共查询到20条相似文献,搜索用时 15 毫秒
1.
M. N. Lioubin M. D. Meier B. H. Ginsberg 《Preparative biochemistry & biotechnology》2013,43(4):303-311
Abstract We have developed a rapid method for producing homogeneous mono-[12]A14 iodoinsulin with high specific activity and yield. After iodination by the lactoperoxidase method, the labeled peptides were applied to a C18 PorasilR prs-column, washed with aqueous buffer to eliminate the free [12]-iodide and placed “in-line” with a C-18 HPLC column; mono-[12]A14 iodoinsulin was then eluted isocratically with 29% aceto-nitrile in 16 minutes. The labeled hormone was extremely stable, and proved suitable for various biological studies. 相似文献
2.
Structure-function studies on red pigment-concentrating hormone, II. The significance of the C-terminal tryptophan amide 总被引:1,自引:0,他引:1
M Christensen J Carlsen L Josefsson 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(8):1051-1060
The significance of the C-terminal tryptophan residue of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) regulating the blanching of the crustacean chromatophores has been investigated. RPCH and a number of analogues that differ only in the C-terminal part of the hormone, have been synthesized and assayed for biological activity on the shrimp Leander adspersus. It has been shown that the indole skeleton of tryptophan is an absolute requirement for the biological activity of the hormone. To provide maximum response the tryptophan must be blocked as the amide. The activity of synthetic [Tyr4]RPCH and adipokinetic hormone (AKH) purified from Schistocerca gregaria has been compared with the activity of synthetic RPCH. 相似文献
3.
The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of 3H2O from either [2-3H]- or [4-3H]estradiol. Hydrogen peroxide added to this system increased the yield of 3H2O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H2O2 but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of 3H from [2-3H]- or [2,4,6,7-3H]- but not [4-3H]- or [6,7-3H]estradiol. It is proposed that the formation of 3H2O from 3H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid. 相似文献
4.
Roma Drozdz Edith Hintermann Heidi Tanner Urs Zumsteg Alex N. Eberle 《Journal of peptide science》1999,5(5):234-242
A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa13,Tyr19]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa13,Tyr19]‐MCH at its Tyr19 residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [125I]‐[d‐ Bpa13,Tyr19]‐MCH with G4F‐7 mouse melanoma cells gave a KD of 2.2±0.2×10−10 mol/l and a Bmax of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [125I]‐[d‐ Bpa13,Tyr19]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [125I]‐[d‐ Bpa13,Tyr19]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [125I]‐[d‐ Bpa13,Tyr19]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
5.
A mixture of 4 purified proteins at equal concentrations was radiolabelled with [125I] and lactoperoxidase and analysed by SDS-polyacrylamide gel electrophoresis. Bovine serum albumin took up 9 times as much label as ovalbumin or lysozyme and 3.3 times as much as α? chymotrypsinogen A. These results suggest that when applying the [125I]- lactoperoxidase technique to labelling unknown mixtures of proteins, such as may exist on the surfaces of cells, caution should be exercised in interpreting the degree of labelling of particular proteins in terms only of surface abundance or accessibility. 相似文献
6.
Characterization of a neutralization-sensitive epitope on the Am 105 surface protein of Anaplasma marginale 总被引:7,自引:0,他引:7
G H Palmer S D Waghela A F Barbet W C Davis T C McGuire 《International journal for parasitology》1987,17(7):1279-1285
Purified immunoglobulin from each of two hybridoma cell lines (ANA 15D2 and ANA 22B1) significantly neutralized the infectivity of 108Anaplasma marginale initial bodies for cattle. Both cell lines produce antibody to the same Am 105 epitope as they inhibited the binding of each other to Am 105 in a competition radioimmunoassay. Complete digestion of Am 105 with proteinase K, pronase, or trypsin prevented monoclonal antibody binding indicating that the epitope was protein in nature rather than surface polysaccharide. In addition, evidence that the neutralization-sensitive epitope was not membrane-protein-bound polysaccharide included: [1] 35S-methionine, but not 3H-glucosamine, was metabolically incorporated into Am 105 during short-term in vitro culture; [2] Am 105 was surface radiolabeled using 125I in a lactoperoxidase mediated reaction, but not labeled using a galactose oxidase-NaB[3H]4 mediated reaction with or without neuraminidase pretreatment; and [3] Am 105 did not bind to concanavalin A, Helix pomatia lectin, peanut lectin, soybean lectin, or wheat germ lectin. 相似文献
7.
