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H Gainer

Publications and source records attributed to H Gainer.

At least 109 records · Page 6Linked to original sources
PubMed1983

Onset of neurophysin self-association upon neurophysin/neuropeptide hormone precursor biosynthesis.

The potential of the common biosynthetic precursor of neurophysin and neuropeptide hormones to self-associate has been assessed by quantitative affinity chromatographic analysis. The precursor form, with the hormone sequence in the amino terminal region and assumed able to interact intramolecularly with the hormone binding site of the neurophysin domain of the folded precursor, exhibits an affinity for neurophysin-agarose which is intermediate between those of unliganded neurophysin and non-covalently hormone-liganded neurophysin. The results lead to a prediction that neurophysin self-association is established upon precursor synthesis and prior to limited proteolysis of the precursor to release mature neurophysin and hormone components. Such self-association could play a role in packaging of the precursor into secretory granules and in regulating subsequent precursor processing events within the granules.

Animals↗
PubMed1983

Dynorphin-A-(1-8) is contained within vasopressin neurosecretory vesicles in rat pituitary.

Dynorphin-A-(1-8), an opioid peptide widely distributed in the rat central nervous system, is present in vasopressin-containing neurosecretory cells terminating in the neural lobe of the pituitary. Electron microscopic immunocytochemistry reveals that dynorphin-A-(1-8) is contained within the same neurosecretory vesicles as vasopressin and vasopressin-associated neurophysin in the neural lobe of the rat. The results indicate that dynorphin may be released in the pituitary concomitantly with vasopressin during the antidiuretic response.

Animals↗
PubMed1983

Calcium-dependent 4-aminopyridine stimulation of protein phosphorylation in squid optic lobe synaptosomes.

When intact synaptosomes were incubated with [gamma-32P]ATP, maximal protein phosphorylation was attained 2 min after the start of incubation. Protein phosphorylation under basal conditions was dependent on external Ca2+, and the dominant peak of phosphorylation was a 50-kd protein. Incubation of intact synaptosomes in the presence of 3-6 mM 4-aminopyridine (4-AP) caused a markedly enhanced phosphorylation of high molecular weight proteins of 90, 100, 130, and 180 kd, with no increase in the 50 or 38 kd proteins. This effect of 4-AP was dependent on external calcium ions in the incubation medium. The 4-AP effect on the high molecular weight proteins was also found in synaptosomal plasma membranes isolated from the synaptosomes. Tetraethylammonium (TEA) ions did not produce this enhancement of phosphorylation.

4-Aminopyridine↗
PubMed1983

Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors.

The squid giant axon and extruded axoplasm from the giant axon were used to study the capacity of axoplasm for phospholipid synthesis. Extruded axoplasm, suspended in chemically defined media, catalyzed the synthesis of phospholipids from all of the precursors tested. 32P-Labeled inorganic phosphate and gamma-labeled ATP were actively incorporated into phosphatidylinositol phosphate, while [2-3H]myo-inositol and L-[3H(G)]serine were actively incorporated into phosphatidylinositol and phosphatidylserine, respectively. Though less well utilized. [2-3H]glycerol was incorporated into phosphatidic acid, phosphatidylinositol, and triglyceride, and methyl-3H]choline and [1-3H]ethanolamine were incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Isolated squid giant axons were incubated in artificial seawater containing the above precursors. The axoplasm was extruded following the incubations. Although most of the product lipids were recovered in the sheath (composed of cortical axoplasm, axolemma, and surrounding satellite cells), significant amounts (4-20%) were present in the extruded axoplasm. With tritiated choline and myo-inositol, the major labeled phospholipids found in both the extruded axoplasm and the sheath were phosphatidylcholine and phosphatidylinositol, respectively. With both glycerol and phosphate, phosphatidylethanolamine was a major labeled lipid in both axoplasm and sheath. These findings demonstrate that all classes of phospholipids are formed by endogenous synthetic enzymes in axoplasm. In addition, we feel that the different patterns of incorporation by intact axons and extruded axoplasm indicate that surrounding sheath cells contribute lipids to axoplasm. A comprehensive picture of axonal lipid metabolism should include axoplasmic synthesis and glial-axon transfer as pathways complementing the axonal transport of perikaryally formed lipids.

Animals↗
PubMed1982

Processing of normal and non-glycosylated forms of toad pro-opiocortin by rat intermediate (pituitary) lobe pro-opiocortin converting enzyme activity.

