The cysteine-proximal aspartates in the Fx-binding niche of photosystem I. Effect of alanine and lysine replacements on photoautotrophic growth, electron transfer rates, single-turnover flash efficiency, and EPR spectral properties.
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The FX electron acceptor in Photosystem I (PS I) is a highly electronegative (Em = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566PsaB and D557PsaB on electron transfer through FX by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566APsaB, D557APsaB, D566KPsaB, and D557KPsaB) or in pairs (D557APsaB/D566APsaB and D557KPsaB/D566APsaB). All mutants except for D557KPsaB/D566APsaB grew photoautotrophically, but the growth of D557KPsaB and D557APsaB/D566APsaB was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557KPsaB and D557APsaB/D566APsaB relative to the wild type and the other mutants. Nevertheless, the rates of NADP+ photoreduction in PS I complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back-reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors FA and FB in PS I complexes from all mutants. The EPR spectrum of FX was identical to that in the wild type in all but the single and double D566APsaB mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PS I rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through FX. (+info)
Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558.
(26/9069)
Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins. (+info)
A sensitive and specific ELISA for plasma pentosidine.
(27/9069)
BACKGROUND: Advanced glycation end products are formed by non-enzymatic glycation and oxidation reaction. Pentosidine is a well-known and characterized structure among them, and has been implicated in the pathogenesis of complications associated with chronic renal failure and long-term dialysis, such as dialysis-related amyloidosis and atherosclerosis. METHODS: We established a highly sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) for plasma pentosidine and applied it to large numbers of plasma samples including haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients. We compared their plasma pentosidine levels determined by the competitive ELISA with those determined by high-performance liquid chromatographic (HPLC) assay currently used. RESULTS: The plasma pentosidine levels determined by the ELISA were correlated well with those determined by sophisticated instrumental HPLC assay, both in non-diabetic and diabetic dialysis patients. Both analyses yielded comparable results, with over 8-fold higher plasma pentosidine levels in HD and CAPD patients, irrespective of the presence or absence of diabetes, as compared to normal subjects and non-uraemic diabetic patients. CONCLUSIONS: The competitive ELISA will provide a rapid and convenient determination of plasma pentosidine content and thus be useful to assess the carbonyl stress in uraemic patients. (+info)
NH4+-induced down-regulation of the Saccharomyces cerevisiae Gap1p permease involves its ubiquitination with lysine-63-linked chains.
(28/9069)
Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces internalization of the general amino acid permease Gap1p and its subsequent degradation in the vacuole. An essential step in this down-regulation is Gap1p ubiquitination through a process requiring the Npi1p/Rsp5p ubiquitin ligase. We show in this report that NPI2, a second gene required for NH4+-induced down-regulation of Gap1p, codes for the ubiquitin hydrolase Doa4p/Ubp4p/Ssv7p and that NH4+-induced Gap1p ubiquitination is strongly reduced in npi2 cells. The npi2 mutation results in substitution of an aromatic amino acid located in a 33-residue sequence shared by some ubiquitin hydrolases of the Ubp family. In this mutant, as in doa4(delta) cells, the amount of free monomeric ubiquitin is at least four times lower than in wild-type cells. Both ubiquitination and down-regulation of the permease can be restored in npi2 cells by over-expression of ubiquitin. In proline-grown wild-type and npi2/doa4 cells overproducing ubiquitin, Gap1p appears to be mono-ubiquitinated at two lysine acceptor sites. Addition of NH4+ triggers rapid poly-ubiquitination of Gap1p, the poly-ubiquitin chains being specifically formed by linkage through the lysine 63 residue of ubiquitin. Gap1p is thus ubiquitinated differently from the proteins targeted by ubiquitination for proteolysis by the proteasome, but in the same manner as the uracil permease, also subject to ubiquitin-dependent endocytosis. When poly-ubiquitination through Lys63 is blocked, the Gap1p permease still undergoes NH4+-induced down-regulation, but to a lesser extent. (+info)
Carbon monoxide stimulates the apical 70-pS K+ channel of the rat thick ascending limb.
