Acceptor substrate selectivity and kinetic mechanism of Bacillus subtilis TagA

Wall teichoic acids (WTAs) are anionic polymers that coat the cell walls of Gram-positive bacteria. Because they are essential for survival or virulence in many organisms, the enzymes involved in the biosynthesis of WTAs are attractive antibiotic targets. The first committed step in the WTA biosynthetic pathway in Bacillus subtilis is catalyzed by TagA, which transfers N-acetylmannosamine (ManNAc) to the C4 hydroxyl of a membrane-anchored N-acetylglucosaminyl diphospholipid (GlcNAc-pp-undecaprenyl, lipid I) to make ManNAc-beta-(1,4)-GlcNAc-pp-undecaprenyl (lipid II). We have previously shown that TagA utilizes an alternative substrate containing a saturated C(13)H(27) lipid chain. Here we use unnatural substrates and products to establish the lipid preferences of the enzyme and to characterize the kinetic mechanism. We report that TagA is a metal ion-independent glycosyltransferase that follows a steady-state ordered Bi-Bi mechanism in which UDP-ManNAc binds first and UDP is released last. TagA shares homology with a large family of bacterial glycosyltransferases, and the work described here should facilitate structural analysis of the enzyme in complex with its substrates.     

Molecular characterization of dopamine D2 receptor isoforms tagged with green fluorescent protein

In this study, a cleavable signal peptide fused to the enhanced green fluorescent protein (EGFP) was tagged to the extracellular N-terminus of the human dopamine D2 receptor short and long isoforms (D2S and D2L). Ligand-binding properties of EGFP-tagged receptors were essentially unchanged in comparison to their respective wild-type receptors. The dopamine-mediated activation of both EGFP-D2 isoforms generated a robust inhibition of adenylyl cyclase type 5 in intact cells. In addition, the receptor density of EGFP-D2S and EGFP-D2L in transfected human embryonic kidney 293 (HEK293) cells was not altered when compared to cells transfected with the untagged D2S and D2L. However, the receptor densities of untagged and EGFP-tagged D2L were significantly lower in comparison to those measured with D2S constructs. Moreover, the receptor density of EGFP-D2S and EGFP-D2L was differentially upregulated in cells treated with antipsychotic drugs. As assessed by confocal microscopy, both EGFP-D2 isoforms were present on the cell surface. Notably, in contrast to the predominant plasma membrane localization of EGFP-D2S, EGFP-D2L was visualized both on the plasma membrane and intracellularly before dopamine exposure. Importantly, EGFP-D2S and EGFP-D2L are robustly internalized after dopamine treatment. Overall, our data suggest a differential intracellular sorting of D2S and D2L.     

Effects of a 1,3-diacylglycerol oil-enriched diet on postprandial lipemia in people with insulin resistance

Postprandial hypertriglyceridemia is common in individuals with insulin resistance, and diets enriched in 1,3-diacylglycerol (DAG) may reduce postprandial plasma triglycerides (PPTGs). We enrolled 25 insulin-resistant, nondiabetic individuals in a double-blind, randomized crossover trial to test the acute and chronic effects of a DAG-enriched diet on PPTG. Participants received either DAG or triacylglycerol (TAG) oil, in food products, for 5 weeks. Fasting lipids, and two separate postprandial tests, one with DAG oil and one with TAG oil, were performed at the end of each 5 week diet period. We found no acute or chronic effects of DAG oil on PPTG. Thus, neither the DAG oil PPTG (h/mg/dl) on a chronic TAG diet [area under the curve (AUC) = 503 +/- 439] nor the TAG oil PPTG on a chronic DAG diet (AUC = 517 +/- 638) was different from the TAG oil PPTG on a chronic TAG diet (AUC = 565 +/- 362). Five weeks of a DAG-enriched diet had no acute or chronic effects on PPTG in insulin-resistant individuals. We suggest further studies to evaluate the effects of DAG on individuals with low and high TG levels.     

Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers

We have hypothesized that ligand-induced binding sites (LIBS), i.e. sites expressed on cell surface receptors only after ligand binding causes the receptor to change shape, mediate subsequent biological events. To test this hypothesis, we have raised monoclonal antibodies that preferentially react with an integrin (platelet glycoprotein (GP) IIb-IIIa) after it bind Arg-Gly-Asp-containing ligands. The 13 anti-LIBS antibodies obtained define at least three distinct GPIIb-IIIa epitopes; one of these epitopes is also expressed following occupancy of another integrin, the vitronectin receptor. Certain of these LIBSs appear to mediate functions, since the antibodies that define them inhibit GPIIb-IIIa-mediated fibrin clot contraction or platelet adhesion to collagen. Nevertheless, none of the anti-LIBS antibodies inhibit binding of the primary ligand, fibrinogen. These data indicate that LIBS may mediate distinct consequences of receptor occupancy.     

The insulin receptor of the turkey erythrocyte. Characterization of the membrane-bound receptor.

The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.     

Synthesis In Vitro of Type 5 Adenovirus Capsid Proteins

Reaction mixtures containing cytoplasmic extracts and ribosomal fractions prepared from KB cells infected with type 5 adenovirus were able to carry out incorporation of amino acids into protein. The in vitro product included proteins which reacted specifically with antisera to adenovirus capsid proteins; in control experiments with extracts from uninfected cells, no reactions with the antisera were found. The viral proteins were synthesized in vitro on small polyribosomes, were released from them, and significant numbers of the free polypeptides were assembled in vitro into multimeric adenovirus capsid structures.