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Biomedical subjects

K E Neet

Publications and source records attributed to K E Neet.

At least 19 recordsLinked to original sources
PubMed1992

The unprocessed C-terminal dipeptide of recombinant beta-nerve growth factor determines three stable forms with distinct biological activities.

The processing of polypeptide neurotrophins in the nervous system is poorly understood. In this paper, we provide information on the effects of C-terminal processing of nerve growth factor. Three forms of recombinant mouse beta-nerve growth factor (rNGF) were produced and isolated from insect cells infected with a recombinant baculovirus. The three purified forms of rNGF exhibited distinct biological activities and differed in their abilities to compete with high affinity binding of mouse beta-nerve growth factor (mNGF). However, they were chemically and structurally indistinguishable from each other. All three forms of rNGF differed from mature mNGF from mouse submaxillary gland in that the C-terminal Arg-Gly dipeptide had not been proteolytically removed. Removal of the C-terminal dipeptide by gamma-NGF peptidase treatment converted the three forms into a single form identical with mature mNGF. The above results demonstrate that a single polypeptide of rNGF, due to the presence of a C-terminal dipeptide, exhibits three stable dimeric protein conformations with distinct biological activities. The apparent lack of gamma-NGF peptidase in the nervous system raises the possibility that the biologically significant form of NGF may differ from mature mNGF; such a difference may be of physiological relevance.

Animals↗
PubMed1992

Equilibrium denaturation studies of mouse beta-nerve growth factor.

Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7.

Animals↗
PubMed1992

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.

Animals↗
PubMed1992

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.

Amino Acid Sequence↗
PubMed1990

A common pentapeptide conformation occurs in viral acid proteases and other proteins.

We found a pentapeptide conformation, termed a type I twist, which has a strikingly high propensity (56%) for aspartic acid in the first position. Type I twists include the active site loops from cellular and viral aspartic proteases, with the catalytic Asp in the first position. Fifteen other type I twists, from non-homologous proteins, were found among high-resolution structures in the Protein Data Bank using a comparison method based on main-chain torsion angles. We propose that the Asp affects electrostatic interactions and thus plays a major structural role in the formation of this recurring motif, in addition to its catalytic role in the aspartic proteases.

Amino Acid Sequence↗
PubMed1990

Demonstration of a slow conformational change in liver glucokinase by fluorescence spectroscopy.

A slow interconversion between two enzyme forms of rat liver glucokinase has been previously inferred from kinetic assay studies. Two different conformations of the enzyme-substrate complex have now been directly demonstrated by an intrinsic fluorescence enhancement in glucokinase upon addition of glucose. The transition between these two conformations upon glucose addition is measurably slow in the presence of glycerol, with a glucose-dependent half-life of 0.5 to more than 10 min. In the presence of 5% glycerol, the forward and reverse isomerization rate constants are 2.6 x 10(-2) s-1 and 8.5 x 10(-4) s-1, respectively. Correspondingly, the overall equilibrium dissociation constant (0.13 mM) is more than 30-fold lower than the first binding step, i.e. the affinity for glucose is greatly increased. This result was also verified by equilibrium titration of the enzyme with glucose. A similar slow transition was analyzed in the presence of 30% glycerol and observed without glycerol. The dilution of stock glucokinase to promote glucose dissociation from the enzyme showed an exponential fluorescence decay, exactly the reverse phenomenon of glucose addition. The deduced rate constant for the reverse reaction coincided with that calculated from the association results. The conformational change is specific for glucose and responsible for the generation of the kinetic cooperativity of this monomeric enzyme, thus playing a regulatory role in the uptake of glucose in liver.

Animals↗
PubMed1990

Observation of a kinetic slow transition in monomeric glucokinase.

Rat liver glucokinase (EC 2.7.1.2) is a monomeric enzyme with positive cooperativity for glucose phosphorylation for which several kinetic mechanisms have been proposed. We have observed a slow kinetic transition when the enzyme is assayed in the presence of 30% glycerol. When the enzyme had been preincubated or stored in 50 mM glucose, the initially rapid activity decayed, via a first-order process, to a new steady-state velocity. The glucose-induced process is reversible since if the enzyme is preincubated without glucose, an initially low activity accelerates over minutes to the same steady-state velocity. This final velocity is independent of the preincubation conditions and is determined solely by the glucose and ATP concentrations in the assay. Possible artifacts which might cause nonlinear progress curves have been ruled out. The transition has a half-time of 2-10 min depending on glucose and ATP concentrations and temperature. In the steady-state kinetics, positive cooperativity occurs with glucose with a Hill coefficient (nH) = 1.3 at high ATP concentrations, approaching unity as the ATP concentration decreases. This pattern is similar to that seen in the linear velocities in the absence of glycerol. Similarly, negative cooperativity with MgATP is seen in the steady-state velocities at low glucose concentrations with the Hill coefficient approaching 1 as the glucose concentrations approach saturation. The initial velocity for enzyme preincubated in high glucose concentration was either Michaelis-Menten as a function of glucose at high MgATP concentration or heterogeneous (nH less than 1, negatively cooperative) at low MgATP concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗
PubMed1990

A comparison of nerve growth factor binding protocols with native and mutant PC12 cells.

