共查询到20条相似文献,搜索用时 15 毫秒
1.
W C Duckworth 《Biochimica et biophysica acta》1976,437(2):518-530
Insulin and glucagon degradation by rat kidney homogenates and subcellular fractions was examined under a variety of conditions including high and low substrate concentrations, at pH 4 and pH 7, with and without glutathione. At high insulin concentration (4.1 - 10(-5) M) insulin degradation by the homogenate was greatest at pH 4 but at low insulin concentration (1 - 10(-10) M) insulin degradation was greatest at pH 7. At either high or low glucagon concentration glucagon degradation by the homogenate was greatest at pH 7. Glutathione at pH 7 stimulated insulin degradation at high insulin concentrations and inhibited insulin degradation at low concentrations; Glucagon degradation at pH 7 was inhibited at both high and low concentrations of glucagon by glutathionemseparation of kidney into cortex and medulla prior to homogenation produced a pattern of insulin and glucagon degradation identical to the whole homogenate but glucagon degradation by the medulla was greater than by the cortex. Examination of degradation by subcellular fractions revealed that at high concentration at neutral pH most insulin was degraded by the 100 000 X g pellet but at low insulin concentrations over 90% of the activity was in the 100 000 X g supernatant; At pH 7, at both high and low concentrations, most glucagon-degrading activity was in the 100 000 X g pellet, although the cytosol also had activity; At pH 4 most degradation occurred in the lysosomal fractions. Separation into cortex and medulla again showed similar distribution of activity as the whole gland with the medulla having more glucagon-degrading activity than the cortex. With low insulin concentrations the cortex 100 000 X g supernatant had higher relative specific activities than the medulla supernatant. Examination of recoveries of enzyme activity revealed that the subcellular fractions consistently had markedly less insulin-degrading activity than the original homogenate. This loss of activity was only discernible when insulin degradation was performed at pH 7 at low substrate concentrations. Comparable losses of glucagon-degrading activity were not seen. 相似文献
2.
Hypocalcemic effect in rats of eel calcitonin was more persistent that that of porcine calcitonin and it was as persistent as that of salmon calcitonin I. Eel calcitonin was more stable than porcine or salmon calcitonin I when incubated in vitro with rat or human serum. Incubation in vitro with rat kidney or liver extract for 1 hour at 37 degrees C caused an almost complete inactivation of porcine calcitonin. On the other hand, both eel and salmon calcitonin I were inactivated less markedly and in the similar manner. The relationship between the hypocalcemic effect of calcitonins and the inactivation is discussed. 相似文献
3.
Preparation and properties of a cell-free system from rat skin that catalyzes sterol biosynthesis 总被引:1,自引:0,他引:1
Homogenates of epidermis from rat skin were centrifuged at 10,000 X g for 20 min. The supernatant fraction ("whole homogenate") catalyzed the demethylation of lanosterol (C(30)) to yield C(27)-sterols. The rate of reaction was measured by the rate of release of (14)CO(2) from the 4-methyl group of lanosterol. Conditions for maximal rates of demethylation were established. Addition of increasing amounts of washed microsomes to a constant amount of substrate resulted in additional release of (14)CO(2), but the release was not proportional to the amount of microsomes. Incubation with increasing amounts of microsomes treated with Triton WR-1339 yielded a proportional rate of release of (14)CO(2). The Triton-treated microsomes were frozen and stored without loss of activity. The rate of formation of (14)CO(2) was constant up to 1 hr of incubation with both Triton-treated microsomes and whole homogenate, for which the K(m) for lanosterol was 5.0 and 3.0 X 10(-5) M, respectively. Other 4-gem-dimethyl sterols were competitive inhibitors, K(i)', 2.0 and 5.5 X 10(-5) M. The enzyme system was inhibited by arsenite. 24,25-Dihydrolanosterol, 24,25-dihydrolanostenone, and squalene were demethylated by the homogenate. The whole homogenate catalyzed the incorporation of mevalonic acid, but not acetic acid, into squalene and sterols. The enzymatic properties of the sterol synthetic system from skin resemble those of similar preparations from rat liver. 相似文献
4.
