Human platelet factor 4 monomer-dimer-tetramer equilibria investigated by 1H NMR spectroscopy

As a function of protein concentration, proton NMR spectra of human platelet factor 4 (PF4) differ. Correlation with low-angle laser light scattering data has allowed identification of concentration-dependent NMR spectral changes to PF4 aggregation, with tetramers being the largest aggregates formed. Well-resolved aromatic ring proton NMR resonances were assigned to Tyr-60, His-I, and His-II in monomer, dimer, and tetramer states. Since Tyr-60 3.5 ring proton resonances are well resolved from state to state, estimation of fractional populations in each state was possible. By varying the PF4 concentration, changes in these populations when plotted according to the Hill equation show a bimolecular mechanism of aggregation which proceeds from monomers to tetramers through a dimer intermediate. Equilibrium constants for dimer association (KD) and tetramer association (KT) have been estimated as a function of pH and ionic strength. At pH 4, where KD and KT approach the same value, resonances associated with all three aggregate states are observed. Lowering the pH shifts the equilibrium to the monomer state, while raising the pH shifts the equilibrium to dimer and tetramer states. Analysis of the pH dependence of KD and KT suggests that electrostatic interactions, probably arising from Glu/Asp and Lys/Arg side chains, play a role in the binding process. Increasing the solvent ionic strength stabilizes the tetramer state especially at low pH, suggesting that intersubunit, repulsive electrostatic interactions probably between/among cationic side chains (Lys/Arg) attenuate the aggregation process. Information based primarily on histidine pKa values and photo-CIDNP 1H NMR data suggests that Tyr-60 and His-I, but not His-II, are significantly affected by the aggregation process.     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (kidney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl-/HCO3- anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in alpha-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (k¯idney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl^?/HCO₃^? anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in α-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t_1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

Platelet-derived growth factor-BB and basic fibroblast growth factor directly interact in vitro with high affinity.

Platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) are potent growth factors active on many cell types. The present study indicates that they directly interact in vitro. The interaction was investigated with overlay experiments, surface plasmon resonance experiments, and solid-phase immunoassays by immobilizing one factor or the other and by steady-state fluorescence analysis. The interaction observed was specific, dose-dependent, and saturable, and the bFGF/PDGF-BB binding stoichiometry was found to be 2:1. K(D)(1) for the first step equilibrium and the overall K(D) values were found to be in the nanomolar and in the picomolar range, respectively. Basic FGF/PDGF-BB interaction was strongly reduced as a function of time of PDGF-BB proteolysis. Furthermore, docking analysis suggested that the PDGF-BB region interacting with bFGF may overlap, at least in part, with the PDGF-BB receptor-binding site. This hypothesis was supported by surface plasmon resonance experiments showing that an anti-PDGF-BB antibody, known to inhibit PDGF-BB binding with its receptor, strongly reduced bFGF/PDGF-BB interaction, whereas a control antibody was ineffective. According to these data, the observed bFGF.PDGF-BB complex formation might explain, at least in part, previous observations showing that PDGF-BB chemotactic and mitogenic activity on smooth muscle cells are strongly inhibited in the presence of bFGF.     

Conformational states of ADP ribosylation factor 1 complexed with different guanosine triphosphates as studied by 31P NMR spectroscopy

Guanine nucleotide binding proteins (GNB-proteins) play an essential role in cellular signaling, acting as molecular switches, cycling between the inactive, GDP-bound form and the active, GTP-bound form. It has been shown that conformational equilibria also exist within the active form of GNB-proteins between conformational states with different functional properties. Here we present (31)P NMR data on ADP ribosylation factor 1 (Arf1), a GNB-protein involved in Golgi traffic, promoting the coating of secretory vesicles. To investigate conformational equilibria in active Arf1, the wild type and switch I mutants complexed with GTP and a variety of commonly used GTP analogues, namely, GppCH(2)p, GppNHp, and GTPγS, were analyzed. To gain deeper insight into the conformational state of active Arf1, we titrated with Cu(2+)-cyclen and GdmCl and formed the complex with the Sec7 domain of nucleotide exchange factor ARNO and an effector GAT domain. In contrast to the related proteins Ras, Ral, Cdc42, and Ran, from (31)P NMR spectroscopic view, Arf1 exists predominantly in a single conformation independent of the GTP analogue used. This state seems to correspond to the so-called state 2(T) conformation, according to Ras nomenclature, which is interacting with the effector domain. The exchange of the highly conserved threonine in position 48 with alanine led to a shift of the equilibrium toward a conformational state with typical properties obtained for state 1(T) in Ras, such as interaction with guanine nucleotide exchange factors, a lower affinity for nucleoside triphosphates, and greater sensitivity to chaotropic agents. In active Arf1(wt), the effector interacting conformation is strongly favored. These intrinsic conformational equilibria of active GNB-proteins could be a fine-tuning mechanism of regulation and thereby an interesting target for the modulation of protein activity.     

Human beta-glucosidase: inhibition by sulphates and purification by affinity chromatography on Dextran-sulphate-Sepharose

The acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from human placenta is inhibited by sulphated macromolecules such as Dextran sulphate or chondroitin sulphate. This inhibition is alleviated by compounds such as crude taurocholate or phospholipids, which are known activators of acid beta-glucosidase. Partially-purified human beta-glucosidase will bind to Dextran sulphate linked to Sepharose 4B and can be eluted with low concentrations of crude sodium taurocholate. This procedure gives a 10-15 fold purification with good yield and has been included in a scheme giving an approx. 4000-fold purification of placental beta-glucosidase. Evidence is presented which suggests that phospholipids bind to beta-glucosidase by both ionic and hydrophobic interactions. The inhibition of enzyme activity caused by sulphated compounds and non-ionic detergents may be attributed to interference with, respectively, the ionic and hydrophobic binding of phospholipid to the enzyme.     

