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J E Estes

Publications and source records attributed to J E Estes.

At least 37 records · Page 2Linked to original sources
PubMed1988

Human platelet-derived growth factor preparations contain a separate activity which potentiates follicle-stimulating hormone-mediated induction of luteinizing hormone receptor in cultured rat granulosa cells: evidence for transforming growth factor-beta.

The ability of platelet-derived growth factor (PDGF) preparations to potentiate FSH-mediated LH receptor induction in rat granulosa cell cultures was shown to be due to a component distinct from PDGF. Purification of heat-treated platelet lysate by carboxymethyl-Sephadex C-50 and Cibacron blue-Sepharose chromatography, followed by Bio-Gel P-60 chromatography, resulted in the separation of two activities: 1) a growth-promoting activity, P60-PDGF, defined on the basis of increased DNA synthesis in BALB/c-3T3 cells, and 2) a differentiation-promoting activity which enhanced FSH-dependent LH receptor induction in granulosa cells. On the basis of electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels, inhibition of tritiated thymidine uptake by epithelial cells, and attenuation of LH/hCG receptor expression in the presence of antitransforming growth factor-beta (anti-TGF beta) immunoglobulin G, the differentiation-promoting component of the preparations appears to be TGF beta. The Bio-Gel fractions that contained TGF beta did not stimulate LH receptor induction of cAMP production in the absence of FSH. PDGF prepared free of TGF beta did not potentiate receptor induction. We conclude, therefore, that the differentiative effects of PDGF previously described in this system are due to TGF beta.

Animals↗
PubMed1987

Conversion of ATP-actin to ADP-actin reverses the affinity of monomeric actin for Ca2+ vs Mg2+.

Monomeric ATP-actin binds Ca2+ 3-4-times more strongly than Mg2+ at pH 8. On conversion of G-ATP-actin to G-ADP-actin, the relative affinity of actin for the divalent cations is reversed, so that Mg2+ is bound 6-times more strongly than Ca2+. The dissociation rate constant of Ca2+ from Ca-ADP-actin is 50-fold higher than that for Ca2+ from Ca-ATP-actin, suggesting that this reversal of divalent cation affinities is due primarily to a higher equilibrium dissociation constant for Ca-ADP-actin. These results demonstrate an interaction between the actin-bound nucleotide and divalent cation or their binding sites.

Actins↗
PubMed1987

Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model.

Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.

Actins↗
PubMed1986

High affinity binding of divalent cation to actin monomer is much stronger than previously reported.

Monomeric actin is known to bind tightly one divalent cation per molecule. We have quantitatively reinvestigated the affinity of actin for Ca++ and Mg++ using the fluorescent Ca++ chelator Quin2 to induce and measure the dissociation of Ca++ from Ca-actin, supporting these studies with measurements using 45Ca. We found that the KD for Ca-actin is actually 1.9 +/- 0.7 nM. Kinetic analysis supported this result and demonstrated a dissociation rate constant (k-) of 0.013 s-1 and an association rate constant (k+) of 6.8 X 10(6)M-1 s-1 for Ca-actin. Competitive binding studies indicated that the binding affinity of actin for Ca++ is 5.4 times that for Mg++, yielding a calculated KD for Mg-actin of about 10 nM. Thus, the tight-binding of divalent cations to actin is 3-4 orders of magnitude stronger than previously thought.

Actins↗
PubMed1986

A kinetic comparison between Mg-actin and Ca-actin.

The kinetics of the elongation reaction in the polymerization of actin containing tightly-bound Mg2+ (Mg-actin) or tightly-bound Ca2+ (Ca-actin) have been studied. The reaction was monitored using the increase in fluorescence intensity of N-(1-pyrenyl)iodoacetamide-labelled monomeric actin as a measure of polymer formation. The actin nucleation reaction was circumvented by the addition of phalloidin-stabilized actin nuclei. Elongation rates were obtained at various actin concentrations and at various temperatures for polymerization induced by the presence of different monovalent and divalent salt concentrations. Values for the relative rate constant of forward polymerization (mk+) for Mg-actin were found to be larger than those for Ca-actin under similar conditions (m = number of polymer ends). The critical actin concentration (Cc) of Mg-actin is lower than the Cc for Ca-actin, as were estimates of the relative rate constant of depolymerization (mk-). The temperature dependence of Cc, mk+ and mk- for Mg-actin was different from that for Ca-actin, further suggesting a difference in monomeric properties due to the type of divalent cation tightly bound to actin. Estimates of the activation enthalpy change for the forward reaction in the G in equilibrium F transformation were similar for both types of actin, but the activation enthalpy change for the depolymerization of Mg-actin was significantly larger than that for Ca-actin.

