Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5-to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5-to-5 phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.

A new spin: The addition of chemically inert perfluoro carboxylic acids (green; see picture) to P450 enzymes results in dramatic activation of their catalytic activity as a result of the conversion of the Fe/heme from a low‐spin to a high‐spin state, and the reduction of the binding‐pocket size. Together these effects allow otherwise inert substrates such as propane and even methane to be oxidized.

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

Ein neuer Dreh: Die Zugabe chemisch inerter Perfluorcarbonsäuren (grün im Bild) zu P450‐Enzymen erhöht deren katalytische Aktivität drastisch. Gründe sind der Übergang des Fe/Häms von einem Low‐ in einen High‐Spin‐Zustand und die Verkleinerung der Bindungstasche. In der Summe hat das zur Folge, dass sonst inerte Substrate wie Propan und sogar Methan oxidiert werden.

No abstract

SummaryHighly purified D-dimer was obtained from plasmin digest of human cross-linked fibrin. After reduction of its disulfide bonds, the γ-γ chain remnant, containing cross-linking site, was then isolated by ion-exchange chromatography on CM-cellulose. Antisera obtained by immunizing rabbits with D-dimer and its γ-γ chain remnant contained a small population of antibodies which specifically reacted with D-dimer. Thus, a specific radioimmunoassay system allowing detection and quantitation of D-dimer in the presence of fibrinogen and monomeric fragment D was made possible.

Two labeled isotopomers of L‐tyrosine, L‐Tyr, have been synthesized using specific properties of the enzymes phenylanine ammonia lyase, PAL, and L‐phenylalanine hydroxylase. In an intermediate step [1‐14C]‐, and [2‐14C]‐L‐phenylalanine, L‐Phe, have been obtained from [1‐14C]‐, and [2‐14C]cinnamic acid, prepared from potassium [14C]cyanate or [2‐14C]malonic acid, and by addition of ammonia in the presence of enzyme PAL. The labeled isotopomers of L‐Phe have been converted into [1‐14C]‐, and [2‐14C]‐L‐Tyr using the enzyme L‐phenylalanine hydroxylase. Copyright © 2001 John Wiley & Sons, Ltd.

The synthesis of four selectively labeled isotopomers of L‐tyrosine, (L‐Tyr), using chemical and enzymatic methods is reported. Four tritium labeled isotopomers of L‐phenylalanine (L‐Phe) – [2‐3H]‐, [2′,6′‐3H2]‐, [3R‐3H]‐ and [3S‐3H]‐ have been synthesized using a combination of chemical and enzymatic methods. The labeled isotopomers of L‐Phe have been converted into [2 ‐3H]‐, [2′,6′‐3H2]‐, [3R‐3H]‐, and [3S‐3H]‐L‐Tyr by using the enzyme L‐phenyl‐alanine 4′‐monooxygenase. Copyright © 2002 John Wiley & Sons, Ltd.