Cerebral glucose metabolism and the glutamine cycle as detected by in vivo and in vitro 13C NMR spectroscopy

We review briefly 13C NMR studies of cerebral glucose metabolism with an emphasis on the roles of glial energetics and the glutamine cycle. Mathematical modeling analysis of in vivo 13C turnover experiments from the C4 carbons of glutamate and glutamine are consistent with: (i) the glutamine cycle being the major cerebral metabolic route supporting glutamatergic neurotransmission, (ii) glial glutamine synthesis being stoichiometrically coupled to glycolytic ATP production, (iii) glutamine serving as the main precursor of neurotransmitter glutamate and (iv) glutamatergic neurotransmission being supported by lactate oxidation in the neurons in a process accounting for 60-80% of the energy derived from glucose catabolism. However, more recent experimental approaches using inhibitors of the glial tricarboxylic acid (TCA) cycle (trifluoroacetic acid, TFA) or of glutamine synthase (methionine sulfoximine, MSO) reveal that a considerable portion of the energy required to support glutamine synthesis is derived from the oxidative metabolism of glucose in the astroglia and that a significant amount of the neurotransmitter glutamate is produced from neuronal glucose or lactate rather than from glial glutamine. Moreover, a redox switch has been proposed that allows the neurons to use either glucose or lactate as substrates for oxidation, depending on the relative availability of these fuels under resting or activation conditions, respectively. Together, these results suggest that the coupling mechanisms between neuronal and glial metabolism are more complex than initially envisioned.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

Organic anion transporting polypeptides (OATP/SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 (Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 (SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C (SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.     

Isolation of a multiprotein complex containing cytochrome b and c1 from Neurospora crassa mitochondria by affinity chromatography on immobilized cytochrome c. Difference in the binding between ferricytochrome c and ferrocytochrome c to the multiprotein complex.

A multiprotein complex which contains in equimolar amounts two cytochromes b (Mr each about 27,000), one cytochrome c1 (Mr 31,000) and six subunits without known prosthetic groups (Mr 8000, 12,000, 14,000, 45,000, 45,000, and 50,000) has been isolated from the mitochondrial membranes of Neurospora crassa by affinity chromatography on immobilized cytochrome c. The chromatographic separation was based upon the specific binding of the complex to ferricytochrome c coupled to Sepharose and its specific release upon conversion of the coupled ferricytochrome c into ferrocytochrome c using ascorbate as a reductant. The chromatography was performed in the presence of the nonionic detergent Triton X-100 at low ionic strengths. A monodisperse preparation of the multiprotein complex was obtained which was used for binding studies with cytochrome c from Neurospora crassa, horse heart and Saccaromyces cerevisiae. At low ionic strength (20 mM Trisacetate) and slightly alkaline pH (pH 7 to 8), more than one molecule of ferricytochrome c were bound to the isolated multiprotein complex with dissociation constants below 1 x 10(-7) M. One of these bindings appeared different from the others, since its high affinity was preserved at an ionic strength at which the affinities of the other bindings decreased. Furthermore, the affinity of only this binding decreased upon reduction of cytochrome c. It is suggested that this binding is at or near the functionally active site(s) of the mulipprotein complex.     

Ring current effects in Adriamycin-flavin mononucleotide complexation as observed by 1H FT NMR spectroscopy

The addition of Adriamycin to a solution containing flavin mononucleotide (FMN) resulted in an upfield shift in the signals of the aromatic ring protons H(6,9) and the 8α, 7α methyl protons of FMN. The chemical shift of the H(6,9) and of the 8α and 7α methyl proton signals of FMN decreased from 7.92, 2.56 and 2.46 ppm, respectively, in the absence of Adriamycin to 7.61, 2.42 and 2.36 ppm, respectively, at 3 mM Adriamycin. Concomitant increases in the linewidth of aromatic and methyl proton siqnals of FMN were also observed. Variable temperature studies over the range of 5 to 43° showed an increase in the chemical shift of both the aromatic and aliphatic proton signals with increasing temperatures. These results suggest that FMN and Adriamycin form a complex via ring-ring stacking.     

Determination of optical purity of 3,5-dimethoxybenzoyl-leucine diethylamide by chiral chromatography and 1H and 13C NMR spectroscopy

(R)-N-3,5-dinitrobenzoyl (DNB) leucine derived chiral selector was used as an HPLC chiral stationary phase for the resolution of various racemic amino acids derivatives. In this study, determination of optical purity of an amino acid derivative was performed by chiral high performance liquid chromatography and 1H and 13C NMR spectroscopy by using the DNB leucine derived chiral selector. The accuracy and precision of each optical purity value are calculated and the data are compared to each other.     

Prediction of affinity and kinetics in biomolecular interactions by affinity chromatography

Computer simulation of affinity chromatography is a valuable tool for accurate prediction of column performance. In our study affinity pairs based on lectin and antibody interactions with carbohydrates have been used as model systems. In this well-characterized system we have demonstrated the usefulness of the simulation approach for determination of affinity and kinetics. These properties are typically difficult to obtain for many weakly interacting molecular species (i.e., when dissociation constants (K(D)) are greater than 10(-5) M). The influence of affinity and kinetics on peak broadening in affinity chromatography has also been investigated.