RESUMEN
Phorbol myristate acetate is known to reproduce the stimulated oxidative activities characteristic of phagocytosis and its initial action is on the cell membrane. In the present study the effect of phorbol myristate acetate on the metabolism of the fatty acyl groups of granulocyte phospholipids was examined and compared with that of phagocytic stimuli. Phorbol myristate acetate stimulated the labeling of phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol by [1-14C]palmitic acid but not by [U-14C]glycerol, whereas starch granules selectively increased the labeling of phosphatidylinositol by both radioactive tracers. Labeled palmitic acid was found at both sn-1- and sn-2-positions of phospholipids and more radioactivity was recovered from the 2-position. The radioactivity at both positions was enhanced in stimulated cells. These data suggest that phorbol myristate acetate increased palmitic acid incorporation into glycerophospholipids by increasing the acylation of the lyso derivatives and that starch granules enhanced the formation of phosphatidylinositol via de novo synthesis and acylation of the lyso derivative as well. Both phorbol myristate acetate and starch granules selectively augmented the incorporation of [1-14C]arachidonic acid into phosphatidylinositol which exhibited the highest specific radioactivity among the phospholipids in control and in stimulated cells. The possible significance of the increased incorporation of arachidonic acid into phosphatidylinositol is discussed.
Asunto(s)
Granulocitos/metabolismo , Forboles/farmacología , Fosfolípidos/sangre , Acetato de Tetradecanoilforbol/farmacología , Animales , Ácido Araquidónico , Ácidos Araquidónicos/sangre , Ácidos Grasos/metabolismo , Glicerol/sangre , Granulocitos/efectos de los fármacos , Cobayas , Cinética , Ácido Palmítico , Ácidos Palmíticos/sangreRESUMEN
A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.
Asunto(s)
Neutrófilos/metabolismo , Fagocitosis , Fosfatidilserinas/biosíntesis , Serina/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Cobayas , Fosfolípidos/biosíntesisRESUMEN
The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2+ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca2+, but not Mg2+, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca2+, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(beta-amino-ethylether)-N,N'-tetraacetic acid. ATP radiophosphorus activity was also enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca2+ fluxes and perturbation of cellular Ca2+ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.
Asunto(s)
Antibacterianos/farmacología , Calcimicina/farmacología , Neutrófilos/metabolismo , Fosfatos/sangre , Fosfolípidos/biosíntesis , Animales , Calcio/farmacología , Glucuronidasa/sangre , Cobayas , Cinética , L-Lactato Deshidrogenasa/sangre , Magnesio/farmacología , Neutrófilos/efectos de los fármacos , Fosfolípidos/sangreRESUMEN
The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.
Asunto(s)
Antibacterianos/farmacología , Calcimicina/farmacología , Glicéridos/sangre , Glicerol/sangre , Ácidos Linoleicos/sangre , Neutrófilos/metabolismo , Ácidos Palmíticos/sangre , Fosfolípidos/sangre , Animales , Cobayas , Cinética , Neutrófilos/efectos de los fármacosRESUMEN
The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.
Asunto(s)
Adenosina Trifosfato/farmacología , Bucladesina/farmacología , Glucuronidasa/metabolismo , Lisosomas/enzimología , Neutrófilos/enzimología , Fosfolípidos/metabolismo , Animales , Citocalasina B , Cobayas , L-Lactato Deshidrogenasa/metabolismo , Lisosomas/efectos de los fármacos , Neutrófilos/efectos de los fármacosRESUMEN
Previous studies showed that interleukin-8 (IL-8) stimulates phospholipase D hydrolysis of phosphatidylcholine to generate phosphatidic acid in human neutrophils. Phosphatidylcholine in these cells contains diacyl, alkylacyl and alkenylacyl subclasses. No studies have examined phospholipase D hydrolysis of the three subclasses of phosphatidylcholine in interleukin-8-stimulated neutrophils. We used a non-radioactive but very sensitive method to assess the relative distribution of the subclasses in phosphatidylethanol, which is derived from phospholipase D activity in ethanol-exposed neutrophils. We present evidence that the relative abundance of diacyl and alkylacyl subclasses in phosphatidylethanol is similar to that in phosphatidylcholine. Alkenylacyl subclass was also detectable in the phosphatidylethanol fraction, albeit as a minor subclass. Our findings suggest that phospholipase D catalyses the hydrolysis of diacyl, alkylacyl and alkenylacyl subclasses of phosphatidylcholine in neutrophils upon IL-8 stimulation.
