Gene/Protein Disease Symptom Drug Enzyme Compound
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In Escherichia coli, expulsion of sodium ions is driven by proton flux via at least two distinct Na+/H+ antiporters, NhaA and NhaB. When the nhaA gene is deleted from the chromosome, the cell becomes sensitive to high salinity and alkaline pH (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302). In the current work we cloned the nhaB gene by complementation of the delta nhaA strain. The gene codes for a membrane protein 504 amino acids long. Hydropathic analysis of the sequence indicates the presence of 12 putative transmembrane helices. NhaB has been specifically labeled with [35S]methionine; it is a membrane protein and displays an apparent M(r) of 47,000, slightly lower than that predicted from its amino acid sequence. Membranes from cells containing multiple dose of nhaB display enhanced Na+/H+ antiporter activity, as measured by the ability of Na+ to collapse a preformed pH gradient or by direct measurement of 22Na+ fluxes. In contrast to NhaA, whose activity increases with pH, NhaB is practically insensitive to pH. Limited homologies with Na+ transporters have been identified.
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PMID:Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli. 131 51

Studies on sphingomyelin metabolism in rat hepatocytes were facilitated by the use of choline-deficient cells which allowed for the rapid labeling of phosphatidylcholine and as a result sphingomyelin. Pulse and pulse-chase studies with [methyl-3H]choline and [methyl-3H]methionine demonstrated that both compounds were effectively used for sphingomyelin biosynthesis and that newly made and pre-existing phosphatidylcholine could be used for sphingomyelin biosynthesis. When hepatocytes were incubated with brefeldin A, there was a 2.4-fold stimulation of the conversion of phosphatidylcholine into sphingomyelin. Since brefeldin A causes collapse of the cis/medial Golgi into the endoplasmic reticulum the stimulation of sphingomyelin biosynthesis could be due to more rapid access of the labeled phosphatidylcholine in the endoplasmic reticulum to sphingomyelin synthase in the collapsed Golgi. Forskolin inhibited the brefeldin A-induced stimulation of sphingomyelin biosynthesis. To investigate whether or not phosphorylation reactions regulate sphingomyelin metabolism, hepatocytes were incubated with okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A. Rather than stimulating sphingomyelin biosynthesis, okadaic acid enhanced the catabolism of sphingomyelin. In contrast, a cyclic AMP analogue and forskolin had no effect on sphingomyelin biosynthesis or catabolism. Surprisingly, other pulse-chase studies demonstrated that okadaic acid stimulated the catabolism of only newly made sphingomyelin. The brefeldin A and okadaic acid effects were independent of lysosomal involvement. Subcellular fractionation studies revealed that brefeldin A and okadaic acid effects were generalized in all sphingomyelin containing membranes. The brefeldin A studies suggest that the rate of transfer of phosphatidylcholine from the endoplasmic reticulum to the Golgi might be limiting for sphingomyelin biosynthesis. The okadaic acid studies indicate that the catabolism of sphingomyelin by a sphingomyelinase is regulated by an unidentified protein kinase and by either protein phosphatase 1 and/or 2A activity in hepatocytes.
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PMID:Stimulation of sphingomyelin biosynthesis by brefeldin A and sphingomyelin breakdown by okadaic acid treatment of rat hepatocytes. 161 52

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.
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PMID:Sequential disassembly of myofibrils induced by myristate acetate in cultured myotubes. 365 56

Cells were microinjected with four mouse monoclonal antibodies that were directed against either alpha- or beta-tubulin subunits, one monoclonal with activity against both subunits, and a guinea pig polyclonal antibody with activity directed against both subunits, to determine the effects on the distribution of cytoplasmic microtubules and 10-nm filaments. The specificities of the antibodies were confirmed by Western blots, solid phase radioimmunoassay, and Western blot analysis of alpha- and beta-tubulin peptide maps. Two monoclonals DM1A and DM3B3, an anti-alpha- and anti-beta-tubulin respectively, and the guinea pig polyclonal anti-alpha/beta-tubulin antibody (GP1T4) caused the 10-nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps; the other monoclonal antibodies had no effect when microinjected into cells. The filament collapsing was observed to be complete at 1.5-2 h after injection. During the first 30 min after injection a few cytoplasmic microtubules near the cell periphery could be observed by fluorescence microscopy. This observation was confirmed by electron microscopy, which also demonstrated assembled microtubules in the juxtanuclear region. By 1.5 h, when most of the 10-nm filaments were collapsed, the complete cytoplasmic array of microtubules was observed. Cells injected in prophase were able to assemble a mitotic spindle, suggesting that the antibody did not block microtubule assembly. Metabolic labeling with [35S]methionine of microinjected cells revealed that total protein synthesis was the same as that observed in uninjected cells. This indicated that the microinjected antibody apparently did not produce deleterious effects on cellular metabolism. These results suggest that through a direct interaction of antibodies with either alpha- or beta-tubulin subunits, 10-nm filaments were dissociated from their normal distribution. It is possible that the antibodies disrupted postulated 10-nm filament-microtubule interactions.
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PMID:10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. 653 4

