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Biomedical subjects

K E Neet

Publications and source records attributed to K E Neet.

At least 55 records · Page 3Linked to original sources
PubMed1987

Nerve growth factor and the concept of neural-epithelial interactions. Immunohistochemical observations in two cases of vasitis nodosa and six cases of prostatic adenocarcinoma.

The so-called perineural invasion is a well-recognized, but poorly understood, phenomenon occurring in vasitis nodosa, a benign epithelial proliferation of the vas deferens; such unusually close epithelial-neural interactions also occur, among others, in benign and malignant conditions of the prostate. In the present study, immunoreactive nerve growth factor was found in the epithelium of these organs. This nerve growth factor is thought to possibly play a role in the process of the so-called perineural invasion.

Adenocarcinoma↗
PubMed1986

Beta nerve growth factor binding to PC12 cells. Association kinetics and cooperative interactions.

The association kinetics of 125I beta nerve growth factor (NGF) binding to the PC12 clonal cell line have been examined in detail at 0.5 and 37 degrees C. These data were examined by utilizing a reversible second-order integrated rate equation, and the results were not consistent with a simple bimolecular process. Two association rates were required to explain the results adequately. At 37 degrees C, the faster component was estimated to have a second-order association rate constant of 1.4 X 10(7) M-1 s-1, while the rate constant for the slower component (3.8 X 10(6) M-1 s-1) was about 4-fold lower. As shown by others, the temperature dependence of the dissociation kinetics indicated that while the rapidly dissociating component was only slightly slowed by lowering the chase temperature to 0.5 degrees C, the second component was slowed by about 270-fold, from 8 X 10(-4) s-1 to 3 X 10(-6) s-1. The binding data that describe the slowly dissociating component were obtained by utilizing this differential temperature dependence and revealed a concave downward Scatchard plot. The binding parameters determined from computer analysis using a nonlinear fitting program (LIGAND) suggest that this component consists of (a) an interacting class of about 4000 sites/cell that have a first stoichiometric steady-state dissociation constant of 65 pM and a second stoichiometric interaction constant of 16 pM, indicative of positively cooperative interactions, and (b) a class of sites consistent with a ratio of sites/Kd of about 11.1 sites/(cell X pM). The steady-state binding results at 37 degrees C indicated only one class of binding sites (155,000 +/- 18,000 sites/cell) that had an apparent Kd of 0.52 +/- 0.03 nM. One class of sites was also observed at 0.5 degrees C, and the receptor concentration was found to be reduced (99,000 +/- 7600 sites/cell) while the Kd was increased (1.7 +/- 0.14 nM). A significant level of positively cooperative interactions was observed frequently at 37 degrees C that was not due to a failure to reach steady-state conditions, internalization, or degradation. Since cooperativity of binding was never observed at 0.5 degrees C, a membrane event may be involved. Determination of the contribution of the different classes of NGF receptors found on PC12 cells to the biological actions of NGF requires a clear understanding of their kinetic properties and their relationship to each other. The studies presented here indicate that their interactions are more complex than previously described.

Adrenal Gland Neoplasms↗
PubMed1986

Inhibition of beta nerve growth factor binding to PC12 cells by alpha nerve growth factor and gamma nerve growth factor.

Pheochromocytoma (PC12) cells have been found to differ from dorsal root ganglionic cells with respect to the modulation of the beta nerve growth factor (beta NGF) binding properties elicited by alpha NGF and gamma NGF. In contrast to our previous results with intact dorsal root ganglionic cells in which only high-affinity binding was blocked, alpha NGF and gamma NGF were found to block competitively all steady-state binding of iodinated beta NGF to PC12 cells at both 37 and 0.5 degrees C. The EC50 that was found for the alpha NGF displacement was 9-10 microM, and the gamma NGF effect had an EC50 of 200 nM, in the predicted range based upon the apparent Kd for dissociation of the alpha beta or the beta gamma complex in solution. The concurrence of the binding EC50 and the Kd for each complex indicates that the formation of alpha beta or beta gamma complexes in solution competes with the process of PC12 receptor binding with 125I-beta NGF. Experiments were carried out examining the dissociation kinetics following the addition of excess unlabeled beta NGF or alpha NGF at both 37 and 0.5 degrees C. Three dissociation components were observed with alpha NGF, in contrast to the two normally found with beta NGF. Lowering the chase temperature to 0.5 degrees C changed the relative contributions made by each component without dramatically changing any of the rate constants. The "slow" receptor was further examined by the dependence on 125I-beta NGF concentration of the slowest component with a chase of either excess alpha NGF or excess gamma NGF at 0.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗
PubMed1985

Effects of tunicamycin on NGF binding and neurite outgrowth in PC12 cells.

