scholarly journals Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

2012 ◽  
Vol 124 (2) ◽  
pp. 200-209 ◽  
Author(s):  
Kurt T. Laha ◽  
Phu N. Tran
Keyword(s):  
Gabaa Receptor ◽  
Binding Pocket ◽  
Surface Expression ◽  
Tyrosine Residues ◽  
Gaba Binding ◽  
Kinetics Of

scholarly journals Multiple Tyrosine Residues Contribute to GABA Binding in the GABAC Receptor Binding Pocket

2012 ◽  
Vol 3 (3) ◽  
pp. 186-192 ◽  
Author(s):  
Sarah C. R. Lummis ◽  
Neil J. Harrison ◽  
Jinti Wang ◽  
Jamie A. Ashby ◽  
Katherine S. Millen ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Pocket ◽  
Tyrosine Residues ◽  
Gabac Receptor ◽  
Gaba Binding

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide

1977 ◽  
Vol 55 (4) ◽  
pp. 424-432
Author(s):  
Bernard R. Glick ◽  
Lewis J. Brubacher
Keyword(s):  
Kinetic Studies ◽  
Peptide Inhibitors ◽  
Tryptophan Residue ◽  
Difference Spectroscopy ◽  
Tyrosine Residues ◽  
Residue Determination ◽  
Chain Cleavage ◽  
Modified Enzyme ◽  
Kinetics Of ◽  
Twofold Decrease

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine, (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH 6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate α-N-benzoyl-L-arginine ethyl ester were studied in the region of pH 3.5–9.0. All substrates (and inhibitors) tested, with the exception of α-N-benzoyl-L-arginine p-nitroanilide, displayed approximately a twofold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is modified is probably Trp-177.

Changes in lipophosphoglycan and gene expression associated with the development ofLeishmania majorinPhlebotomus papatasi

Parasitology ◽  
1995 ◽  
Vol 111 (3) ◽  
pp. 275-287 ◽  
Author(s):  
E. M. B. Saraiva ◽  
P. F. P. Pimenta ◽  
T. N. Brodin ◽  
E. Rowton ◽  
G. B. Modi ◽  
...  
Keyword(s):  
Differential Expression ◽  
Fold Increase ◽  
Surface Expression ◽  
Side Chain ◽  
Surface Coat ◽  
Natural Vector ◽  
Metacyclic Promastigotes ◽  
Kinetics Of

SUMMARYStage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course ofL. majorinfection in a natural vector,Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics invitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promatigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesisin vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression invitro, also showed selective expression by promastigotes in the fly, and was used inin situhybridization studies to demonstrate the positioning of metacyclics in the anterior gut.

PLASMINOGEN ACTIVATION BY SINGLE-CHAIN UROKINASE (scu-PA) IN FUNCTIONAL ISOLATION - DEMONSTRATION OF A NOVEL MECHANISM

1987 ◽  
Author(s):  
V Ellis ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Human Kidney ◽  
Binding Pocket ◽  
Moderate Activity ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Irreversible Inhibitor ◽  
Peptide Substrates ◽  
Sds Page ◽  
Catalytic Constant ◽  
Kinetics Of

The kinetics of the activation of Glu- and Lys- plasminogen by single-chain urokinase (pro-urokinase) derived from the transformed human kidney cell line, TCL-598, has been studied and compared with two-chain urokinase (UK). Plasminogen activation was determined by the change in fluorescence polarization of fluorescein-labelled aprotinin (Trasylol), an essentially irreversible inhibitor of plasmin. This methodology allows plasmin production by scu-PA to be measured in functional isolation, with no interfering generation of two-chain UK. scu-PA was found to activate plasminogen to plasmin with Michaelis-Menten type kinetics. The Km for this reaction was determined as 70µM, with a catalytic constant of 2.25 min-l. The generation of two-chain plasmin was confirmed by reduced SDS-PAGE. Plasminogen activation by UK was found to have a similar Km but the kcat was 16-fold higher, at 36.0 min-l. This is in contrast to the amidolytic activity of scu-PA which was less than 0.2% that of UK. The activation of scu-PA to UK by plasmin was also characterized. Using these data it is possible to calculate the theoretical rate of plasminogen activation by scu-PA, in the absence of aprotinin when UK will be generated by plasmin action. The calculated rate was in good agreement with that determined experimentally when using the chromogenic substrate, S-2251. These data demonstrate that scu-PA has properties which distinguish it from conventional serine protease zymogens. There is a lack of activity against peptide substrates (and also DFP) demonstrating the inaccessibility of the substrate binding pocket. However, there is moderate activity against plasminogen suggesting that plasminogen may be acting as both an effector and a substrate for scu-PA.

scholarly journals Invariant chain and DM edit self-peptide presentation by major histocompatibility complex (MHC) class II molecules.

