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J Bryan

Publications and source records attributed to J Bryan.

At least 181 records · Page 10Linked to original sources
PubMed1985

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes.

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F-actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.

Actins↗
PubMed1985

Mitogen-like monoclonal anti-actin antibodies.

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.

Absorption↗
PubMed1984

Platelet activation induces the formation of a stable gelsolin-actin complex from monomeric gelsolin.

We have studied the interactions between gelsolin and actin in crude extracts from activated and unactivated platelets and in mixtures of purified platelet gelsolin and muscle actin. Extracts were prepared using 10 mM EGTA from human platelets treated either with 100 microM aspirin and 2.5 mM tetracaine to retard activation or with the calcium ionophore A23187 to effect activation. The extracts were fractionated by gel filtration on Sephadex G-150 or by sedimentation on sucrose gradients and then analyzed using anti-gelsolin immunoblots and actin filament nucleation assays. The nucleation activity in both extracts was associated with gelsolin. The activity in the extracts from unactivated platelets sedimented with an S value of 5.2 and had an Mr = 90,000. The activity in the extracts prepared with EGTA from activated platelets sedimented at 6.8 S and had an Mr = 130,000. We have shown previously that the Mr = 130,000 species is an EGTA-stable binary complex of one actin and one gelsolin. Transient exposure of the extracts from unactivated platelets to 100 microM Ca2+ and subsequent fractionation in EGTA-containing buffers demonstrated that the formation of the binary complex occurs in the presence of Ca2+. Fractionation in the presence of 100 microM Ca2+ demonstrated higher order complexes including a ternary complex with a sedimentation constant of 8.2 S and an Mr = 165,000. Sedimentation and gel filtration experiments using purified platelet gelsolin and rabbit skeletal muscle actin demonstrated that formation of the EGTA-stable binary complex required Ca2+. At least one additional actin is bound to the binary complex in the presence of Ca2+, but is not sufficiently stable to be purified when EGTA is added. The results suggest that gelsolin exists either as a monomer or perhaps as a weak complex with actin in unactivated platelets but complexes tightly with actin during the transient Ca2+ rise that occurs during activation.

Actins↗
PubMed1984

Actin-gelsolin interactions. Evidence for two actin-binding sites.

We have used a fluorescence enhancement of actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-actin) to study the interactions between rabbit skeletal muscle G-actin and either purified platelet gelsolin or a 130-kDa binary complex of platelet actin and gelsolin that is stable in EGTA and can be purified from human platelets. We have delineated four binding reactions. The exchange of Mg2+ for Ca2+ on the divalent cation-binding site of NBD-actin gives a small fluorescence increase. Binding of monomeric NBD-actin to the binary complex results in a 2.5-fold increase in the emission at 530 nm in the presence of Ca2+ and a 2-fold increase in the presence of EGTA. Titration experiments show that, under nonpolymerizing conditions, one additional actin is bound to the 130-kDa species to form a ternary complex. This binding is Ca2+-sensitive. Purified gelsolin does not appear to bind to NBD-actin in the presence of EGTA, as determined by fluorescence enhancement, gel filtration, or sedimentation measurements, but the addition of Ca2+ promotes rapid binding with a 1.6-1.7-fold enhancement of the emission intensity. A comparison of the relative fluorescence yields/NBD-actin molecule for a binary complex of gelsolin and one NBD-actin, a ternary complex of gelsolin and two NBD-actin molecules, and a ternary complex with an unlabeled actin in the EGTA-stable site and an NBD-actin in the second site indicates that the first NBD-actin, in the EGTA-stable site, does not give a fluorescence increase on binding but the second one does. Finally, we have demonstrated that one molecule of 45Ca2+ is "trapped" when the binary complex is formed and cannot be removed by EGTA. A summary model for these reactions is presented that indicates the interaction between actin and gelsolin is not a freely reversible Ca2+-controlled reaction.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases. Purification of the human erythrocyte proteins without the use of activating regulatory ligands.