Asunto(s)
Glicerofosfolípidos/biosíntesis , Interleucina-8/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ácidos Fosfatidicos/biosíntesis , Cromatografía en Capa Delgada , Citocalasina B/farmacología , Diglicéridos/análisis , Etanol/farmacología , Humanos , Ácidos Fosfatidicos/análisis , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Transducción de Señal , Factores de TiempoRESUMEN
Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.
Asunto(s)
Ácido Aspártico , Somatostatina/química , Succinimidas/análisis , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión/métodos , Focalización Isoeléctrica , Espectrometría de Masas , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Somatostatina/aislamiento & purificación , Porcinos , TripsinaRESUMEN
In the present study, a simple tripeptide alkylamine, syndyphalin 33 (SD33, Tyr-DMet (O)-Gly-methylphenethylamide) was shown to stimulate somatotropin (GH) secretion in sheep, hogs and rats following peripheral administration. Intravenous (i.v.) administration of SD33 at doses of 0.05, 0.1 and 0.2 mumol/kg stimulated a significant increase in circulating GH levels in sheep within 5 minutes post-injection. This response was not attenuated following repeated i.v. injections of SD33 (0.05 /mmol/kg) administered at 2 hour intervals. In addition, plasma GH levels were significantly stimulated following either subcutaneous (s.c.) or oral administration of SD33 in hogs and rats. Subcutaneous administration of SD33 at doses of 0.5, 1.0 and 2.0 mumol/kg stimulated a significant increase in plasma GH concentrations within 30 minutes of injection in both species. Oral administration of SD33 at 1.0, 10 or 100 mumol/kg in rats resulted in a significant elevation in plasma GH levels which peaked at 30 minutes post-gavage. In the pig, circulating GH levels were significantly increased within 30 minutes post-ingestion and remained elevated for at least 2 hours at the 2.0 mumol/kg dose level. The ability of naloxone to block SD33-stimulated GH secretion suggests that this peptide acts via mu opiate receptors.
Asunto(s)
Hormona del Crecimiento/metabolismo , Oligopéptidos/farmacología , Administración Oral , Animales , Inyecciones Intravenosas , Inyecciones Subcutáneas , Masculino , Naloxona/farmacología , Oligopéptidos/antagonistas & inhibidores , Hipófisis/efectos de los fármacos , Ratas , Ratas Endogámicas , Ovinos , PorcinosRESUMEN
Central administration of NPY (1-36) potently increases food intake and it has been hypothesized that biological activities of NPY are related to its ability to form an alpha-helix, represented by the fragment NPY (14-31). In this experiment the necessity of N-terminal fragments for increasing food intake was evaluated. Two-h fasted male rats were administered 0, 0.2, 1.0 or 5.0 nmol NPY (1-36) or NPY fragments in 5 microliters saline ICV and intake of lab chow pellets was measured for 22 h. Fragments containing all or part of the polyproline-like helix [NPY (1-8)] antiparallel to the alpha-helix dose-relatedly increased food intake for 4 hours after injection. Five nmol NPY (1-36) and NPY (2-36) increased 4-hour food intake 486 and 219%, respectively (p less than 0.05). Fragments excluding the first 8 amino acids but including all of the alpha-helix also increased food intake, but the response was much reduced. Five nmol NPY (9-36) and NPY (14-36) increased 4-hour food intake 128% (p = 0.02) and 62% (NS), respectively. When all or part of the alpha-helix was excluded, no activity was detected, i.e., NPY (21-36) and NPY (32-36). Substitution of dPro for lPro in position 2 increased potency but not efficacy of NPY since food intake was increased at the 0.2 and 1.0 but not 5.0 nmol doses and the percent increase was not more than to 5 nmol NPY (1-36). Thus the maximum food intake response to NPY requires both C-terminal and N-terminal fragments as well as the alpha-helix.