The effects of inhibition of glycosylation on nerve growth factor (NGF) binding and neurite outgrowth response of PC12 cells have been examined. Exposure to tunicamycin (1-10 micrograms/ml) for 24-36 hr eliminates the rapidly dissociating component of NGF binding and decreases the proportion of PC12 cells capable of elaborating neurites in a dose-dependent manner. These decreased cellular responses are probably due to an underglycosylation of the NGF receptor, since the effects of tunicamycin are correlated with a decrease in 3H-fucose incorporation rather than a general decline in cellular metabolism as measured by viability and protein synthesis. These results suggest that carbohydrate side chains are important for the function and/or orientation of the NGF receptor in PC12 cells and that the rapidly dissociating component of NGF binding may be associated with a minimum concentration of functional receptors per cell required for the full biologic response.

Adrenal Gland Neoplasms↗
PubMed1984

Subunit interactions of 7 S nerve growth factor. Gamma-esterase activity, rates, and conformational changes during reassociation.

The gamma-esterase activity of 7 S nerve growth factor (NGF) is depressed relative to free subunit because of constraints within the oligomer. 4 M urea causes a reversible dissociation of 7 S NGF and concomitant increase in esterase activity. The gamma-esterase activity with tosyl-arginyl-methyl-ester increased between 1 and 4 M urea and was inhibited at higher concentrations. Upon dilution of 7 S NGF from 6 M urea, the esterase activity was initially identical with that of the similarly treated gamma-subunit but decayed rapidly (10 min) to approach that of the native 7 S NGF. In the presence of 100 microM EDTA the activity was higher, but still required the same length of time to reach a constant depressed value. Upon dilution of 7 S NGF from 2 M NaCl the rapid decay was not observed and the activity remained constant at about the same level as the equilibrium values for the urea-treated and renatured 7 S NGF. A red shift in the fluorescence maximum and a concomitant increase in quantum yield occurred for the 7 S in 8 M urea. Dilution into buffer resulted in a rapid decay and return to native 7 S fluorescence but not when EDTA was included. Both the decay in esterase activity and in fluorescence intensity upon dilution from urea were first order reactions with a rate constant of about 8 X 10(-3) s-1 suggesting that both methods were measuring a unimolecular renaturation process. Renaturation from NaCl or recombination of isolated subunits was much more rapid, indicating a simple combination of subunits in a native conformation. The circular dichroism spectra of urea and NaCl-treated 7 S NGF were different, and spectra of the renatured species with and without EDTA differed. Reassociation to a conformationally different 7 S NGF probably occurs in the presence of EDTA. The results emphasize the role of zinc in 7 S NGF formation and the influence of conformational changes in the effect of beta-NGF on the activity of the gamma-esterase in 7 S NGF.

Animals↗
PubMed1983

6-(p-toluidinyl)naphthalene-2-sulfonic acid as a fluorescent probe of yeast hexokinase: conformational states induced by sugar and nucleotide ligands.