Methods were developed to adequately extract, separate and, without the use of NaF as stabilizing agent, purify to better than 90% purity human erythrocyte Ns and Ni, the stimulatory and inhibitory guanine nucleotide- and Mg-binding regulatory components of adenylyl cyclases, as well as a protein containing Mr = 35,000 subunits. On the basis of a functional assay for Ns, it was purified about 5,000-fold from starting washed erythrocyte membranes with a yield of about 10%. A typical purification yields from 60 units of outdated human blood, between 500 and 1,000 micrograms of pure Ns, and a similar amount of Ni. Pure Ns and Ni contain each at least one alpha and one beta subunit (Northup, J.K., Sternweis, P.C., Smigel, M.D., Schleifer, L.S., Ross, E.M., and Gilman, A.G. (1980) Proc. Natl. Acad. Sci. U.S.A. 74, 6516-6520; Codina, J., Hildebrandt, J.D., Iyengar, R., Birnbaumer, L., Sekura, R.D., and Manclark, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 77, 4276-4280). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at varying acrylamide concentrations yielded Mr values of 42,000 and 40,000 for the alpha subunits of Ns and Ni, and of 35,000 for the beta subunits of Ns and Ni. Two-dimensional thin layer analysis of tryptic peptides obtained from digesting 125I-labeled subunits of Ns and Ni confirmed the finding of Manning, D., and Gilman, A.G. (1983) J. Biol. Chem. 258, 7059-7063) that while their alpha subunits are clearly different, their beta subunits are the same. Hydrodynamic analysis of the molecular weights of the nondenatured proteins showed behavior consistent with Mr = 95,500 for Ns, the same for Ni, and Mr = 40,000 for the protein containing the Mr = 35,000 beta subunit. Sedimentation coefficients and Stokes radii of the purified Ns were indistinguishable from those of Ns activity present in initial cholate extracts from human erythrocyte membranes. Further, the overall kinetics with which Ns activity in cholate extracts and Ns activity in the purified protein reconstituted the Ns-deficient adenylyl cyclase system of cyc- S49 cells was also indistinguishable. We conclude that we have purified the native unactivated form of Ns, and by serendipity the Ni, as well as a protein containing the 35 kDa beta subunit of Ns and Ni.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1983

Purification and characterization of a gelsolin-actin complex from human platelets. Evidence for Ca2+-insensitive functions.

Extracts of human platelets contain a 90,000-Da protein that is retained by DNase I-agarose in the presence of Ca2+. The 90-kDa protein, tightly complexed with platelet actin, can be eluted from DNase I-agarose by ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA). The platelet 90-kDa protein is immunologically related to rabbit macrophage gelsolin. The 90-kDa protein-actin complex was purified from platelet extracts using DEAE-Sephacel, Sephadex G-200, and hydroxyapatite and is stable in EGTA and 0.8 M KCl. The purified complex will modulate the assembly of fluorescently labeled 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole-actin in the presence of both Ca2+ and EGTA. In addition, the complex affects the low shear viscosity of F-actin solutions in the presence of both Ca2+ and EGTA. Finally, the complex increases the critical concentration for actin assembly about 4-fold. The results are consistent with a strong preferential binding to or capping of the barbed end of actin filaments by the complex in either Ca2+ or EGTA.

Actins↗
PubMed1983

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid.

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.

Actins↗
PubMed1983

Isolation and characterization of actin from Entamoeba histolytica.

Actin has been identified and purified partially from trophozoites of Entamoeba histolytica HMI-IMSS by a procedure that minimizes proteolysis. In cellular extracts, Entamoeba actin would copolymerize with muscle actin, but would not bind to DNase I or form microfilaments. Fractionation of the extracts by DEAE-cellulose and Sephadex G-150 chromatography yielded a purified actin that would copolymerize with rabbit skeletal muscle actin or polymerize alone into long filaments at 24 degrees C upon addition of 100 mM KC1 and 2 mM MgCl2. These filaments are not cold-stable and will depolymerize at 4 degrees C in 1 or 2 h. Entamoeba actin filaments bind phallotoxin with the same affinity as muscle actin and decorate with rabbit skeletal muscle heavy meromyosin. Entamoeba actin filaments activate the Mg2+ ATPase of heavy meromyosin to the same Vmax as muscle actin, but the Kapp is 2.8 times higher. Entamoeba actin is a single species with a slightly higher molecular weight than muscle actin (45,000) and a more acidic pI (5.4). The purified actin does not bind to DNase I, produce inhibition of the enzymatic activity, or block the binding of muscle actin. Comparison of the peptides obtained by limit digest with protease V8 from Staphylococcus aureus shows sequences with common mobility between alpha-actin and Entamoeba actin, but additional peptides are present which may account for the different properties of the Entamoeba actin. Finally, in vitro translation of mRNA from trophozoites produces a single polypeptide equivalent to the molecule purified from Entamoeba extracts.

