Immobilization of alpha1-acid glycoprotein for chromatographic studies of drug-protein binding II. correction for errors in association constant measurements

A new method for the immobilization of α₁-acid glycoprotein (AGP) in high-performance liquid chromatography (HPLC) columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R- or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R- and S-propranolol and HPLC columns containing immobilized AGP.     

Rac1 activation inhibits E-cadherin-mediated adherens junctions via binding to IQGAP1 in pancreatic carcinoma cells

Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. The two isoproteins of Nck are functionally redundant in many aspects and differ in only few amino acids that are mostly located in the linker regions between the interaction modules. Nck proteins connect receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. Thereby, Nck regulates activation-dependent processes during cell polarisation and migration and plays a crucial role in the signal transduction of a variety of receptors including for instance PDGF-, HGF-, VEGF- and Ephrin receptors. In most cases, the SH2 domain mediates binding to the phosphorylated receptor or associated phosphoproteins, while SH3 domain interactions lead to the formation of larger protein complexes. In T lymphocytes, Nck plays a pivotal role in the T cell receptor (TCR)-induced reorganisation of the actin cytoskeleton and the formation of the immunological synapse. However, in this context, two different mechanisms and adapter complexes are discussed. In the first scenario, dependent on an activation-induced conformational change in the CD3ε subunits, a direct binding of Nck to components of the TCR/CD3 complex was shown. In the second scenario, Nck is recruited to the TCR complex via phosphorylated Slp76, another central constituent of the membrane proximal activation complex. Over the past years, a large number of putative Nck interactors have been identified in different cellular systems that point to diverse additional functions of the adapter protein, e.g. in the control of gene expression and proliferation.     

End-joining long nucleic acid polymers

Many experiments involving nucleic acids require the hybridization and ligation of multiple DNA or RNA molecules to form a compound molecule. When one of the constituents is single stranded, however, the efficiency of ligation can be very low and requires significant individually tailored optimization. Also, when the molecules involved are very long (>10 kb), the reaction efficiency typically reduces dramatically. Here, we present a simple procedure to efficiently and specifically end-join two different nucleic acids using the well-known biotin–streptavidin linkage. We introduce a two-step approach, in which we initially bind only one molecule to streptavidin (STV). The second molecule is added only after complete removal of the unbound STV. This primarily forms heterodimers and nearly completely suppresses formation of unwanted homodimers. We demonstrate that the joining efficiency is 50 ± 25% and is insensitive to molecule length (up to at least 20 kb). Furthermore, our method eliminates the requirement for specific complementary overhangs and can therefore be applied to both DNA and RNA. Demonstrated examples of the method include the efficient end-joining of DNA to single-stranded and double-stranded RNA, and the joining of two double-stranded RNA molecules. End-joining of long nucleic acids using this procedure may find applications in bionanotechnology and in single-molecule experiments.     

Complete transformation of 1,1,1-trichloroethane to chloroethane by a methanogenic mixed population

A methanogenic mixed population in a packed-bed reactor completely transformed 1,1,1-trichloroethane (10 μM) to chloroethane by a cometabolic process. Chloroethane was not further transformed. Acetate and methanol served as electron donors. Complete transformation of 1,1,1-trichloroethane to chloroethane only occurred when sufficient electron donor was fed into the reactor. Otherwise, besides chloroethane, 1,1-dichloroethane was also found as a product. The products of 1,1,1-trichloroethane transformation also depended on the type of electron donor present. With acetate, the degree of dechlorination was higher, i.e. more 1,1,1-trichloroethane was transformed to chloroethane than with methanol. In an enrichment culture obtained from the reactor contents, 1,1,1-trichloroethane was only transformed to 1,1-dichloroethane and was not further metabolized. Methanol, acetate, formate, ethanol, 2-propanol, trimethylamine and H₂, but not dimethylamine and methylamine, served as electron donors for 1,1,1-trichloroethane transformation by this enrichment culture. Both nitrate and nitrite inhibited 1,1,1-trichloroethane transformation; while nitrate completely inhibited 1,1,1-trichloroethane dechlorination, some conversion did occur in the presence of nitrite. The product(s) of this conversion remain unknown, since no chlorinated hydrocarbons were detected. Received: 19 June 1998 / Received revision: 14 September 1998 / Accepted: 17 September 1998     

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin

Biointeraction studies based on high performance affinity chromatography were used to investigate the binding of human serum albumin (HSA) to two major phenytoin metabolites: 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). This was initially examined by conducting self-competition zonal elution experiments in which m-HPPH or p-HPPH were placed in both the mobile phase and injected sample. It was found that each metabolite had a single major binding site on HSA. Competitive zonal elution experiments using l-tryptophan, warfarin, digitoxin, and cis-clomiphene as site-selective probes indicated that m-HPPH and p-HPPH were interacting with the indole-benzodiazepine site of HSA. The estimated association equilibrium constants for m-HPPH and p-HPPH at this site were 3.2 (+/-1.2)x10(3) and 5.7 (+/-0.7)x10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Use of these metabolites as competing agents for injections of phenytoin demonstrated that m-HPPH and p-HPPH had direct competition with this drug at the indole-benzodiazepine site. However, the use of phenytoin as a competing agent indicated that this drug had additional negative allosteric interactions on the binding of these metabolites to HSA. These results agreed with previous studies on the binding of phenytoin to HSA and its effects on the interactions of HSA with site-selective probes for the indole-benzodiazepine site.     

An RNA toolbox for single-molecule force spectroscopy studies

Precise, controllable single-molecule force spectroscopy studies of RNA and RNA-dependent processes have recently shed new light on the dynamics and pathways of RNA folding and RNA-enzyme interactions. A crucial component of this research is the design and assembly of an appropriate RNA construct. Such a construct is typically subject to several criteria. First, single-molecule force spectroscopy techniques often require an RNA construct that is longer than the RNA molecules used for bulk biochemical studies. Next, the incorporation of modified nucleotides into the RNA construct is required for its surface immobilization. In addition, RNA constructs for single-molecule studies are commonly assembled from different single-stranded RNA molecules, demanding good control of hybridization or ligation. Finally, precautions to prevent RNase- and divalent cation-dependent RNA digestion must be taken. The rather limited selection of molecular biology tools adapted to the manipulation of RNA molecules, as well as the sensitivity of RNA to degradation, make RNA construct preparation a challenging task. We briefly illustrate the types of single-molecule force spectroscopy experiments that can be performed on RNA, and then present an overview of the toolkit of molecular biology techniques at one's disposal for the assembly of such RNA constructs. Within this context, we evaluate the molecular biology protocols in terms of their effectiveness in producing long and stable RNA constructs.