The fluorescent dye 6-(p-toluidinyl)naphthalene-2-sulfonic acid (2,6-TNS) has been shown to be a sensitive and nonperturbing probe of conformational states of yeast hexokinase. The binding of sugar ligands to hexokinase induced conformational states of the enzyme which could be distinguished by monitoring 2,6-TNS fluorescence and correlated well with their behavior during the catalytic reaction. The binding of five-carbon sugar inhibitors such as lyxose induced a conformational state of hexokinase that demonstrated a small quenching of 2,6-TNS fluorescence but an increased ability to bind metal-ligands when compared to free enzyme. The binding of good sugar substrates such as glucose produced a conformational state of hexokinase which demonstrated a large enhancement (37%) of bound 2,6-TNS fluorescence. This glucose-induced conformational state had an increased ability to bind metal-ATP ligands; however, the relative changes in the dissociation constants for the various metal-ATP ligands differ from those observed with hexokinase in the presence of lyxose. Hence, the lyxose-induced conformational state of hexokinase was concluded to be significantly different from the glucose-induced conformational state. The binding of poor sugar substrates such as 5-thioglucose induced a conformational state of hexokinase similar to the conformational state induced by glucose, but with a smaller enhancement of 2,6-TNS fluorescence (15%) and a lesser ability to increase the affinity for metal-ATP ligands. The six-carbon inhibitor with a bulky group on the 2-position, N-acetylglucosamine, gave minimal changes in 2,6-TNS fluorescence and effects on metal-nucleotide binding. These conformational states are interpreted in terms of the closure of the cleft between the two domains observed by X-ray crystallography. The binding of A1ATP to free hexokinase was not observed at concentrations up to 100 microM, which is consistent with the kinetic properties reported for this metal-ATP ligand. Although both CrATP and A1ATP have been reported to produce a slow burst-type transient in the progress curve of hexokinase, only CrATP demonstrated slow changes in 2,6-TNS fluorescence, indicating that the conformational state of hexokinase induced by A1ATP is different from the conformational state induced by CrATP.

Fluorescent Dyes↗
PubMed1983

Association equilibria and reacting enzyme gel filtration of yeast hexokinase.

The association-dissociation of yeast hexokinase has been re-examined and the size of the reacting form of the enzyme has been investigated under a variety of conditions. Both Sephacryl S-200 and high performance liquid chromatography on Bio-Sil have been employed with continuous effluent monitoring under reacting and nonreacting conditions. The reacting enzyme was monomeric under all conditions, suggesting that the dimer is not an important catalytic species in normal assays. The reacting enzyme remained as a monomer under the following conditions: 0.1-15 micrograms/ml loading concentration, from 30 to 100 mM ionic strength, with 2 mM citrate, with 50% D2O, and at 160 atm hydrostatic pressure. The dissociation constant for the nonreacting hexokinase was 22 (+/- 2) micrograms/ml (uncorrected for 5-fold dilution) in 100 mM triethanolamine, pH 6.5, and 25 degrees C. Glucose or MgATP had dissociative effects under all conditions studied, but MgATP was much less effective and only slightly more effective than an equivalent ionic strength. NaCl, between 30 and 80 mM, promoted dissociation, with a concomitant conformational change suggested by nonlinearity of log-log plots. The extent of dissociation with MgCl2 was slightly greater than an equivalent NaCl ionic strength and the shape of the association curves suggested the formation of an elongated dimer in the presence of MgCl2. The conclusion is made that hexokinase is monomeric under most assay conditions and that the dissociation is predominantly the result of the glucose interaction. High performance liquid chromatography has been shown to be a useful method of assessing the association state of enzymes under both reacting and nonreacting conditions.

Chromatography, Gel↗
PubMed1983

Interconversions between different sulfhydryl-related kinetic states in glucokinase.

Rat liver glucokinase (EC 2.7.1.2) undergoes two distinct sulfhydryl-related reversible kinetic transitions. During normal assays in the presence of both substrates but without added reducing agents, the activity decays ("kappa" decay) over time to a new steady-state rate. The half-time for this decay is essentially constant at glucose levels from 2 to 200 mM and averages 6.2 +/- 2 min. Glucokinase in this kappa steady state displays an increased Km for glucose but has the same Vmax as normal, sulfhydryl-activated glucokinase. The kappa form does not itself exhibit kinetic cooperativity with glucose. In contrast, glucokinase incubated with neither glucose nor sulfhydryl reagents decays (mu decay) to a form whose Vmax is near zero. The t 1/2 for this transition is about 0.5 min at 0 or very low (0.5 mM) glucose concentrations. For both decays, incubations of enzyme with intermediate levels of reducing agents give steady-state mixtures of activated and either kappa and/or mu forms, depending on conditions during the decay. Enzyme at intermediate stages of the kappa decay displays an unchanged Vmax, intermediate (increased relative to activated enzyme) glucose S0.5 values, and diminished glucose cooperativity. In contrast, enzyme at intermediate steady-state mixtures of activated and mu forms has a normal glucose S0.5 and cooperativity but a diminished Vmax from the activated states. The enzyme at any stage of each decay may be fully reactivated by the addition of sulfhydryl reducing agents such as dithiothreitol, dithioerythritol, glutathione, or mercaptoethanol. A model is proposed to account for this complex behavior in glucokinase kinetics which proposes different enzymatic states (kappa and mu) locked in by sulfhydryl oxidation of different conformations dictated by glucose concentration. These sulfhydryl-related transitions may be important in regulation of glucokinase activity, since glucokinase is very sensitive (at least 20-fold differential activity) to concentrations of glutathione within the physiological range, perhaps allowing the normally variable glutathione levels or cytosolic redox potential to modify the rate of uptake and storage of blood glucose through control of glucokinase activity.