《生物化学与生物物理学报:生物膜》2001,1510(1-2):378-400
Incubation of the isolated H+-ATPase from chloroplasts, CF0F1, with 2-azido-[α-32P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-32P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-32P]ATP was possible. CF0F1 with non-covalently bound 2-azido-[α-32P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF0F1. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F0-part and the F1-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385. 相似文献
8.
Lars G. Öberg Karl Gustav Paul 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,842(1):30-38
The lactoperoxidase-catalyzed transformations of penta-, 2,3,4,6,-tetra-, 2,4,6,-tri, 2,4,-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol: H2O2 ranged from 1:1 for the tri- and tetrachlorophenol at pH 7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (ν0) was only slightly influenced by changes in [H2O2] ? 5 μM. ν0 responded to [chlorophenol] according to the empirical expression ν0 = [lactoperoxidase]·(k1[chlorphenol] + k2[chlorophenol]2). The constant k1 was found to be 5.8 · 105, 1.8 · 106, 1.9 · 106 M?1 · s?1 for the protonated forms of penta-, tetra- and trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 μM for H2O2 and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g peroxidase-containing tissue, possibly with deposition of the products. 相似文献
9.
Mizuki Yamamoto Yuko Terada Takayasu Motoyama Munehisa Shibata Tsutomu Saito 《Bioscience, biotechnology, and biochemistry》2020,84(7):1497-1500
ABSTRACT γ-glutamyl peptides have been suggested to impart kokumi properties to foods by activating human calcium-sensing receptor (hCaSR). In this study, the relationship between γ-glutamyl peptide structure and hCaSR activity was systematically analyzed using γ-[Glu](n=0-4)-α-[Glu](n=0-3)-Tyr. Our results suggest that N-terminal [Glu]3 moiety is very important for hCaSR activities of γ-glutamyl peptides. 相似文献
10.
We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated.The LETS protein was also labelled with [14C]glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with [3H]fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium. 相似文献
11.
T. Valcana 《Neurochemical research》1981,6(7):743-753
In this study we have examined whether rat brain nuclear thyroid hormone receptors bind T4 or metabolites of T4 and whether there is a developmental change in brain T4 metabolism and binding. Developing animals were injected with trace [125I]3,5-tetraiodothyronine ([125I]T4) and after sacrifice brain nuclear and cytoplasmic fractions were examined to determine whether their radioactivity was represented by the injected [125I]T4 or any of its metabolites. Of the radiothyronines specifically bound to the nucleus, 90% was found to be triiodothyronine ([125I] T3) and 10% was [125I]T4. Of the cytoplasmic, protamine sulfate-precipitable fraction, 40% was [125I]T4 and 60% [125I]T3. Inasmuch as the percentage of [125I] T3 found in plasma during the same postinjection interval was similar to that present as contaminant of the injected material, it was concluded that brain [125I] T3 derives from local monodeiodination of T4 to T3. The main developmental change observed was a marked decline in the total cytoplasmic and nuclear [125I] T4 uptake. However, with development, the T3/T4 ratio remained constant in the nuclear fraction while it decreased in the cytoplasmic fraction. It is concluded that although T3, deriving from monodeiodianation of T4, is the main form of thyroid hormone that regulates brain development by its binding to brain nuclear receptors, the fact that T4 is the most available from during the critical period makes it, indirectly, very important to brain development. Further, the decline observed with development in T4 uptake and monodeiodination to T3, may contribute to the concomitantly declining role of thyroid hormones on brain tissue. 相似文献
12.
Surface label experiments using the galactose oxidase-[3 H] -borohydride technique reveal that cells from drug-resistant Chinese hamster ovary clones possess a surface carbohydrate component of apparent molecular weight 165,000 which is absent from wild-type cells. The component may also be demonstrated by [14C] glucosamine incorporation but not by [3 H] leucine incorporation or by the lactoperoxidase surface labeling reaction. 相似文献
13.