1996 ◽  
Vol 184 (5) ◽  
pp. 1747-1753 ◽  
Author(s):  
J F Katz ◽  
C Stebbins ◽  
E Appella ◽  
A J Sant
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
Mhc Class Ii ◽  
Binding Pocket ◽  
Surface Expression ◽  
Class Ii ◽  
Invariant Chain ◽  
Generalized Function ◽  
Major Histocompatibility ◽  
Histocompatibility Complex

We have studied the consequences of invariant chain (Ii) and DM expression on major histocompatibility complex (MHC) class II function. Ii has a number of discrete functions in the biology of class II, including competitive blocking of peptide binding in the endoplasmic reticulum and enhancing localization in the endocytic compartments. DM is thought to act primarily in endosomes to promote dissociation of the Ii-derived (CLIP) peptide from the class II antigen-binding pocket and subsequent peptide loading. In this study, we have evaluated the functional role of Ii and DM by examining their impact on surface expression of epitopes recognized by a large panel of alloreactive T cells. We find most epitopes studied are influenced by both Ii and DM. Most strikingly, we find that surface expression of a significant fraction of peptide-class II complexes is extinguished, rather than enhanced, by DM expression within the APC. The epitopes antagonized by DM do not appear to be specific for CLIP. Finally, we found that DM was also able to extinguish recognition of a defined peptide derived from the internally synthesized H-2Ld protein. Thus, rather than primarily serving in the removal of CLIP, DM may have a more generalized function of editing the array of peptides that are presented by class II. This editing can be either positive or negative, suggesting that DM plays a specifying role in the display of peptides presented to CD4 T cells.

Kinetics of CXCR4 and CCR5 up-regulation and human immunodeficiency virus expansion after antigenic stimulation of primary CD4+ T lymphocytes

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1853-1856
Author(s):  
Reinhard Maier ◽  
Marı́a Matilde Bartolomé-Rodrı́guez ◽  
Corinne Moulon ◽  
Hans Ulrich Weltzien ◽  
Andreas Meyerhans
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Lymphocytes ◽  
Chemokine Receptors ◽  
Surface Expression ◽  
Antigenic Stimulation ◽  
Productive Infection ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Stimulation Of

The chemokine receptors CCR5 and CXCR4 are coreceptors for the human immunodeficiency virus (HIV) and determine the cell tropism of different HIV strains. Previous studies on their regulation were performed under conditions of unspecific T-lymphocyte stimulation and provided conflicting results. To mimick physiologic conditions, highly purified primary Staphylococcus enterotoxin B (SEB)-reactive CD4 T lymphocytes were stimulated in the presence of autologous antigen-presenting cells and the kinetics of CCR5 and CXCR4 surface expression and HIV replication were studied. Both chemokine receptors were transiently up-regulated with maximal expression at day 3 after stimulation. The stimulated T cells were equally susceptible to productive infection with R5-and X4-tropic virus strains. Thus, antigenic stimulation of T cells promotes efficient replication of both, T cell-tropic and macrophage-tropic HIV.

Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

1972 ◽  
Vol 50 (12) ◽  
pp. 1282-1296 ◽  
Author(s):  
A. Kurosky ◽  
T. Hofmann
Keyword(s):  
Nitrous Acid ◽  
Conformational State ◽  
Serine Proteinases ◽  
Amino Groups ◽  
Model Compounds ◽  
Third Order ◽  
Tyrosine Residues ◽  
Reactive Groups ◽  
Primary Amino ◽  
Kinetics Of

The kinetics of the reaction of nitrous acid at 4° and pH 4.0 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the HONO concentration showing that N2O3 was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pK values. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate of nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by spectrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for α-amino groups, ε-amino groups, and other residues in proteins were compared. The conformational state of the N-terminal residues in serine proteinases, as revealed from their reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially α-amino groups and indole rings.

scholarly journals Phosphorylation and internalization of gp130 occur after IL-6 activation of Jak2 kinase in hepatocytes.

1994 ◽  
Vol 5 (7) ◽  
pp. 819-828 ◽  
Author(s):  
Y Wang ◽  
G M Fuller
Keyword(s):  
Protein Synthesis ◽  
Cell Surface ◽  
De Novo ◽  
Cell Types ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Protein Synthesis Inhibitors ◽  
Different Cell Types ◽  
Kinetics Of

Recent evidence has shown that members of the Jak kinase family are activated after IL-6 binds to its receptor complex, leading to a tyrosine phosphorylation of gp130, the IL-6 signal-transducing subunit. The different members of the IL-6 cytokine subfamily induce distinct patterns of Jak-Tyk phosphorylation in different cell types. Using monospecific antibodies to gp130, Jak2 kinase, and phosphotyrosine, we investigated the kinetics of IL-6 stimulation of members of this pathway in primary hepatocytes. Our findings show that Jak 2 is maximally activated within 2 min of exposure to IL-6, followed by gp130 phosphorylation that reaches its peak in another 2 min then declines to basal level by 60 min. In vitro phosphorylation experiments show that activated Jak 2 is able to phosphorylate both native gp130 and a fusion peptide containing its cytoplasmic domain, demonstrating gp130 is a direct substrate of Jak 2 kinase. Experiments designed to explore the cell surface expression of gp130 show that > or = 2 h are required to get a second round of phosphorylation after the addition of more cytokines. This finding suggests that activated gp130 is internalized from the cell surface after IL-6 stimulation. Additional experiments using protein synthesis inhibitors reveal that new protein synthesis is required to get a second cycle of gp130 phosphorylation indicating gp130 must be synthesized de novo and inserted into the membrane. These findings provide strong evidence that down regulation of the IL-6 signal in hepatocytes involves the internalization and cytosol degradation of gp130.

Synthetic site-directed ligands

1989 ◽  
Vol 323 (1217) ◽  
pp. 495-509 ◽  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Opioid Peptides ◽  
Binding Pocket ◽  
Tyrosine Residues ◽  
Chemotactic Peptides ◽  
Binding Cavity ◽  
False Floor ◽  
Type Site ◽  
Main Cavity

Complexes of nucleotides, peptides and arom atic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. Alter tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04- 01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the ‘native’ protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Meg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the ε-DNP ring near the floor of the main cavity and the α-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with both DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Meg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.

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