Animals↗
PubMed1982

Tryptophan residues of the gamma subunit of 7S nerve growth factor: intrinsic fluorescence, solute quenching, and N-bromosuccinimide oxidation.

The environment of tryptophan residues of the gamma subunit derived from the 7S nerve growth factor has been studied by intrinsic fluorescence, solute quenching, and oxidation with N-bromosuccinimide (NBS). The native protein has a fluorescence emission maximum of 345 nm with a tryptophan quantum yield of 0.10. A red shift in the emission maximum occurs between 3 and 6 M urea; a 20% increase in quantum yield occurred at 7-8 M urea. NBS (20-24 mol of NBS/mol of protein) completely oxidized one tryptophan of the gamma subunit, causing a 70% quenching of the intrinsic fluorescence, a complete loss of esterolytic activity toward synthetic substrates, an inability to combine with alpha and beta subunits to re-form the 7S complex, and a shift of about 12% of the structure from beta strands to random coil as determined by circular dichroism. About 80% of the fluorescing tryptophans are accessible to quenching by acrylamide but not to potassium iodide. These results suggest the presence of an exposed tryptophan contributing greater than or equal to 70% of the native fluorescence, located near a negative charge, critical to the esterase activity and possibly to interaction with the beta subunit to re-form 7S NGF.

Bromosuccinimide↗
PubMed1982

Modulation of the neuronal binding of the beta subunit of nerve growth factor (NGF) by the alpha-NGF subunit.

The effect of the alpha subunit of the 7S-NGF on the binding of beta-NGF to its two classes of sites on target cells has been studied. The presence of microM concentrations of alpha-NGF causes the displacement of 125I-beta-NGF from one class of sites on dissociated dorsal root ganglia neurons from stage E9 chicken embryos. At 0.1 nM 125I-beta-NGF, increasing alpha-NGF concentrations produce a monotonic displacement curve with half-maximal displacement occurring at 10 microM alpha-NGF. The affinity and number of sites of the 125I-beta-NGF displaced by alpha-NGF are similar to those of beta-NGF that binds to the higher affinity (site I) receptors. The binding to the lower affinity class of sites (site II) is not affected by concentrations of alpha-NGF up to 30 microM. This modulation of 125I-beta-NGF binding does not occur with equivalent concentrations of serum albumin. No detectable neuronal binding of 125I-alpha-NGF was found, suggesting that the mechanism does not involve direct competition for receptor sites. The dissociation constant for the alpha-beta complex is in the microM range, and formation of this complex in solution can thus compete with the process of 125I-beta-NGF binding to neurons. A model accounting for these observations includes binding of the alpha-beta complex to the lower affinity but not to the higher affinity sites. We conclude that there are differences in the specificity of the two classes of receptors.

Animals↗
PubMed1980

Inhibition of biological activity of mouse beta-nerve growth factor by monoclonal antibody.

A continuous hybrid cell line was derived that secretes monoclonal antibody capable of inhibiting the biological activity of mouse beta-nerve growth factor (beta-NGF). Results obtained with monovalent fragments indicate that the monoclonal antibody inhibits activity by interfering with the direct interaction between beta-NGF and the cell membrane receptor rather than by precipitating the dimeric form of beta-NGF. This monoclonal antibody binds to an antigenic determinant common to mouse beta-NGF, snake venom (Naja naja) beta-NGF, and human beta-NGF. These antibodies should provide specific molecular probes for a variety of studies of nerve growth factor including its tissue distribution and mechanism of action.

Animals↗