Biomedical subjects

E Azoulay

Publications and source records attributed to E Azoulay.

At least 91 records · Page 5Linked to original sources

PubMed1975

Membrane reconstitution in chl-r mutants of Escherichia coli K 12. VII. Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles.

Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them. The purification of the soluble enzyme is described. The purified protein gives a single band in 7.5% polyacrylamide gel electrophoresis. The molecular weight is estimated to be 350 000. The enzyme is cold-labile, Mg-2+ dependent, insensitive to inhibition by N, N'-dicyclohexylcarbodiimide and specific for ATP and ADP. Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg-2+ are able to incorporate the purified ATPase only in the presence of 2-6 mM Mg-2+. ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants. There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles. The kinetics of incorporation have been studied. ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increased in proportion with the peak of 1.18 particles. These kinetics have a hyperbolic pattern. In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.

Adenosine Triphosphatases↗

PubMed1975

Membrane reconstitution in chl-r mutants of Escherichia coli K 12. VIII. Purification and properties of the FA factor, the product of the chl B gene.

The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted. The purified protein gives a single band in 10% sodium dodecylsulfate/polyacrylamide gel electrophoresis. The molecular weight is estimated to be 35 000. This protein, that we have named "FA factor", does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl. The addition of purified FA protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the "complementation reaction", i.e. the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function. FA protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme. The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase. It is shown that FA factor is incorporated only into the particles of intermediate density. In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate. Protein FA can be released from either of these membranes (native or reconstituted) by removing Mg-2+ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of FA protein does not exert any effect on the level of the nitrate reductase activity.

Bacterial Proteins↗

PubMed1975

Membrane reconstitution in chl-r mutants of Escherichia coli K 12. IX. Part played by phospholipids in the complementation process.

The supernatant extracts of the chl A and chl B mutants of Escherichia coli K 12, the phospholipids of which are labeled by growth in 32 P or [2- 3H]glycerol media, contain 20 times more radioactivity than the supernatant extract of the wild-type strain grown under the same conditions. We have observed that, after complementation, 80% of the radioactivity previously contained by Extracts A and B is incorporated into reconstituted particles. The chromatography of 3H-labeled Extract B on DEAE-cellulose and followed by gel filtration of radioactive fractions on Sephadex G-200 has shown that the phospholipids of Extract B are only bound to soluble proteins and not to fragments of membranes; it can be assumed that they have been solubilized in the form of a lipid-protein complex by cell breakage. When Extracts A and B are treated by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) before being mixed together, an inhibition of the reconstitution of nitrate reductase activity which is proportional to the phospholipase C concentration and the length of treatment is observed. The analysis of lipids and phospholipids of particles (Peak I, Peak II and Peak III) formed during complementation and reconstituted nitrate reductase shows that their phospholipid contents (phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylserine) and especially that of Peak II (d equals 1.18) are closely related to that of native particles from the wild-type strain. These results allow one to propose a hypothesis explaining the mechanism involved in complementation.

Cell Membrane↗

PubMed1975

YATP value in Candida tropicalis grown on n-alkanes, fatty acids, and acetate.

The amount of ATP produced during n-alkane, fatty acid, or acetate metabolism in Candida tropicalis has been established from the P/O ratios measured on isolated mitochondria, yield on substrate and carbon balance. For these three kinds of substrates YATP value has been found to be close to 4, although Ysub on acetate is very different from those found with n-alkanes or fatty acids.

Acetates↗

PubMed1975

Dependence of 2,3-DPG and oxygen affinity of haemoglobin on sex and pregnancy in the guinea-pig.

Oxygen half-saturation of blood (P50), 2,3-diphosphoglycerate concentration (2,3-DPG) and Bohr effect were determined in male, and nonpregnant and pregnant female guinea pigs, according to a randomized block design. P50 was significantly higher in the female group (26.3 Torr plus or minus 0.22 SEM) than in the male group (24.8 Torr plus or minus 0.26 SEM) and was significantly lower in both these groups than in the pregnant group (27 Torr plus or minus 0.35 SEM). This difference in oxygen affinity was explained by differences in 2,3-DPG: 1.08 plus or minus 0.02 SEM in males, 1.24 plus or minus 0.03 in non-pregnant females and 1.34 plus or minus 0.03 mol/mol HB in pregnant females P50, 2,3-DPG and haemoglobin concentrations were significantly correlated for the ensemble of the 3 groups. There was no significant difference in Bohr effect between the 3 groups.

Adenosine Triphosphate↗

PubMed1974