A comparison has been made of various methods for measuring binding of nerve growth factor (NGF) to PC12 cells in suspension, on plates, and by a combination of the two. Results indicated that the extensive washing in the plate binding assay removed some cell surface ligand, underestimated the fast receptor binding, and overestimated the proportion of internalized ligand. In addition, the binding and internalization by a nonresponding PC12 mutant cell line has been studied. The nonresponding mutants had fewer total NGF receptors (10-50%) than normal cells in any binding assay. However, when measured in the suspension assay, the mutant cells showed both fast and slow binding receptors, in proportion approximately equivalent to those found on native PC12 cells. The PC12 nonresponders in suspension were also found to internalize and degrade low levels of NGF, in proportion to their reduced receptor number. Different results concerning PC12 wild type and mutant cells that have been reported in the literature may be due to the particular binding assay protocol that was used.

Adrenal Gland Neoplasms↗
PubMed1989

The regulatory kinetic properties of porcine hepatic glucokinase.

Porcine hepatic glucokinase (ATP: D-hexose 6-phosphotransferase EC 2.7.1.1) has been purified by a modification of the procedure for its purification from rats. However, difficulties were encountered with endogenous proteases and the reliability of a source for porcine livers. The molecular weight has been determined to be 60,400 +/- 1,400 by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. The enzyme has been characterized kinetically. The parameter values, S0.5 (glucose) and Hill coefficient (nH) are 2.4 mM and 1.9 respectively under sulfhydryl-reducing conditions. The enzyme undergoes the two sulfhydryl-related decays of its activity previously observed in the enzyme isolated from rat (Tippett PS, Neet KE: Arch Biochem Biophys 222:285-298, 1983). The enzyme is inhibited by palmitoyl-CoA, Ki (apparent) = 1.0 microM, nH = 1.8; this concentration of inhibitor is significantly below its critical micelle concentration. Physically and kinetically glucokinase isolated from pig is similar to the enzyme isolated from rat. The porcine system provides a second source for isolation and further characterization of this important and unusual enzyme.

Allosteric Regulation↗
PubMed1989

The lack of a role for protein kinase C in neurite extension and in the induction of ornithine decarboxylase by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) causes pheochromocytoma cells (PC12) to undergo a number of physiological changes which mimic the differentiated neuronal cell, including neurite extension. We have examined protein kinase C (Ca2+/phospholipid-dependent enzyme) as a potential signaling mechanism in NGF-stimulated neurite outgrowth and induction of the enzyme ornithine decarboxylase. Phorbol 12-myristate 13-acetate (PMA) can activate protein kinase C and induce ornithine decarboxylase in PC12 cells with kinetics which are similar to those of NGF induction, but only to levels about 10-fold lower. The induction of ornithine decarboxylase by both NGF and PMA is inhibited by cycloheximide and actinomycin D suggesting that both agents increase enzyme activity by increasing gene transcription. The evidence presented here, however, shows that the induction produced by the two agents is through two different pathways. First, maximal induction by NGF is increased when PMA is included in the media showing that the two effects are synergistic. Second, NGF does not cause induction of ornithine decarboxylase in the mutant PC12nnr5 cell line (Green, S.H., Rydel, R.E., Connolly, J.L., and Greene, L.A. (1986) J. Cell Biol. 103, 1967-1978) while added PMA does produce an induction of the enzyme. Finally, when protein kinase C is down-regulated by incubating PC12 cells with PMA in serum-containing or serum-free medium for 24 h, the induction by PMA is completely inhibited, while the NGF induction is not affected. A recent study (Hall, F.L., Fernyhough, P., Ishii, D.N., and Vulliet, P.R. (1988) J. Biol. Chem. 263, 4460-4466) using sphingosine inhibition concluded that protein kinase C was required for NGF-stimulated neuritogenesis. In contrast, results presented here show that down-regulation of protein kinase C also has no effect on NGF-mediated neurite extension in PC12 cells grown in serum-free medium. Our data demonstrate that induction of ornithine decarboxylase and formation of neurites in PC12 cells by NGF does not require a protein kinase C-mediated pathway.

Adrenal Gland Neoplasms↗
PubMed1989

Comparing short protein substructures by a method based on backbone torsion angles.