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We have investigated the expression of heme oxygenase (HO) in the rat kidney and the effects of HO-dependent heme metabolites on the apical 70-pS K+ channel in the thick ascending limb (TAL). Reverse transcriptase-PCR (RT-PCR) and Western blot analyses indicate expression of the constitutive HO form, HO-2, in the rat cortex and outer medulla. Patch-clamping showed that application of 10 microM chromium mesoporphyrin (CrMP), an inhibitor of HO, reversibly reduced the activity of the apical 70-pS K+ channel, defined by NPo, to 26% of the control value. In contrast, addition of 10 microM magnesium protoporphyrin had no significant effect on channel activity. HO involvement in regulation of the apical 70-pS K+ channel of the TAL, was further indicated by the addition of 10 microM heme-L-lysinate, which significantly stimulated the channel activity in cell-attached patches by 98%. The stimulatory effect of heme on channel activity was also observed in inside-out patches in the presence of 0.5-1 mM reduced nicotinamide adenine dinucleotide phosphate. This was completely abolished by 10 microM CrMP, suggesting that a HO-dependent metabolite of heme mediated the effect. This was further supported by exposure of the cytosolic membrane of inside-out patches to a carbon monoxide-bubbled bath solution, which increased channel activity. Moreover, carbon monoxide completely abolished the effect of 10 microM CrMP on the channel activity. In contrast, 10 microM biliverdin, another HO-dependent metabolite of heme, had no effect. We conclude that carbon monoxide produced from heme via an HO-dependent metabolic pathway stimulates the apical 70-pS K+ channel in the rat TAL. (+info)
The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status.
(30/9069)
The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status. (+info)
An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity.
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Sphingolipid activator proteins are small glycoproteins required for the degradation of sphingolipids by specific lysosomal hydrolases. Four of them, called saposins, are encoded by the prosaposin gene, the product of which is proteolytically cleaved into the four mature saposin proteins (saposins A, B, C, D). One of these, saposin B, is necessary in the hydrolysis of sulphatide by arylsulphatase A where it presents the solubilised substrate to the enzyme. As an alternative to arylsulphatase A deficiency, deficiency of saposin B causes metachromatic leukodystrophy. We identified a previously undescribed mutation (N215K) in the prosaposin gene of a patient with metachromatic leukodystrophy but with normal arylsulphatase A activity and elevated sulphatide in urine. The mutation involves a highly conserved amino acidic residue and abolishes the only N-glycosylation site of saposin B. (+info)
Postexercise net protein synthesis in human muscle from orally administered amino acids.
(32/9069)
We examined the response of net muscle protein synthesis to ingestion of amino acids after a bout of resistance exercise. A primed, constant infusion of L-[ring-2H5]phenylalanine was used to measure net muscle protein balance in three male and three female volunteers on three occasions. Subjects consumed in random order 1 liter of 1) a mixed amino acid (40 g) solution (MAA), 2) an essential amino acid (40 g) solution (EAA), and 3) a placebo solution (PLA). Arterial amino acid concentrations increased approximately 150-640% above baseline during ingestion of MAA and EAA. Net muscle protein balance was significantly increased from negative during PLA ingestion (-50 +/- 23 nmol. min-1. 100 ml leg volume-1) to positive during MAA ingestion (17 +/- 13 nmol. min-1. 100 ml leg volume-1) and EAA (29 +/- 14 nmol. min-1. 100 ml leg volume-1; P < 0.05). Because net balance was similar for MAA and EAA, it does not appear necessary to include nonessential amino acids in a formulation designed to elicit an anabolic response from muscle after exercise. We concluded that ingestion of oral essential amino acids results in a change from net muscle protein degradation to net muscle protein synthesis after heavy resistance exercise in humans similar to that seen when the amino acids were infused. (+info)