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1.
Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase
In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP
adenosine triphosphate
- -AR
-adrenergic receptors
- AC
adenylyl cyclase
- DHA
dihydroalprenolol
- DPH
diphenylhexatriene
- [Ca2+]i
Ca2+ concentration inside proteoliposomes
- [Ca2+]o
Ca2+ concentration outside proteoliposomes
- cAMP
cyclic adenosine monophosphate
- DTT
Dithiothreitol
- FS
fluorescein sulfonate
- Gs
Stimulatory GTP-binding protein
- GTP
guanosine triphosphate
- GTPS
guanosine 5-O-(3-thiotriphosphate)
- kDa
kilodalton
- SDS
sodium dodecyl sulfate
- Tris
N-tris(hydroxymethyl)aminomethane 相似文献
2.
Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum 总被引:2,自引:0,他引:2
B. Ewa Snaar-Jagalska Peter J. M. Van Haastert 《Molecular and cellular biochemistry》1990,92(2):177-189
cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS
Adenosine 5-0-(3-Thiotriphosphate)
- GTPS
Guanosine 5-0-(3-thiotri-phosphate)
- (Sp)-cAMPS
Adenosine 3,5-monophosphorothioate-Sp-isomer
- dcAMP
2-deoxyadenosine 3,5-monophosphate
- Hepes
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- DTT
Dithiothreitol
- buffer A
10 mM KH2PO4/Na2HPO4, pH 6.5
- buffer B
40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7 相似文献
3.
Dong-Hyun Kim Norihiro Azuma Hideyuki Tanaka Choemon Kanno 《Glycoconjugate journal》1998,15(4):361-369
The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd 相似文献
4.
Prof. Rita Pascolini P. Ceccarelli A. M. Gargiulo S. Lorvik 《Cell and tissue research》1985,239(2):443-445
Summary In the testis of Esox lucius at the time of spermiation, activity of cyclic adenosine 3,5-monophosphate (cAMP) was immunocytochemically localized at the level of the Sertoli cells. In these cells adenylate cyclase activity was also ultracytochemically demonstrated by using adenylyl imidodiphosphate as a substrate. Reaction products of adenylate cyclase were primarily detectable on the basal and adluminal plasma membranes and on the surface of protrusions of the cell body into the lumen. 相似文献
5.
The present studies were undertaken to examine if the impaired vascular function observed in diabetes is attributed to the altered levels of G-protein. Diabetes was induced in Sprague Dawley rats by a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg body wt) and after a period of 5 days, the aorta were used for adenylyl cyclase activity determination and protein quantification. A temporal relationship between the expression of Gi proteins and development of diabetes was also examined on day 1, 2, 3, 4 and 5 of injection of STZ. Blood glucose levels were significantly increased from day 1 in STZ-rats as compared to their counterpart control rats and reached to about 20 mM on 3rd day and 30 mM on 5th day. The expression of Gi-2 and Gi-3 proteins as determined by immunoblotting techniques was decreased by about 70 and 50% respectively in aorta from STZ rats compared to the control rats after 5 days of treatment, whereas 40% decrease in Gi-2 and Gi-3 was observed after 3rd day of STZ injection. On the other hand, the expression of Gs was unaltered in STZ rats. In addition, the stimulatory effect of cholera toxin (CT) on GTP-mediated stimulation of adenylyl cyclase was not different in STZ as compared to the control group. However, the stimulatory effects of isoproterenol, glucagon, NaF and FSK on adenylyl cyclase activity were significantly enhanced in STZ rats as compared to control rats, whereas basal adenylyl cyclase activity was significantly lower in STZ-rats as compared to control rats. In addition, GTPS inhibited FSK-stimulated adenylyl cyclase activity in concentration-dependent manner (receptor-independent functions of Gi) in control rats which was completely attenuated in STZ-rats. In addition, receptor-mediated inhibitions of adenylyl cyclase by angiotensin II, oxotremorine, atrial natriuretic peptide (ANP99–126) and C-ANP4–23 were also attenuated (receptor-dependent functions of Gi) in STZ-rats. These results indicate that aorta from diabetic rats exhibit decreased levels of cAMP and decreased expression of Gi. The decreased expression of Gi may be responsible for the altered responsiveness of adenylyl cyclase to hormonal stimulation and inhibition in STZ-rats. It may thus be suggested that the impaired adenylyl cyclase-Gi protein signaling may be one of the possible mechanisms responsible for the impaired vascular functions in diabetes. 相似文献
6.