Actins↗
PubMed1985

PDGF stimulates transient phosphorylation of 180,000 dalton protein.

Cell-free extracts of platelet-derived growth factor (PDGF) treated, density-arrested, quiescent BALB/c-3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P-40 solubilized cell preparations that had been incubated with [gamma-32P]ATP. When quiescent BALB/c-3T3 cell cultures were incubated at 37 degrees C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3-min exposure of the intact cells to PDGF, which was maximal after 10-15 minutes and had diminished by 30-60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4 degrees C; however, in contrast to PDGF exposure at 37 degrees C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4 degrees C. When cells exposed to PDGF at 4 degrees C were transferred to 37 degrees C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37 degrees C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation of PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF-stimulated phosphorylation activity in relation to the cell cycle and growth regulation.

Animals↗
PubMed1984

Bound-cation exchange affects the lag phase in actin polymerization.

The delay or lag phase at the onset of polymerization of actin by neutral salt is generally attributed to an actin nucleation reaction. However, when nucleation is circumvented by the use of phalloidin-stabilized nuclei, a lag phase persists when Ca2+-containing actin is polymerized with MgCl2. Pretreatment of actin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and/or Mg2+ shortens or eliminates this lag phase, suggesting that exchange of the actin-bound divalent cation occurs during this nucleation-independent lag phase. Measurement of the actin-bound cation initially and after brief incubation with EGTA/Mg2+ directly verifies that Mg2+ has replaced Ca2+ as the actin-bound cation, producing a highly polymerizable Mg2+-actin species. Bound-cation exchange prolongs the lag phase in actin polymerization and probably explains what has been termed the monomer activation step in actin polymerization.

Actins↗
PubMed1984

Macrophage-derived growth factor for fibroblasts and Interleukin-1 are distinct entities.

P388D1, a mouse macrophagelike cell line, was adapted to grow continuously in an unsupplemented, serum-free culture medium and continued to elaborate substances that were mitogenic for quiescent mouse fibroblasts (BALB/c 3T3 cells) and for thymocytes suboptimally stimulated with lectins. We have previously described [37] the fibroblast mitogenic activity as a macrophage-derived competence factor (MDCF). Serum-free, macrophage-conditioned culture medium was concentrated 1,000-fold by a combination of ultrafiltration (hollow fiber) and lyophilization. Concentrates of medium were subjected to gel filtration (Sephadex G-75 or G-150), and the fractions were assayed for mitogenic activity (MDCF) on density-arrested BALB/c 3T3 cells and for Interleukin-1 (IL-1) activity in suboptimally stimulated (Con A) mouse thymocytes. The apparent molecular weight (MW) of MDCF activity was estimated at 56,000 daltons, whereas the peak of IL-1 chromatographed at an apparent MW of 14-16K daltons. There was no detectable IL-1 activity in the MDCF fractions and no detectable MDCF in the IL-1 fractions. These data indicate that P388D1 cells produce both MDCF and IL-1 activities under continuous serum-free conditions and that the two activities are not identical. Stimulation of responsive mononuclear phagocytes with lipopolysaccharide and/or lymphokine-rich supernates resulted in a differential modulation of MDCF and IL-1 activities. Finally, antibody-purified IL-1 had no significant ability to stimulate DNA synthesis in quiescent fibroblasts at concentrations that were mitogenic for thymocytes. However, IL-1 did augment the mitogenic activity of suboptimal amounts of platelet-derived growth factor (PDGF), another competence factor. Further studies revealed that neither the generation nor the activity of MDCF was modulated by the presence of various inhibitors of proteolytic enzymes.

Animals↗
PubMed1983

The tightly bound divalent cation regulates actin polymerization.

