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1.
We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the α-subunits of the stimulatory and inhibitory G-proteins of AC (G sα and G i−2α) in cultured prolactin-producing rat pituitary adenoma cells (GH 3 cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G sα levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G sα levels by more than 50% at all time-points. G i−2α levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G s protein α-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways. 相似文献
2.
In the present paper, the modulation of the basolateral membrane (BLM) Na +-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na +-ATPase activity in a dose-dependent manner (from 10 −11 to 10 −5 M), with maximal effect obtained at 10 −7 M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT 2 receptor: The effect of both polypeptides is completely reversed by 10 −8 M PD 123319, a selective AT 2 receptor antagonist, but is not affected by either (10 −12–10 −5 M) losartan or (10 −10–10 −7 M) A779, selective antagonists for AT 1 and AT (1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10 −9 M guanosine 5′- O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10 −10 M guanosine 5′- O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G s protein in the isolated basolateral membrane fraction; (4) (10 −10–10 −6 M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity. 相似文献
3.
The uptake of the neuroactive sulphur amino acids
-cysteine sulphinate,
-cysteate,
-homocysteine sulphinate and
-homocysteate was investigated in astrocytes cultured from the prefrontal cortex; in neurons, cultured from cerebral cortex; and, in granule cells, cultured from cerebellum. It was shown that each amino acid acted as a substrate for a plasma membrane transporter in both neurons and astrocytes. Astrocytes and neurons exhibited a high-affinity uptake for
-cysteine sulphinate and
-cysteate with Km values ranging from 14–100 μM, and a low-affinity uptake for
-homocysteine sulphinate and
-homocysteate, with Km values ranging from 225–1210 μM. The uptake of all transmitter candidates studied was partially sodium-dependent. This sodium-dependency was most evident at low (< 100 μM) concentrations of each substrate. The apparent uptake measured in the absence of sodium was included as a component in corrections made for non-saturable influx. With the exception of
-cysteine sulphinate, uptake of each sulphur amino acid was greatest in astrocytes, with Vmax values ranging between 15–32 nmol min −1 mg −1 cell protein. Moreover, the uptake of each sulphur amino acid in cerebellar granule cells ( Vmax values ranging between 10–25 nmol min −1 mg −1 cell protein) was consistently greater than that in cerebral cortex neurons ( Vmax values ranging between 1.5–6 nmol min −1 mg −1 cell protein). 相似文献
4.
We investigated the restoration of [Ca 2+] i in fura-2-loaded human platelets following discharge of internal Ca 2+ stores in the absence of external Ca 2+. After stimulation by thrombin [Ca 2+] i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca 2+] i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca 2+] i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca 2+ efflux, perhaps via protein kinase C actions on a plasma membrane Ca 2+ pump. 相似文献
6.
Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l −1). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml −1 LDH and 0.8 U ml −1 LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC10 of 800 nM (213 μg l −1) and a minimum inhibition detectable ( MID) limit of 650 nM (173 μg l −1). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml −1 LDH, 0.8 U ml −1 LOD and 0.1 U ml −1 GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC10 limit was 150 nM (39.9 μg l −1) and the MID was 100 nM (26.6 μg l −1). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring. 相似文献
7.
We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O 2−) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O 2− was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [ 14C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O 2− in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O 2− or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm 2. Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O 2− (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O 2−, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca 2+ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca 2+ from internal stores. Thus, COM-induced mitochondrial O 2− requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O 2− production, which is probably regulated by Δψ m. 相似文献
8.