Seven regions of the subunit of human insulin receptor (HIR) were synthesized and examined for their ability to bind radioiodinated insulin. A peptide representing one of these regions (namely, residues 655–670) exhibited a specific binding activity for insulin. In quantitative radiometric titrations, the binding curves of125I-labeled insulin to adsorbents of peptide 655–670 and of purified placental membrane were similar or superimposable. The binding of radioiodinated insulin to peptide or to membrane adsorbents was completely inhibited by unlabeled insulin, and the inhibition curves indicated that the peptide and the membrane on the adsorbents had similar affinities. Synthetic peptides that were shorter (peptide 661–670) or longer (peptide 651–670) than the region 655–670 exhibited lower insulin-binding activity. It was concluded that an insulin-binding region in the HIR subunit resides within residues 655–670. The results do not rule out the possibility that other regions of the subunit may also participate in binding of HIR to insulin, with the region described here forming a face within a larger binding site. 相似文献
7.
L. A. Kuznetsova 《Journal of Evolutionary Biochemistry and Physiology》2002,38(4):371-391
Various aspects of mechanisms of the adenylyl cyclase (AC)* activity regulation are considered in the review. Variants of modulation of various AC isoform activity by G-proteins (s- and i subunits, -dimer), Ca2+-calmodulin, phosphorylation by PKA, PKC or Ca2+-CM-activated kinases are presented. Evidence is presented that AC functions as a signaling integrator in the cell by providing propagation and amplification of many both extracellular (with participation of hormones) and intracellular signals (interaction between Ca2+ and AC). 相似文献
8.
Ribonuclease activity dependent cytotoxicity of Asp fl,a major allergen of A. fumigatus 总被引:1,自引:0,他引:1
Vasoactive peptides such as angiotensin II (AII), atrial natriuretic peptide (ANP) and vasopressin play an important role in the regulation of blood pressure. We have recently shown an augmentation of Gi levels in heart and aorta from genetic and experimentally-induced hypertensive rats, which may be attributed to the increased levels of vasoactive peptides. We have therefore investigated the effect of AII and ANP on the expression of G-proteins (Gi and Gs) in cultured vascular smooth muscle cells (VSMC) and their relationship with adenylyl cyclase activity. Exposure of VSMC with AII resulted in the augmentation of the levels of Gi-2 and Gi-3 proteins and Gi-2 and Gi-3 mRNA and not of Gs as determined by immunoblotting and Northern blotting techniques respectively. However, the stimulatory effects of N-ethylcarboxamide adenosine (NECA) and isoproterenol on adenylyl cyclase was diminished by AII treatment, whereas the inhibitory effects of AII and C-ANP4-23 were completely attenuated. On the other hand, pretreatment of the cells with C-ANP4-23 resulted in the reduction of the levels of Gi-2 and Gi-3 and not of Gs. The inhibitory responses of adenylyl cyclase to C-ANP4-23 and AII were also attenuated and the stimulatory effects of GTPgS and other agonists were significantly augmented. These data indicate that AII and ANP modulate the expression of Gia protein in a different manner. It may be suggested that the enhanced levels of Gi protein observed in hypertension may be attributed to the augmented levels of AII and not to ANP. 相似文献
9.
Summary Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot.125I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an heterodimeric insulin receptor complex from the 22 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional heterodimeric insulin receptor complex. (ii) the heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the 22 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the heterodimers.Abbreviations used are ATP
adenosine 5-triphosphate
- DTT
dithiothreitol
- SDS
sodium dodecyl sulfate
- DSS
disuccinimidyl suberate
- NEM
N-ethylmaleimide
- IGF-I
insulin-like growth factor-I
- EDTA
ethylenediaminetetraacetic acid
- HEPES
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid 相似文献
10.
Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of 3Jcp coupling constants. 相似文献
11.
In this paper we use a dynamical systems approach to prove the existence of a unique critical value c
* of the speed c for which the degenerate density-dependent diffusion equation u
ct
= [D(u)u
x
]
x
+ g(u) has: 1. no travelling wave solutions for 0 < c < c
*, 2. a travelling wave solution u(x, t) = (x - c
*
t) of sharp type satisfying (– ) = 1, () = 0 *; '(*–) = – c
*/D'(0), '(*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c
*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions. 相似文献
12.
