is a potent inhibitor for the anion permeability of red blood cells membrane, inhibited the permeability of sarcoplasmic reticulum for anions such as Cl-, Pi and methanesulfonate, while it slightly increased that for cations and neutral molecules such as Na+, K+, choline and glycerol. Binding of 5 mumol SITS/g protein was necessary for the inhibition of anion permeability. These results suggest the existence of a similar anion transport system in sarcoplasmic reticulum membrane as revealed in red blood cell membrane. 相似文献
14.
During the process of transformation cells bind its own DNA but little or no foreign DNA. This specificity for recognition of DNA was studied by cloning Haemophilus DNA in . Haemophilus DNA fragments were cloned using plasmid pBR322 as a vector. The fragment cH7 cloned in pBR322 was found to be homologous to Haemophilus DNA and shown to bind irreversibly to competent Haemophilus cells. The fact that cH7 isolated from lacks Haemophilus modification leads to the conclusion that modification does not play a role in the uptake mechanism. Uptake specificity is a function of recognition sequences that reside in DNA itself. 相似文献
15.
Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions. 总被引:3,自引:2,他引:1
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A Weston M G Brown H R Perkins J R Saunders G O Humphreys 《Journal of bacteriology》1981,145(2):780-787
Plasmid deoxyribonucleic acid (DNA) was tightly bound to cells of Escherichia coli at 0 degrees C in the presence of divalent cations. During incubation at 42 degrees C, 0.1 to 1% of this DNA became resistant to deoxyribonuclease. Deoxyribonuclease-resistant DNA binding and the ability to produce transformants became saturated when transformation mixtures contained 1 to 2 micrograms of plasmid NTP16 DNA and about 5 X 10(8) viable cells. Under optimum conditions, between 1 and 2 molecule equivalents of 3H-labeled NTP16 DNA per viable cell became deoxyribonuclease resistant. Despite this, only 0.1 to 1% of viable cells became transformed by saturating amounts of the plasmid. The results suggest that transport of DNA across the inner membrane is a limiting step in transformation. After transformation the bulk of labeled plasmid DNA remained associated with outer membranes. However, in vitro assays indicated that plasmid DNA would bind equally well to preparations of inner or outer membranes provided divalent cations were present to preparations of inner or outer membranes provided divalent cations were present. Divalent cations promoted differing levels of binding to isolated inner and outer membranes in the order Ca2+ much greater than Ba2+ greater than Sr2+ greater than Mg2+. This parallels their relative efficiencies in promoting transformation. Binding of plasmid DNA was greatly reduced when outer membranes were treated with trypsin; this suggests that protein components may be required for the binding or transport of DNA (or both) during transformation. 相似文献
16.
A nuclear factor requires Zn2+ to bind a regulatory MRE element of the mouse gene encoding metallothionein-1 总被引:3,自引:0,他引:3
The metal ion requirement of nuclear proteins for binding to the metal regulatory element d(MREd) of the mouse gene encoding metallothionein-1 was investigated using an in vitro exonuclease III footprinting assay. The specific DNA-binding activity of the factor was inactivated by the chelating agents, EDTA and 1,10-phenanthroline. Binding activity was restored by Zn2+, but not by Cd2+. These results show that Zn2+ ions are a required component for specific in vitro DNA binding of the MREd-binding protein. 相似文献
17.
Large macromolecules can be introduced into cultured mammalian cells using erythrocyte membrane vesicles 总被引:1,自引:0,他引:1
Hideo Sugawa Tsuyoshi Uchida Yoshihiro Yoneda Masahiro Ishiura Yoshio Okada 《Experimental cell research》1985,159(2):410-418
Plasmid 6.4 kbp DNA, 14 kbp DNA, lambda phage particles, all of which contained herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, or IgM molecules, were mixed with erythrocyte membranes and treated with neutral detergent. The transparent mixture was diluted with phosphate-buffered saline (PBS), followed by centrifugation to collect membrane vesicles containing the large macromolecules. 10-15% of 6.4 kbp, 3% of 14 kbp, 4-7% of the lambda phage particles and 14.5% of IgM were trapped within erythrocyte membrane vesicles. The membrane vesicles containing these molecules were fused with L cells, or rat F2408#20 cells, both of which are deficient in thymidine kinase activity. In each case, transformants were obtained. 2 X 10(5) - 7 X 10(5) phage PFU or 1.5 X 10(6) - 8 X 10(7) DNA molecules were required to obtain one transformant from L cells, but 2-3 X 10(7) phage PFU or 2 X 10(9) - 1 X 10(10) DNA molecules were required for one transformant from rat cells. Number of colonies which transiently expressed TK genes in L cells was also determined by autoradiography. The ratio of stable transformants to colonies positive for transient expression in cells treated with low doses of DNA or lambda phage was 46-68%. The transformation efficiency of human fibroblast cells by pSV2-gpt DNA trapped in erythrocyte membrane vesicles was less than that of L cells by HSV-TK DNA, but almost the same as that of rat cells by HSV-TK DNA. 相似文献
18.
