Biomedical subjects

M Criado

Publications and source records attributed to M Criado.

At least 73 records · Page 4Linked to original sources

PubMed1982

Effects of lipids on acetylcholine receptor. Essential need of cholesterol for maintenance of agonist-induced state transitions in lipid vesicles.

The effects of lipids on the maintenance of characteristic functional properties of the acetylcholine receptor during the course of reconstitution into lipid vesicles were studied by following the kinetics of agonist-induced state transitions. The requirements for successful preservation of these properties could be dissected into two components: (a) adequate nature and concentration of lipids during detergent solubilization; (b) correct lipid environment during reincorporation into lipid vesicles by the cholate dialysis procedure. Optimal bulk lipid concentrations and lipid:cholate ratios for preserving state transitions during solubilization were studied by using both crude soybean lipids and pure synthetic phospholipids. The latter class of lipids was found to be unsuitable substitutes for the crude soybean lipids, irrespective of their polar head group and/or fatty acyl chain, even when detergent:lipid ratios as high as 1:1 (w/w) were employed. Addition of cholesteryl hemisuccinate was able to make up this deficiency, attaining preservation of acetylcholine receptor state transitions at cholate:steroid ratios of about 6:1 (w/w). The presence of steroid decreased the amount of protein solubilized. The correct choice of lipid type was also essential to the reincorporation step, and higher concentrations of lipid were required--about 20 mg/mL for soybean lipids. Pure phospholipids at similar concentrations, however, were unable to maintain the state transitions. Again, steroid (40-46% cholesteryl hemisuccinate/mol of total lipid) provided the adequate conditions at the reincorporation stage and enhanced the amount of protein reincorporated into the vesicles. A large (70-90%) percentage of the receptor was reincorporated with the correct vectorial sidedness. No specificity could be detected for the phospholipid polar head or alkyl chain in relation to any of these findings. The effect of the protein on the physical state of the lipids in the reconstituted vesicles was studied by diphenylhexatriene fluorescence depolarization. The results may be interpreted as a disordering of the acyl chains in the gel state and an ordering in the liquid-crystalline state in the presence of protein, accompanied by shifts in the transition temperatures of the pure phospholipids to lower values.

Animals↗

PubMed1982

Purification and chemical characterization of a W2 protein from brain myelin.

Starting from a pellet of beef brain myelin insoluble in chloroform/methanol (2:1, vol/vol)(Wolfgram protein fraction), a pure W2 protein with apparent molecular weight of 52,000 was isolated by a simple preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. A comparative chemical analysis was carried out between purified W2 and a standard tubulin. Glutamic acid and arginine were the N-terminals detected. Similar peptide maps and amino acid composition were also found in both proteins. Immunological cross-reactivity was detected when W2 protein was tested against antitubulin serum. These results suggest that W2 protein could have a tubulin-like protein nature that is associated with the myelin membrane and could play a role in the myelination process.

Amino Acids↗

PubMed1982

Method for lyophilizing brain proteolipid preparations that increases subsequent solubilization by detergents.

A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X-100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X-100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed.

Animals↗

PubMed1981

Action of detergents and pre- and postsynaptic localization of 3H-naloxone binding in synaptosomal membranes. A structural approach.

3H-naloxone specific binding was carried out on synaptosomal membranes isolated from basal ganglia of the cat brain. A high- and a low-affinity site with Kd1 = 3.7 nM and Kd2 = 35 nM having B max 1 = 79 pmole/g protein and B max 2 = 224 pmole/g protein were found. The Hill number for the high- and low-affinity sites were, respectively, 1.01 and 0.86. Digitonin and Triton X-100 had an inhibitory effect on the binding at concentrations between 10(-5) and 10(-1)% (w/v). Deoxycholate and Nonidet P-40 also inhibited the binding of 3H-naloxone, but at 10(-4)% produced a 50% enhancement. After the binding to membranes, the 3H-naloxone receptor complex is stable to the action of Triton X-100 and dissociates slowly. In membranes bound with 10 nM 3H-naloxone and then submitted to 0.1-0.2% Triton X-100, in which only the presynaptic membrane disintegrates, the specific radioactivity is decreased. With a more drastic treatment that disintegrates the postsynaptic membrane, the 3H-naloxone binding to synaptosomal membranes is almost completely abolished. These results suggest that opiate receptors may be localized both pre- and postsynaptically in central synapses.

