NG-Hydroxy-L-arginine, [15N]-NG-hydroxy-L-arginine, and NG-hydroxy-NG- methyl-L-arginine were used as mechanistic probes of the initial step in the reaction catalyzed by nitric oxide synthase isolated from murine macrophages. NG-Hydroxy-L-arginine was found to be a substrate for nitric oxide synthase with a Km equal to 28.0 microM, yielding nitric oxide and L-citrulline. NADPH was required for the reaction and (6R)-tetrahydro-L-biopterin enhanced the initial rate of nitric oxide formation. The stoichiometry of NG-hydroxy-L-arginine loss to L-citrulline and nitric oxide (measured as nitrite and nitrate) formation was found to be 1:1:1. NG-Hydroxy-L-arginine was also observed in small amounts from L-arginine during the enzyme reaction. Studies with [15N]-NG-hydroxy-L-arginine indicated that the nitrogen in nitric oxide is derived from the oxime nitrogen of [15N]-NG-hydroxy-L- arginine. NG-Hydroxy-NG-methyl-L-arginine was found to be both a reversible and an irreversible inhibitor of nitric oxide synthase, displaying reversible competitive inhibition with K(i) equal to 33.5 microM. As an irreversible inhibitor, NG-hydroxy-NG-methyl-L-arginine gave kinact equal to 0.16 min-1 and KI equal to 26.5 microM. This inhibition was found to be both time- and concentration-dependent as well as showing substrate protection against inactivation. Gel filtration of an NG-hydroxy-NG-methyl-L-arginine-inactivated nitric oxide synthase failed to recover substantial amounts of enzyme activity.
We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.
The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.
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