An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes delta t, the root mean square difference in phi and psi torsion angles over a small number of amino acids (typically 3-5). Using this algorithm, large numbers of protein substructure comparisons were feasible. The parameter delta t was sensitive to variations in local protein conformation, and it correlates with delta r, the root mean square deviation in atomic coordinates. Values for delta t were obtained that define similarity thresholds, which determine whether two substructures are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of delta t due to measurement noise from comparisons of independently refined coordinates of the same protein. A sample distribution of delta t from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons. Unlike methods based on C alpha atoms alone, delta t was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologous proteins by delta t showed that the active site torsion angles are highly conserved. The delta t method was applied to the alpha-chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.

Algorithms↗
PubMed1989

Stoichiometry of slow binding of palmitoyl-CoA to liver glucokinase.

The interaction of palmitoyl-CoA with porcine glucokinase was studied by the gel permeation technique. The finding that glucokinase "bound" up to 60 molecules was unexpected from the specific inhibition of rat glucokinase by long chain acyl-CoA (Tippett & Neet, J. Biol. Chem. (1982) 287, 12839-12845). Sephacryl S-200 gel filtration in the presence of palmitoyl-CoA demonstrated a protein peak without enzyme activity that was eluted earlier than the active enzyme peak, indicating a large molecular weight shift for the inactivated enzyme form and confirming a large number (greater than or equal to 30) of associated palmitoyl-CoA molecules. The binding was also verified by analyzing the absorption characteristics of the inactivated enzyme peak. In the presence of glycerol, the size of the inactivated peak greatly decreased, but the separation between the two peaks remained unchanged. Therefore, the amphiphile bound predominantly to the inactive enzyme and not to the active form, suggesting that the rapid inhibitory interactions between palmitoyl-CoA and glucokinase previously observed are specific. Parallel enzyme activity studies showed that in the time range of the column experiments (4-20 h), both the rat and pig enzyme were greatly inactivated (greater than 90%) in the presence of palmitoyl-CoA (15 microM) in the absence of glycerol. This slow inactivation is different from the immediate specific inhibition previously reported and depends on both enzyme and palmitoyl-CoA concentrations. The presence of up to 20% glycerol slowed this inactivation process. These results demonstrated that even below the critical micelle concentration, partial inactivation of glucokinase occurs in the presence of palmitoyl-CoA over a long period of time.

Acyl Coenzyme A↗
PubMed1989

Nerve growth factor in human amniotic and cerebrospinal fluid.

Nerve growth factor (NGF) is a polypeptide hormone involved in development of the sympathetic and central nervous systems. The detection and measurement of NGF in clinical samples would be useful in evaluating its role in various disease states. In this report, NGF activity and protein levels have been investigated in human amniotic fluid and cerebrospinal fluid samples. In amniotic fluid, NGF activity was found at levels ranging from less than 10 pM to nanomolar. The activity in all samples was blocked by polyclonal and monoclonal antibodies to mouse NGF. The finding of NGF in clinically obtainable samples raises the possibility of correlating NGF levels with a variety of disorders in which changes in NGF levels or activity have been implicated.

Adult↗
PubMed1988

Expression of the cDNA for mouse beta-nerve growth factor protein in Escherichia coli.

The cDNA coding for the mature beta-nerve growth factor (beta-NGF) has been cloned into a plasmid expression vector, pAS1, and expressed in Escherichia coli. The cDNA fragment in pAS1 is under the control of strong phage transcriptional and translational initiation elements that provide for regulated expression of cloned genes in E. coli. The protein, produced in bacteria at a level of about 0.0005-0.1% of cell protein, was purified by ammonium sulfate precipitation and ion exchange chromatography. The recombinant NGF was biologically active in the PC12 neurite outgrowth assay, and formed a band at Mr of about 11,000 to 12,000, when electrophoresed on sodium dodecyl sulfate-polyacrylamide gel and Western-blotted.

Animals↗
PubMed1988

Internalization of nerve growth factor by PC12 cells. A description of cellular pools.

Binding and internalization of nerve growth factor (NGF) by responsive cells is a complex process. We have incubated rat pheochromocytoma cells (PC12) with 125I-NGF at 37 degrees C and measured the association of ligand after removal of subsets of bound ligand by different methods. Chase with unlabeled NGF at either 4 or 37 degrees C, acid stripping, nonionic detergent stability, and combinations of these protocols were utilized. These variations of the binding assay were able to distinguish ligand bound to fast versus slow cell surface receptors, NGF bound to slow receptors at the cell surface versus cell interior, and soluble ligand versus cytoskeletally attached NGF. Quantitative and temporal relations among five cellular pools were defined. Experiments with the inhibitors chloroquine, cytochalasin B, and colchicine defined pools of NGF in terms of the route through the cell from the plasma membrane to the lysosome. Chloroquine caused accumulation of NGF only in the pool that was not associated with the cytoskeleton, implicating the involvement of this pool in supplying ligand to the lysosome. Results with cytochalasin B and colchicine suggest that both microfilaments and microtubules are involved in pathways leading to NGF degradation. A semiquantitative model for the movement of NGF through the cell is presented based on these observations.

Adrenal Gland Neoplasms↗
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↗