Summary (1) Our earlier studies indicate a downsteam regulatory role of the β-adrenergic receptor (β-AR) system in thyroid hormone
induced differentiation and maturation of astrocytes. In the present study we have investigated the contributions of the subtypes
of β-AR in the above phenomenon. (2) Primary astrocyte cultures were grown under thyroid hormone deficient as well as under
euthyroid conditions. [125I]Pindolol ([125I]PIN) binding studies showed a gradual increase in the specific binding to β2-AR when observed at 5, 10, 15, and 20 days under both cultural conditions. Thyroid hormone caused an increase in binding
of [125I]PIN to β2-AR compared to thyroid hormone deficient controls at all ages of astrocyte culture. (3) Saturation studies using [125I]PIN in astrocyte membranes prepared from 20-day-old cultures showed a significant increase in the affinity of the receptors
(K
D) in the thyroid hormone treated cells without any change in receptor number (B
max). (4) β2-AR mRNA levels were measured by real-time PCR during ontogenic development as well as during exposure of 10-day-old hypothyroid
cultures to normal levels of thyroid hormone for 2, 6, 12, and 24 h. None of the conditions caused any significant change
in the β2-adrenergic receptor mRNA levels when compared with corresponding hypothyroid controls. (5) Over expression of β2-AR cDNA in hypothyroid astrocytes caused morphological transformation in spite of the absence of thyroid hormone in the medium.
(6) Taken together, results suggest thyroid hormone causes a selective increase in [125I]PIN binding to β2-AR due to increase in receptor affinity, which may lead to maturation of astrocytes. 相似文献
14.
Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of pKa = 4.75 in intestinal peroxidase and pKa = 3.5 in lactoperoxidase. The apparent pKa (pKa′) increased with the increase in chloride concentration. The pKa′ vs log[Cl?] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (pK = 2.7). Although the dissociation constant (Kd) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of logKd vs log[Cl?] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron. 相似文献
15.
An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19] -MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmocstrategy and Polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD ) was 1.18 × 10?10 M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as α-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors. 相似文献
16.
Gabriele Gaus Lewis H. Kleinholz Günter Kegel Rainer Keller 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1990,160(4):373-379
Summary Red-pigment-concentrating hormone (RPCH) has been isolated from nerve tissue of six decapod crustaccan species. The primary structure of three of the six hormones, i.e., those ofCancer magister, Carcinus maenas andOrconectes limosus, was determined by manual microsequencing as: pELNFSPGW-NH2. This sequence is identical to that of RPCH fromPandalus borealis, the only previously known sequence of a crustacean RPCH. The other three hormones fromLiocarcinus puber, Nephrops norvegicus, andPacifastacus leniusculus could not be characterized completely. However, amino acid compositions, the presence of N-terminal pGlu, and the blocked N-terminal ends are in accordance with the primary structure established for the other three RPCHs. We suggest that all six peptides have the same amino acid sequence. These results indicate that RPCH, which is likely to be related to the peptides of the AKH family in insects, is highly conserved among crustacean species. This is in remarkable contrast to the high degree of molecular evolution exemplified by the many different AKH-like peptides among insect species.Abbreviations
AKH
adipokinetic hormone
-
Aufs
absorption units full scale
-
BSA
bovine serum albumin
-
ECH
erythrophore concentrating hormone (=RPCH)
-
EDTA
ethylenediamine-tetra-acetic acid
-
HOAc
acetic acid
-
HPLC
high pressure liquid chromatography
-
O.D.
optical density
-
PDH
pigment dispersing hormone
-
PGAP
pyroglutamate aminopeptidase
-
RPCH
red-pigment-concentrating hormone (=ECH)
-
SOG
suboesophageal ganglion
-
TFA
trifluoroacetic acid 相似文献
17.