The influence of glycosylation of a prohormone, pro-opiocortin, on its processing by intermediate (pituitary) lobe converting enzyme activity in vitro was studied. [3H]-arginine-labeled glycosylated and non-glycosylated pro-opiocortins were isolated from untreated, and tunicamycin treated toad neurointermediate lobes, respectively, after pulse-labeling in [3H]-arginine containing incubation media. These labeled precursors were then incubated at 37 degrees C in the presence of pro-opiocortin converting enzyme activity derived from rat intermediate lobe (pituitary) secretory granule lysates. The rates of conversion of the glycosylated and nonglycosylated pro-opiocortins to smaller peptide products, in vitro, were similar. Analysis of the peptide products by immunoprecipitation with ACTH and beta-endorphin antisera, and subsequent electrophoresis on acid-urea gels, indicate a comparable processing in vitro of the two forms of pro-opiocortin substrate. The only difference was that the normally glycosylated peptide products derived from glycosylated pro-opiocortin (i.e., 13K ACTH, 21K ACTH, and the 16K glycopeptide) differed in their gel electrophoretic mobilities from their counterparts derived from nonglycosylated prohormone, in a manner consistent with the absence of carbohydrate on the latter's peptides. These data show that glycosylation of the prohormone does not influence its processing in vitro by the converting enzyme activity.

Animals↗
PubMed1982

Characterization of pro-opiocortin-converting activity in purified secretory granules from rat pituitary neurointermediate lobe.

Lysates of secretory granules from rat pituitary neurointermediate lobes were incubated with [3H]arginine- or [3H]phenylalanine-labeled toad pro-opiocortin. The processed products formed were identified by immunoprecipitation with adrenocorticotropin (ACTH) and endorphin antisera and by migration behavior on acid/urea/polyacrylamide gels. Pro-opiocortin was cleaved by the proteolytic activity in the secretory granule fraction to approximately 21,000 Mr ACTH, approximately 13,000 Mr ACTH, alpha-melanotropin, 16,000 Mr NH2-terminal glycopeptide, beta-lipotropin, and an endorphin-related peptide. Characterization of this pro-opiocortin-converting activity shows that it (i) is present in membrane and soluble fractions of the granule lysates, (ii) has a pH optimum of 5.0, (iii) appears to cleave at pairs of basic amino acid residues in the precursor, and (iv) is inhibited by leupeptin, pepstatin A, and p-chloromercuribenzoate but not diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, or EDTA. These inhibitor studies suggest that the converting-enzyme activity is due to an acid thiol, arginyl protease, distinct from any known cathepsin B-like activity.

Adrenocorticotropic Hormone↗
PubMed1982

Proopiocortin-converting enzyme activity in bovine neurosecretory granules.

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.

Adrenocorticotropic Hormone↗
PubMed1982

Distribution of calcium-activated protease activity and endogenous substrates in the squid nervous system.

Calcium-activated protease (CAP) activity was studied in various neural tissues of the squid using endogenous (neurofilament protein) and exogenous ([14C]casein) substrate assays. Both assays demonstrated a significant CAP activity in perikarya from stellate ganglia, in axoplasm extruded from the giant axon, and in squid retinal fibers. The endogenous protein substrates in the perikarya and axons were 60,000 and 200,000 daltons, respectively. The Km for the CAP degradation of [14C] casein in axoplasm was about 2 x10-6 m. In contrast, both assays detected no CAP activity nor endogenous substrate in nerve terminals (synaptosomes from squid optic lobe). The absence of both CAP activity and endogenous substrate in nerve endings suggests that the axonal neurofilaments are degraded by CAP at the axon-nerve ending junction, followed by an autoinactivation of the CAP. Consistent with this hypothesis is that exposure of axoplasmic CAP to calcium leads to a rapid degradation of axonal neurofilament protein (t 1/2 less than 2 min) and a slower inactivation of the CAP (t 1/2 = 90 min). Axonal CAP requires a relatively high concentration of CA2+ sensitivity form of CAP found in other tissues.

Animals↗
PubMed1981

Time course of appearance and release of [35S]cysteine labelled neurophysins and peptides in the neurohypophysis.

[35S]cysteine injected adjacent to the supraoptic nuclei (SON) of rats is rapidly incorporated into two macromolecular (both about 20,000 Daltons) common precursors of arginine vasopressin (AVP) and its associated neurophysin, and oxytocin (OT) and its neurophysin. Conversion of the larger precursor proteins to the smaller peptides appears to occur intragranularly during axonal transport to the neurohypophysis. The labelled products of this conversion (neurophysin, AVP, and OT) are released, in a Ca2+-dependent manner, from the posterior pituitary in response to depolarization by veratridine. Both the rates of biosynthesis and of processing of the precursors are greatly increased by increased functional activity (i.e. secretion) of the hypothalamo--neurohypophysial system.

Animals↗
PubMed1981

Multiple-rate components of axonally transported proteins in the hypothalamo-neurohypophysial system of the rat.

The transport of labeled proteins from the hypothalamus to the neurohypophysis following 35S-methionine injection into the rat supraoptic nucleus was studied using a unique approach adapted for the study of short-axon systems. Multiple-rate components to those found in other neuronal systems were demonstrated. Neurosecretory vesicle-containing proteins (e.g., neurophysins) were transported at fast rates (greater than 120 mm/day), whereas the cytoskeletal protein, actin, moved principally in the slow component of transport. Two-dimensional gel electrophoresis was used to analyze the diverse patterns of labeled proteins found in the various rate components of axonal transport in this system.

Actins↗
PubMed1981

Membrane-associated cytoskeletal proteins in squid giant axons.

Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.

Animals↗