Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of alcohol on albumin synthesis was studied in the isolated perfused rabbit liver. Carbonate-(14)C was used to label the intracellular arginine pool which serves as the precursor of both the carbon of urea and the guanido carbon of arginine in albumin. The control group synthesized albumin at a rate of 33 mg/100 g of wet liver weight during 2.5 hr of perfusion. When alcohol, 220 mg/100 ml, was added to the perfusate, albumin synthesis decreased to between 7 and 11 mg, less than one-third the control rate. The addition of 10 mM tryptophan to perfusates containing alcohol prevented most of the inhibitory effects and albumin synthesis increased to average 24 mg. Further, the addition of alcohol to the perfusate decreased the hepatic protein/DNA ratio from 70 to 54 and the RNA/DNA ratio from 2.3 to 1.8, changes equivalent to those seen after a 24 hr fast. The addition of tryptophan to the perfusate prevented these findings in both instances. Endoplasmic membrane-bound polysomes were examined for aggregation. Alcohol decreased the quantity of heavier aggregates. Reaggregation occurred when tryptophan was added but quantitative changes in albumin synthesis could not be related to the degree of reaggregation.
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PMID:Alcohol-induced depression of albumin synthesis: reversal by tryptophan. 556 88

Beta-2 microglobulin (beta 2M) is a 12,000 dalton protein associated with membrane-bound cell surface antigens. Variants of beta 2M, beta 2MA and beta 2MB, were first detected by Michaelson et al. (Immunogenetics 11, 93-95, 1980). An improved method was used to purify beta 2MA and beta 2MB from BALB/c and C57BL/6 mouse livers, respectively. Reproducible yields of 10% were obtained. The purifications were accomplished by a 3 M sodium thiocyanate (NaSCN) extraction of a crude membrane fraction, an acid precipitation step, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose and CM-cellulose in that order. The elution profile of beta 2MA and beta 2MB on the ion-exchange columns was found to be different, indicating the presence of structural changes. beta 2MA was found to be more acidic (pI = 7.35) than beta 2MB (pI = 7.68) by isoelectric focusing in gels. Complete sequence analysis of beta 2MA and partial sequence analysis of beta 2MB (61 of 99 residues) were performed by automated Edman degradation of the intact chain and of the overlapping peptides obtained by: (a) tryptic cleavage at arginines after acetimidation of lysine side chains, (b) BNPS-skatole cleavage at tryptophan residues and (c) hydroxylamine cleavage at asparagine-glycine linkages. A comparison of the primary structure of beta 2MA to the partial amino acid sequence obtained for beta 2MB revealed a single amino acid substitution (aspartic acid for alanine at position 85) that accounts for the differences in biochemical properties observed.
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PMID:Purification and characterization of mouse beta-2 microglobulin: allelic variants from two different strains. 617 66

The transport inhibitor, eosin 5-maleimide, reacts specifically at an external site on the membrane-bound domain of the anion exchange protein, Band 3, in the human erythrocyte membrane. The fluorescence of eosin-labeled resealed ghosts or intact cells was found to be resistant to quenching by CsCl, whereas the fluorescence of labeled inside-out vesicles was quenched by about 27% at saturating CsCl concentrations. Since both Cs+ and eosin maleimide were found to be impermeable to the red cell membrane and the vesicles were sealed, these results indicate that after binding of the eosin maleimide at the external transport site of Band 3, the inhibitor becomes exposed to ions on the cytoplasmic surface. The lifetime of the bound eosin maleimide was determined to be 3 ns both in the absence and presence of CsCl, suggesting that quenching is by a static rather than a dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence of erythrocyte membranes was also investigated using anion transport inhibitors which do not appreciably absorb light at 335 nm. Eosin maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence. Covalent labeling of red cells by either eosin maleimide or BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused an increase in the susceptibility of membrane tryptophan fluorescence to quenching by CsCl. The quenching constant was similar to that for the quenching of eosin fluorescence and was unperturbed by the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced a large change in the relative magnitude of the tryptophan emission. The tryptophan residues that can be quenched by CsCl appear to be different from those quenched by eosin or BIDS and are possibly located on the cytoplasmic domain of Band 3. The results suggest that a conformational change in the Band 3 protein accompanies the binding of certain anion transport inhibitors to the external transport site of Band 3 and that the inhibitors become exposed on the cytoplasmic side of the red cell membrane.
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PMID:Evidence that inhibitors of anion exchange induce a transmembrane conformational change in band 3. 618 90

The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.
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PMID:The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements. 621 52

Infrared and tryptophan fluorescence spectra of practically all sufficiently stable functional complexes of a highly purified preparation of membrane-bound (Na+, K+)-dependent ATPase have been measured. The formation of any functional complex was not accompanied by any considerable change of either shape or position of the tryptophan fluorescence spectrum. Only in the presence of adenine nucleotides was there a small decrease of fluorescence intensity (by 5-8%), which apparently results from a change of the sample light scattering. Analysis of the results obtained leads to the conclusion that the environment of no more than one or a few tryptophan residues may differ in all the (Na+, K+)-ATPase complexes studies. A comparison of infrared protein spectra in the region of amide I band showed that at any wavenumber the differences between them did not exceed 3% of the maximum absorption. This means that no more than 3% of protein peptide groups can change their conformation upon transition between the enzyme functional states. These results, obtained by two independent techniques, allow us to conclude that even if changes of the internal protein structure occur during the working cycle of this transport system, if they have an extremely local character.
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PMID:Lack of gross protein structure changes in the working cycle of (Na+, K+)-dependent adenosinetriphosphatase. Evidence from infrared and intrinsic fluorescence spectroscopy data. 625 Aug 25

