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(1/1810) Phospholipid hydroperoxide cysteine peroxidase activity of human serum albumin.

Human serum albumin (HSA) reduced the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-l-3-phosphatidylcholine (PLPC-OOH) to the corresponding hydroxy-derivative with a high apparent affinity (Km=9. 23+/-0.95 microM). Removal of bound lipid during purification increased this activity. At physiological concentration, HSA reduced the phospholipid hydroperoxide in the absence of a cofactor. However, in the presence of a cofactor (reductant), the rate of the reaction was increased. All of the major aminothiols in plasma could act as reductants, the best being the most abundant, cysteine (Km=600+/-80 microM). For every nanomole of PLPC-OOH reduced by HSA, 1.26 nmol of cystine was formed, indicating a reaction stoichiometry of 1 mol PLPC-OOH to 2 mol cysteine. We used chemical modification to determine which amino acid residues on HSA were responsible for the activity. Oxidation of thiol group(s) by N-ethylmaleimide led to a reduction in the rate of activity, whereas reduction of thiols by either dithiothreitol or the angiotensin-converting enzyme inhibitor, captopril, increased the activity. Both N-ethylmaleimide-modified HSA and dithiothreitol-treated HSA exhibited increased apparent affinities for PLPC-OOH. For a range of preparations of albumin with different modifications, the activity on PLPC-OOH was dependent on the amount of free thiol groups on the albumin (correlation coefficient=0.91). Patients with lowered albumin concentrations after septic shock showed lowered total plasma thiol concentrations and decreased phospholipid hydroperoxide cysteine peroxidase (PHCPx) activities. These results therefore show for the first time that HSA exhibits PHCPx activity, and that the majority of the activity depends on the presence of reduced thiol group(s) on the albumin.  (+info)

(2/1810) The covalent attachment of polyamines to proteins in plant mitochondria.

Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases.  (+info)

(3/1810) Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase.

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.  (+info)

(4/1810) Extraction of erythrocyte membrane proteins by sulfhydryl inhibitors.

Human red cell membrane proteins were extracted by incubation of the ghost with hypotonic phosphate buffer (pH 7.4), N-ethylmaleimide and p-hydroxy-mercuribenzoate. In paroxysmal nocturnal hemoglobinuria (PNH), hereditary spherocytosis (HS) and hereditary elliptocytosis, the amount of proteins extracted by these procedures was significantly less than the amount extractable from the ghost of normal and aplastic anemia red cells. Polypeptide patterns of red cell membranes in these hematological disorders were essentially similar to those of normal ghosts. Analysis of the supernatant by SDS polyacrylamide gel electrophoresis revealed that this reduction was mainly due to the reduced amount of peripheral proteins extracted. The extraction of peripheral proteins by sulfhydryl reagents was accompanied by shape changes resulting in the formation of membrane vesicles, suggesting an important role of peripheral proteins in the maintenance of ghost shape. It is also suggested that qualitative abnormalities of peripheral proteins such as altered reactivity to sulfhydryl reagents and/or strong binding to the membrane are present in PNH, HS and hereditary elliptocytosis red cells.  (+info)

(5/1810) Non-specific action of methoxamine on Ito, and the cloned channels hKv 1.5 and Kv 4.2.

The alpha1-adrenoceptor agonist methoxamine acted independently of receptor activation to reduce Ito and the sustained outward current in rat ventricular myocytes, and hKv 1.5 and Kv 4.2 cloned K+ channel currents. Two hundred microM methoxamine reduced Ito by 36% in the presence of 2 microM prazosin, and by 37 and 38% after preincubation of myocytes with either N-ethylmaleimide or phenoxybenzamine (n=6). The EC50 values at +60 mV for direct reduction of Ito, hKv 1.5, and Kv 4.2 by methoxamine were 239, 276, and 363 microM, respectively, with Hill coefficients of 0.87-1.5. Methoxamine accelerated Ito and Kv 4.2 current inactivation in a concentration- and voltage-dependent manner. Apparent rate constants for methoxamine binding and unbinding gave Kd values in agreement with EC50 values measured from dose-response relations. The voltage-dependence of block supported charged methoxamine binding to a putative intracellular site that sensed approximately 20% of the transmembrane electrical field. In the presence of methoxamine, deactivating Kv 4.2 tail currents displayed a distinct rising phase, and were slowed relative to control, such that tail current crossover was observed. These observations support a dominant mechanism of open channel block, although closed channel block could not be ruled out. Single-channel data from hKv 1.5 patches revealed increased closed times with blank sweeps and decreased burst duration in the presence of drug, and a reduction of mean channel open time from 1.8 ms in control to 0.4 ms in 500 microM methoxamine. For this channel, therefore, both open and closed channel block appeared to be important mechanisms for the action of methoxamine.  (+info)

