排序方式: 共有16条查询结果,搜索用时 0 毫秒
1.
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes. 相似文献
2.
Kamerzell TJ Joshi SB McClean D Peplinskie L Toney K Papac D Li M Middaugh CR 《Protein science : a publication of the Protein Society》2007,16(6):1193-1203
The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed. 相似文献
3.
The most impressive regressions obtained to date with the use of chemotherapy are in metastatic choriocarcinoma of females. In the management of Hodgkin''s disease, leukemia, and lymphoma, chemotherapy is a useful and accepted form of therapy. In many cases radiation and chemotherapy are interdependent. In disseminated carcinomas arising in the lung, ovary or breast, there is less likelihood of significant improvement with the use of chemotherapy, but if the disease is not amenable to radiotherapy, useful palliation can be obtained at times.While newer chemotherapeutic agents for cancer, notably the pyrimidine analogues, have a broader spectrum of antitumor effects than others investigated earlier, they should be regarded as providing a valuable research stimulus in the continued search for more effective and less toxic agents. 相似文献
4.
Isaac M Slassi M Xin T Arora J O'Brien A Edwards L MacLean N Wilson J Demschyshyn L Labrie P Naismith A Maddaford S Papac D Harrison S Wang H Draper S Tehim A 《Bioorganic & medicinal chemistry letters》2003,13(24):4409-4413
A novel series of highly potent human 5-HT1D agonists, dimethyl-{2-[6-substituted-indol-1-yl]-ethyl}-amine, was synthesized. Structure–activity relationship (SAR) investigation revealed 4-[1-(2-dimethylamino-ethyl)-1H-indol-6-yl]-tetrahydro-thiopyran-4-ol, 11b (ALX-2732), as a potent (Ki=2.4 nM) agonist at the human 5-HT1D receptor with good selectivity over the other serotonin receptor subtypes. This compound demonstrated favorable in vitro metabolic stability in human and rat liver microsomes and was found to be orally bioavailable in rats (Fpo=51%). 相似文献
5.
D I Papac K R Thornburg E E Büllesbach R K Crouch D R Knapp 《The Journal of biological chemistry》1992,267(24):16889-16894
Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors. 相似文献
6.
Jones AJ Papac DI Chin EH Keck R Baughman SA Lin YS Kneer J Battersby JE 《Glycobiology》2007,17(5):529-540
To understand how the carbohydrate moieties of a recombinant glycoprotein affected its pharmacokinetic (PK) properties, the glycan distribution was directly assessed from serial blood samples taken during PK studies in cynomolgus monkeys and humans. The protein studied was an immunoadhesin (lenercept), containing an Fc domain from human immunoglobulin G (IgG-1) and two copies of the extensively glycosylated extra cellular domain of tumor necrosis factor receptor p55. The protein was recovered in pure form using a dual column, immunoaffinity-reversed-phase high-performance liquid chromatography method. The glycans were released and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Alternatively, trypsin was used to obtain glycopeptides, and these were analyzed by MALDI-TOF. The composition versus time profiles show that the distribution of glycans in the Fc domain was not altered over 10 days of circulation, consistent with their sequestration in the interior of the protein. However, the glycan composition in the receptor domain was changed dramatically in the first 24 h and then remained relatively constant. Analysis of the acidic glycans (derived exclusively from the receptor domain) showed that, in the rapid initial phase of clearance, glycans carrying terminal N-acetylglucosamine (tGlcNAc) were selectively cleared from the circulation. This phenomenon occurred similarly in humans and cynomolgus monkeys. Sialic acid content and terminal galactose showed only small changes. These data confirm the correlation of tGlcNAc and half-life of the molecule, and support the hypothesis that the mannose receptor (which can also bind tGlcNAc) causes the variable clearance of this molecule. 相似文献
7.
