Expression, purification, characterization, and site-directed mutagenesis of phosphorylase kinase [upsilon] subunit

The overall aim was to elucidate the substrate specificity and regulatory properties of the catalytic subunit of phosphorylase kinase (PhK) to better understand how this enzyme works. I have expressed the PhK [gamma] subunit (full-length and seven truncated forms) in E. coli. One of the truncated forms of [gamma], [gamma][subscript]1-300 has a 2-fold higher specific activity than the full-length [gamma], suggesting that an autoinhibitory domain(s) is located at the C-terminus of [gamma], [gamma][subscript]301-386. The truncated [gamma][subscript]1-300 purified to homogeneity has several properties similar to full-length [gamma], including its substrate specificity and metal ion response. Therefore, [gamma][subscript]1-300 was used as a model enzyme to probe structure-function relationships of PhK [gamma] subunit. Charge to alanine and charge reversal scanning mutations were used to locate substrate and metal ion binding sites of [gamma]. The secondary structures of mutant proteins were evaluated by FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy). Those mutant proteins with similar secondary structures compared to wild-type [gamma][subscript]1-300 were further characterized. Two mutant proteins, E111K and E154R, were shown to be involved in substrate, pseudosubstrate, and metal ion binding. Using these two mutants, we demonstrated that E[superscript]111 binds to the P-3 site (K[superscript]11) and E[superscript]154 interacts with the P-2 site (Q[superscript]12) of phosphorylase b. Based on results with these two mutants and others, it is suggested that the second metal ion binding site of [gamma] is between the D[superscript]168FG loop and E[superscript]111--KPE[superscript]154N loop similar to the second metal ion binding site in cAMP-dependent protein kinase. Two synthetic peptides, PhK 13 ([gamma][subscript]302-326) and PhK 5 ([gamma][subscript]342-366), corresponding to two calmodulin binding regions of [gamma] were used as inhibitors. Based on studies of inhibition mechanisms of [gamma][subscript]1-300 and both mutant proteins, we suggest the inhibition mechanism of PhK 13 is through a pseudosubstrate mechanism and PhK 5 is via an inhibitory mechanism. The active site region of [gamma] is proposed to have similarities to calmodulin-binding site based on these data and other speculations. Because PhK and [gamma][subscript]1-300 can phosphorylate seryl and tyrosyl residues dependent on the metal ions, new substrates and pathways might exist for this enzyme beyond the known glycogenolysis cascade. Two proteins, which were phosphorylated on tyrosine in muscle cell extracts either directly or indirectly by PhK, raise this possibility

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit.

Journal ArticleUsing site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154)

An Economic Analysis of Stream Restoration in an Urban Watershed: Austin, Texas.

By 2006, the U.S. government has spent $15 billion to address the degradation of urban streams, including erosion of stream banks, disconnection of rivers from the floodplain, and disturbance of surface runoff pathways. Bank stabilization is one of the most prevalent restoration activities in urban stream restoration. Unfortunately, most stream restoration projects have been undertaken without a pre- or post-evaluation of the impact of stream restoration on real value in the area. All restoration projects beg the question: Did the money spent on the project result in greater benefits to stream stability as well as to adjacent properties? The Walnut Creek watershed, located in Austin, Texas, has experienced varying stages of urbanization since the 1990s. One of the streams, the Walnut Creek tributary, was restored in 2003. The purpose of this study is to assess the impact of stream restoration on housing values. We applied the hedonic pricing method to evaluate the changes in housing value associated with housing and environmental characteristics. Repeat ground photography was utilized to assess stream restoration activities at spatial and temporal scales. Our results suggest that the stream restoration project resulted in significant positive impacts on housing values in the periods of restoration (8.3%) and restoration adjustment (10.7%). However, the project did not enhance the values of houses on the floodplain. In addition, results show that erosion had continuous negative impacts on housing values. Overall, the restoration project contributed to the greater benefits during the restoration adjustment period right after restoration by an increase of 1% of the average housing value for each property on the restoration site. In this study, the benefits of stream restoration project were minimal since bank stabilization was the main activity considered in this stream restoration project. Nevertheless, restoration enhances the stability of the stream banks, minimizes erosion problems, and presents an enhanced aesthetic beauty of the stream in Austin, Texas

