A spectrophotometric study of Penicillium brevicompactum RNAase complex formation with adenine nucleotides]

Spectrophotometric study of extracellular Pen. brevicompactum RNAse interaction with adenyl nucleotides and constituents have been carried out. It is states that: 1) complex RNAse--nucleotide is formed by association of one enzyme molecule with one nucleotide molecule; 2) all the nucleotide components base sugar and phosphate, take part in the formation of this complex; 3) for the effective association of this complex it is necessary to have a high correspondence (complementaryty) of nucleotide geometric configuration to the space composition of the active site of RNAse.     

Visualizing rank deficient models: a row equation geometry of rank deficient matrices and constrained-regression

Situations often arise in which the matrix of independent variables is not of full column rank. That is, there are one or more linear dependencies among the independent variables. This paper covers in detail the situation in which the rank is one less than full column rank and extends this coverage to include cases of even greater rank deficiency. The emphasis is on the row geometry of the solutions based on the normal equations. The author shows geometrically how constrained-regression/generalized-inverses work in this situation to provide a solution in the face of rank deficiency.     

Ribonuclease: a spectrophotometric assay using acridine orange--RNA complex

Bovine pancreatic RNase can be accurately and sensitively assayed with RNA-acridine orange complex (1:1, wt/wt) as substrate. The enzyme is optimally assayed in 0.05 m sodium acetate buffer, pH 5.0, at 37°C with a RNA concentration of 0.300 mg/ml. The acridine orange released after 30 min incubation can be determined, after filtration, with a simple visible light spectrophotometer at 492 nm. The increase in absorbance at 492 nm was linear for final RNase concentrations of 0.05–0.6 μg/ml. The assay is applicable to crude cell-free extracts, but RNase T₁ was inactive in this assay.     

Diffusion NMR study of complex formation in membrane-associated peptides

Pulsed-field-gradient nuclear magnetic resonance (PFG-NMR) is used to obtain the true hydrodynamic size of complexes of peptides with sodium dodecyl sulfate SDS micelles. The peptide used in this study is a 19-residue antimicrobial peptide, GAD-2. Two smaller dipeptides, alanine–glycine (Ala–Gly) and tyrosine–leucine (Tyr–Leu), are used for comparison. We use PFG-NMR to simultaneously measure diffusion coefficients of both peptide and surfactant. These two inputs, as a function of SDS concentration, are then fit to a simple two species model that neglects hydrodynamic interactions between complexes. From this we obtain the fraction of free SDS, and the hydrodynamic size of complexes in a GAD-2–SDS system as a function of SDS concentration. These results are compared to those for smaller dipeptides and for peptide-free solutions. At low SDS concentrations ([SDS] ≤ 25 mM), the results self-consistently point to a GAD-2–SDS complex of fixed hydrodynamic size R = (5.5 ± 0.3) nm. At intermediate SDS concentrations (25 mM < [SDS] < 60 mM), the apparent size of a GAD-2–SDS complex shows almost a factor of two increase without a significant change in surfactant-to-peptide ratio within a complex, most likely implying an increase in the number of peptides in a complex. For peptide-free solutions, the self-diffusion coefficients of SDS with and without buffer are significantly different at low SDS concentrations but merge above [SDS] = 60 mM. We find that in order to obtain unambiguous information about the hydrodynamic size of a peptide-surfactant complex from diffusion measurements, experiments must be carried out at or below [SDS] = 25 mM.     

The role of GlcNAc in formation and function of extracellular matrices

N-acetylglucosamine (GlcNAc) is an abundant hexose that as a monomer or as part of macromolecules plays multiple roles in eukaryotic cells. Especially as a residue of oligo- and polysaccharides and conjugated with lipids and proteins, GlcNAc contributes to the function and architecture of extracellular matrices. Several human disorders caused by mutations in genes coding for enzymes dealing with GlcNAc have been identified. However, molecular aspects of their clinical picture are laborious to investigate and are mostly addressed in yeast and vertebrate cultured cells that as unicellular or artificial systems ultimately do not allow conclusive deductions for complex organisms. An excellent model system to study the biology of GlcNAc in a multi-cellular organism is the genetically, biochemically and physiologically manipulable fruitfly Drosophila melanogaster that despite the evolutionary distance shares basic features of GlcNAc biology with humans. The aim of this review is to summarise the use of GlcNAc both in mammals and in Drosophila by highlighting the molecular consequences of perturbing GlcNAc function.     

The Wilcoxon signed rank test for paired comparisons of clustered data

The Wilcoxon signed rank test is a frequently used nonparametric test for paired data (e.g., consisting of pre- and posttreatment measurements) based on independent units of analysis. This test cannot be used for paired comparisons arising from clustered data (e.g., if paired comparisons are available for each of two eyes of an individual). To incorporate clustering, a generalization of the randomization test formulation for the signed rank test is proposed, where the unit of randomization is at the cluster level (e.g., person), while the individual paired units of analysis are at the subunit within cluster level (e.g., eye within person). An adjusted variance estimate of the signed rank test statistic is then derived, which can be used for either balanced (same number of subunits per cluster) or unbalanced (different number of subunits per cluster) data, with an exchangeable correlation structure, with or without tied values. The resulting test statistic is shown to be asymptotically normal as the number of clusters becomes large, if the cluster size is bounded. Simulation studies are performed based on simulating correlated ranked data from a signed log-normal distribution. These studies indicate appropriate type I error for data sets with > or =20 clusters and a superior power profile compared with either the ordinary signed rank test based on the average cluster difference score or the multivariate signed rank test of Puri and Sen. Finally, the methods are illustrated with two data sets, (i) an ophthalmologic data set involving a comparison of electroretinogram (ERG) data in retinitis pigmentosa (RP) patients before and after undergoing an experimental surgical procedure, and (ii) a nutritional data set based on a randomized prospective study of nutritional supplements in RP patients where vitamin E intake outside of study capsules is compared before and after randomization to monitor compliance with nutritional protocols.     