Animals↗

PubMed1981

Blood levels of middle molecules and their effects on motor nerve conduction velocity.

This paper presents the data from 53 patients with end stage renal disease and under regular dialysis treatment. The haemodialysis scheme consists fundamentally in short dialysis with coil dialyzers. The correlations between motor nerve conduction velocity and the patients' residual renal function, HD dose, and serum level of middle molecules are analyzed. At the same time, the influence of RRF and HH dose on the serum level of MM is studied. From the assembled data, several deductions can be established: Uremic neuropathy is fundamentally influenced by the MM and the RRF; the HD dose also exerts some influence on the MNCV, but to a much lesser degree and through dialysables substances different from the MM; the influence of RRF on the MNCV is mainly due to the modification induced by this variable in the serum level of MM.

Humans↗

PubMed1980

Inhibition by local anesthetics, phentolamine and propranolol of [3H]quinuclydinyl benzylate binding to central muscarinic receptors.

[3H]Quinuclydinyl benzylate ([3H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with KD 0.25 nM, Hill number 0.89 and Bmax 114 pmol/g protein. The local anesthetics procaine, tetracaine and dibucaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 microM, inhibited [3H]QNB binding with Ki varying between 9 microM for procaine and 80 microM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [3H]QNB binding sites. Neither 1 mM Ca2+ nor Mg2+ affected [3H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [3H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action and local anesthetics on synaptic transmission is discussed.

Anesthetics, Local↗

PubMed1980

Protection by atropine of the inhibition caused by Triton X-100 on central muscarinic receptors.

Synaptosomal membranes from cat cerebral cortex were labelled with [3H]-quinuclidinyl benzylate ([3H]QNB). There was shown to be a single type of binding site with Kd = 0.34 nM, Bmax = 2.2 nmol/g protein and Hill No. = 1.01. Triton X-100 at 5 x 10(-4)% inhibited the specific binding of [3H]QNB and the inhibition was almost complete at 10(-2)%. Treatment with 2.5 x 10(-6) M atropine, followed by centrifugation washings protected the receptor site from the inhibitory action of the detergent. The protection afforded by other cholinergic drugs was less effective. The use of this technique has confirmed the results of our previous work on the possible pre- and postsynaptic location of central muscarinic receptors. These findings open the possibility for protection of other detergent-sensitive receptors and for their isolation and purification as well-defined macromolecules.

Animals↗

PubMed1980

Action of detergents on 3H-dihydroergokriptine binding and localization of alpha-adrenoceptors in synaptosomal membranes.

3H-dihydroergokriptine (3H-DHE) binding was carried out in synaptosomal membranes from basal ganglia of the cat. A single type of binding site with Kd 3.7 nM, Hill number = 0.95 and Bmax = 1000 pmol/g protein was found. 3H-DHE bound to alpha-adrenoceptors and not to serotonin or dopamine receptors. At very low concentrations, some detergents enhanced binding, but at higher concentrations of those used (Triton X-100, Nonidet P-40, deoxycholate and digitonin), inhibited the binding of 3H-DHE. After binding to the membrane protein, the 3H-DHE-receptor complex was stable to the action of Triton X-100. At concentrations of Triton X-100. At concentrations of Triton X-100 (0.1--0.2%). in which only the presynaptic membrane disintegrated, the 3H-HDE specific radioactivity was reduced. With a more drastic treatment that disintegrated the postsynaptic membrane, 3H-DHE binding was further reduced. These results suggest that alpha-adrenergic receptors may be localized at both the pre- and postsynaptic membranes of central synapses.

Animals↗

PubMed1980

The use of p-toluene sulfonate to dissolve synaptosomal membrane proteins into organic solvents.

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Amino Acids↗

PubMed1980

Cholinergic binding to the receptor proteolipid from Torpedo electroplax separated by ion exchange chromatography.

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Acetylcholine↗

PubMed1979

Pre- and postsynaptic localization of central muscarinic receptors.

3H-Quinuclidinyl benzylate and dimethyl 14C-d-tubocurarine were used to localize muscarinic and nicotinic receptors in synaptosomal membranes of the rat cerebral cortex. The results obtained, after the action of different concentrations of Triton X-100, suggest that, while nicotinic receptors are postsynaptic, the muscarinic ones are both pre- and postsynaptic.

Animals↗

PubMed1973