This report describes a convenient method for the rapid and efficient
release of N-linked oligosaccharides from low microgram amounts of
glycoproteins. A 96-well MultiScreen assay system containing a
polyvinylidene difluoride (PVDF) membrane is employed to immobilize
glycoproteins for subsequent enzymatic deglycosylation. Recombinant
tissue-type plasminogen activator (rt-PA) is used to demonstrate the
deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled
the recovery of a sufficient amount of N-linked oligosaccharides released
enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5
microgram rt-PA for subsequent analysis by matrix-assisted laser
desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The
immobilization of rt-PA to the PVDF membrane did not sterically inhibit the
PNGaseF-mediated release of oligosaccharides from rt-PA as determined by
tryptic mapping experiments. Comparison of the oligosaccharides released
from 50 micrograms of rt-PA by either the 96-well plate method or by a
standard solution digestion procedure showed no significant differences in
the profiles obtained by high-pH anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD). Both neutral and sialylated
oligosaccharide standards spiked into wells were recovered equally as
determined by HPAEC-PAD. One advantage of this approach is that reduction
and alkylation can be performed on submicrogram amounts of glycoproteins
with easy removal of reagents prior to PNGaseF digestion. In addition, this
method allows 60 glycoprotein samples to be deglycosylated in 1 day with
MALDI-TOF or HPAEC-PAD analysis being performed on the following day.
相似文献
8.
Vijay Kumar D Hoarau C Bursavich M Slattum P Gerrish D Yager K Saunders M Shenderovich M Roth BL McKinnon R Chan A Cimbora DM Bradford C Reeves L Patton S Papac DI Williams BL Carlson RO 《Bioorganic & medicinal chemistry letters》2012,22(13):4377-4385
Efforts to optimize biological activity, novelty, selectivity and oral bioavailability of Mps1 inhibitors, from a purine based lead MPI-0479605, are described in this Letter. Mps1 biochemical activity and cytotoxicity in HCT-116 cell line were improved. On-target activity confirmation via mechanism based G2/M escape assay was demonstrated. Physico-chemical and ADME properties were optimized to improve oral bioavailability in mouse. 相似文献
9.
Elevated ammonium concentrations in the medium of cultivated cells have
been shown to increase the intracellular levels of uridine-5'-diphospho-
N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N-
acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994). These sugar
nucleotides are substrates for glycosyltransferases in the glycosylation
pathway. In our experiments, recombinant Chinese hamster ovary cells
producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated
under controlled cell culture conditions in the presence of different
ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added
exogenously to the cell culture media to determine if ammonium was
incorporated into UDP-GlcNAc and cytidine-5'-
monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently
incorporated into GP1-IgG as N-linked glycans. The intracellular pools of
UDP-activated hexosamines (UDP-GNAc) were followed during the time course
of the experiment. To assess the extent of15NH4+incorporation into the
glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by
protein A chromatography. Enzymatically released N- glycans were then
analyzed by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry. N-Glycans synthesized in the presence of15NH4Cl revealed an
N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da. These results
indicate that 60-70% of the total nitrogen containing monosaccharides had
incorporated15N. Presumably,15NH4+was incorporated into GlcNAc and N-
acetylneuraminic acid as proposed earlier (Ryll et al., 1994). This might
be a universal and previously not described reaction in mammalian cells
when exposed to nonphysiological but in cell culture commonly found
concentrations of ammonium. The data presented here are of significance for
glycoprotein production in mammalian cell culture, since it has been shown
previously that elevated levels of UDP- activated hexosamines affect
N-glycan characteristics such as branching and degree of amino sugar
incorporation. In addition, our results demonstrate that isotope labeling
in combination with MALDI-TOF-MS can be used as an alternate tool to
radioactive labeling of sugar substrates in metabolic studies.
相似文献
10.
S Weikert D Papac J Briggs D Cowfer S Tom M Gawlitzek J Lofgren S Mehta V Chisholm N Modi S Eppler K Carroll S Chamow D Peers P Berman L Krummen 《Nature biotechnology》1999,17(11):1116-1121
We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns. 相似文献