The three nitric-oxide synthases differ in their kinetics of tetrahydrobiopterin radical formation, heme-dioxy reduction, and arginine hydroxylation

The nitric-oxide synthases (NOSs) make nitric oxide and citrulline from l-arginine. How the bound cofactor (6R)-tetrahydrobiopterin (H4B) participates in Arg hydroxylation is a topic of interest. We demonstrated previously that H4B radical formation in the inducible NOS oxygenase domain (iNOSoxy) is kinetically coupled to the disappearance of a heme-dioxy intermediate and to Arg hydroxylation. Here we report single turnover studies that determine and compare the kinetics of these transitions in Arg hydroxylation reactions catalyzed by the oxygenase domains of endothelial and neuronal NOSs (eNOSoxy and nNOSoxy). There was a buildup of a heme-dioxy intermediate in eNOSoxy and nNOSoxy followed by a monophasic transition to ferric enzyme during the reaction. The rate of heme-dioxy decay matched the rates of H4B radical formation and Arg hydroxylation in both enzymes. The rates of H4B radical formation differed such that nNOSoxy (18 s(-1)) > iNOSoxy (11 s(-1)) > eNOSoxy (6 s(-1)), whereas the lifetimes of the resulting H4B radical followed an opposite rank order. 5MeH4B supported a three-fold faster radical formation and greater radical stability relative to H4B in both eNOSoxy and nNOSoxy. Our results indicate the following: (i) the three NOSs share a common mechanism, whereby H4B transfers an electron to the heme-dioxy intermediate. This step enables Arg hydroxylation and is rate-limiting for all subsequent steps in the hydroxylation reaction. (ii) A direct correlation exists between pterin radical stability and the speed of its formation in the three NOSs. (iii) Uncoupled NO synthesis often seen for eNOS at low H4B concentrations may be caused by the slow formation and poor stability of its H4B radical.     

Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.     

Phospholipids inhibit lipopolysaccharide (LPS)-induced cell activation: a role for LPS-binding protein

The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood. It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP). Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS. Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity. The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-alpha release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, we show that LBP is a target for the inhibitory function of phospholipids.     

Membrane chromatography of DNA: conformation-induced capacity and selectivity

The chromatographic purification of biological macromolecules requires a novel approach to overcome some of the pore size limitations of commercially available resins. Membrane adsorbers offer the potential for better resolution as well as productivity. Sharp peaks are gained by the rapid exchange rate with the adsorbing membranes associated with the convective flow path, in contrast to the pore diffusion requirement for resin exchange. The resolution advantage is preserved even when the very short bed heights of membranes are exploited for the purpose of exceptionally high flow rates and productivity.Breakthrough experiments were used to assess the membrane dynamic loading capacities of flexible macromolecules using supercoiled (SC) DNA as a model system. In contrast to reports for smaller biomolecules such as proteins and antibodies, the dynamic capacity for DNA was found to be highly dependent on flow rates and concentrations. Increasing flow rates induced DNA elongation, which increased the surface coverage and, in turn, lowered the capacity. Increasing concentrations beyond C*, the overlap concentration, led to exclusion-volume interactions, which reduced the size of DNA and increased the membrane adsorber capacity. In the chromatographic mode, membranes with a strongly positive charge were able to resolve various isoforms of DNA, surpassing the capabilities of analogous chromatographic resins. In this study, we found that the convective-flow-induced-structural behavior of DNA is responsible for the resolution in separation.     

Identification of an extracellular domain within the human PiT2 receptor that is required for amphotropic murine leukemia virus binding

Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.     

Genetic analysis of the cytoplasmic dynein subunit families

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.     

Spontaneous processing of abstract categorical information in the ventrolateral prefrontal cortex

In various aspects of linguistic analysis and human cognition, some forms of observed variation are ignored in the service of handling more abstract categories. In the absence of training, rhesus discriminate between different types of vocalizations based on the information conveyed as opposed to their acoustic morphologies. We hypothesized that neurons in the ventrolateral prefrontal cortex (vPFC), an area involved in auditory-object processing, might be involved in this spontaneous categorization. To test this hypothesis, we recorded vPFC activity while rhesus listened to vocalizations conveying information about food and non-food events. Results showed between, but not within category discrimination. That is, vPFC neurons discriminated between vocalizations associated with food versus non-food events but not within the class of food calls associated with differences in quality. These results indicate that the vPFC plays a significant role in spontaneously processing abstract categorical information.     

An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach

This protocol describes a method that we developed to adapt the tandem affinity purification (TAP) approach for use in mammalian cells. The protocol involves fusing a protein of interest with a tandem tag consisting of two FLAG tags (FF) followed by two protein-A immunoglobulin G (IgG) binding domains (ZZ). The protocol improves upon previously published TAP approaches by employing FLAG in place of calmodulin binding peptide (CBP) with resulting higher recovery during purification. In addition, we use a bicistronic expression system that ensures recovery of stably transfected cell lines expressing easily detectable levels of the protein of interest. A method is also presented for generating cytoplasmic and nuclear extracts, which extends use of this protocol to identify protein-protein interactions occurring specifically in the cytoplasm or nucleus. This protocol facilitates the preparation of partially purified recombinant protein and identification of protein-protein interactions in mammalian cell culture models. The protocol can be completed in 34 h.     

Integrating large-scale genotype and phenotype data

With the completion of the Human Genome Project, a new emphasis is focusing on the sequence variation and the resulting phenotype. The number of data available from genomic studies addressing this relationship is rapidly growing. In order to analyze these data as a whole, they need to be integrated, aggregated and annotated in a timely manner. The Pharmacogenetics and Pharmacogenomics Knowledge Base PharmGKB; () assembles and disseminates these data and their associated metadata that are needed for unambiguous identification and replication. Assembling these data in a timely manner is challenging, and the scalability of these data produce major challenges for a knowledge base such as PharmGKB. However, it is only through rapid global meta-annotation of these data that we will understand the relationship between specific genotype(s) and the related phenotype. PharmGKB has confronted these challenges, and these experiences and solutions can benefit all genome communities.