THE CHANGE OF KNEE KINEMATICS AFTER ANTERIOR CRUCIATE LIGAMENT DEFICIENCY AND RECONSTRUCTION DURING LANDING

The purpose of this study were to evaluate the different of knee kinematics analysis after ACLD and ACLR during landing performances. The participants were instructed to finish counter moment jump (CMJ) with arms free 5 times as hard as possible with Vicon motion system and two force platforms. The ACLD showed a significant less knee flexion degree at the peak vertical GRF compared with others. Our founding was similar to the present studies; the impulse during landing among three groups was almost the same, but the RF EMG showed lower after two ACL groups, especially in ACLD

POINeT: protein interactome with sub-network analysis and hub prioritization

<p>Abstract</p> <p>Background</p> <p>Protein-protein interactions (PPIs) are critical to every aspect of biological processes. Expansion of all PPIs from a set of given queries often results in a complex PPI network lacking spatiotemporal consideration. Moreover, the reliability of available PPI resources, which consist of low- and high-throughput data, for network construction remains a significant challenge. Even though a number of software tools are available to facilitate PPI network analysis, an integrated tool is crucial to alleviate the burden on querying across multiple web servers and software tools.</p> <p>Results</p> <p>We have constructed an integrated web service, POINeT, to simplify the process of PPI searching, analysis, and visualization. POINeT merges PPI and tissue-specific expression data from multiple resources. The tissue-specific PPIs and the numbers of research papers supporting the PPIs can be filtered with user-adjustable threshold values and are dynamically updated in the viewer. The network constructed in POINeT can be readily analyzed with, for example, the built-in centrality calculation module and an integrated network viewer. Nodes in global networks can also be ranked and filtered using various network analysis formulas, i.e., centralities. To prioritize the sub-network, we developed a ranking filtered method (S3) to uncover potential novel mediators in the midbody network. Several examples are provided to illustrate the functionality of POINeT. The network constructed from four schizophrenia risk markers suggests that EXOC4 might be a novel marker for this disease. Finally, a liver-specific PPI network has been filtered with adult and fetal liver expression profiles.</p> <p>Conclusion</p> <p>The functionalities provided by POINeT are highly improved compared to previous version of POINT. POINeT enables the identification and ranking of potential novel genes involved in a sub-network. Combining with tissue-specific gene expression profiles, PPIs specific to selected tissues can be revealed. The straightforward interface of POINeT makes PPI search and analysis just a few clicks away. The modular design permits further functional enhancement without hampering the simplicity. POINeT is available at <url>http://poinet.bioinformatics.tw/</url>.</p

Dynamic Antenna Alignment Control in Microwave Air-Bridging for Sky-Net Mobile Communication Using Unmanned Flying Platform

This paper presents a preliminary study on establishing a mobile point-to-point (P2P) microwave air-bridging (MAB) between Unmanned Low Altitude Flying Platform (ULAFP) and backhaul telecommunication network. The proposed Sky-Net system relays telecom signal for general mobile cellphone users via ULAFP when natural disaster sweeps off Base Transceiver Stations (BTSs). Unlike the conventional fix point microwave bridging application, the ULAFP is cruising on a predefined mission flight path to cover a wider range of service. The difficulty and challenge fall on how to maintain antenna alignment accurately in order to provide the signal strength for MAB. A dual-axis rotation mechanism with embedded controller is designed and implemented on airborne and ground units for stabilizing airborne antenna and tracking the moving ULAFP. The MAB link is established in flight tests using the proposed antenna stabilizing/tracking mechanism with correlated control method. The result supports backbone technique of the Sky-Net mobile communication and verifies the feasibility of airborne e-Cell BTS