The polymerization characteristics of Ca++-actin and Mg++-actin were studied by measuring initial rates of polymerization upon addition of phalloidin-stabilized nuclei and neutral salt. Under conditions where the effects of divalent cation exchange were minimized, CaCl2 and MgCl2 were found to be equally effective in polymerizing actin. Mg++-actin was found to nucleate and polymerize more readily than Ca++-actin, having a forward rate constant about twice that of Ca++-actin under a variety of polymerizing conditions. The critical concentration for Ca++-actin is approximately 20 times that for Mg++-actin under equivalent conditions. These data imply that the polymer of Mg++-actin must be more stable than that of Ca++-actin, having a depolymerization rate constant about 10 fold lower. Since Mg++ is probably the tightly-bound cation in vivo, whereas Ca++-actin has been more widely studied in vitro, it would appear that actin in its physiological state is probably more polymerizable and more stable in the polymer form than previously considered.

Actins↗
PubMed1983

Actin-induced reticuloendothelial phagocytic depression as mediated by its interaction with fibronectin.

Circulating fibronectin, also known as opsonic alpha 2 surface binding glycoprotein or cold-insoluble globulin, modulates phagocytosis of tissue debris, fibrin microaggregates, and gelatin-coated colloids by the reticuloendothelial (RE) system. Opsonically active fibronectin has an actin binding site and a demonstrated in vitro affinity for actin. Since actin potentially released into blood and tissue fluids following tissue injury could complex with fibronectin, the present study evaluated the effect of actin on plasma opsonic activity and Kupffer cell phagocytosis. Intravenous injection of actin did not acutely decrease plasma immunoreactive fibronectin levels although fibronectin levels increased at 6, 12, and 24 hr postinjection. However, intravenous actin injection did depress RE phagocytic activity in vivo as measured by decreased blood clearance of test colloid and impaired hepatic uptake of colloid particles as well as retention of the particles in the circulation. In vitro, preincubation of plasma with actin depressed the opsonic activity of plasma with respect to its ability to support phagocytosis, but such treatment of plasma did not alter the detection of fibronectin by immunoassay. Utilizing purified fibronectin with demonstrated opsonic activity, it was also observed that actin interaction with fibronectin would block its biological ability to enhance phagocytosis. This effect appeared to be mediated at the humoral level, since no direct depressant effect of actin on Kupffer cell function was observed. Thus, actin, if released into the blood following injury, may contribute to bioassayable opsonic fibronectin deficiency and phagocytic dysfunction, but this disturbance would remain undetectable by immunoassay of fibronectin levels.

Actins↗
PubMed1981

Mechanism of action of phalloidin on the polymerization of muscle actin.

Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.

Actins↗
PubMed1980

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells.

Platelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine blood. Bovine platelet-poor plasma had properties similar to those previously described for human platelet-poor plasma; e.g., it would (a) support the growth of virally transformed but not normal BALB/c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.

Animals↗
PubMed1980

Effect of shock on calcium accumulation by cardiac sarcoplasmic reticulum.

Earlier work has suggested that the decreased cardiac performance in shock is due to negative inotropic substances that alter the Ca++ metabolism of the myocardium. We investigated this possibility by examining the Ca++-dependent ATPase activity and the Ca++-uptake ability of cardiac sarcoplasmic reticulum (microsomes) from normal hearts and endotoxin-shocked hearts. Both the ATPase activity and the accumulation of Ca++ by microsomes from shocked hearts were lower than for microsomes from normal hearts. In the presence of serum from shocked dogs, the ATPase activities and Ca++-uptake of both microsome preparations were reduced from those measured in modified Krebs-Henseleit, but this was not considered significant because the same extent of depression was seen with serum from normal dogs. These findings could not attribute the depressed ATPase activities and Ca++-uptake abilities to a shock-induced circulating cardio-depressant material but did demonstrate that calcium metabolism in cardiac tissue was altered in the shock state.

Adenosine Triphosphatases↗
PubMed1978

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.

Actins↗
PubMed1978

Epstein-Barr virus-associated thymidine kinase.

Superinfection of Raji cells with Epstein-Barr virus induced a new thymidine kinase that was distinguishable from both adult and fetal kinases of the host cell by discontinuous electrophoresis on polyacrylamide gels and glycerol gradients.

Burkitt Lymphoma↗