J. F. Leatherland† K. Ogasawara‡ M. S. Rahman‡ R. Renaud H. Yamashiro§¶ A. Takemura‡ 《Journal of fish biology》2003,62(6):1414-1434
Testicular and ovarian fragments of the protogynous Pacific wrasse Haliochoeres trimaculatus were incubated in vitro with [3H]pregnenolone ([3H]P5), [3H]17‐hydroxyprogesterone ([3H]17OHP4), non‐radioactive (nr) 17β‐oestradiol (nrE2) or nrP5 to identify the major gonadal steroidogenic pathways and steroid products in females and in the two male variants of this species, the terminal phase (TP) and initial phase (IP) males. Both testis and ovarian tissues exhibited 7 hydroxylase activity resulting in the formation of 7α‐hydroxypregnenolone (7OHP5) from [3H]P5, and many HPLC peaks were identified as products of testicular (c. 29) and ovarian (c. 23) steroidogenesis, and only c. 50% of these metabolites co‐eluted with authentic reference standards; only very small amounts of conjugated steroid were synthesized from any of the precursors. [3H]P5 was converted by testis mainly to 7αOHP5, and two unknown steroids, whereas [3H]17OHP4 metabolism gave rise to [3H]17,20β‐dihydroxy‐4‐pregnen‐3‐one (DHP), 11‐ketotestosterone (11KT), and two unknown steroids. For ovarian tissues, [3H]17OHP4 and [3H]P5 were metabolized to form E2, oestrone (E1), androstenedione (A4), 20α‐ and 20β‐dihydroprogesterone (20αDHP and 20βDHP), 7αOHP5 (from [3H]P5) and a major unknown. The HPLC steroid profiles for testis incubations for IP and TP males were similar, however, the steroidogenic response of the testis of TP males to human chorionic gonadotrophin, in vitro (determined by hormone assay), was significantly higher than that of IP males. 相似文献
18.
A.A. Licata W.Y. Au J. Vera F.C. Bartter 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,582(1):59-66
We investigated some effects of prostaglandin E1 on the metabolism of rat parathyroid glands using a culture system containing basal Eagle's medium supplemented with 5–10% heat-inactivated rat serum. Rat parathyroid glands incorporate [3H]fucose and 14C-labeled amino acids into cellular glycoproteins and secrete some of these into the culture medium. Gel filtration chromatography separates these glycoproteins into three classes, the smallest of which (peak 3) is secreted with immunoreactive parathyroid hormone. In cultures of 48 h, prostaglandin E1 (1 μg/ml) specifically inhibits the secretion of peak 3 and of parathyroid hormone but has no effect on the incorporation of [3H]-fucose, 14C-labeled amino acids, or [3H]uridine into parathyroid glands. Cytochalasin B inhibits the secretion of parathyroid hormone and the incroporation of isotopic fucose and amino acids. Cortisol stimulates incorporation of [3H]fucose and the secretion of parathyroid hormone even in the presence of inhibitory doses of prostaglandin E1. It is concluded that, in organ culture, prostaglandin E1 inhibits the secretion of parathyroid hormone and of a specific glycoprotein the function of which may be related to the secretion of the hormone. 相似文献
19.
A rapid method for the preparation of (125I)alpha-bungarotoxin 总被引:2,自引:0,他引:2
Electrofocusing followed by chromatography on Sephadex G-50 proved to be a fast method for the purification of the neurotoxin, α-bungarotoxin (α-BGT) from the venom of the krait (Bungarus multicinctus). The purity of the isolated α-BGT was checked by electrofocusing and ion-exchange chromatography. Blockade of the binding of [3H]ACh to the acetylcholine receptor in a membrane preparation of Torpedo electroplax was a convenient biochemical assay to identify the α-neurotoxin polypeptide. Iodination, accomplished by lactoperoxidase attached to Sepharose 4B, was simple and rapid, with a 60% recovery of [125I]α-BGT. Biological activity of the α-BGT was determined by its toxicity to mice as well as by autoradiography of [125I]α-BGT which proved its localization in the endplates of the mouse diaphragms. 相似文献
20.
The involvement of CL-proteins in the formation of lipoprotein A+ during adipokinetic hormone action has been investigated using radiolabelling experiments. Injected [3H]-CL-proteins associate rapidly with lipoprotein A+ during its formation. Both [3H]-CL-proteins and [3H]-Ayellow are liberated from [3H]-A+ during its natural degradation in the haemolymph (when adipokinetic hormone action is declining). It appears that [3H]-CL-proteins bind reversibly to A+, since they are easily displaced in vivo and in vitro by competing concentrations of non-labelled CL-proteins. It is suggested that Ayellow is an integral component of the A+ lipoprotein complex, whereas CL-proteins may play only a relatively minor part in its structural organisation. Possible functions of the binding of CL-proteins to A+ are discussed. 相似文献