Asunto(s)
Encéfalo/fisiología , Conducta Alimentaria/fisiología , Neuropéptido Y/fisiología , Fragmentos de Péptidos/fisiología , Secuencia de Aminoácidos/genética , Secuencia de Aminoácidos/fisiología , Animales , Masculino , Datos de Secuencia Molecular , Neuropéptido Y/genética , Fragmentos de Péptidos/genética , Mapeo Peptídico , Ratas , Ratas Endogámicas , PorcinosRESUMEN
This study extended the earlier finding that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) promotes arachidonic acid incorporation into neutrophil phosphatidylinositol (PI) and phosphatidylcholine (PC). In the present study the effect of PAF on fatty acid uptake by human neutrophils and the incorporation of extracellular linoleic acid and palmitic acid into phospholipids were investigated. Incubation of 10(-7) M PAF with neutrophils and radiolabeled arachidonic acid or linoleic acid or palmitic acid for 1-10 min resulted in an increased rate of loss of label from the incubation medium. PAF stimulated the incorporation of linoleic acid and palmitic acid most significantly into PI and PC. The magnitude of stimulation was greater in PI than in PC for the incorporation of linoleic acid, and vice versa for the incorporation of palmitic acid. The positional distribution of linoleic acid and palmitic acid in PI and PC and the mass of these phospholipids were not altered in PAF-stimulated neutrophils. An increased incorporation of all three fatty acids into both diacyl and alkylacyl species of PC was demonstrated after a two minute incubation of cells with PAF. While more radioactivity was recovered in the diacyl species, the magnitude of increase of radioactivity in the alkylacyl species was more pronounced than that in the diacyl species of PC. These results suggest that both increased fatty acid uptake and increased available lysophospholipids may be contributory to the increased phospholipid acylation induced by PAF.
Asunto(s)
Neutrófilos/metabolismo , Fosfolípidos/metabolismo , Factor de Activación Plaquetaria/fisiología , Ácidos Araquidónicos/metabolismo , Humanos , Hidrólisis , Cinética , Ácidos Linoleicos/metabolismo , Ácidos Palmíticos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositoles/metabolismoRESUMEN
Docosahexaenoic acid was not only acylated into phospholipids but also into triacylglycerols by intact human neutrophils. The distribution of radiolabeled docosahexaenoic acid among individual phospholipids was dependent on the incubation time. [1-14C]Docosahexaenoic acid at all concentrations (1 to 8 microM) was acylated mainly into phosphatidic acid after 1-2 min incubation, and the radioactivity of phosphatidic acid started to decline after a longer period of incubation, suggesting the participation of docosahexaenoyl-phosphatidic acid in the synthesis of other glycerolipids. It was acylated primarily into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) after a 2-hr incubation. The labeled phosphatidic acid may be rapidly deacylated and the 22:6(n-3) moiety is then reacylated into other lysophospholipids. The low levels of [14C]22:6(n-3) in 1,2-diacylglycerol suggest that the deacylation-reacylation cycle may be a major pathway in the formation of [14C]22:6(n-3)-PC and -PE in intact neutrophils. This n-3 fatty acid was a relatively poor substrate for acylation into phosphatidylinositol as compared to arachidonic acid and eicosapentaenoic acid. However, the patterns of distribution of all three polyunsaturated fatty acids among the diacyl- and ether-linked class compositions of PC and PE were similar. These data suggest the potential of increasing the content of docosahexaenoic acid of membrane lipids in neutrophils by dietary supplement of this fatty acid.
