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G Benga

Publications and source records attributed to G Benga.

At least 19 recordsLinked to original sources
PubMed1996

Quantitation of the water channel protein aquaporin (CHIP28) from red blood cell membranes by densitometry of silver stained polyacrylamide gels.

A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.

Acrylic Resins↗
PubMed1995

A Fourier-transform infrared spectroscopic investigation of the hydrogen-deuterium exchange and secondary structure of the 28-kDa channel-forming integral membrane protein (CHIP28).

Fourier-transform infrared spectroscopy (FTIR) has been employed to investigate the structural properties of the 28-kDa channel-forming integral membrane protein (CHIP28) present in phospholipid vesicles suspended in aqueous media. This study reports the FTIR spectra of this membrane protein present in H2O and 2H2O. The secondary structure of the protein was determined and found to consist of 36% alpha-helical and 42% beta-sheet structures. These results are in close agreement with the results of a previous CD study [Van Hoek, A. N., Wiener, M., Bicknese, S., Miercke, L., Biwersi, J. & Verkman, A. S. (1993) Biochemistry 32, 11,847-11,856]. However, the results differ from those given in an FTIR analysis by the same workers who recorded FTIR spectra of the CHIP28 protein in a dehydrated state. An unusually high extent of hydrogen-deuterium exchange of the peptide groups of this protein occurs. The magnitude of the spectral changes observed upon exposure of the protein to 2H2O is greater than has been observed with any other membrane protein previously studied. Thus, over 80% of the peptide groups exchange within 5 min and the amide I band maximum shifts to low frequency by approximately 20 cm-1. This high hydrogen-deuterium exchange observed with the CHIP28 protein is consistent with the presence of an aqueous pore within the protein structure.

Aquaporin 1↗
PubMed1995

Amino acid composition of rat and human liver microsomes in normal and pathological conditions.

The amino acid composition of proteins from liver microsomes has been studied in rats and in human subjects with normal liver, with obstructive jaundice or liver cirrhosis. The pattern of the amino acid composition of microsomes appeared to be species-specific. Phenylalanine, threonine, serine, proline, histidine and [aspartic acid plus asparagine] were increased, while alanine, tyrosine, glycine and arginine were decreased in the human compared to the rat microsomes. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in leucine and phenylalanine could be noticed, while in the case of liver cirrhosis amino acid composition was markedly changed.

Amino Acids↗
PubMed1995

Diffusional water permeability of mammalian red blood cells.

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (Pd) and of the activation energy (Ea,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of Pd, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and "large" kangaroos (Macropus giganteus and Macropus rufus) had Pd values approximately 5 x 10(-3) cm/s at 25 degrees and 7 x 10(-3) cm/s at 37 degrees C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had Pd values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of Ea,d was in most cases correlated with the values of Pd. A value of Ea,d approximately 26 kJ/mol was found for the RBCs from humans and the species having similar Pd values. Low values of Ea,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of Pd. The highest values of Ea,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of Ea,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that Pd in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.

Animals↗
PubMed1994

Water channels in membranes.

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (Ea,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had Pd values around 5 x 10(-3) cm/s at 25 degrees C, 7 x 10(-3) cm/s at 37 degrees C with Ea,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the Pd values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of Ea,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylamide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.

Animals↗
PubMed1993

NMR studies of diffusional water permeability of red blood cells from macropodid marsupials (kangaroos and wallabies).

1. The water permeabilities of the red blood cell (RBC) membranes of five species of macropodid marsupials were monitored by using a Mn(2+)-doping 1H nuclear magnetic resonance (NMR) technique. 2. Since this appears to be the first time that this type of measurement at 400 MHz for 1H has been reported, an analysis of instrumental parameters influencing the estimated value of the water exchange time (Te), and of the diffusional water permeability (Pd), was performed on samples of human RBC. 3. It was found that a short interpulse delay in the Carr-Purcell-Meiboom-Gill pulse sequence had to be used (around 100 microseconds) to avoid an underestimation of the relaxation times, that occurred as the result of molecular diffusion through non-uniform local magnetic fields in and around the cells. 4. There were no significant differences, in the water permeabilities of the RBC membranes, between the five species (Macropus rufogriseus, M. parma, M. eugenii, M. parryi and Wallabia bicolor). 5. There were also no significant differences between various colonies of M. eugenii living in different habitats. 6. The average values of Pd were 9.1 x 10(-3) cm/sec at 24.6 degrees C, 10.4 x 10(-3) cm/sec at 30 degrees C, 12.6 x 10(-3) cm/sec at 37 degrees C, and 14.7 x 10(-3) cm/sec at 42.1 degrees C; these were more than twice as high as those for human RBC. 7. In agreement with the high water permeability the RBC of macropodids displayed a relatively low activation energy of water diffusion across their membranes, approximately 21 kJ/mol, compared with 25 kJ/mol for human RBC.

Animals↗
PubMed1993

Comparative nuclear magnetic resonance studies on water diffusional permeability of red blood cells from mice and rats.

1. The diffusional water permeability (P) of mouse and rat red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following exposure to various concentrations of p-chloromercuribenzene sulfonate (PCMBS). 2. There were no significant differences in the water permeability of mouse and rat RBCs, the values of P being around 6 x 10(-3)/sec at 20 degrees C and 11 x 10(-3) cm/sec at 37 degrees C. 3. Systematic studies of the effects of PCMBS on water diffusion indicated that the maximal inhibition is reached in 60 min at 20 degrees C with 1 mM PCMBS for the mouse RBCs and with 2 mM PCMBS for the rat RBCs. 4. The values of maximal inhibition ranged from 55-57% at 37 degrees C and reached values around 70% at 10 degrees C. 5. The degree of inhibition increased as the temperature of measurement decreased, regardless of PCMBS concentration and incubation time. 6. The basal permeability to water of mouse RBCs was estimated as 1.8 x 10(-3) cm/sec at 20 degrees C and 4.6 x 10(-3) cm/sec at 37 degrees C, and that of rat RBCs as 2.2 x 10(-3) cm/sec at 20 degrees C and 4.2 x 10(-3) cm/sec at 37 degrees C. 7. In both species the activation energy was around 27 kJ/mol and reached values over 40 kJ/mol after incubation with PCMBS in the conditions of maximal inhibition of water diffusion.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from sheep and cow.