Actins↗
PubMed1983

Identification of G actin-binding proteins in rat tissues using a gel overlay technique.

Actin-binding proteins were assayed in various tissues using an 125I-actin overlay procedure. Four major G actin-binding proteins of 90000, 65000, 58000 and 40000 Mr have been identified. The 90K protein is present in all tissues and binds labelled actin in a calcium-sensitive manner with binding increasing 3-4-fold in the presence of Ca2+. The distribution of the 58K and 65K protein which are not Ca2+-sensitive was more variable. These proteins were present in different ratios in different tissues. 125I-actin binding to all four actin-binding proteins is specific and can be displaced by preincubation of the gels with unlabelled actin. The interaction of actin with these proteins does not appear to involve ionic forces, since binding is not diminished by varying the salt concentration. Skeletal muscle glycolytic enzymes, the lens crystallins and the histones also bind 125I-actin. This binding cannot be displaced by preincubation with unlabelled actin and is presumably non-specific. The calcium sensitivity of two highly purified actin-binding proteins, the 90K human platelet protein and villin was compared using 125I-actin. The platelet 90K protein binds actin at less than 10(-7) M free calcium, but detectable binding to villin does not occur below 10(-6) M free calcium. The ubiquity of these actin-binding proteins is clear and we conclude that the calcium-sensitive 90K actin-binding protein in all of these tissues is the same as the platelet protein.

Actins↗
PubMed1982

R68.45 mediated chromosomal gene transfer in Agrobacterium tumefaciens.

A large plasmid enables its host Agrobacterium tumefaciens to cause tumorous condition in a wide variety of dicotyledonous plants[see Ooms et al. Gene 14:33--50 (1981) )). The location and role of chromosomal genes in this phenomenon are not known. As the first stage in studying this aspect, a project was initiated to investigate the chromosomal genetics of the bacterium. R68.45, a P group plasmid, was chosen as a transmission agent. After a preliminary assessment it was decided to use C58 as a standard strain to carry out the mapping. The plasmid itself, as judged by the presence of antibiotic markers, appears to be stable in A. tumefaciens; its ability to promote chromosomal mobilisation, however, remains only in 60--80% transconjugants. Good Agrobacterium donors are capable of transferring chromosomal genes at a frequency varying between 10(-5) to 10(-6) per recipient. The recombinants are stable even under non-selective conditions. A linear linkage map consisting of 16 markers was built using coinheritance frequencies obtained from 21 four-point crosses.

Chromosomes, Bacterial↗
PubMed1981

A DNase I binding/immunoprecipitation assay for actin.

An actin assay which employs the competition between labeled and unlabeled rabbit skeletal muscle actin for DNase I has been developed. Iodination of actin by the method of Bolton and Hunter results in the incorporation of approximately 0.5 mol of 125-iodine/incorporation of approximately 0.5 mol of 125-iodine/mol of actin. This 125I-actin retained the ability to bind to DNase I and inhibit enzymatic activity. The 125I-actin-DNase complex can be precipitated by the addition of a monospecific rabbit antibody to DNase I. The efficiency of this immunoprecipitation step is improved by the use of a second sheep anti-rabbit gamma-globulin. Using this immunoprecipitation assay, there is a linear displacement of the DNase I-bound 125I-actin by rabbit skeletal muscle actin standards or by the actin present in tissue and cell extracts. Using 17.5 ng of DNase I and approximately 500 pg of 125I-actin, 50% inhibition of binding was obtained with 23 ng of unlabeled actin. Reducing the amount of DNase I to 2 ng results in 50% inhibition of binding with 4 ng of unlabeled actin and an increase in the estimated sensitivity of the assay from 1.7 to 0.24 ng. The slopes of the displacement curves generated with both vertebrate and invertebrate non-muscle actins are parallel to rabbit skeletal muscle actin. This observation indicates approximately equal actin-DNase I binding affinities and suggests a high degree of conservation of the actin-DNase I binding site. The assay is useful for measuring the pools of F- and G-actin in a wide range of cells.