Asunto(s)
Ácidos Grasos Insaturados/sangre , Neutrófilos/metabolismo , Fosfolípidos/sangre , Radioisótopos de Carbono , Ácidos Docosahexaenoicos , Humanos , Técnicas In Vitro , Cinética , Fosfolípidos/biosíntesis , Técnica de Dilución de Radioisótopos , Triglicéridos/biosíntesis , Triglicéridos/sangreRESUMEN
The present study showed that platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF), but not lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) rapidly (within 15 sec) stimulated the incorporation of both [1-14C]arachidonate and [1-14C]docosahexaenoate into phosphatidylinositol (PI) and phosphatidylcholine (PC) in human neutrophils. Concomitantly, it inhibited the formation of labeled phosphatidic acid from both fatty acids. The magnitude of stimulation (percentage of control) was greater in PI than in PC for the incorporation of arachidonate and vice versa for the incorporation of docosahexaenoate. It reached a maximum at 10(-7) M and started to decline at 10(-6) M. Extracellular Ca2+ was not essential for the action of PAF on phospholipid acylation. The distribution of labeled arachidonate in the molecular species of PC was not altered by PAF after 1 min incubation, suggesting that the increased formation of arachidonyl-PC during the early stage of neutrophil-PAF interaction was not originated from the added PAF. No measurable changes in the mass of each phospholipid were detected in neutrophils challenged by PAF from 15 sec to 2 min. The data suggest that the increased incorporation of extracellular fatty acids into PI and PC elicited by PAF may be secondary to increased deacylation of these phospholipids, and the magnitude of stimulation reflects the specificity of acyltransferase catalyzing the acylation of lysoPI and lysoPC by fatty acyl-CoA.
Asunto(s)
Neutrófilos/metabolismo , Fosfolípidos/sangre , Factor de Activación Plaquetaria/farmacología , Acilación , Ácido Araquidónico , Ácidos Araquidónicos/sangre , Calcio/farmacología , Humanos , Neutrófilos/efectos de los fármacos , Fosfatidilcolinas/sangre , Fosfatidilinositoles/sangreRESUMEN
The present study demonstrated that the patterns of the incorporation of [1-14C] arachidonic acid and [1-14C] eicosapentaenoic acid into individual phospholipids by polymorphonuclear leukocytes were similar. However, human leukocytes exhibited higher activity than guinea pig peritoneal leukocytes in the formation of arachidonoyl- and eicosapentaenoyl-phosphatidic acid. Cells from both origins showed a decrease of label in phosphatidylcholine accompanied by an increase of label in phosphatidylethanolamine after a longer period (30-120 min) of incubation, suggesting that part of the arachidonoyl or eicosapentaenoyl moiety in phosphatidylethanolamine may be derived from that of phosphatidylcholine. The observed difference between human cells and elicited cells in the timecourse of the incorporation of both fatty acids into phosphatidylcholine and phosphatidylethanolamine appears to be due to different contents of the diacyl and ether-linked class compositions of these phospholipids in cells from different origins. Both labeled fatty acids were incorporated more rapidly into the diacyl-linked class, but were retained to a greater extent in alkylacyl-phosphatidylcholine and alkenylacyl-phosphatidylethanolamine. The data suggest that, in addition to alkylacylphosphatidylcholine and phosphatidylinositol, alkenylacyl-phosphatidylethanolamine may be an important endogenous source of arachidonic acid and eicosapentaenoic acid in stimulated human leukocytes.
