Fractionation of rat-liver-chromatin nonhistone proteins into two groups with different metabolic rates.

In the pH interval 10.5-11.8, 70% of the nonhistone proteins normally present in rat liver chromatin were dissociated. The rest remained complexed with DNA even at pH 13. Dodecylsulfate-polyacrylamide gel electrophoresis revealed that the majority of the high-molecular-weight nonhistone proteins together with a few characteristic fractions with molecular weights of 40 000-60 000 remained in the alkali-resistant group. L-[14C]Leucine pulse-labelling experiments showed that the specific radioactivity of the alkali-labile nonhistone proteins was 2-3 times higher than that of the alkali-resistant nonhistone proteins, which, in turn, had the same specific radioactivity as that of the histones. The same held true for chromatin from regenerating rat liver. In the course of a 21-day chase the specific radioactivity of the alkali-labile nonhistone proteins gradually decreased and finally became 3 times lower than that of the alkali-resistant nonhistone proteins. On the contrary, the ratio of the specific radioactivities of the alkali-resistant nonhistone proteins and of the histones to the specific radioactivity of DNA remained constant during the chase. A conclusion can be drawn that a fraction of liver nonhistone proteins exists which is alkali-resistant and is conserved in chromatin like histones.     

Identification of two lithocholic acid-binding proteins. Separation of ligandin from glutathione S-transferase B.

1. Two lithocholic acid-binding proteins in rat liver cytosol, previously shown to have glutathione S-transferase activity, were resolved by CM-Sephadex chromatography. 2. Phenobarbitone administration resulted in induction of both binding proteins. 3. The two proteins had distinct subunit compositions indicating that they are dimers with mol.wts. 44 000 and 47 000. 4. The two lithocholic acid-binding proteins were identified by comparing their elution volumes from CM-Sephadex with those of purified ligandin and glutathione S-transferase B prepared by published procedures. Ligandin and glutathione S-transferase B were eluted separately, as single peaks of enzyme activity, at volumes equivalent to the two lithocholic acid-binding proteins. 5. Peptide 'mapping' revealed structural differences between the two proteins.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Interaction of G-protein-coupled receptors with synaptic scaffolding proteins

The calcium-independent receptors for latrotoxin (CIRL1-CIRL3) constitute a family of seven-transmembrane receptors with an unsually large N-terminal extracellular domain which comprises several motifs usually found in cell adhesion molecules. By yeast two-hybrid screening, we have identified the intracellular C-termini of CIRL1 and CIRL2 as interaction partners of the PDZ domain of the proline-rich synapse-associated protein (ProSAP)/somatostatin receptor-interacting protein (SSTRIP) family of postsynaptic proteins (SSTRIP, ProSAP1 and ProSAP2, also known as shank1-shank3 respectively). Overlay assays indicate that the ProSAP1/shank2 PDZ domain in particular interacts strongly with the C-terminus of CIRL1 and CIRL2. Co-immunoprecipitation of ProSAP1 and CIRL1 (but not CIRL2) from rat brain extracts indicates that this interaction also occurs in vivo in rat brain. The known postsynaptic localization of ProSAP1, as well as our observation that CIRL1 (but not CIRL2) is enriched in postsynaptic density preparations from the rat brain, suggests that CIRL1 is localized pre- as well as post-synaptically in the central nervous system.     

Phospholipase B from the plasma membrane of Saccharomyces cerevisiae. Separation of two forms with different carbohydrate content

Two forms of phospholipase B could be solubilized from the plasma membrane of Saccharomyces cerevisiae, separated by gel filtration with Sephacryl S-300 and identified by SDS-polyacrylamide gel electrophoresis as glycoproteins of the apparent molecular weights of about 220 000 (phospholipase B1) and 145 000 (phospholipase B2). The enzymes are very similar in respect to their catalytic properties. Both forms converted lysophosphatidylcholine to diacylphosphatidylcholine and unesterified fatty acids. The carbohydrate content of the glycoproteins could be reduced by treatment with endoglycosidase H and HF. By incubation of phospholipase B1 and phospholipase B2 with endoglycosidase H from Streptomyces griseus, one main protein with an apparent Mr of 67 000 and the same residual carbohydrate content was obtained. Treatment with HF reduced phospholipase B1 and phospholipase B2 to proteins with an apparent Mr of 52 000 and 67 000, respectively. These results could indicate that the two forms are similar in respect to their protein moieties. An antiserum raised in mice against phospholipase B2 showed no crossreactivity with phospholipase B1 as detected by immunoblot analysis. The reactivity of phospholipase B2 was diminished or abolished by progressive removal of carbohydrate. These results were taken as indications for differences in the carbohydrate component of the two enzyme forms.     