4β-Phorbol 12-myrisate 13-acetate (PMA), a tumour-promoting phorbol ester, and 1-oleoly-2-acetylglycerol (OAG), a synthetic diacylglycerol, induced an inhibition of muscarinic and 1-adrenergic receptor-mediated stimulation of PIP 2 breakdown and IPs accumulation in both rabbit retinal slices and primary retinal cultures. Furthermore, an increase in [Ca 2+] i, mediated by activation of these receptors in 3–5 and 25–30 day old rabbit retinal cultures, was also inhibited by PMA. Neither PMA nor OAG had an effect on the serotonin-mediated PIP 2 breakdown, IPs accumulation or Ca 2+ mobilization. Although A23187 also stimulated IPs formation by acting directly on phospholipase C, PMA had no effect. Maximal inhibition of the carbachol- and noradrenaline-mediated responses was achieved with a 15 min preincubation with PMA at concentrations of 0.1 and 0.01 μM in retinal slices and primary retinal cultures, respectively. Neither PMA nor OAG influenced the basal levels of phosphoinositides, IPs or [Ca 2] i. In addition, the inactive phorbol ester, 4-phorbol 12,13-didecanoate, had no effect on any of the agonist-induced responses. Staurosporine, a potent inhibitor of protein kinase C, significantly attenuated the inhibitory effects exerted by PMA and OAG. These results suggest that calcium- and phospholipid-dependent protein kinase, which is activated by either PMA or OAG, exert inhibitory effects on muscarinic and 1-adrenergic responses. This modulatory feedback “down regulation” role by PKC does not, however, affect serotonergic mediated responses, and thus exhibits a certain selectivity about the site of action. The possible mechanism(s) by which PKC induces its actions are discussed. 相似文献
9.
Recently, it has been shown that PKA-mediated phosphorylation of the β 2-adrenergic receptor (β 2-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G s and increases its affinity for G i. Here we demonstrate that, like the β 2-AR, the β 1-AR is also capable of “switching” its coupling from G s to G i in a PKA-dependent manner. The β 1-AR is capable of activating adenylate cyclase via G s, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β 1-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G i/G o, and to the PKA inhibitor, H-89. β 1-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G s-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β 1-AR, like the β 2-AR, can undergo PKA-dependent “G s/G i switching”. 相似文献
11.
A series of aliphatic and aromatic trifluoromethyl ketones has been tested as inhibitors of the antennal esterases of the Egyptian armyworm Spodoptera littoralis, by evaluation of the extent of hydrolysis of [1- 3H]-( Z,E)-9, 11-tetradecadienyl acetate (1), a tritiated analog of the major component of the sex pheromone. The most active compounds with a long chain aliphatic structure were 3-octylthio-1,1,1-trifluoropropan-2-one (2) (IC 50 0.55 μM) and 1,1,1-trifluorotetradecan-2-one (4) (IC 50 1.16 μM). The aromatic compounds were generally less potent inhbitors than the coressponding aromatic ones, although β-naphthyltrifuloromethyl ketone (10) exhibited a remarkable inhibitory activity (IC 50 7.9 μM). Compounds 2, 4 and 10 exhibit a competitive inhibition with K i values of 2.51×10 −5 M, 2.98×10 −5 M and 2.49×10 −4 M, respectively. Some of the trifluoromethyl ketones tested were slow-binding inhibitors and compounds 2 and 10 are described as inhibitors of the antennal esterases of a moth for the first time. 相似文献
12.
Platelet-activated factor (PAF) (
), formyl-methionyl-leucyl-phenylalanine (fMPL) (
), phorbol 12-myristate 13 acetate (PMA) (
), opsonized zymosan (OPZ) (0.01–1 mg/ml) were potent stimuli to superoxide generated by guinea-pig peritoneal macrophages. Superoxide generation by low (≤ −8M) concentrations but not high (≥−7M) concentrations of PAF or fMLP were attenuated by rolipram (100 μM) in the presence of 1 μM prostaglandin E 2 (PGE 2). That stimulated by PMA or OPZ, however, was unaffected. At 1μM, staurosporine was a potent inhibitor of superoxide generation stimulated by both fMLP and PAF but was without effect on that stimulated by OPZ. Superoxide generation stimulated by fMLP, PAF and OPZ was inhibited by 100 μM mepacrine. We conclude that superoxide generation stimulated by the chemoattractants fMLP and PAF involves a cyclic AMP regulated and cyclic AMP independent process. The cyclic AMP independent process is mediated by protein kinase C. Although protein kinase C seems a central element in the respiratory burst stimulated by fMLP, PAF and PMA that stimulated by OPZ bypasses this mechanism. Phospholipase A 2 however, represents a common stage in the signal transduction pathway. 相似文献
13.