Purification of pork renal cortex membranes yielded a particulate adenylate cyclase retaining good sensitivity to stimulation by parathyroid hormone and glucagon and a modest but significant response to porcine calcitonin. Treatment of this partially purified membrane fraction with 0.5% Lubrol PX and 5 mM NaF released adenylate cyclase activity into a fraction which was not sedimented by centrifugation for 20 min at 37,000 X g or for 2 hours at 100,000 X g and passed through a Millipore filter (0.22 mum pore). This solubilized adenylate cyclase was stimulated by porcine calcitonin and NaF but not by parathyroid hormone or glucagon. On gel filtration (Sephadex G-200) in the presence of 1mM dithiothreitol and 5mM NaF, the major portion of the adenylate cyclase activity eluted with the void volume of the column and showed 2.0-fold stimulation with 10 muM calcitonin. Binding of 125I-labeled porcine calcitonin was demonstrated in the 37,000 X g and the 100,000 X g supernatants. From 74 to 86% of the observed binding could be blocked by the addition of unlabeled porcine calcitonin to the reaction mixture. Addition of salmon calcitonin, parathyroid hormone, or glucagon blocked only 12 to 18% of the binding. The dose-response curves for inhibition of binding of iodinated calcitonin by unlabeled calcitonin and the activation of adenylate cyclase by the hormone each showed 50% maximal effect at a concentration between 4.5 and 8 muM porcine calcitonin and maximal effect at a concentration between 33 and 66 muM porcine calcitonin. 相似文献
5.
Nag M 《Indian journal of experimental biology》2004,42(9):941-944
Drugs with efficacy in psychiatric disorders affect the function of central neurotransmitter amines, which are inactivated primarily by monoamine oxidase (MAO). Effect of these drugs on the two types of MAO (MAO-A and MAO-B) has been studied in rat brain. The result showed that chlorpromazine (CPZ) and imipramine (IMI) at concentrations of 1x10(-2), 5x10(-3) and 2.5x10(-3) M inhibited rat brain mitochondrial MAO-A activity in vitro by 82, 50, 39 and 86, 74, 38 %, respectively. CPZ at concentrations of 5x10(-3), 2.5x10(-3), 1x10(-3) M inhibited rat brain mitochondrial MAO-B activity in vitro by 83, 55, 39 %, respectively, while IMI at concentrations of 5x10(-4), 2.5x10(-4), 1x10(-4) M inhibited the in vitro enzyme activity by 43, 35, 21 %, respectively. Lithium at concentration of 5x10(-3) M could not either inhibit MAO-A or MAO-B in the mitochondrial fraction of rat brain. 相似文献
6.
