Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes.

We have isolated and sequenced cDNA clones containing the entire coding region of both the chloroplast and cytosolic versions of phosphoglycerate kinase from wheat. Comparison of these sequences reveals a higher than expected level of similarity between the nucleic acids and encoded proteins. Analysis of this data in relation to that for phosphoglycerate kinase sequences of mammals, prokaryotes and yeasts suggests that the wheat genes have recombined. This has resulted in the chloroplast and cytosolic kinases being more similar to each other than would be expected if the chloroplast enzyme had evolved directly from that of a prokaryotic progenitor.     

Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K _D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K _D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K _D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K _D in the 10^?8 M range and (ii) a kinetically rapid, low-affinity interaction with K _D in the 10^?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K _D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.     

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.     

Kinase array design, back to front: biaryl amides

New kinase inhibitors can be found by synthesis of targeted arrays of compounds designed using system-based knowledge as well as through screening focused or diverse compounds. Most array strategies aim to add functionality to a fragment that binds in the purine subpocket of the ATP-site. Here, an alternative pharmacophore-guided array approach is described which set out to discover novel purine subpocket-binding groups. Results are shown for p38alpha and cFMS kinase, for which multiple distinct series with nanomolar potency were discovered. Some of the compounds showed potency in cell-based assays and good pharmacokinetic properties.     

Specificity of Pyrrolysyl-tRNA Synthetase for Pyrrolysine and Pyrrolysine Analogs

Pyrrolysine, the 22nd amino acid, is encoded by amber (TAG = UAG) codons in certain methanogenic archaea and bacteria. PylS, the pyrrolysyl-tRNA synthetase, ligates pyrrolysine to tRNA^Pyl for amber decoding as pyrrolysine. PylS and tRNA^Pyl have potential utility in making tailored recombinant proteins. Here, we probed interactions necessary for recognition of substrates by archaeal PylS via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays. Replacement of the methylpyrroline ring of pyrrolysine with cyclopentane indicated that solely hydrophobic interactions with the ring-binding pocket of PylS are sufficient for substrate recognition. However, a 100-fold increase in the specificity constant of PylS was observed with an analog, 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid (2Thf-lys), in which tetrahydrofuran replaced the pyrrolysine methylpyrroline ring. Other analogs in which the electronegative atom was moved to different positions suggested PylS preference for a hydrogen-bond-accepting group at the imine nitrogen position in pyrrolysine. 2Thf-lys was a preferred substrate over a commonly employed pyrrolysine analog, but the specificity constant for 2Thf-lys was 10-fold lower than for pyrrolysine itself, largely due to the change in K_m. The in vivo activity of the analogs in supporting UAG suppression in Escherichia coli bearing genes for PylS and tRNA^Pyl was similar to in vitro results, with l-pyrrolysine and 2Thf-lys supporting the highest amounts of UAG translation. Increasing concentrations of either PylS substrate resulted in a linear increase in UAG suppression, providing a facile method to assay bioactive pyrrolysine analogs. These results illustrate the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyrrolysine and provide a promising new series of easily synthesized pyrrolysine analogs that can serve as scaffolds for the introduction of novel functional groups into recombinant proteins.     

The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses

Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin.     

ENU‐induced mutant allele of Dnah1, ferf1, causes abnormal sperm behavior and fertilization failure in mice

Given attention to both contraception and treatment of infertility, there is a need to identify genes and sequence variants required for mammalian fertility. Recent unbiased mutagenesis strategies have expanded horizons of genetic control of reproduction. Here we show that male mice homozygous for the ethyl‐nitroso‐urea‐induced ferf1 (fertilization failure 1) mutation are infertile, producing apparently normal sperm that does not fertilize oocytes in standard fertilization in vitro fertilization assays. The ferf1 mutation is a single‐base change in the Dnah1 gene, encoding an axoneme‐associated dynein heavy chain, and previously associated with male infertility in both mice and humans. This missense mutation causes a single‐amino‐acid change in the DNAH1 protein in ferf1 mutant mice that leads to abnormal sperm clumping, aberrant sperm motility, and the inability of sperm to penetrate the oocyte's zona pellucida; however, the ferf1 mutant sperm is competent to fertilize zona‐free oocytes. Taken together, the various mutations affecting the DNAH1 protein in both mouse and human produce a diversity of phenotypes with both subtle and considerable differences. Thus, future identification of the interacting partners of DNAH1 might lead to understanding its unique function among the sperm dyneins.     

Understanding the enzymology of fibrinolysis and improving thrombolytic therapy

Cardiovascular disease is responsible for 17 million deaths per year but acute myocardial infarction and stroke can be treated with thrombolytics ("clot busters"), which are plasminogen activators. However, despite many years of study and huge investment from the pharmaceutical industry, clinical trials of new drugs have often been disappointing. Part of the problem may be our incomplete understanding of the regulation of plasminogen activation in vivo. We have developed precise in vitro methods and with the application of computer simulations, we hope to improve our understanding of plasminogen activation to facilitate improvements in thrombolytic therapy.     

The photoreaction of active-site-methylated bacteriorhodopsin: an investigation using static and time-resolved infrared difference spectroscopy

The photoreaction of active-site-methylated, permethylated bacteriorhodopsin has been investigated by static and time-resolved UV-vis and infrared difference spectroscopy. Additional information on the isomeric composition of the initial state and of photoproducts was obtained by retinal extraction and subsequent HPLC analysis. The data show that the dark-adapted state contains only all-trans-retinal. Prolonged illumination produces a metastable state which contains essentially only 9-cis-retinal and which decays back to the dark-adapted initial state within 8 h. The time-resolved infrared difference spectra clearly demonstrate that laser flash excitation produces an intermediate that has all the characteristics of the L intermediate. It is demonstrated that the methyl group at the Schiff base nitrogen introduces a steric hindrance with the protein which inhibits a photoreaction at 80 K, but which allows the generation of an L-like intermediate at room temperature and 173 K.