The formation at 37 C of a nondissociable receptor-estradiol complex

The receptor-estradiol complex formed in rat uterine cytosol when heated at 37 C converts from a dissociable to a nondissociable form. The conversion is best observed in cytosols pretreated with charcoal at 0 C which renders the subsequently formed receptor-estradiol complexes thermostable at 37 C. In the presence of dithiothreitol the heated complex remains dissociable. Tamoxifen does not form nondissociable complexes with the estradiol receptor. It is proposed that the nondissociable form of the receptor complex is a required phase in the mechanism of estradiol action.     

The TatBC complex formation suppresses a modular TatB-multimerization in Escherichia coli

Twin-arginine translocation (Tat) systems allow the translocation of folded proteins across biological membranes of most prokaryotes. In proteobacteria, a TatBC complex binds Tat substrates and initiates their translocation after recruitment of the component TatA. TatA and TatB belong to one protein family, but only TatB forms stable complexes with TatC. Here we show that TatB builds up TatA-like modular complexes in the absence of TatC. This TatB ladder ranges from about 100 to over 880 kDa with 105+/-10 kDa increments. TatC alone can form a 250 kDa complex which could be a scaffold that can recruit TatB to form defined TatBC complexes.     

The study of complex formation of human recombinant HSP70 with tumor-associated peptides

Molecular chaperones of the HSP70 family (70 kDa heat shock proteins) are involved in presentation of exogenous antigenic peptides by antigen-presenting cells (APC). HSP70 complexes with tumor-associated peptides are powerful immunotherapeutic agents, inducing an adaptive cytotoxic T-cell mediated immune response. Several clinical trials have shown that HSP-based autological anticancer vaccines possess high efficiency and safety. However, sometimes it is impossible to isolate sufficient amount of such vaccines and so human recombinant HSP are used for in vitro loading with tumor-associated peptides. In this study we have investigated binding of two human recombinant proteins HSP70_HYB and HSC70 with antigenic peptides of different origin and optimized conditions for complex formation. The proposed method for complex formation increases the repertoire of HSP70 bound peptides compared with in vivo formed complexes.     

Optical biosensor study of ternary complex formation in a cytochrome P4502B4 system

The optical biosensor method was used for the revelation of ternary complexes, formed by the full-length NADPH-cytochrome P450 reductase (d-Fp) and cytochromes P4502B4 (d-2B4) and b5 (d-b5) in the course of their interactions within the reconstituted d-2B4-containing system. Based on the lack of competition between d-b5 and d-Fp for the binding sites on immobilized 2B4 (3) as well as on the analysis of data obtained in the three proteins' dissociation reactions, the possibility of formation of ternary complexes through the interactions between membranous hydrophobic fragments of proteins was substantiated. All the complexes obtained were productive.     

The role of Ia in the formation of a T cell antigen-recognition complex

We have evaluated the ability of a peptide-specific, I-Ak-restricted murine T hybridoma to bind its Ag in the presence and absence of class II MHC molecules. The restricting Ia molecule, when supplied as a plasma membrane preparation of I-Ak-expressing APC, specifically increases the avidity of the Ag-binding complex by lengthening its t1/2, without affecting the rate at which the complex is formed. Experiments using mutated I-Ak molecules indicate that the ability of a mutant Ia species to present Ag is distinct from its ability to stabilize the Ag-recognition complex, suggesting that T cell stimulation depends not only upon stabilization of Ag-TCR-Ia complexes, but also upon distinct Ia-influenced conformational signals.     

The ribosome-associated complex antagonizes prion formation in yeast

Abstract

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.     

The ribosome-associated complex antagonizes prion formation in yeast

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.     

Amphotericin B-sterol complex formation and competition with egg phosphatidylcholine: a monolayer study

The conditions of formation of amphotericin B-cholesterol or -ergosterol complexes in monolayers are investigated by the penetration into a monolayer of egg phosphatidylcholine/sterol of 14C-labelled N-fructosyl-amphotericin B dissolved in the aqueous subphase. An increase of both surface pressure and radioactivity as a function of concentration are observed simultaneously while a 'saturation' effect occurs only for the surface pressure. The experiments are not accurate enough to make conclusions about the number of actually penetrated amphotericin B molecules. Therefore, the existence of an amphotericin B-sterol complex was evidenced from a study of surface pressure area per molecule isotherm. The results indicate that a complex with a 2:1 stoichiometry is formed and that the amphotericin B-ergosterol interaction is larger than the amphotericin B-cholesterol interaction. The complex is dissociated by addition of egg phosphatidylcholine due to a competition between egg phosphatidylcholine and amphotericin B for sterol.