Analogy with the isolable oxo cluster [Fe 3(CO) 9(μ 3-O)] 2−, which is structurally interesting and synthetically useful, prompted the present attempt to synthesize its ruthenium analog. Although the high reactivity of [Ru 3(CO) 9(μ 3-O)] 2− (I) prevented its isolation, the reaction of this species with [M(CO) 3(NCCH 3)] +, where M = Mn or Re, yields [PPN][MRu 3(CO) 12(η 2-μ 3-NC(μ-O)CH 3] −. The high nucleophilicity of the oxo ligand in [Ru 3(CO) 9(μ 3-O)] 2− (I) appears to be responsible for the conversion of acetonitrile to an acetamidediato ligand and for the instability of I. The crystal structure of [PPN][MnRu 3(CO) 12(η 2-μ 3-NC(μ-O)CH 3)]] reveals a hinged butterfly array of metal atoms in which the acetamidediato ligand bridges the two wings with μ 3-N bonding to an Mn and two Ru atoms, and μ-O bonding to an Ru atom. 相似文献
14.
1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives. 2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[14C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria. 3. 3. High affinity-fuscin binding sites (Kd = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations. 4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents; 5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (Ki in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (Ki = 5–10 μM); (c) the transport systems of phosphate (Ki ≈ 20 μM) and of glutamate (Ki = 3–5 μM); (d) the ADP transport, indirectly (Ki ≈ 10 μM). 6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH− carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier. 7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria. 8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation. 9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.
Abbreviations: DTNB, 5,5′-dithio-bis-(2-nitrobenzoic acid); PCMB, p-chloromercuribenzoate 相似文献
15.
The phosphinoalkenes Ph 2P(CH 2) nCH=CH 2 ( n= 1, 2, 3) and phosphinoalkynes Ph 2P(CH 2) n C≡CR (R = H, N = 2, 3; R = CH 3, N = 1) have been prepared and reacted with the dirhodium complex (η−C 5H 5) 2Rh 2(μ−CO) (μ−η 2−CF 3C 2CF 3). Six new complexes of the type (ν−C 5H 5) 2(Rh 2(CO) (μ−η 1:η 1−CF 3C 2CF 3)L, where L is a P-coordinated phosphinoalkene, or phosphinoalkyne have been isolated and fully characterized; the carbonyl and phosphine ligands are predominantly trans on the Rh---Rh bond, but there is spectroscopic evidence that a small amount of the cis-isomer is formed also. Treatment of the dirhodium-phosphinoalkene complexes with (η−CH 3C 5H 4)Mn(CO) 2thf resulted in coordination of the manganese to the alkene function. The Rh 2---Mn complex [(η−C 5H 5) 2Rh 2(CO) (μ−η 1:η 1−CF 3C 2CF 3) {Ph 2P(CH 2) 3CH=CH 2} (η−CH 3C 5H 4)Mn(CO) 2] was fully characterized. Simi treatment of the dirhodium-phosphinoalkyne complexes with Co 2(CO) 8 resulted in the coordination of Co 2(CO) 6 to the alkyne function. The Rh 2---Co 2 complex [(η−C 5H 5) 2Rh 2(CO) (μ−η 1:η 1−CF 3C 2CF 3) {Ph 2PCH 2C≡CCH 3}Co 2(CO) 2], C 37H 25Co 2F 6O 7PRh 2, was fully characteriz spectroscopically, and the molecular structure of this complex was determined by a single crystal X-ray diffraction study. It is triclinic, space group
( Ci1, No. 2) with a = 18.454(6), B = 11.418(3), C = 10.124(3) Å, = 112.16(2), β = 102.34(3), γ = 91.62(3)°, Z = 2. Conventional R on | F| was 0.052 fo observed ( I > 3σ( I)) reflections. The Rh 2 and Co 2 parts of the molecule are distinct, the carbonyl and phosphine are mutually trans on the Rh---Rh bond, and the orientations of the alkynes are parallel for Rh 2 and perpendicular for Co 2. Attempts to induce Rh 2Co 2 cluster formation were unsuccessful. 相似文献
16.