Asunto(s)
Ácidos Araquidónicos/sangre , Ácidos Grasos Insaturados/sangre , Neutrófilos/metabolismo , Fosfolípidos/sangre , Animales , Ácido Araquidónico , Líquido Ascítico , Ácido Eicosapentaenoico , Cobayas , Humanos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Factores de TiempoRESUMEN
The signal transduction pathway of leukotriene B4 involves phospholipase D activation in cytochalasin B-primed neutrophils, but leukotriene B4 stimulation of increased phosphatidic acid mass in neutrophils has not been demonstrated. Employing the NIH Image program, we have examined the effect of leukotriene B4 on phosphatidic acid mass in human neutrophils incubated with or without cytochalasin B. Our results show that 0.15 microM leukotriene B4 without cytochalasin B was capable of increasing phosphatidic acid mass in neutrophils by 2-fold after 5 s, 2.5-fold after 1 min, and 2-fold after 5 min incubation. Leukotriene B3, leukotriene B4, and leukotriene B5 were equipotent stimuli for phosphatidic acid mass elevation. Leukotriene B4 induced phosphatidylethanol formation at the expense of phosphatidic acid in cells preincubated with 0.25-1% ethanol, indicating phospholipase D activation. Cytochalasin B enhanced leukotriene B4 stimulation of phosphatidic acid mass elevation and phosphatidylethanol formation. There were no measurable changes in 1,2-diglyceride mass after 5 s, but a 1.7-fold increase occurred after 1 min and declined thereafter. Leukotriene B4 stimulation of [3H]glycerol incorporation into phosphatidic acid, diglyceride and phosphatidylinositol was detectable after a 1-min incubation, suggesting increased de novo synthesis of these lipids. These results suggest that leukotriene B4 stimulation of phospholipase D activity contributes to part of the early increased phosphatidic acid mass and that combined actions of stimulated phospholipases C and D, and de novo phosphatidic acid synthesis contribute to the total increased phosphatidic acid mass.
Asunto(s)
Leucotrieno B4/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ácidos Fosfatidicos/metabolismo , Citocalasina B/farmacología , Diglicéridos/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Técnicas In Vitro , Leucotrieno B4/análogos & derivados , Ácidos Fosfatidicos/biosíntesis , Fosfolipasa D/metabolismo , Transducción de SeñalRESUMEN
The present study was undertaken to test the hypothesis that leukotriene B4 (LTB4) may promote extracellular fatty acid incorporation into neutrophil choline glycerophospholipids (PC) to replenish phospholipids after deacylation. Incubation of human neutrophils with LTB4 (1.5 to 150 nM) for 1 to 5 min resulted in increased fatty acid incorporation into phosphatidylinositol (PI), diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and alkylacyl-GPC. The magnitude of stimulation (percentage of control) of fatty acid incorporation appears to reflect increased activity of the acyltransferase catalyzing acylation of the respective lysophospholipids. LTB4 stimulation of arachidonic acid incorporation into PI was greater than into PC, whereas the stimulation of palmitic acid incorporation into PC was greater than into PI. LTB4 stimulated phosphatidic acid labeling by palmitic acid but not by arachidonic acid. LTB4 and 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (cPAF) exhibited a similar stimulatory effect on fatty acid incorporation into the PC fraction. Phosphate analysis could not detect changes in the mass of PI or of PC in neutrophils exposed to LTB4 or cPAF. The results suggest that increased fatty acid incorporation into phospholipids in LTB4-activated neutrophils reflects activation of phospholipase A2 and acyltransferases as well as of de novo phospholipid synthesis.