Growth cones are powerful amplifiers for signals from the microenvironment. Their collapse can be triggered by cell surface components of myelin and brain membranes, as well as by soluble ligands, including neurotransmitters. GAP-43 is a protein concentrated on the inner surface of the growth cone membrane. Assayed in isolation, it interacts with the heterotrimeric protein, G(o), and in oocytes it amplifies the effects of ligand-triggered G protein activation. We wished to examine whether GAP-43 serves to amplify signals at the growth cone. The G(o) stimulating region of GAP-43 is encoded in the 10 amino acids (MLCCMRRT-KQ) of the first exon. We examined the effect of this peptide upon chick dorsal root ganglion growth cone collapse and neurite retraction triggered by brain membranes or myelin, as well as by serotonin. We find that application of the GAP-43 1-10 peptide amplifies the effects of all three ligands. The amplification is greater when GAP-43 1-10 is injected intracellularly. Peptides with amino acid substitutions for the two cysteine residues manifest parallel changes in growth cone collapse and G(o) stimulation. In particular, tyrosine or methionine substitutions cause the peptide to inhibit G(o) and to block induced growth cone collapse. The GAP-43 peptides therefore regulate the sensitivity of growth cones to extrinsic signals. The modified peptides serve as a starting point for the design of reagents to enhance CNS regeneration.
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PMID:Ligand-induced growth cone collapse: amplification and blockade by variant GAP-43 peptides. 764 8

Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.
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PMID:Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis. 783 May 60

Analysis of the three-dimensional structure of class A beta-lactamases suggests that deformation of the substrate binding site can be produced by changes in the hydrophobicity of residue 69 behind the beta-sheet and by outward movement of the B3 beta-strand by introduction of a non-glycine residue at position 242 on the B4 beta-strand. By site-directed mutagenesis Met69-IleGly242-Cys, a double mutant, of the OHIO-1 beta-lactamase, was constructed. The minimum inhibitory concentrations (MICs) of the double mutant compared with the wild type and each single mutant revealed an increased susceptibility to beta-lactams. Met69-IleGly242Cys hydrolyzed cephaloridine (Km = 213 microM) but had Km > 500 microM for other beta-lactams tested including cefotaxime, and demonstrated a higher apparent Ki for inhibitors (clavulanate Ki = 500 microM sulbactam = 434 microM, and tazobactam = 70 microM). In a competition experiment with cephaloridine, the apparent Ki values for penicillin and cefotaxime remained low, 21 microM and 0.7 microM, respectively. Since Ile is twice as hydrophobic as Met, the Met69-Ile mutation may result in partial collapse of the oxyanion hole. This would also increase the distance between Arg-244 and the carboxyl of clavulanic acid. The Gly242-Cys mutation opens the lower portion of the active site to bulky R groups of cephalosporins. Although these two mutations result in a catalytically impaired enzyme, they can be used to model the complementary role of two distinct residues, neither of which interacts directly with beta-lactam substrates or inhibitors.
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PMID:Complementary roles of mutations at positions 69 and 242 in a class A beta-lactamase. 787 79

The neuronal growth-associated protein GAP-43 is expressed maximally during development and regeneration, and is enriched at the cytosolic surface of the growth cone membrane. GAP-43 can activate the GTP-binding protein G(o) which is also a major component of the growth cone membrane. These findings have led to the hypothesis that GAP-43 might modulate neurite outgrowth by altering G-protein activity. Here we define the sequence requirements for GAP-43 amino terminal peptide stimulation of G(o), and test these peptides as potential modulators of neurite outgrowth. The first 10 amino acids of GAP-43, Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln, stimulate G(o). Substitutions at particular residues reveal that cys3, cys4, arg6, and lys9 are critical, but arg7 is not. Both the GAP-43(1-10) peptide and the G-protein-activating peptide mastoparan induce growth cone collapse and inhibit neurite extension from embryonic chick dorsal root ganglion and retinal neurons. This is likely to be mediated by G-proteins: pertussis toxin blocks the inhibition, and mutant peptides that do not activate G(o) do not alter outgrowth. In contrast to the case with embryonic chick dorsal root ganglion cells, neurite outgrowth from N1E-115 neuroblastoma cells is stimulated by GAP-43(1-10). This is probably also a G-protein-mediated event because it is blocked by pertussis toxin, because the sequence requirements match those for G(o) stimulation, and because mastoparan stimulates outgrowth from these cells. The longer GAP-43(1-25) peptide does not alter neurite outgrowth unless the cells are permeabilized, suggesting an intracellular site of action. These data identify a novel set of compounds that modulate neurite outgrowth, and also support the notion that GAP-43 can alter neurite extension by modulating pertussis toxin-sensitive G-protein activity in the growth cone.
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PMID:GAP-43 amino terminal peptides modulate growth cone morphology and neurite outgrowth. 808 50

Initiation of protein folding by light can dramatically improve the time resolution of kinetic studies. Here we present an example of an optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are closer to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.
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PMID:Fast events in protein folding initiated by nanosecond laser photolysis. 826 38

Atomic force microscopy (AFM) was used to image ribosomes and ribosomal subunits (60S, 40S and native 40S ribosomal subunits) isolated from rat liver. A variety of topographic images were obtained directly and found to be consistent with models established by other biophysical methods. In addition, the ternary complex of eIF-2 x GTP x Met-tRNA(i) and the 43S preinitiation complex have been discerned by AFM directly. Detailed information about the binding sites for eIF-1A, eIF-2, eIF-3, and Met-tRNA(i) on the 40S ribosomal subunit was derived from the AFM images. Finally, factors which may give rise to artifactual images, namely, convolution of the AFM tip on ribosomes, surface tension collapse effect and dehydration, are discussed. This work demonstrates that AFM is useful for imaging ribosomes and translational complexes and provides valuable information that can be used to complement other well-established techniques.
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PMID:Topography of ribosomes and initiation complexes from rat liver as revealed by atomic force microscopy. 919 Oct 23


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