(6/1810) Nitric oxide inhibits L-type Ca2+ current in glomus cells of the rabbit carotid body via a cGMP-independent mechanism.

Previous studies have shown that nitric oxide (NO) inhibits carotid body sensory activity. To begin to understand the cellular mechanisms associated with the actions of NO in the carotid body, we monitored the effects of NO donors on the macroscopic Ca2+ current in glomus cells isolated from rabbit carotid bodies. Experiments were performed on freshly dissociated glomus cells from adult rabbit carotid bodies using the whole cell configuration of the patch-clamp technique. The NO donors sodium nitroprusside (SNP; 600 microM, n = 7) and spermine nitric oxide (SNO; 100 microM, n = 7) inhibited the Ca2+ current in glomus cells in a voltage-independent manner. These effects of NO donors were rapid in onset and peaked within 1 or 2 min. In contrast, the outward K+ current was unaffected by SNP (600 microM, n = 6), indicating that the inhibition by SNP was not a nonspecific membrane effect. 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO; 500 microM), an NO scavenger, prevented inhibition of the Ca2+ current by SNP (n = 7), whereas neither superoxide dismutase (SOD; 2,000 U/ml, n = 4), a superoxide scavenger, nor sodium hydrosulfite (SHS; 1 mM, n = 7), a reducing agent, prevented inhibition of the Ca2+ current by SNP. However, SNP inhibition of the Ca2+ current was reversible in the presence of either SOD or SHS. These results suggest that NO itself inhibits Ca2+ current in a reversible manner and that subsequent formation of peroxynitrites results in irreversible inhibition. SNP inhibition of the Ca2+ current was not affected by 30 microM LY 83, 583 (n = 7) nor was it mimicked by 600 microM 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP; n = 6), suggesting that the effects of NO on the Ca2+ current are mediated, in part, via a cGMP-independent mechanism. N-ethylmaleimide (NEM; 2.5 mM, n = 6) prevented the inhibition of the Ca2+ current by SNP, indicating that SNP is acting via a modification of sulfhydryl groups on Ca2+ channel proteins. Norepinephrine (NE; 10 microM) further inhibited the Ca2+ current in the presence of NEM (n = 7), implying that NEM did not nonspecifically eliminate Ca2+ current modulation. Nisoldipine, an L-type Ca2+ channel blocker (2 microM, n = 6), prevented the inhibition of Ca2+ current by SNP, whereas omega-conotoxin GVIA, an N-type Ca2+ channel blocker (1 microM, n = 9), did not prevent the inhibition of Ca2+ current by SNP. These results demonstrate that NO inhibits L-type Ca2+ channels in adult rabbit glomus cells, in part, due to a modification of calcium channel proteins. The inhibition might provide one plausible mechanism for efferent inhibition of carotid body activity by NO.  (+info)

(7/1810) Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys.

The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].  (+info)

(8/1810) A Cys-less variant of the bacterial ATP binding cassette protein MalK is functional in maltose transport and regulation.

The cysteine residues of the ABC protein MalK from Salmonella typhimurium maltose transport system (C40, C350, C360) were consecutively replaced by serines. Cys-less MalK was fully functional in maltose transport in vivo. Moreover, the activity of MalK as a repressor of other maltose-regulated genes was also retained. The absence of cysteine residues in the purified protein was verified by its failure to react with fluorescein-5-maleimide. In contrast to purified wild-type MalK, the ATPase activity of the C40S variant was insensitive to inhibition by N-ethylmaleimide.  (+info)