Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Interaction of the serum amyloid A proteins with phospholipid

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.     

Asymmetric labeling of amino lipids in liposomes.

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.     

Cooperativity in nucleosomes assembly on supercoiled pBR322 DNA.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.     

Expression of acetylcholinesterase during visual system development in Drosophila

As in other insects acetylcholine (ACh) and acetylcholinesterase (AChE) function in synaptic transmission in the central nervous system of Drosophila. Studies on flies mutant for AChE indicate that in addition to its synaptic function of inactivating acetylcholine, this neural enzyme is required for normal development of the nervous system (J.C. Hall, S.N. Alahiotis, D.A. Strumpf, and K. White, 1980, Genetics 96, 939-965; R.J. Greenspan, J.A. Finn, and J.C. Hall, 1980, J. Comp. Neurol. 189, 741-774). In order to understand what role AChE may play in neural development, it is necessary to know, in detail, where and when the enzyme appears. The use of monoclonal antibodies to localize AChE in the developing visual system of wild type Drosophila has yielded the novel observation that AChE appears in photoreceptor (retinula) cells 4-6 hr after they differentiate and 3 to 4 days before they are functional. Three days later the staining in the cell body of these cells is reduced. Because retinula cells have no functional connections at the time when AChE is first detected, AChE can not be performing its standard synaptic function. Subsequent to the reduction of AChE in the retinula cells, midway through the pupal stage, the enzyme accumulates rapidly in the neuropils of the optic lobes of the brain. Thus, there is a biphasic accumulation of AChE in the developing visual system with the enzyme initially being expressed in the retinula cells and accumulating later in the optic lobes.     

Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes.

Trypanosoma cruzi trans-sialidase (TS) is a recently described enzyme which transfers alpha(2-3)-linked sialic acid from host-derived sialylated glycoconjugates to parasite surface molecules [Schenkman et al. (1991) Cell, 65, 1117]. We report here on the ability of TS to transfer sialic acid from donor sialyl-alpha(2-3)lactose to sialidase-treated sheep and human erythrocytes. Up to approximately 50% resialylation of both desialylated red cells could be attained. Resialylation of desialylated sheep erythrocytes restores their resistance to lysis by human complement. This ascribes a possible biological role for T. cruzi TS and demonstrates directly that sialic acid is solely responsible for preventing alternative pathway activation of human complement by sheep erythrocytes.     

Protein 4.1 is involved in a structural thermotropic transition of the red blood cell membrane detected by a spin-labeled stearic acid

Proteins involved in a structural transition in red blood cell membranes detected at 8 +/- 1.5 degrees C by a stearic acid spin-label have been investigated. Calcium loading of red blood cells with ionophore A23187 caused the disappearance of the 8 degrees C transition. Protein 4.1 appears to be the most susceptible protein to Ca2+ treatment. Antibodies specific for spectrin, band 3 (43K cytoplasmic domain), and protein 4.1 have been utilized as specific probes to modify membrane thermotropic properties. The 8 degrees C transition was eliminated by anti-4.1 protein antibodies but was not modified by the other antibodies. To further characterize the protein(s) involved in the transition, ghosts were subjected to sequential extraction of skeletal proteins. The extraction of band 6, spectrin, and actin did not modify the 8 degrees C transition. In contrast, high-salt extraction (1 M KCl) of spectrin-actin-depleted vesicles, a procedure that extracts proteins 2.1 and 4.1, was able to eliminate the 8 degrees C transition. Rebinding of purified protein 4.1 to the high salt extracted vesicles restored the 8 degrees C transition. These results indicate the involvement of protein 4.1 in the transition and suggest a functional membrane association of this protein. The binding of protein 4.1 to the membrane seems to contribute significantly to the thermotropic properties of red blood cells.     

Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells.

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.     

Cord blood plasma lipoproteins inhibit mitogen-stimulated lymphocyte proliferation

Lipoproteins were isolated from adult plasma and the umbilical cord blood plasma of newborn infants and were compared for their capacity to inhibit mitogen-stimulated [3H]thymidine uptake of adult peripheral blood mononuclear cells in vitro. Relative to the comparable adult lipoproteins, cord blood low density lipoproteins and high density lipoproteins inhibited mitogen stimulation at twofold to fourfold lower total protein concentrations. Apoproteins AI, B, and E were quantitated by radioimmunoassay of each of the adult and cord blood lipoprotein fractions. A strong correlation was observed between inhibitory activity and the amount of apoprotein E in the cord blood low and high density lipoproteins. Further evidence that lipoproteins containing apoprotein E accounted for the difference in suppressive activity of cord blood low and high density lipoproteins relative to the adult lipoproteins was obtained by selective removal of the apoprotein E-containing lipoproteins by using immunoaffinity chromatography or heparin-agarose adsorption. The results indicated that cord blood lipoproteins containing apoprotein E in association with apoproteins AI or B are capable of suppressing lymphocyte proliferation in vitro.