Estimates of the net equilibrium binding constants for [(H2O)(NH3)5RuII]2+, [Cl(NH3)5RuIII]2+, cis-[(H2O)2(NH3)4RuII]2+ and cis-[Cl2(NH3)4RuIII]+ with apotransferrin (Tf) and holotransferrin (Fe2Tf) suggests that RuIII, but not RuII complexes bind with a higher affinity to the iron binding sites. Several other presumably histidyl imidazole sites bind with approximately the same affinity (Keff = 10(2) to 10(3) M(-1) to both RuII and RuIII. Compared to HeLa cells, an order of magnitude higher level of nuclear DNA binding ([Ru]DNA/[P]DNA) was required to achieve the same level of toxicity in Jurkat Tag cells, which probably relates to the substantially higher levels of cis-[Cl2(NH3)4Ru]+ needed to inhibit 50% of the cell growth in the Jurkat Tag cell line. Against Jurkat Tag cells, the toxicity of the pentaammineruthenium(III) group is enhanced by approximately two orders of magnitude upon binding primarily to the Fe-sites in apotransferrin, whereas the toxicity of the tetraammineruthenium(III) moiety is only marginally increased. Binding to Fe2Tf does not increase the toxicity of either group. Significant dissociation over 24 h of the ammineruthenium(III) ions from apotransferrin requires reduction to RuII. 相似文献
19.
The effect of Mg2+ on the binding of the Streptococcus pneumoniae single-stranded DNA binding (SSB) proteins, SsbA and SsbB, to various dT(n) oligomers was examined by polyacrylamide gel electrophoresis. The results were then compared with those that were obtained with the well characterized SSB protein from Escherichia coli, SsbEc. In the absence of Mg2+, the results indicated that the SsbEc protein was able to bind to the dT(n) oligomers in the SSB(35) mode, with only two of the four subunits of the tetramer interacting with the dT(n) oligomers. In the presence of Mg2+, however, the results indicated that the SsbEc protein was bound to the dT(n) oligomers in the SSB(65) mode, with all four subunits of the tetramer interacting with the dT(n) oligomers. The SsbA protein behaved similarly to the SsbEc protein under all conditions, indicating that it undergoes Mg2+ -dependent changes in its DNA binding modes that are analogous to those of the SsbEc protein. The SsbB protein, in contrast, appeared to bind to the dT(n) oligomers in an SSB(65)-like mode in either the presence or the absence of Mg2+, suggesting that it may not exhibit the pronounced negative intrasubunit cooperativity in the absence of Mg2+ that is required for the formation of the SSB(35) mode. Additional experiments with a chimeric SsbA/B protein indicated that the structural determinants that govern the transitions between the different DNA binding modes may be contained within the N-terminal domains of the SSB proteins. 相似文献
20.
Activation affects access to the platelet receptor for adhesive glycoproteins 总被引:4,自引:1,他引:3
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B S Coller 《The Journal of cell biology》1986,103(2):451-456
Blood platelets have a receptor for macromolecular adhesive glycoproteins, located on a heteroduplex membrane glycoprotein complex (GPIIb/IIIa) that only becomes "exposed" when platelets are activated. Binding of the adhesive glycoproteins, in particular fibrinogen, to the receptor is required for platelet aggregation, which in turn is required to arrest bleeding. A murine monoclonal antibody whose rate of binding to the receptor is affected by platelet activation was both cross-linked and fragmented to assess the effects of changes in molecular size on its rate of binding to unactivated and activated platelets. The results indicate that small molecules can bind more rapidly to the receptors on unactivated platelets than can large molecules and that activation involves a conformational and/or microenvironmental change that permits the large molecules to bind more rapidly. 相似文献