Z Lojda 《Histochemistry》1979,64(2):205-221
A histochemical method for the demonstration of a brush border endopeptidase is described based on results of biochemical and histochemical experiments. The substrate of choice is Glut-Ala-Ala-Ala-MNA which displays a very good localization ability and suitable kinetic properties. Km estimated in rat kidney homogenate amounts to 2.35 X 10(-4) M. pH optimum of this endopeptidase associated with the brush border membrane is in the alkaline range. The activity is dependent on the buffer used. In phosphate and cacodylate buffers of pH 7.2 about 30% lower activity in rat kidney and about 25% lower activity in rat small intestine than in Tris-HCl buffer of the same pH was found. The most suitable diazonium salt for the detection "in situ" is Fast Blue B. It inhibits the endopeptidase activity of rat kidney by about 85% at pH 7.2 AND BY ABOUT 55% AT PH 6.0. The best results are obtained in cryostat sections adherent to semipermeable membranes treated with chloroform-acetone before the incubation. A microdensitometric evaluation of the reaction product is possible and results are in good agreement with those of the biochemical determination. When Suc-Ala-Ala-Ala-INA is used as substrate hexazonium-p-rosaniline is the most suitable coupling agent although it inhibits more than Fast Blue B. The reaction using acylated trialanyl naphthylamides as substrates runs in two steps. Endopeptidase sets free Ala-NA which is attacked by aminopeptidase M. Aminopeptidase M is not reaction rate or localization limiting factor because its activity in the brush border is very high and the enzyme is anchored to the cell membrane very closely to endopeptidase. In homogenates of rat kidney and jejunal mucosa the endopeptidase activity was inhibted by EDTA (2X10(-3) M) by 75% in the kidney and by 68% in the jejunum, by DFP (10(-3) M) by 41% in the kidney and by 35% in the intestine, by Mn2+ (5X10(-3) M) by 25% in the kidney and by 30% in the intestine. No inhibition was exerted by E 600. In sections the results were similar. 1,10-phenanthroline (10(-2) M) caused a substantial inhibition. Endopeptidase activity was detected in the brush border of cells of proximal convuluted tubules of the kidney and in the brush border of differentiated enterocytes of the small intestine. In the same species enterocytes display a lower activity than kidney tubular cells. There are species differences in the distribution pattern of endopeptidase in the kidney. In the rabbit and man the positive reaction occurs in the whole cortex. It is distributed unevenly, however. In the rat the tubules of the inner cortex display a very high activity. In the outer cortex straight portions react strongly. In the rabbit kidney cells of the parietal layer of Bowman's capsule display a weak reaction as well. No sex differences were found in the distribution pattern of endopeptidase in the rat kidney. In the intestine of all species examined a proximo-distal gradient was found... 相似文献
7.
Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme 总被引:2,自引:0,他引:2
Elaine E. Kaufman Thomas Nelson David Miller Noam Stadlan 《Journal of neurochemistry》1988,51(4):1079-1084
An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction. 相似文献
8.
It has been reported that prostacyclin (PGI2) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI2 is inactivated under physiological conditions it has not been possible to demonstrate specific PGI2 binding to the rat stomach. Therefore a stable PGI2 analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE2 nor 6 keto PGF1 alpha displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 X 10(-11) M and 7.1 X 10(-8) M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI2 was less potent than unlabelled Iloprost in displacing 3H-Iloprost from its binding site. The addition of PGE2 to the incubation medium resulted in an increase in 3H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI2-like agents but not for either PGE2 or 6 keto PGF1 alpha. 相似文献
9.
A specific receptor for gastrin I has been demonstrated in the rat stomach fundus. Specific binding of 125I-labelled gastrin I was localised to particles sedimenting between 250--20 000 X g. Saturation of binding sites occurred with a gastrin concentration of 10(-11) M in an assay system containing 0.6--1.7 mg/ml of homogenate protein. Gastrin binding was shown to be reversible, temperature- and pH-dependent, and susceptible to tryptic digestion. Electron microscopic and enzymatic studies showed the binding fraction to contain predominantly mitochondria. Preincubation of the homogenate with 10(-8) M cholecystokinin or secretin inhibited gastrin binding to a greater extent than an equimolar concentration of pentagastrin. Cimetidine, a histamine receptor antagonist, did not affect binding of gastrin to the receptor. 相似文献
10.
The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat 总被引:1,自引:0,他引:1
Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase. 相似文献
11.
Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone. 相似文献
12.
K Akiyama H I Yamamura B C Wilkes W L Cody V J Hruby A M Castrucci M E Hadley 《Peptides》1984,5(6):1191-1195
alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues. 相似文献
13.