Madhu B. Anand-Srivastava 《Molecular and cellular biochemistry》1996,157(1-2):163-170
The present studies were undertaken to examine if adenylyl cyclase activity and the levels of G-proteins (Gs and Gi) are altered in cardiovascular tissues in hypertension. Adenylyl cyclase activity and its responsiveness to stimulatory and inhibitory hormones as well as the expression of G-proteins (Gs and Gi) were determined at protein and mRNA levels by using specific antibodies and cDNA probes in hearts and aorta from 12 week old spontaneously hypertensive rats (SHR) and their age-matched control Wistar Kyoto (WKY) rats. The stimulatory effects of guanine nucleotides, isoproterenol, glucagon etc. on adenylyl cyclase activity were decreased in SHR rats as compared to the WKY rats, whereas, the inhibitory hormones inhibited enzyme activity to a grater extent in SHR rats as compared to WKY rats. Furthermore, the levels of Gi-2 and Gi-3 proteins and Gi-2 and Gi-3 mRNA as determined by immunoblotting and Northern blotting techniques respectively were higher in SHR as compared to WKY rats. However, the levels of Gsa were unaltered in SHR. To further investigate if these alterations are the cause or effect of hypertension, the SHRs at various ages of the development of blood pressure (3–5 days, 2, 4 and 8 weeks) and their age-matched WKY were used for G-protein expression and adenylyl cyclase activity. The increased expression of Gi–2 and Gi–3 protein and mRNA levels in hearts and aorta were observed as early as in 2-weeks old SHR as compared to WKY, when the blood pressure was still normal. However, the levels of Gs in SHR were not different from WKY rats. In addition, the altered responsiveness of adenylyl cyclase to hormone stimulation and inhibition was also observed as early as in 2 week old SHR. These results suggest that the increased expression of Gi–2 and Gi–3 and decreased levels of cAMP precedes the development of blood pressure and may be one of the contributing factors in the pathogenesis of hypertension.Abbreviations NECA
N-ethylcarboxamideadenosine
- Iso
Isoproterenol
- Glu
Glucagon
- ANF
atrial natriuretic factor
- AII
angiotensin II
- PT
pertussis toxin
- CT
cholera toxin
- FSK
forskolin
- GTPS
guanosine 5-[-thio]triphosphate
- Gs
stimulatory guanine nucleotide regulatory protein
- Gi
inhibitory guanine nucleotide regulatory protein
- WKY
WistarKyoto rats
- SHR
spontaneously hypertensive rats
The work presented in this report was supported by grants from Medical Research Council of Canada and Quebec Heart FoundationM.B.A-S is a recipient of the Medical Research Council Scientist Award from the Medical Reserch Council of Canada. 相似文献
13.
S. Frillingos M. Frangou-Lazaridis K. Seferiadis J. D. Hulmes Y. -C. E. Pan O. Tsolas 《Molecular and cellular biochemistry》1991,108(1):85-94
Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT
Prothymosin
- T1
Thymosin 1
- MLR
Mixed Lymphocyte Response
- HPLC
High Performance Liquid Chromatography
- RIA
Radioimmunoassay
- B
Aspartic acid or Asparagine
- Z
Glutamic acid or Glutamine 相似文献
14.
Noriaki Inamura Saburo Sone Akio Okubo Eiji Kunishige Mie Nakanishi Takeshi Ogura 《Cancer immunology, immunotherapy : CII》1989,28(3):164-170
Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [125I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL
interleukin
- IFN
interferon
- TNF
tumor necrosis factor 相似文献
15.
Stable isotope and radiocarbon compositions of methane emitted from tropical rice paddies and swamps in Southern Thailand 总被引:1,自引:0,他引:1
Fumiko Nakagawa Naohiro Yoshida Atsuko Sugimoto Eitaro Wada Takahito Yoshioka Shingo Ueda Pisoot Vijarnsorn 《Biogeochemistry》2002,61(1):1-19
Stable isotopes (13C, D) and radiocarbon weremeasured in methane bubbles emitted from rice paddies and swamps in southernThailand. Methane emitted from the Thai rice paddies was enriched in13C (mean 13C; –51.5 ±7.1 and–56.5 ± 4.6 for mineral soil and peat soil paddies,respectively)relative to the reported mean value of methane from temperate rice paddies(– 63 ± 5). Large seasonal variation was observed in13C(32) in the rice paddies, whereas variationinD was much more smaller (20), indicating that variation in13C is due mainly to changes in methane production pathways.Values of 13C were lower in swamps (–66.1 ±5.1)than in rice paddies. The calculated contribution of acetate fermentation from13C value was greater in rice paddies (mineral soils:62–81%, peat soils: 57–73%) than in swamps (27–42%). Din methane from Thai rice paddies (–324± 7 (n=46)) isrelativelyhigher than those from 14 stations in Japanese rice paddies ranging from–362 ± 5 (Mito: n=2) to –322 ± 8(Okinawa: n=3), due tohigher D in floodwaters. 14C content in methane produced fromThai rice paddies (127±1 pMC) show higher 14Cactivity compared with previous work in paddy fields and those from Thai swamps(110±2 pMC). 相似文献
16.