Paired assessment of volatile anesthetic concentrations with synaptic actions recorded in vitro

The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4 +/- 0.2 mM isoflurane and were diluted to desired "nominal" concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.     

Organization of synaptic junction proteins

Most of the polypeptides of isolated brain synaptic junction preparations are cross-linked by disulfide bonds; these bonds are readily reformed following reduction by β-mercaptoethanol, suggesting that other, non-covalent interactions may hold the polypeptides in close contact. When synaptic junctions were treated with β-mercaptoethanol in conjunction with a large variety of reagents known to disrupt certain types of non-covalent bonds, however, the polypeptides could still be cross-linked. Furthermore, virtually all of these species remained associated with β-mercaptoethanol-reduced membrane in the presence of 0.05 N NaOH or 0.5% Triton X-100, although each of these solvents extracts large amounts of protein from extra-junctional membrane, which has a composition apparently similar to that of synaptic junctions.These results indicate that any non-covalent interactions existing among synaptic junction proteins are inaccessible to treatments at the surface of the membrane, being located within the lipid bilayer or perhaps at points of contact with sub-membranous arrays such as the post-synaptic density. Under special conditions, the polypeptides of extra-junctional membrane can also be induced to form disulfide cross-links among one another, and these bonds can likewise be rapidly reformed following reduction, giving this membrane properties similar to that of the junction. This suggests a model by which stabilized junctional membrane may be formed from fluid extra-junctional membrane during synaptogenesis.     

Linear discrete models with different time scales

Aggregation of variables allows to approximate a large scale dynamical system (the micro-system) involving many variables into a reduced system (the macro-system) described by a few number of global variables. Approximate aggregation can be performed when different time scales are involved in the dynamics of the micro-system. Perturbation methods enable to approximate the large micro-system by a macro-system going on at a slow time scale. Aggregation has been performed for systems of ordinary differential equations in which time is a continuous variable. In this contribution, we extend aggregation methods to time-discrete models of population dynamics. Time discrete micro-models with two time scales are presented. We use perturbation methods to obtain a slow macro-model. The asymptotic behaviours of the micro and macro-systems are characterized by the main eigenvalues and the associated eigenvectors. We compare the asymptotic behaviours of both systems which are shown to be similar to a certain order.     

Evidence for two different gas vesicle proteins and genes in Halobacterium halobium.

Most halobacteria produce gas vesicles (GV). The well-characterized species Halobacterium halobium and some GV+ revertants of GV- mutants of H. halobium produce large amounts of GV which have a spindlelike shape. Most other GV+ revertants of H. halobium GV- mutants and other recently characterized halobacterial wild-type strains possess GV with a cylindrical form. The number of intact particles in the latter isolates is only 10 to 30% of that of H. halobium. Analysis of GV envelope proteins (GVPs) by electrophoresis on phenol-acetic acid-urea gels showed that the GVP of the highly efficient GV-producing strains migrated faster than the GVP of the low-GV-producing strains. The relative molecular mass of the GVP was estimated to be 19 kilodaltons (kDa) for high-producing strains (GVP-A) and 20 kDa for low-producing strains (GVP-B). Amino acid sequence analysis of the first 40 amino acids of the N-terminal parts of GVP-A and GVP-B indicated that the two proteins differed in two defined positions. GVP-B, in relation to GVP-A, had Gly-7 and Val-28 always replaced by Ser-7 and Ile-28, respectively. These data suggest that at least two different gvp genes exist in H. halobium NRL. This was directly demonstrated by hybridization experiments with gvp-specific DNA probes. A fragment of plasmid pHH1 and a chromosomal fragment of H. halobium hybridized to the probes. Only a chromosomal fragment hybridized to the same gyp probes when both chromosomal and plasmid DNAs from the low-GV-producing halobacterial wild-type strains SB3 and GN101 were examined. These findings support the assumption that GVP-A is expressed by a pHH1-associated gvp gene and GVP-B by a chromosomal gvp gene.     