Actins↗
PubMed1981

Isolation of calcium-dependent platelet proteins that interact with actin.

Low Ca2+ extracts of platelets rapidly form an actin gel when warmed to 25 degrees C. The addition of Ca2+ has three effects. At Ca/EGTA = 0.4, the gel begins to contract. Increasing the Ca2+ concentration increases the rate of contraction and reduces the amount of actomyosin gel. Between Ca/EGTA = 0.4 and 0.5, a protease is activated that selectively degrades polypeptides with molecular weight greater than the myosin heavy chain. At Ca/EGTA = 1, about 70% of the total actin is nonsedimentable. Addition of excess EGTA produces the rapid formation of an actomyosin gel, which is not readily solubilized by re-addition of calcium. Using DNAase l-Sepharose chromatography, we have isolated a protein fraction whose binding to actin is Ca2+ -dependent. This fraction contains a major polypeptide with a molecular weight of 90,000. This fraction increases the rate of development of high sheer viscosity, but lowers the final value if Ca2+ is present. This decrease in viscosity is due to the generation of shorter filaments. In the presence of Ca2+, this protein(s) selectively blocks the addition of actin monomers to the barbed end of glutaraldehyde-fixed S1-decorated actin fragments and will nucleate assembly of filaments. We speculate that this protein(s) may serve as a Ca2+ -dependent nucleation site in situ.

Actins↗
PubMed1981

Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels.

Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.

Actins↗
PubMed1980

Redistribution of actin and fascin in sea urchin eggs after fertilization.

Following fertilization, the sea urchin egg cortex undergoes a structural change involving the assembly and organization of actin filaments into microvilli. Antifascin localizes this actin cross-linking protein in the microvilli of the fertilized egg cortex but no organized staining is present in the unfertilized cortex. Determination of the actin content of eggs using the DNAase I inhibition assay indicates that actin is about 1.4% of the total protein. Approximately 90% of this actin is soluble in low calcium isotonic extracts of unfertilized eggs while only 60-65% can be recovered in identical extracts of fertilized eggs. Similar measurements for fascin using a radioimmunoassay indicate this molecule represents about 0.3% of the total egg protein, essentially all of which is recovered in low calcium isotonic extracts of unfertilized eggs. After fertilization only 65-70% of this actin cross-linking protein is in the soluble phase. These results demonstrate a markedly different solubility for actin and fascin after fertilization, when the indirect immunofluorescence staining localizes fascin in the microvilli, and are consistent with the idea that fascin organizes newly polymerized actin filaments into the microvillar cores. A consideration of the amounts of actin and fascin incorporated into the cortex after fertilization and the number of microvilli on the egg surface indicates that the measured values are sufficient to account for the observed microvillar elongation.

Actins↗
PubMed1980

Yellow fever in the Gambia, 1978--1979: entomological aspects and epidemiological correlations.

An entomological survey was conducted in the Gambia in January 1979, during the last phase of a yellow fever (YF) outbreak which began during the previous rainy season. In the dry conditions which prevailed in January, Aedes aegypti was the only YF vector present. Two YF virus strains were isolated from females of this mosquito species caught in a village of western Gambia, where active human cases were documented. The ae. aegypti breeding sites were exclusively of the domestic type. Larval indices varied greatly from place to place, but generally appeared to correlate with the incidence of disease. A better understanding of the conditions that prevailed at the onset and during the early phase of the epidemic will require further entomological investigations. Nevertheless, it appears probable that initial transmission as by sylvatic vectors such as the Ae. furcifer-taylori group and possibly others such as Ae. luteocephalus, Ae. metallicus, and Ae. vittatus. As the outbreak progressed, interhuman transmission by Ae. aegypti also occurred, and this mixed epidemiological pattern later gave way to transmission by Ae. aegypti only when sylvatic vector populations declined in the dry season. We speculate that a prolongation of the rainy season during 1976--1978 was important in the origin of the outbreak. The relationship of this epidemic to the established focus of sylvatic YF in southeastern Senegal is discussed. The Gambian outbreak is considered the result of a recent northwesterly extension of the YF Emergence Zone.

Aedes↗