A new method is described to evaluate contact between fluorophors and lipids in model membranes. This method utilizes a nitroxide spin-labeled phosphatidylcholine to quench th fluorescence from a variety of membrane-bound molecules by a static process. The distance dependence of the fluorescence quenching is analyzed in terms of simple models. The analysis shows that quenching of diphenylhexatriene, p-terphenyl, and molecules containing tryptophan arises only from spin-labeled phospholipid that is in contact with the fluorophor.
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PMID:Fluorescence quenching in model membranes. 1. Characterization of quenching caused by a spin-labeled phospholipid. 626 7

The nature of the propranolol binding site of the beta-adrenergic receptor has been examined by utilizing the intrinsic fluorescence of propranolol as a probe. Additionally, the spatial relationship between the propranolol binding site and membrane tryptophan has been examined by utilizing I-quenching of intrinsic tryptophan fluorescence, chemical modification of membrane tryptophan, and singlet-singlet energy transfer between membrane-bound propranolol and tryptophan. Propranolol, at concentrations consistent with specific beta-receptor binding, protected approximately 42% of the membrane tryptophan fluorescence from I-quenching. Further, in the presence of propranolol, the apparent quenching constant (kq) was altered from 3.6 to 21.8 M-1. Reaction of the membrane fragments with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent I) in the presence and absence of propranolol indicated that low concentrations of propranolol protected approximately 45% of the membrane tryptophan from the reagent. The singlet-singlet energy transfer from tryptophan to propranolol was determined by sensitized emission. The distance between these two species was found to be less than 20 A. These results have been interpreted to indicate that propranolol, when bound to the beta-adrenergic receptor, is situated such that its naphthyl moiety is inserted into a tryptophan-rich hydrophobic pocket of the receptor.
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PMID:Fluorescence studies of the beta-adrenergic receptor topology. 626 86

Degradation of CCK-4 and -8 by purified synaptic membranes was followed by measuring the fluorescence of tryptophan released from the peptides after separation of degradation products by HPLC. For enkephalins and related fragments, the release of tyrosine was monitored using the same method. Kinetics of hydrolysis of CCK-like peptides indicated a rapid processing of CCK-4 and a slower breakdown of CCK-8 (with a greater resistance of the sulfated form of CCK-8 as compared to the unsulfated peptide). Leu- and met-enkephalins were degraded at the same rate while their N-terminal tri- and dipeptides were hydrolysed more slowly. When CCK-4 or CCK-8 were incubated in the presence of leu-enkephalin, a dose-dependent inhibition of the release of tryptophan was observed. Enkaphalin fragments do not modify the kinetics of degradation of CCK-4. The degradation of leu-enkephalin was inhibited in a dose-dependent manner by the presence of CCK-related peptides in the medium. After solubilization of membrane-bound enzymes by Triton X-100 followed by chromatography on DEAE cellulose, five peaks of CCK-4 degrading activity were detected (two minor and three major peaks). With enkephalin as substrate, five peaks were also observed; the three major activities were the same as those detected for CCK-4.
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PMID:Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. I. Evidence for competition with enkephalins for in vitro common degradation pathways. 628 89

Fluorescence quenching is the loss of fluorescence intensity which is observed when a fluorescent molecule or group interacts with another molecule or group, called the quencher. By use of tryptophan residues of proteins, together with specific probe molecules, quenching can be applied to problems of biological and model membrane structure. Quenching interactions are short range (less than 50 A) so that structure on the scale of molecular dimensions can be examined. This review summarizes the recent applications of fluorescence quenching by spin (nitroxide)-labeled molecules to problems of membrane structure, including determination of the distance of membrane-bound molecules from the membrane surface, the strength of lipid-protein interactions and the strength of protein-protein interactions within membranes. The unique advantages and the limitations of this powerful method are examined.
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PMID:Investigation of membrane structure using fluorescence quenching by spin-labels. A review of recent studies. 628 77

Fructose 1,6-bisphosphate aldolase [EC 4.1.2.13] in rat liver was found to be bound to the intracellular membraneous structures such as microsomes and nuclear membranes when the animals were fasted for 48 hr or administered tryptophan. Upon refeeding the rats the aldolase was released into the cytosol. The membrane-bound aldolase was almost inactive, showing about 50-fold larger Km and a smaller Vmax (37%) as compared with those of the free enzyme. The enzyme was released cooperatively from the membrane by exposure to fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate or dihydroxyacetone phosphate at low concentrations. Apparent desorption constants (Kd, concentrations necessary for 50% desorption of enzyme) for fructose 1,6-bisphosphate of the enzymes bound to microsomes, mitochondria and nuclei were estimated to be 8 X 10(-5), 6.1 X 10(-6), and 4.8 X 10(-6)M, respectively, at pH 7.3. With the microsome-bound enzyme Kd values of 3.9 X 10(-4), 4.1 X 10(-4), 2.7 X 10(-3), 1.1 X 10(-2) and 2.0 X 10(-2) M were obtained for glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, fructose 1-phosphate, fumarate, and KCl, respectively. Strong cooperativities were observed in the enzyme desorption by the substances which showed large Kd values.
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PMID:Membrane-bound fructose 1,6-bisphosphate aldolase: catalytic activity and mechanisms of desorption. 738 24


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