1. The diffusional water permeability (p) of sheep and cow red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS). 2. There were no significant differences in the water permeability of sheep and cow RBCs, the values of p being around 3 x 10(-3) cm/sec at 20 degrees C and 5 x 10(-3) cm/sec at 37 degrees C. 3. Systematic studies of the effects of PCMBS on water diffusion indicated that in both species the maximal inhibition is reached in 60-90 min at 37 degrees C with 1 mM PCMBS. 4. The degree of inhibition increased as the temperature of measurement decreased, regardless of PCMBS concentration and incubation time. 5. The values of maximal inhibition ranged from 60-70% at 20 degrees C to 50-60% at 37 degrees C in the case of sheep RBCs, and from 45-55% at 20 degrees C to 40-50% at 37 degrees C in the case of cow RBCs. 6. The basal permeability to water of sheep RBCs was estimated as 1.0 x 10(-3) cm/sec at 20 degrees C and 2.2 x 10(-3) cm/sec at 37 degrees C, and that of cow RBCs as 1.6 x 10(-3) cm/sec at 20 degrees C and 2.7 x 10(-3) cm/sec at 37 degrees C. 7. In both species the activation energy of water diffusion was around 23 kJ/mol in control cells and reached values of around 30 kJ/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V--Rabbit (Oryctolagus cuniculus).

1. The diffusional water permeability (Pd) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS). 2. The values of Pd were around 6.3 x 10(-3) cm/sec at 15 degrees C, 7.0 x 10(-3) cm/sec at 20 degrees C, 8.0 x 10(-3) cm/sec at 25 degrees C, 9.1 x 10(-3) cm/sec at 30 degrees C and 10.7 x 10(-3) cm/sec at 37 degrees C. 3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37 degrees C with 0.5 mM PCMBS. 4. The values of maximal inhibition were around 71-74% at all temperatures. 5. The basal permeability to water was estimated as 1.6 x 10(-3) cm/sec at 15 degrees C, 2.0 x 10(-3) cm/sec at 20 degrees C, 2.4 x 10(-3) cm/sec at 25 degrees C, 2.6 x 10(-3) cm/sec at 30 degrees C, and 3.1 x 10(-3) cm/sec at 37 degrees C. 6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion. 7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart. 8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.

Amino Acids↗
PubMed1992

Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Water transport in human red cells: effects of 'non-inhibitory' sulfhydryl reagents.

The water diffusional permeability of human red blood cells following exposure to various sulfhydryl group (SH) reagents have been studied using a nuclear magnetic resonance technique. Exposure of red blood cells up to 12 mM N-ethylmaleimide (NEM) or 10 mM 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNE) alone does not affect water diffusion. In contrast, when DTNB treatment follows a preincubation of the cells with NEM, a small (18% at 37 degrees C) but significant inhibition of water permeability occurs. The NEM and DTNB treatment of the cells caused no change of the cell shape and volume or of the cell water volume. Consequently, the inhibition observed after NEM and DTNB treatment has a real significance.

Cell Membrane Permeability↗
PubMed1991

Amino acid composition of human liver mitochondrial membranes in normal and pathological conditions.

The amino acid composition of proteins from liver mitochondrial membranes has been studied in patients with normal liver, with biliary diseases and fatty liver, with obstructive jaundice or liver cirrhosis. A characteristic pattern of the amino acid composition in patients with normal liver has been found. In the mitochondrial membranes of patients with fatty liver tryptophan and lysine were decreased while [aspartic acid plus asparagine] and [glutamic acid plus glutamine] were increased compared to their counterpart in the normal liver. In patients with obstructive jaundice of short duration (less than two months) only a slight decrease in methionine content was found, while in the case of liver cirrhosis amino acid composition was markedly changed.

Amino Acids↗
PubMed1990

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique.

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7 X 10(-3) cm s-1 at 10 degrees C, 1.2 X 10(-3) cm s-1 at 15 degrees C, 1.4 X 10(-3) cm s-1 at 20 degrees C, 1.8 X 10(-3) cm s-1 at 25 degrees C, 2.1 X 10(-3) cm s-1 at 30 degrees C and 3.5 X 10(-3) cm s-1 at 37 degrees C. The mean value of the activation energy of water diffusion (Ea,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS--inhibited erythrocytes. The values of P and Ea,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.

4-Chloromercuribenzenesulfonate↗
PubMed1989

Studies of water permeability and proteins of erythrocyte membranes in patients with Duchenne muscular dystrophy.

The characteristics of water permeability of erythrocytes from 54 Duchenne muscular dystrophy (DMD) patients and age-matched controls have been determined by a pulse nuclear magnetic resonance (NMR) technique. A decreased permeability of erythrocyte membrane in DMD was definitely found at all temperatures between 15 and 42 degrees C, with normal values for the activation energy of water diffusion. No differences between DMD and control subjects in the pattern of erythrocyte membrane polypeptides separated by two-dimensional electrophoresis could be detected. The findings are discussed in relation to the molecular mechanisms of water diffusion across erythrocyte membrane and the problem of erythrocyte membrane abnormalities in DMD. A new interpretation of erythrocyte membrane alterations is proposed based on the recent findings regarding the molecular pathology of DMD.

Adolescent↗