Metathesis of [(η 3:η 3−C 10H 16)Ru(Cl) (μ−Cl)] 2 (1) with [R 3P) (Cl)M(μ-Cl)] 2 (M = Pd, Pt), [Me 2NCH 2C 6H 4Pd(μ-Cl)] 2 and [(OC) 2Rh(μ-Cl)] 2 affords the heterobimetallic chloro bridged complexes (η 3:η 3-C 10H 16) (Cl)Ru(μ-Cl) 2M(PR 3)(Cl) (M = Pd, Pt), (η 3:η 3-C 10H 16) (Cl)Ru(μ-Cl) 2PdC 6H 4CH 2NMe 2 and (η 3:η 3-C 10H 16) (Cl)Ru(μ-Cl) 2Rh(CO) 2, respectively. Complex 1 reacts with [Cp *M(Cl) (μ-Cl)] 2 (M = Rh, Ir), [ p-cymene Ru(Cl) (μ-Cl] 2 and [(Cy 3P)Cu(μ-Cl)] 2 to give an equilibrium of the heterobimetallic complexes and of educts. The structures of (η 3:η 3-C 10H 16)Ru(μ-Cl) 2Pd(PR 3) (Cl) ( R = Et, Bu) and of one diastereoisomer of (η 3:η 3-C 10H 16)Ru(μ-Cl) 2IrCp *(Cl) were determined by X-ray diffraction. 相似文献
17.
The rates of respiratory O 2 uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O 2 (gDW) −1 h −1), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O 2 (gDQ) −1 h −1). The rates for the shoots of bryophytes (53–66 μmol O 2 (gDW) −1 h −1) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O 2 (gDW) −1 h −1) were slightly higher than those of most angiosperm stems, but lower than those for most leaves. These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo. 相似文献
18.
This study examines the effect of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], 24,25-dihydroxyvitamin D 3 [24,25(OH) 2D 3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9- cis retinoic acid and all- trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH) 2D 3 or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9- cis retinoic acid and 10 nM 1,25(OH) 2D 3 or 10 nM KH 1060, and 1 μM 9- cis retinoic acid and 10 nM 1,25(OH) 2D 3 or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9- cis retinoic acid, alone or combined with 10 nM 1,25(OH) 2D 3 or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9- cis retinoic acid and 10 nM 1,25(OH) 2D 3 or EB 1089. The levels of the c- myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9- cis retinoic acid, alone or combined with 10 nM 1,25(OH) 2D 3 or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9- cis retinoic acid and 10 nM 1,25(OH) 2D 3 or 10 nM EB 1089 resulted in a synergistic c- myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma. 相似文献
19.
The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7- 3H 2,4- 14C]estradiol, 7-ethoxycoumarin, and [N-methyl- 3H 3]cocaine. 6-Hydroxy[7- 3H,4- 14C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6- 3H label was established. The initial rate kinetics of the 6-hydroxylation gave K m of 4.3 μM, V max of 4.02 nmol min −1mg −1, and turnover rate of 0.27 min −1. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K m of 200 μM, V max of 12.5 nmol min −1mg −1 and turnover rate of 1.06 min −1. The N-demethylation of cocaine was analysed by the 3H-release method, giving K m of 670 μM, V max of 4.76 nmol min −1mg −1, and turnover rate of 0.49 min −1. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min −1mg −1 ( n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min −1. 相似文献
20.
The binding of [ 3H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a Kd of 400 ± 82 nM and a Bmax of 23.7 ± 6.7 pmol/mg protein. The second had a Kd of 2.4 ± 0.2 μM and a Bmax of 96.3 ± 16.7 pmo/mg protein. Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [3]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed. 相似文献
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