Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and F0-F1 complex from Rhodospirillum rubrum

PPi driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PPiase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze-thaw procedure. In the presence of ADP, Mg2+, Pi and PPi the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein-1 X min-1, at 20 degrees C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N'-dicyclohexylcarbodiimide.     

Glyceraldehyde-3-phosphate dehydrogenase activity studied under physiological conditions with a linear assay.

An assay method for glyceraldehyde-3-phosphate dehydrogenase in which none of the primary products accumulate and which gives linear kinetics under physiological conditions has been developed. It is based on the use of the 1,3-diphosphoglycerate produced by the enzyme for the formation of NADPH, while the NADH produced is recycled with an auxiliary system. Revised Km values at pH 7.4 for the muscle (rabbit and rat) enzyme are: glyceraldehyde-3-P, 50 μM; NAD, 100 μM; Pi, 10 mM. The rat erythrocyte enzyme gave similar values except for glyceraldehyde-3-P which was 300 μM. Cooperativity for NAD⁺ tends to be positive but is a variable parameter.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [³⁵S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.     

DARPin-Assisted Crystallography of the CC2-LZ Domain of NEMO Reveals a Coupling between Dimerization and Ubiquitin Binding

NEMO is an integral part of the IκB kinase complex and serves as a molecular switch by which the NF-κB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IκB kinase activation in the NF-κB signaling pathway.     

Identification of a novel calcium binding protein from bovine brain

A novel Ca²⁺ binding protein, named caligulin, was extracted from the heat-treated 100 000 × g supernatant of bovine brain and purified to electrophoretic homogeneity. The apparent M_r of caligulin determined on sodium dodecyl sulfate polyacrylamide gels was 24 000. Analysis by gel filtration chromatography indicated an apparent M_r of 33 000, suggesting a monomeric protein. Amino acid composition data demonstrated the presence of 25% acidic residues, 12% basic residues and 10% leucine. In the presence of 1 mM MgCl₂ and 0.15 M KCl, caligulin bound 1 mol Ca²⁺/mol protein with half-maximal binding at about 0.2 μM Ca²⁺.     

Modulation of 25-hydroxyvitamin D3-24-hydroxylase by aminophylline: a cytochrome P-450 monooxygenase system.

The enzymatic activity of human erythrocyte pyruvate kinase was found to decrease on incubation of the purified enzyme with red blood cell ghosts, ATP and cAMP. If $\text{[}{}^{32}\text{P]γATP}$ was used radioactivity was found associated with the protein after gel electrophoresis. Various effectors protected the enzyme against phosphorylation. Treatment of the modified enzyme with a protein phosphatase restored enzymatic activity and also caused the loss of the radioactive label. Modification of the pyruvate kinase in this way altered the affinity of the enzyme for one of its substrates (phosphoenolpyruvate), but the binding of the other substrate (ADP) was unaffected.     

The binding of benz(a)anthracene derivatives to glyceraldehyde-3-phosphate dehydrogenase and mammary tissue following exposure to laboratory light.

7,12-Dimethylbenz(a)anthracene (DMBA) and 7-methoxymethyl-12-methylbenz(a)anthracene (MeO-DMBA) are converted to a number of products during short exposures in aqueous suspension to laboratory illumination. The mixture of products binds to glyceraldehyde-3-phosphate dehydrogenase (GPDH) while inhibiting its activity but there is no apparent relationship between the binding and inhibition of enzyme activity. There is little, or no, binding or enzyme inhibition when the compounds are protected from light. 7-Bromomethyl-12-methylbenz(a)anthracene (Br-DMBA) binds to GPDH whether photoactivated or not but enzyme inhibition depends upon light exposure. The binding of light-exposed DMBA by surviving rat mammary tissue was five-times greater than with the unchanged hydrocarbon. Binding of MeO-DMBA products also occurred after light exposure but not in the dark.