The object of this study was to determine whether binding components for pregnenolone, analogous to those described in the adrenal cortex of guinea pigs and rats, were present in the porcine adrenal. A binding component for pregnenolone in the cytosolic fraction of porcine adrenal was demonstrated by sucrose density gradient centrifugation. It banded maximally at 9.6% sucrose (w/w) compared to 12.2% and 12.4% sucrose (w/w) for the plasma-binding component and serum albumin, respectively. At a pregnenolone concentration of 1 X 10(-5) M, specific cytosolic binding of 1 X 10(-8) M [3H]pregnenolone was decreased by 42%. The fractions from sucrose gradients which bound pregnenolone maximally contained 3 beta-hydroxysteroid dehydrogenase-isomerase. The cytosolic supernatant of porcine adrenal gland was resolved by chromatography on hydroxyapatite into eleven fractions, four of which bound added pregnenolone and three of which displayed enzymatic activity. Electrophoretic analysis of the enzymatically active fractions in polyacrylamide gel showed that two of them were of heterogeneous composition, whereas the third, most enzymatically active, fraction consisted principally of one band of high molecular weight. 相似文献
14.
The porcine LH/hCG receptor. Characterization and purification 总被引:4,自引:0,他引:4
J Wimalasena P Moore J P Wiebe J Abel T T Chen 《The Journal of biological chemistry》1985,260(19):10689-10697
Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method. 相似文献
15.
S Moorkerjea 《Canadian journal of biochemistry》1979,57(1):66-71
Microsomal membranes were solubilized by incubation with lysolecithin which caused considerable release of galactosyl- and N-acetylglucosaminyl-transferase into a high-speed supernatant fraction. With a critical concentration of lysolecithin (2.5 mg/10 mg protein in 1 mL microsome suspension), there was a maximal binding of radioactive lysolecithin to the sediment fraction obtained after high-speed centrifugation. Increase of lysolecithin concentration (above 2.5 mg/mL) in the incubation mixture caused a progressive release of the enzymes into the supernatant fraction. Lysolecithin binding to the membrane was greatly inhibited by 1 M NaCl, and high salt concentration also inactivated galactosyltransferase in the sediment, suggesting an electrostatic interaction between lysolecithin and membrane enzyme. In contrast, high NaCl concentration had no inhibitory effect on the enzyme activity in the sediment when the fraction was prepared by treatment with Triton X-100. Lysolecithin-treated microsomal sediment and supernatant galactosyltransferase was inactivated by oleoyllysophosphatidic acid but not by palmitoyllysophosphatidic acid or egg yold lysophosphatidic acid. Triton X-100 treated microsomal fractions were also similarly affected by different species of lysophosphatidic acid. The results suggested a similarity of interactions of lysophosphatidic fatty acyl species with lysolecithin and Triton-treated galactosyltransferase. 相似文献
16.
Amelia Fiorilli Bruno Venerando Carola Siniscalco Eugenio Monti Roberto Bresciani Luigi Caimi Augusto Preti Guido Tettamanti 《Journal of neurochemistry》1989,53(3):672-680
A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle
D W Schwertz J B Halverson J W Palmer H Feinberg 《Archives of biochemistry and biophysics》1987,253(2):388-398
Phosphoinositide-specific phospholipase C (PI-PLC) activity was investigated in the rat heart ventricle. Incubation of ventricle homogenate or 100,000g supernatant fraction with [3H]myoinositol or [3H]arachidonate-labeled phosphatidylinositol in the presence of Ca2+ resulted in a decrease in phosphatidylinositol with a concomitant increase in water-soluble [3H]inositol phosphate or [3H]diglyceride, respectively. Total overt homogenate PI-PLC activity could be accounted for in the supernatant fraction. Neutral, zwitterionic, cationic, or anionic detergents did not unmask membrane-associated activity. While cytosolic phospholipase C was active against a pure phosphatidylinositol substrate in the presence of Ca2+, no hydrolytic activity was detected when phosphatidylinositol was presented as a component (4-5%) of a mixture of phospholipids. However, addition of deoxycholate to the incubation mixture (pH 6.5, Ca2+ 10(-3) M) containing mixed phospholipids resulted in the exclusive hydrolysis of inositol phospholipids. Ventricular supernatant phospholipase C-mediated phosphatidylinositol degradation has a sharp pH optimum at 5.5 and a specific requirement for Ca2+. Activity is maximal at 1 to 2 X 10(-3) M Ca2+, with inhibition occurring at higher levels. Under optimized conditions phosphatidylinositol is hydrolyzed at a rate of 20-25 nmol/min/mg protein. Multivalent cations inhibit Ca2+-dependent PI-PLC activity while monovalent cations and anions have no effect. There is no apparent selectivity for specific fatty acid moieties on phosphatidylinositol. Soluble PI-PLC is inhibited by sulfhydryl reagents, neomycin, mepacrine, trifluoperazine, and propranolol. Chlorpromazine, dibucaine, and tetracaine exert a biphasic influence, stimulating at lower and inhibiting at higher concentrations. 相似文献
18.
