PubMed Health⌕ Search

Biomedical subjects

W A Frazier

Publications and source records attributed to W A Frazier.

At least 91 records · Page 5Linked to original sources
PubMed1976

Subcellular localization of nerve growth factor receptors. Thirteen-day chick embryo brain.

The subcellular location of the specific binding sites of nerve growth factor (NGF) as judged by binding of the 125I-labeled protein in 13-day chick embryo brain, has been examined. The homogenized tissue was separated into four fractions, P1, P2, P3, and S, by differential centrifugation. Fraction P2, which contained the majority of the specific 125I-NGF binding, was further separated by discontinuous sucrose density gradient centrifugation into three fractions. Fraction B contains many synaptosome-like structures, which, when derived from adult brain, result from the shearing off and resealing of synaptic terminals. This fraction contained 65% of the specific 125I-NGF binding of P2. Following hypoosmotic lysis by water, Fraction B was separated into seven fractions, O, D, E, F, G, H, and I, by discontinuous sucrose density gradient centrifugation. The specific 125I-NGF binding was localized with the denser fractions, G, H, and I, with about a 10-fold purification as compared to the original homogenate. However, only 65% of the binding of Fraction B was found in the sum of the tertiary fractions, indicating that some loss of specific binding accompanied the lysis. By means of marker enzymes and macromolecules, as well as electron microscopy, it was determined that the distribution of cellular components of embryonic tissue in this fractionation technique is very similar to that observed for adult brain tissue. Thus, the properties of the NGF receptors determined in whole brain, which are remarkably similar to those found in peripheral neurons, are the properties of the receptors that appear to be located in the developing synaptosomal structures.

Animals↗
PubMed1976

Subcellular localization of nerve growth factor receptors. Developmental correlations in chick embryo brain.

The subcellular distribution of receptors for nerve growth factor (NGF), as assayed by specific binding of the 125I-labeled protein, during the embryonic development of chick brain has been examined. Utilizing differential centrifugation and discontinuous sucrose density gradient centrifugation, it was observed that the majority of the specific NGF receptors are localized in the crude (P2) and purified (B) synaptosomal fractions at all stages of brain development examined. However, during early stages (day 6 to 8) a significant portion of the receptors are also found in the P1 fraction, consistent with their location on dense subcellular structures. Significant 125I-NGF binding is observed in this period of development before the appearance of neurochemical markers (choline acetyltransferase, dopamine-beta-hydroxylase, and 125I-alpha-bungarotoxin binding) which are associated with synaptosomes. The latter stages of development, particularly days 13 to 18, show parallel rapid increases in all neurochemical markers and 125I-NGF binding consistent with the association of nearly all late appearing NGF receptors with synaptosomes. These two apparent classes of NGF receptors may have distinct functional roles in the embryonic development of the chick brain.

Animals↗
PubMed1976

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.

Amino Acid Sequence↗
PubMed1976

Comparison of the nerve growth factor proteins from cobra venom (Naja naja) and mouse submaxillary gland.

The nerve growth factors (NGF's) isolated from cobra venom (Naja naja) and mouse submaxillary gland are closely related proteins. They are structurally similar in that about 60% of their amino acid residues are identical (Hogue-Angeletti, R. A., Frazier, W. A., Jacobs, J. W., Niall, H.D., and Bradshaw, R. A. (1976), Biochemistry, preceding paper in this issue). They are functionally similar in that they both elicit maximum neurite outgrowth from chick embryonic dorsal root ganglia at the same protein concentration. However, the extent of the response is not as great with Naja naja NGF. The cobra NGF has an affinity close to that of mouse NGF for a major proportion of the specific NGF receptors on cells from dissociated embryonic dorsal root ganglia. Despite these similarities there are differences which can be detected between the two proteins. High concentrations of Naja naja NGF will not displace approximately 20% of the binding of mouse NGF to specific NGF receptors. Moreover, Naja naja NGF shows limited cross-reactivity with antiserum to mouse NGF in a competition radioimmunoassay, consistent with the extent of its amino acid sequence homology with mouse NGF. Naja naja NGF does not interact with the alpha- and gamma-subunits of 7S NGF to form a high molecular weight complex. In this behavior it resembles a modified form of mouse NGF which, like Naja naja NGF, lacks COOH-terminal arginine residues.

Amino Acids↗
PubMed1975

Purification and comparison of two developmentally regulated lectins from Dictyostelium discoideum. Discoidin I and II.

When cells of the cellular slime mold Dictyostelium discoideum differentiate from a nonsocial amoeboid form to a cohesive, aggregating form, they synthesize a lectin-like protein called discoidin, which is present on the cell surface. It is now reported that discoidin consists of two distinct lectins, designated discoidin I and discoidin II, which, although similar in some respects, differ in their electrophoretic mobilities, isoelectric points, subunit molecular weights, amino acid compositions, tryptic peptide maps, the erythrocyte species which they agglutinate, and the sensitivity of their agglutination activity to inhibition by monosaccharides. Furthermore, discoidins I and II differ in their developmental regulation as evidenced by the distinct time courses of their appearance during differentiation.

Amino Acids↗
PubMed1973

Interaction of nerve growth factor with surface membranes: biological competence of insolubilized nerve growth factor.

Nerve growth factor was insolubilized by covalent attachment to Sepharose beads. Nerve growth factor-Sepharose was biologically active in both the neurite outgrowth assay for nerve growth factor and in preserving responsive neurons in vitro. Modification of the bioassay to detect solubilized activity of nerve growth factor and histological examination of ganglia treated with nerve growth factor-Sepharose revealed that nerve growth factor-Sepharose prepared by reaction in 6 M guanidine hydrochloride released negligible amounts of solubilized nerve growth factor activity. These observations extend the previously noted correlations on the structure and function of nerve growth factor and insulin to include the primary action of these two proteins. Thus nerve growth factor, like insulin, appears to express its biological activity by first binding to a receptor on the surface membrane of responsive cells.

Animals↗
PubMed1973

Mechanism of action of nerve growth factor and cyclic AMP on neurite outgrowth in embryonic chick sensory ganglia: demonstration of independent pathways of stimulation.

The suggested role of adenosine 3':5'-cyclic monophosphate as the "second messenger" in the neurite outgrowth from chick embryonic sensory ganglia mediated by nerve growth factor was examined. Although N(6),O(2)-dibutyryl adenosine 3':5'-cyclic monophosphate induces fiber outgrowth at concentrations of 1-5 mM, this response is morphologically distinct from that produced by nerve growth factor, is pH dependent, is mimicked by sodium butyrate, and does not occur in sympathetic ganglia. In addition, nerve growth factor does not alter the amounts of intracellular cyclic AMP during incubations up to 24 hr and does not stimulate adenylate cyclase in broken-cell preparations. Addition of theophylline, an inhibitor of phosphodiesterase, causes increases in intracellular levels of cyclic AMP but does not affect fiber outgrowth. These observations indicate that the nerve growth factor response is not mediated through cyclic AMp and that stimulation of sensory ganglia by exogenous cyclic AMP derivatives is probably of limited physiological significance. These findings are also compatible with the developing hypothesis, based on structural similarities, that nerve growth factor and insulin exert their effects on their respective responsive tissues by related mechanisms.

Adenylyl Cyclases↗