Insulin-like growth factors I and II are unable to form and maintain their native disulfides under in vivo redox conditions.
Insulin-like growth factor (IGF) I does not quantitatively form its three native disulfide bonds in the presence of 10 mM reduced and 1 mM oxidized glutathione in vitro [Hober, S. et al. (1992) Biochemistry 31, 1749-1756]. In this paper, we show (i) that both IGF-I and IGF-II are unable to form and maintain their native disulfide bonds at redox conditions that are similar to the situation in the secretory vesicles in vivo and (ii) that the presence of protein disulfide isomerase does not overcome this problem. The results indicate that the previously described thermodynamic disulfide exchange folding problem of IGF-I in vitro is also present in vivo. Speculatively, we suggest that the thermodynamic disulfide exchange properties of IGF-I and II are biologically significant for inactivation of the unbound growth factors by disulfide exchange reactions to generate variants destined for rapid clearance. (+info)
Influence of a new antiulcer agent, ammonium 7-oxobicyclo (2, 2, 1) hept-5-ene-3-carbamoyl-2-carboxylate (KF-392) on gastric lesions and gastric mucosal barrier in rats.
Antiulcer effects of KF-392 were studied in several experimental gastric ulcer models in rats. It was found that KF-392 given orally at 1.0 to 5.0 mg/kg had a marked suppression on the developments of Shay ulcer as well as the aspirin-, stress-, and reserpine-induced gastric lesions. The influence of KF-392 on gastric mucosal barrier was also studied. A back diffusion of H+ into the gastric mucosa and a fall of transmucosal potential difference were induced with KF-392 given orally at the above mentioned doses. KF-392 given s.c. at 5.0 mg/kg showed no inhibition of Shay ulcer and no induction of back diffusion of H+ into the gastric mucosa. (+info)
Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles.
Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release. (+info)
Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation.
It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin. (+info)
A novel role for carbonic anhydrase: cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes.
1. The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton-coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy-SNARF-1 fluorescence in combination with whole-cell microspectrofluorimetry or confocal microscopy. 2. In Hepes-buffered Tyrode solution, application of the dipeptide Phe-Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole. 3. In the presence of CO2/HCO3--buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less. 4. Following application of the carbonic anhydrase inhibitor acetazolamide (100 microM) in the presence of CO2/HCO3- buffer, addition of Phe-Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole. 5. We conclude that acid influx due to proton-coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration. (+info)
Nonsaturable entry of neuropeptide Y into brain.
Neuropeptide Y (NPY) is found and is active both in the periphery and brain, but its crossing of the blood-brain barrier (BBB) in either direction has not been measured. We used multiple time-regression analysis to determine that radioactively labeled NPY injected intravenously entered the brain much faster than albumin, with an influx constant of 2.0 x 10(-4) ml. g. -1. min-1. However, this rate of entry was not significantly changed by injection of 10 microgram/mouse of excess NPY, by leptin, or by food deprivation. HPLC showed that most of the NPY entering the brain was intact, and capillary depletion with and without washout showed that the NPY did not remain bound to endothelial cells or associated with vascular elements. Perfusion in a blood-free solution eliminated binding to serum proteins as an explanation for the lack of saturation. Efflux of labeled NPY from the brain occurred at the same rate as albumin, reflecting the normal rate of reabsorption of cerebrospinal fluid. Thus NPY can readily enter the brain from blood by diffusion across the BBB. (+info)
A novel strategy for the preparation of liposomes: rapid solvent exchange.
During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed. (+info)
Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.
The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions. (+info)