The concentrations of PGE, PGF, and 6-keto-PGF1 alpha were increased in rat seminal vesicle tissue following mating activity. Likewise, synthesis of PGE and PGF was stimulated by epinephrine (3 X 10(-7) to 3 X 10(-6) M) in tissues and media from in vitro incubations of intact rat seminal vesicles. The in vitro stimulation was inhibited by phentolamine, an alpha-adrenoreceptor blocking agent. Carbamylcholine (2 X 10(-6) M) and bradykinin (1 X 10(-6) M) had no effect on PGE or PGF synthesis, even though both compounds stimulated contractility of the rat seminal vesicle at these concentrations. These data suggest that mating and adrenergic stimulation increase prostaglandin synthesis in the rat seminal vesicle, probably through an alpha-adrenergically mediated mechanism. 相似文献
19.
Identification of aminopeptidase M as an enkephalin-inactivating enzyme in rat cerebral membranes 总被引:6,自引:0,他引:6
Two membrane-bound enkephalin-hydrolyzing aminopeptidase activities were partially purified from rat brain membranes. The first, which represents 90% of the total activity, was highly sensitive to both puromycin (Ki = 1 microM) and bestatin (Ki = 0.5 microM). The second was inhibited much more by bestatin (Ki = 4 microM) than by puromycin (Ki = 100 microM). The latter puromycin-insensitive aminopeptidase was found to resemble aminopeptidase M purified from rat kidney brush border membranes. Both displayed the same purification pattern and the same kinetic constants of substrates and inhibitors, and both were similarly inactivated by metal chelating agents. Moreover, antibodies raised in rabbits against rat kidney aminopeptidase M inhibited the aminopeptidase activities of both kidney and brain puromycin-insensitive enzymes at similar dilutions, while the brain puromycin-sensitive aminopeptidase activity was not affected. Thus, aminopeptidase M (EC 3.4.11.2) was found to occur in brain, and the role of this enzyme in inactivating endogenous enkephalins released from their neuronal stores is suggested. 相似文献
20.
Actions of recombinant interleukin 1, interleukin 2, and interferon-gamma on bone resorption in vitro 总被引:23,自引:0,他引:23
Human peripheral blood cells, when cultured in vitro, release bone-resorbing factors, which have been called osteoclast-activating factors (OAF) but remain unidentified. We showed previously that a monocyte product, similar to interleukin 1 (IL 1), is a powerful stimulator of bone resorption in vitro. However, the possibility remained that other immune cell products may contribute to OAF activity. We have therefore tested three recombinant cytokines; IL 1, interleukin 2 (IL 2), and interferon-gamma (IFN-gamma) for their activity in a neonatal mouse bone resorption assay. We report here that purified recombinant murine IL 1 is a potent and powerful stimulator of bone resorption in vitro, active over a concentration range of 0.14 to 33 U/ml (1.3 X 10(-12) to 3.1 X 10(-10) M). IL 1-stimulated bone resorption was unaffected by cyclooxygenase inhibition but was inhibited by calcitonin and IFN-gamma. IL 2 had no effect on bone resorption. 相似文献