Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them, PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts. Upon extraction with a buffer containing urea, the recombinant enzyme was p...     

Multioperational Oxidase Biosensors Based on Carbosilane Dendrimers with Interacting Ferrocenes

The bioelectrocatalytical properties and kinetic characteristics of new oxidase biosensors based on two different carbosilane dendrimers are described. The best glucose biosensor developed displayed, in an ascorbate interference free work potential interval, a strictly linear range from 0 to 4.0 mM, a detection limit of 40,6 μM and a response time less than 3 s. The lactate biosensor displayed a linear range from 0 to 0.8 mM, a detection limit of 0.73 µM and a response time less than 2 s. The apparent Michaelis–Menten constants were calculated to be 4.39 mM and 2.08 mM respectively, according to Lineweaver–Burk equation.     

Immobilized enzyme reactors in proteomics

Fast, efficient characterization of proteins is becoming one of the hottest topics in the bioanalytical community, especially for large-scale proteomic studies. As an attractive approach, protein digestion by enzymes supported on various matrices (referred to as immobilized enzyme reactors, IMERs) has recently attracted much attention.In this article, we present a critical overview of some highly efficient IMERs and related analytical systems. We give major coverage to applications of IMERs in proteomic analysis, including protein-expression profiling, characterization of proteins with post-translational modifications, and protein quantification. We also comment on promising trends for IMERs in proteomics.     

超声波对固定化酶活性的影响

本文介绍了超声波对固定化酶的影响，同时对作用过程中的影响因素进行了阐述，并探讨了超声波影响固定化酶的可能机理。     

Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase

Bisphosphonates, known for their effectiveness in the treatment of osteoporosis, inhibit bone resorption via mechanisms that involve binding to bone mineral and cellular effects on osteoclasts. The major molecular target of nitrogen-containing bisphosphonates (N-BPs) in osteoclasts is farnesyl diphosphate synthase (FPPS). N-BPs likely inhibit this enzyme by mimicking one or more of the natural isoprenoid lipid substrates (GPP/DMAPP and IPP) but the mode of inhibition is not established. The active site of FPPS comprises a subsite for each substrate. Kinetic studies with recombinant human FPPS indicate that both potent (risedronate) and weak (NE-58051) enzyme inhibitors compete with GPP for binding to FPPS, however, binding to this site does not completely explain the difference in potency of the two inhibitors, suggesting that a second binding site may also be a target of bisphosphonate inhibition. Using the docking software suite Autodock, we explored a dual inhibitor binding mode for recombinant human FPPS. Experimental support for dual binding is suggested by Dixon plots for the inhibitors. N-BPs may inhibit by binding to both the GPP and a second site with differences in potency at least partly arising from inhibition at the second site.     

Direct immobilization in poly(dimethylsiloxane) for DNA, protein and enzyme fluidic biochips

A new arraying method is presented based on the properties of poly(dimethylsiloxane) (PDMS) polymer to entrap beads bearing biologically active compounds. It is shown that such beads could be spotted and dried at the surface of a poly(vinyl chloride) master and subsequently transferred at the PDMS interface by direct moulding of the polymer on the mask. Moreover, the use of the PDMS-assisted-immobilization enables the development of either a low density array (100 spots) or a micro-channel biochip with a direct incorporation of the sensing element in a fluidic system for the quantitative detection of enzyme substrates, antigens and oligonucleotides, depending on the immobilized sensing element. All biochip formats were revealed by a chemiluminescent reaction detected with a charge coupled device camera.As a result, arrays of beads bearing active enzymes, antibodies and oligonucleotides were successfully obtained and enabled the achievement of biochips for the chemiluminescent detection of enzyme substrates, protein antigens and oligonucleotides sequence with detection limit of 1 μM, 1.5×10⁷ molecules and 10⁸ molecules, respectively.