Hans Eberhard Fischer Klaus Fürste 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1964,34(1):22-27
Zusammenfassung In zunehmendem Maße werden anisoploideBeta-Rübensorten angebaut, deren zytologische Kontrolle zwecks Feststellung der Genomstufenprozentanteile recht arbeitszeitaufwendig ist. Übereinstimmend mit polnischen Autoren wurde festgestellt, daß die Hypokotylfarbe ein geeigneter Markierungsfaktor für die einzelnen Genomstufen darstellt. Kreuzt man tetraploide Pflanzen, die ein grünes Hypokotyl besitzen, mit diploiden Pflanzen, die ein rosa Hypokotyl aufweisen, so erhält man von dem tetraploiden Partner tetraploide grüne und triploide hellbraune, von dem diploiden Partner diploide rosa und triploide hellbraune Nachkommenschaften. Die in bezug auf die Hypokotylfarbe heterozygoten Pflanzen kann man demnach von den homozygot grünen und homozygot rosa Individuen unterscheiden. Die Kreuzung diploid grünxtetraploid rosa ist für diese Zwecke nicht brauchbar, da sich die triploiden Heterozygoten mit einem grünen und zwei rosa Allelen in der Hypokotylfarbe nicht deutlich von den homozygoten rosa Pflanzen abheben. Auf die Bedeutung dieser Markierungsmöglichkeit für bestimmte Forschungsprobleme, die Züchtung und die Saatgutkontrolle wird hingewiesen. 相似文献
17.
N. N. Smirnov 《Hydrobiologia》1966,27(3-4):337-343
Summary The differences in the structure of the trunk limbs allow to outline three sections of Chydoridae (see table I and fig. 1), coinciding with the sections distinguished according to the structure of the head pores.
Chydoridae (Cladocera)
Chydoridae (. ), , .相似文献
18.
Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat
Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [35S]GTPS with a relatively high affinity (Kd10nM). The binding was saturable and specific as it was indicated by the displacement of bound [35S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [35S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [35S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [35S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- G
protein hetero-trimeric GTP binding protein with , , subunits
- Gn
protein GTP binding protein detected on nitrocellulose blots
- GTPS
guanosine 5-[-thio]triphosphate
- IAA
3-indoleacetic acid
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis 相似文献
19.
Lakhu M. Keshvara Elizabeth M. Newton A. Heather Good Ole Hindsgaul Monica M. Palcic 《Glycoconjugate journal》1992,9(1):16-20
ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min. 相似文献
20.
Michael Böhm 《Molecular and cellular biochemistry》1995,147(1-2):147-160
Alterations of receptor-G-protein-regulated adenylyl cyclase activity have been suggested to represent an important alteration leading to contractile dysfunction in the failing human heart. Recent experiments suggest that the 1-adrenoceptor(1AR) density and mRNA levels are reduced, while 2-adrenoceptors and stimulatory G-proteins are unchanged (mRNA and protein level). Functional assays demonstrated that the catalyst of the adenylyl cyclase is not different between failing and nonfailing myocardium. Inhibitory G-proteins are increased (pertussis toxin substrates, protein and mRNA) and correlate to the reduced inotropic effects of -adrenoceptor agonists and of CAMP-PDE inhibitors. Gi-coupled m-cholinoceptors and A1-adrenergic receptors are unchanged in density and affinity. Stimulation of these receptors resulted in an unchanged antiadrenergic effect on force of contraction. In conclusion, a downregulation of 1-AR and an increase of Gi have been observed as signal transduction alteration in failing human myocardium. These alterations are due to alterations of gene expression in the failing heart and are related to a defective regulation of force of contraction in heart failure. 相似文献