Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers

BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t((1/2))) of 11-14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.     

Liposomal forms of rhenium cluster compounds: enhancement of biological activity

Liposomal formulations of dinuclear cluster rhenium (Re) compounds were used in biochemical trials. Interaction of liposomal forms of some Re compounds with red blood cells in experiments in vitro showed strong cell-stabilizing properties. In the models of tumor growth and hemolytic anemia in vivo, liposomal forms had better therapeutic effects in comparison with their solutions. The process of formation of liposomes of cluster Re compounds with different organic ligands was investigated by the method of electronic absorption spectra and mechanism of their interactions with lipids is proposed. Encapsulation of cluster Re compounds to lipid coating may have activation significance for the quadruple Re-Re bond.     

3-isobutylmethylxanthine inhibits hepatic urea synthesis: protection by agmatine

We previously showed that agmatine stimulated hepatic ureagenesis. In this study, we sought to determine whether the action of agmatine is mediated via cAMP signaling. A pilot experiment demonstrated that the phosphodiesterase inhibitor, 3-isobutylmethylxanthine (IBMX), inhibited urea synthesis albeit increased [cAMP]. Thus, we hypothesized that IBMX inhibits hepatic urea synthesis independent of [cAMP]. We further theorized that agmatine would negate the IBMX action and improve ureagenesis. Experiments were carried out with isolated mitochondria and (15)NH(4)Cl to trace [(15)N]citrulline production or [5-(15)N]glutamine and a rat liver perfusion system to trace ureagenesis. The results demonstrate that IBMX induced the following: (i) inhibition of the mitochondrial respiratory chain and diminished O(2) consumption during liver perfusion; (ii) depletion of the phosphorylation potential and overall hepatic energetic capacity; (iii) inhibition of [(15)N]citrulline synthesis; and (iv) inhibition of urea output in liver perfusion with little effect on [N-acetylglutamate]. The results indicate that IBMX directly and specifically inhibited complex I of the respiratory chain and carbamoyl-phosphate synthase-I (CPS-I), with an EC(50) about 0.6 mm despite a significant elevation of hepatic [cAMP]. Perfusion of agmatine with IBMX stimulated O(2) consumption, restored hepatic phosphorylation potential, and significantly stimulated ureagenesis. The action of agmatine may signify a cascade effect initiated by increased oxidative phosphorylation and greater ATP synthesis. In addition, agmatine may prevent IBMX from binding to one or more active site(s) of CPS-I and thus protect against inhibition of CPS-I. Together, the data may suggest a new experimental application of IBMX in studies of CPS-I malfunction and the use of agmatine as intervention therapy.     

The butyrylcholinesterase knockout mouse is obese on a high-fat diet

Butyrylcholinesterase (BChE) inactivates the appetite stimulating hormone octanoyl-ghrelin. The hypothesis was tested that BChE−/− mice would have abnormally high body weight and high levels of octanoyl-ghrelin. It was found that BChE−/− mice fed a standard 5% fat diet had normal body weight. However, BChE−/− mice fed a diet containing 11% fat became obese. Their obesity was not explained by increased levels of octanoyl-ghrelin, or by increased caloric intake, or by decreased exercise. Instead, a role for BChE in fat utilization was suggested.     

Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines

Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.     

A survey of the year 2006 literature on applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a fast and robust method to determine the energetics of association reactions in solution. The changes in enthalpy, entropy and heat capacity that accompany binding provide unique insights into the balance of forces driving association of molecular entities. ITC is used nowadays on a day-to-day basis in hundreds of laboratories. The method aids projects both in basic and practice-oriented research ranging from medicine and biochemistry to physical chemistry and material sciences. Not surprisingly, the range of studies utilizing ITC data is steadily expanding. In this review, we discuss selected results and ideas that have accumulated in the course of the year 2006, the focus being on biologically relevant systems. Theoretical developments, novel applications and studies that provide a deeper level of understanding of the energetic principles of biological function are primarily considered. Following the appearance of a new generation of titration calorimeters, recent papers provide instructive examples of the synergy between energetic and structural approaches in biomedical and biotechnological research.     

Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis

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