Comparison of fractionation of groundnut proteins by two different methods

The salt-soluble proteins of groundnut meal were fractionated by precipitation with (NH₄)₂SO₄ by increasing the (NH₄)₂SO₄ saturation in steps of 10%. The sharp separation into arachin and conarachin claimed by earlier workers was not achieved, as protein was precipitated at each stage from 20 to 100% saturation with (NH₄)₂SO₄. The fractions so obtained were examined by disc electrophoresis on polyacrylamide gel and the amino acid compositions were determined by ion-exchange chromatography. Differences in both electrophoretic pattern and amino acid composition were found. The protein precipitated by CaCl₂ solution was similar in yield, nitrogen content, electrophoretic pattern, and amino acid composition to the fraction precipitating at 10–20% (NH₄)₂SO₄ saturation. The main differences in amino acid composition of the various fractions precipitated by (NH₄)₂SO₄ were found in the amino acids cystine, methionine, and lysine, which increased with increase in (NH₄)₂SO₄ saturation. The electrophoretic pattern and amino acid composition of “conarachin” varied according to the method of preparation.     

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.     

Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography.

Membrane vesicles from the envelope of Escherichia coli were separated by electrophoresis through dilute agarose and by sizing chromatography through Sephacryl S-1000. These techniques revealed that proteins were associated with different subsets of vesicles. In particular, dilute agarose electrophoresis clearly separated the inner membrane penicillin-binding proteins (PBPs) into different vesicle groups. Vesicles containing PBPs 4, 6, 7, and 8 migrated rapidly through agarose; vesicles with PBPs 1a, 1b, 2, 3, and 5 eluted later. With the exception of PBP 4, which migrated with PBPs 1 through 5, chromatography through Sephacryl S-1000 was able to distinguish the same two vesicle sets, though the extent of separation was poorer than with agarose. The existence of these associations among vesicles and proteins suggests that there is an organization to the inner membrane of E. coli which is not observed when membrane vesicles are separated solely on the basis of density in sucrose gradients.     

Visualizing recycling of synaptic vesicle proteins

The use of modern techniques (in particular, novel fluorescence markers of a few molecular participants of the exo-and endocytotic processes, including pH-sensitive agents, immuno-electron and laser-scanning microscopy) allows experimenters to visualize different stages of recycling of synaptic vesicle proteins. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 350–351, July–October, 2007.     

Relationship between sequence determinants of stability for two natural homologous proteins with different folds

In the Cro protein family, an evolutionary change in secondary structure has converted an alpha-helical fold to a mixture of alpha-helix and beta-sheet. P22 Cro and lambda Cro represent the ancestral all-alpha and descendant alpha+beta folds, respectively. The major structural differences between these proteins are at the C-terminal end of the domain (residues 34-56), where two alpha-helices in P22 Cro align with two beta-strands in lambda Cro. We sought to assess the possibility that smooth evolutionary transitions could have converted the all-alpha structure to the alpha+beta structure through sequences that could adopt both folds. First, we used scanning mutagenesis to identify and compare patterns of key stabilizing residues in the C-terminal regions of both P22 Cro and lambda Cro. These patterns exhibited little similarity to each other, with structurally important residues in the two proteins most often occurring at different sequence positions. Second, "hybrid scanning" studies, involving replacement of each wild-type residue in P22 Cro with the aligned wild-type residue in lambda Cro and vice versa, revealed five or six residues in each protein that strongly destabilized the other. These results suggest that key stability determinants for each Cro fold are quite different and that the P22 Cro sequence strongly favors the all-alpha structure while the lambda Cro sequence strongly favors the alpha+beta structure. Nonetheless, we were able to design a "structurally ambivalent" sequence fragment (SASF1), which corresponded to residues 39-56 and simultaneously incorporated most key stabilizing residues for both P22 Cro and lambda Cro. NMR experiments showed SASF1 to stably fold as a beta-hairpin when incorporated into the lambda Cro sequence but as a pair of alpha-helices when incorporated into P22 Cro.     

The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides

Coated vesicles have been purified in the past on the basis of their remarkably homogeneous structure, not their function. We have succeeded in isolating two subpopulations of bovine brain coated vesicles that carry specific "cargoes," in this case two synaptic vesicle membrane polypeptides (Mr = 95,000 and 65,000). Monoclonal antibodies that recognize cytoplasmic domains of these polypeptides can penetrate the clathrin coat and recognize them on the outer surface of the coated vesicle membrane. An immunoadsorption technique could therefore be used to fractionate coated vesicles on the basis of their membrane composition. The subpopulations have the normal complement of conventional coated vesicle proteins. Exclusive, however, to the subpopulations that carry synaptic vesicle polypeptides are two new coated vesicle polypeptides (Mr = 38,000 and 29,000).