Two classes of receptors for '25I-labeled nerve growth factor in chick embryonic dorsal root neurons have been observed. One type is associated with the plasma membrane (or microsomal fraction) and can be completely solubilized by Triton X-100. These receptors display the nonsaturable binding isotherms and curvilinear Scatchard plots previously reported for nerve growth factor receptors in whole cells. The second class of binding sites is located in the nucleus, firmly bound to chromatin. These receptors are not solubilized by detergent, show saturable binding, and yield linear Scatchard plots of the type associated with a single class of binding sites of high affinity. The presence of the two receptor types suggests a bimodal mechanism of action for nerve growth factor. Nerve growth factor (NGF) is an insulin-like protein (1) that induces the morphologic and metabolic differentiation of sympathetic and sensory neurons in vertebrates (2). However, the responsiveness of these two tissues is quite different. Sensory neurons bind and are stimulated by NGF only during a relatively narrow period of embryonic development (2-4). In contrast, the sympathetic cells retain sensitivity, even in adult animals (5), although the nature of the response changes during different stages of development.The types of cellular processes effected by NGF are also variable. With low levels of hormone (about 1-10 ng), both sensory and sympathetic neurons produce the characteristic neurite outgrowth with accompanying general stimulation of anabolic metabolism and neurotubule polymerization (2, 6).At higher concentrations (about 1-10,tg), the specific induction of enzymes involved in nonadrenergic neurotransmitter synthesis is observed (7) as well as such effects as regeneration of adrenergic fibers in brain (8), inhibition of the biosynthesis of mucopolysaccharide in chrondrocytes (9), and stimulation of the temporal conversion of cell surface adhesive specificity in embryonic optic tectal cells (10).The responses associated with the low concentrations of NGF appear to be mediated through receptors bound to cell surface membranes. NGF covalently linked to Sepharose beads under conditions that prevent the release of soluble NGF produces a stimulation of neurite outgrowth from embryonic dorsal root sensory ganglia indistinguishable morphologically from that of the unbound hormone (11). By using 125I-labeled NGF (125I-NGF), these receptors have been shown to possess an affinity of approximately 1010 liters/mol (12-14), corresponding to the concentration range required for activity. However, the specific binding is a nonsaturable process with lower affinity (about 2 X 107 liters/mol) of binding sites at higher concentrations of labeled NGF (12). The range of affinities observed results from negatively cooperative interactions among a single class of receptor molecules, analogous to that observed for in- sulin receptors (15), rather than from receptor heterogeneity.Recently, it has also been demonstrated that NGF can be taken up by th...
Two forms of rat brain cytosolic diacylglycerol kinase (EC2.7.1.107) were separated by heparin-agarose column chromatography. These forms, designated DGK-I and DGK-II, were not interconvertible as determined by rechromatography. DGK-I and DGK-II had respective molecular masses of 88 and 180 kDa, as measured by Sepharose 6B chromatography. Both forms preferred diacylglycerol over monoacylglycerol and were insensitive to R59022. DGK-II, but not DGK-I, was activated by an activator substance prepared from chicken egg yolk. DGK-II was activated by a rat brain cytosolic activator and was exclusively sensitive to 5'-AMP-mediated inactivation. Further studies revealed that these two forms had the following distinct characteristics: (a) substrate specificity, (b) inhibition by heparin, (c) sensitivity to lysine-containing polyamino acids, and (d) responses to different phospholipids. In general, DGK-II was more responsive to various inhibitors and activators, making it a prime candidate for a regulatable enzyme.
Saturable and specific binding sites for 5-[3H]hydroxytryptamine (5-HT, serotonin) characterized by a KD of 3.5-4.5 nM were detected in the chick embryo brain and were shown to develop linearly as a function of age, weight, and protein content. Saturation and displacement studies using unlabeled 5-HT as the displacing ligand suggested a single population of binding sites. However, displacement studies using 5-methoxytryptamine, lysergic acid diethylamide (LSD), 2-bromo-lysergic acid diethylamide (BOL), methysergide, and spiperone as competing ligands suggested the existence of subclasses of [3H]5-HT binding sites because the Hill coefficients were less than unity. When compared with the reported [3H]5-HT binding sites (5-HT1) in the rat forebrain, the IC50 values of the competing ligands were similar. However, the Hill coefficients for LSD and methysergide were less than unity which suggested that the [3H]5-HT binding sites in the chick embryo brain may be more similar to those found in rat spinal cord than rat forebrain. To study [3H]5-HT binding site regulation and development, various serotonergic compounds were injected into the chorioallantoic fluid of the eggs at different times during embryonic development. Multiple pretreatments with d,l-5-hydroxytryptophan, 5-HT, or BOL were found to have no significant effects on either the affinity (KD) or number (Bmax) of specific [3H]5-HT binding sites. Multiple pretreatments with p-chlorophenylalanine were found to increase the Bmax of specific [3H]5-HT binding by 23% (p less than 0.01) whereas multiple pretreatments with LSD were found to decrease the Bmax of specific binding by 45% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
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