Determination of the number and relative position of tryptophan residues in various albumins.
(17/3557)
A technique is described by which both the numbers of tryptophan residues and their approximate locations in the peptide chain of a protein can be determined by cleavage with N-bromosuccinimide followed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The number of new peptide bands appearing in the gel is a function of the number of tryptophan residues, and the relative migration of the bands permits calculation of peptide molecular weights and an estimation of the positions of the tryptophan residues in the peptide chain. The technique uses a sample of about 0.5 mg and is suitable for any protein that contains a small number of tryptophan residues. These are the very specimens that are difficult to assay accurately for tryptophan by spectrophotometric or colorimetric methods. Tryptophan residues which are within about 20 residues of the ends of the peptide chain or of each other would not be detected. The specificity of the cleavage with N-bromosuccinimide was ascertained by utilizing human serum albumin, which is known to have a single tryptophan residue at position 214. The technique was then applied to a comparative study of the numbers and locations of tryptophans in the serum albumins of 16 species, namely 11 mammals, three birds and two amphibians. The number of tryptophan residues were confirmed by an independent colorimetric method. All of the mammalian albumins contained a tryptophan residue near position 213. The three avian albumins examined have no tryptophan. Frog and toad albumins contained two tryptophan residues, which appear to be situated at different positions from those in mammalian albumins. (+info)
The synthesis and hydrolysis of long-chain fatty acyl-coenzyme A thioesters by soluble and microsomal fractions from the brain of the developing rat.
(18/3557)
1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids. (+info)
Evidence for the involvement of IgE-basophil system in acute serum sickness.
(19/3557)
The role of the basophils in acute serum sickness of rabbits was examined by monitoring daily the absolute number of basophils before, during and after the disease period. After antigen (bovine serum albumin, BSA) elimination, levels of serum IgE and in vitro basophil degranulation in the presence of BSA were determined. The results showed that the onset of glomerular lesions depends upon the simultaneous occurrence of circulating immune complexes greater than 19 S and of an in vivo basophil depletion--probably equivalent to degranulation--reaching 70% of the pre-disease number. Post-disease antigen-dependent in vitro degranulation of the basophils and levels of serum IgE anti BSA did not prove to be good indexes of basophil sensitization. Our data suggest that basophils are instrumental at early stages of the deposition of immune complexes, most probably through their sensitization by membrane-bound IgE antibodies. (+info)
A quantitative study of pinocytosis and lysosome function in experimentally induced lysosomal storage.
(20/3557)
The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis. (+info)
Interaction of peroxynitrite and hydrogen peroxide with dinitrosyl iron complexes containing thiol ligands in vitro.
(21/3557)
The interaction of peroxynitrite with thiolate dinitrosyl iron complexes (DNIC) has been examined and compared with the interaction with H2O2. Peroxynitrite oxidized DNIC containing various thiolate ligands--cysteine, glutathione, and bovine serum albumin. Analysis of the oxidation suggested a two-electron reaction and gave third-order rate constants of (9.3 +/- 0.5).109 M-2.sec-1 for DNIC with BSA, (4.0 +/- 0.3).108 M-2.sec-1 for DNIC with cysteine, and (1. 8 +/- 0.3).107 M-2.sec-1 for DNIC with glutathione at 20 degrees C and pH 7.6. Peroxynitrite was more reactive towards DNIC than towards sulfhydryls. Addition of sodium dithionite after the reaction led to significant restoration of the EPR signal of DNIC with cysteine. The reaction of glutathione DNIC with H2O2 was about 600 times slower than with ONOO- and not reversed by sodium dithionite. Thus peroxynitrite, in contrast to hydrogen peroxide, changes the pool of nitrosocompounds which can be responsible for interconversion, storage, and transportation of nitric oxide in vivo. (+info)
The potent platelet inhibitory effects of S-nitrosated albumin coating of artificial surfaces.
(22/3557)
OBJECTIVES: We studied the antithrombotic effect of coating glass, collagen and metal stent surfaces with bovine serum albumin (BSA) covalently modified to carry S-NO functional groups denoted (pS-NO-BSA). METHODS: Video-enhanced light microscopy was used to visualize canine blood platelet adhesion and aggregation in a parallel plate glass chamber. Platelet adhesion was observed for 60 min on glass, glass coated with BSA, glass coated with pS-NO-BSA, collagen I (CO) surface, CO coated with BSA and CO coated with pS-NO-BSA. We also coated Palmaz-Shatz (P-S) stents with pS-NO-BSA. Coated and uncoated stents were then immersed in porcine platelet-rich plasma for two min and the platelet cyclic GMP level was measured. In six anesthetized pigs, coated and uncoated stents were placed in the carotid arteries and [111In]-labeled platelets were circulated for 2 h. The stented arteries were then removed and placed in a gamma well counter. RESULTS: There was significantly less platelet attachment, adhesion and aggregation on the pS-NO-BSA coated surfaces compared with the BSA coated and uncoated surfaces. The pS-NO-BSA coating increased the platelet cGMP levels to 5.9+/-0.7 pmoles/10(8) platelets compared with 2.7+/-0.9 pmoles/10(8) platelets for control (p < 0.01). The average gamma ray count from [111In]-labeled platelets that attached to the coated stents was 90,000+/-42,000/min and 435,000+/-290,000/min for the uncoated stents (p < 0.01). CONCLUSIONS: The pS-NO-BSA coating of thrombogenic surfaces reduces platelet adhesion and aggregation, possibly by increasing the platelet cGMP. This inhibitory effect appears to be a consequence of the direct antiplatelet actions of NO combined with the antiadhesive properties of albumin. (+info)
MHC class I-restricted presentation of maleylated protein binding to scavenger receptors.
(23/3557)
Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs. (+info)
Liver uptake and hepato-biliary transfer of galactosylated proteins in rats are determined by the extent of galactosylation.
(24/3557)
The effect of molecular mass and surface density of galactose residues on hepatic uptake and subsequent biliary excretion of galactosylated proteins was investigated in rats. Several proteins with different molecular weights (15-70 kDa) and different numbers of galactose units were synthesized and radiolabeled with 111In. Galactosylated proteins were administered i.v. to anaesthetized rats and samples of plasma and bile were collected for 3 h. Liver was harvested at the end of the experiments and the radioactivity of all samples was measured. Galactosylated proteins accumulated primarily in the liver and 2-10% of the administered dose appeared in the bile, mainly in undegraded form. The hepatic uptake clearance (Cl liver) and biliary excretion rate constant (kbile) of galactosylated proteins were calculated. No direct effect of molecular weight was observed, however, on increasing the galactose density, Cl liver increased from about 4 to 400 ml/h whereas kbile gradually decreased from about 0.057 to 0.007 (h-1). In conclusion, both hepatic uptake and biliary excretion of galactosylated proteins were found to be affected by the extent of galactosylation. (+info)