Asunto(s)
Ácidos Araquidónicos/sangre , Leucotrieno B4/farmacología , Neutrófilos/metabolismo , Ácidos Palmíticos/sangre , Fosfolípidos/sangre , Acilación , Ácido Araquidónico , Humanos , Técnicas In Vitro , Cinética , Neutrófilos/efectos de los fármacos , Ácido PalmíticoRESUMEN
The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Somatomedinas/fisiología , Secuencia de Aminoácidos , Animales , Bovinos , División Celular , Humanos , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/aislamiento & purificación , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor II del Crecimiento Similar a la Insulina/aislamiento & purificación , Datos de Secuencia Molecular , Músculos/citología , Músculos/efectos de los fármacos , Músculos/fisiología , Ratas , SolubilidadRESUMEN
Erythropoietin (Ep) is the peptide growth factor whose actions on the erythroid progenitor cell induce terminal differentiation. However, the intracellular signaling system that is activated by Ep is poorly understood. Our previous studies have implicated the lipoxygenase metabolites of arachidonic acid in the actions of Ep. In this study, we report an early (30 s to 5 min) increase in levels of two lipoxygenase metabolites: leukotriene B4 (LTB4; 3- to 5-fold) and 12-hydroxyeicosatetraenoic acid (12-HETE; 2-fold). These responses were blocked by an antibody to Ep, by lipoxygenase inhibitors, or by 1,6-di[O-(carbamoyl)cyclohexanone oxime]hexane (RHC80267), an inhibitor of diacylglycerol (DAG) lipase. RHC 80267 also significantly inhibited Ep-mediated proliferation. Ep induced the release of [3H]arachidonic acid from cellular phospholipids at 5 min and also increased DAG accumulation at 1 min with a maximum increase of 68.2% over control seen at 30 min. No increase in levels of inositol trisphosphate or phosphatidic acid was observed in response to Ep. Taken together, these data suggest that the signal transduction pathway of the Ep receptor includes the activation of phospholipases A2 and C, resulting in the liberation of DAG and arachidonate and the subsequent formation of LTB4 and 12-HETE.
Asunto(s)
Fosfolipasas A/metabolismo , Receptores de Superficie Celular/fisiología , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animales , Ácido Araquidónico/metabolismo , Células Cultivadas , Diglicéridos/metabolismo , Activación Enzimática , Eritropoyetina/farmacología , Feto/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/metabolismo , Lipoproteína Lipasa/metabolismo , Hígado/citología , Hígado/enzimología , Fosfolípidos/metabolismo , Receptores de EritropoyetinaRESUMEN
Two major dimers are generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin (bST). These dimers represent the major part of the inactive high molecular weight species that are formed in this process. The structures of the two dimers are unambiguously determined by peptide mapping using trypsin, thrombin cleavage, and selective DTT reduction experiments. Results indicate that the formation of both dimers involves the large disulfide loop cysteines. The latter-eluting dimer from RP-HPLC, previously reported as a large loop concatenated dimer, was revised to be an antiparallel disulfide-linked dimer. On the other hand, the first eluting dimer is a concatenane in which two monomers are held together by the interlocking of the two large disulfide loops.
Asunto(s)
Hormona del Crecimiento/química , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Mapeo Peptídico , Polímeros , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
The first twenty-nine amino acids of human Growth Hormone Releasing Factor (hGRF) possess a distinct amphiphilic character. This is seen as twisted hydrophobic and hydrophilic bands in the helical net projection. Four amidated analogs were designed by optimizing amphiphilic and helical potentials of the native sequence. These designed analogs, with up to eight-amino acid changes, were tested in sheep via intravenous injection. The growth hormone-stimulating activities of the analogs were significantly higher when compared to bovine Growth Hormone Releasing Factor (bGRF44-NH2). This suggests that the amphiphilic conformation of GRF(1-29) is important to the receptor.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Dicroismo Circular , Hormona del Crecimiento/sangre , Humanos , Conformación Proteica , Ovinos , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Garlic has been used in herbal medicine for thousands of years. While garlic oil contains many components and has been widely studied, the pharmacology of pure allicin, a constituent of garlic oil, is not well understood. We report that allicin inhibits human platelet aggregation in vitro without affecting cyclooxygenase or thromboxane synthase activity or cyclic adenosine monophosphate (AMP) levels. Allicin does not alter the activity of vascular prostacyclin synthase. However, it inhibits ionophore A23187-stimulated human neutrophil lysosomal enzyme release. In vivo allicin dilates the mesenteric circulation of the cat independent of prostaglandin release or a beta adrenergic mechanism.