N itric oxide is a secondary messenger involved in the cGMP cascade, vasodilation, and a known inhibitor of platelet aggregation. S-nitrosothiols (RSNOs) are formed via nitrosation of thiols under aerobic conditions (1). Apart from being cellular sources of NO, they also prolong its half-life (2, 3). RSNOs are involved in signaling pathways, immune responses, and the actions of nitrovasodilating compounds (4-7). Therefore, a study involving the effects and transport of S-nitroso-BSA (BSA-NO) into live cells is of utmost physiological importance and the center of pharmacological interest (8).Many indirect, discontinuous fluorescent, electrochemical, and colorometric assays have been developed for RSNOs (8, 9). However, none of these are conducive to measuring the transport of RSNO-bound NO into live cells, in vitro. The probe for NO influx presented here is N-dansylhomocysteine (DnsHCys). The fluorescence of this compound is completely quenched on its S-nitrosation, yielding N-dansyl-S-nitrosohomocysteine (DnsHCysNO). Here we show that DnsHCysNO is a direct fluorogenic substrate for protein disulfide isomerase (PDI).PDI acts as a chaperone molecule in the endoplasmic reticulum where it catalyses protein thiol exchange reactions. Hotchkiss et al. (10) have reported that PDI is also secreted by endothelial cells as well as deposited on the cell surface. Recent studies have presented indirect evidence for the involvement of cell-surface PDI (csPDI) in the influx of 12). Stamler and coworkers (13) also have shown that the export of intracellular NO from red blood cells to be facilitated by S-nitrosation of the cysteine residues in the hemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Membranebound PDI may catalyze the formation of AE1-SNO and the subsequent export of cytosolic NO.Here, the extracellular RSNO-dependent quenching of the DnsHCys fluorescence was shown to be a csPDI-dependent process with the aid of antisense-mediated underexpression of PDI and the sense-mediated overexpression of PDI in HT1080 fibrosarcoma cells as well as with a cell-impermeant inhibitor that reacts with vicinal dithiols. In addition, N-dansylhomocystine (DnsHCys 2 ) is shown to be a sensitive intracellular probe for the kinetic characterization of csPDI in human umbilical vein endothelial cells (HUVECs), hamster lung fibroblasts, and HT1080 fibrosarcoma cells. Based on this data, a mechanism for csPDI-meditated intracellular S-nitrosation, by extracellular RSNOs, has been proposed. Materials and Methods Synthesis of S-Nitrosoglutathione (GSNO).Glutathione (GSH, Sigma) was dissolved in ice-cold 0.5 M HCl. Equimolar sodium nitrite was added and the reaction was carried out in the dark at 4°C for 40 min. The pH of the reaction mixture was adjusted to 7.0 and crystallized by the slow addition of cold acetone. BSA-NO was synthesized by using the above-mentioned protocol (3). Synthesis of DnsHCysNO.HCysNO was prepared by treating HCys (Sigma) with acidified nitrite. Dansylation was carried out in 0.1 M phosphate buffer (pH ...

Key Points Platelet-derived ERp57 plays an important role in physiologic platelet function and thrombosis. ERp57 directly interacts with αIIbβ3 in regulating its function.

S-Nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NO x probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The K m values estimated for GSNO were 65 ؎ 5 M and 40 ؎ 10 M for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO ⅐ . A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His 160 a-domain or His 589 a-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO-and O 2 -rich environment, PDI can form N 2 O 3 in its hydrophobic domains. This "NO-charged PDI" can perform intra-and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol. Protein disulfide isomerase (PDI)1 was identified about 40 years ago (1). Although large levels of this enzyme are found in the endoplasmic reticulum, PDI is secreted from cells in which it associates electrostatically with the cell surface (2, 3). One of the most studied functions of PDI is its ability to catalyze isomerization and rearrangement of disulfide bonds in the endoplasmic reticulum, contributing to a proper folding of nascent proteins (4). Cell surface PDI was initially discovered in platelets (5), in which it plays a dual role in integrin-mediated adhesion and aggregation (6, 7), RSNO-mediated platelet inhibition, and GSNO denitrosation (8).S-Nitrosothiols (RSNOs) are known nitric oxide (NO) donors. RSNOs range in size from low molecular weight, such as cysteine-NO and homocysteine-NO, to high molecular weight nitrosated proteins, such as serum albumin-NO. They are known to prolong NO half-life (9) and to act as a NO transport system in a cellular environment (10 -12).An additional novel...

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

Some aspects of the physiological role of NO may be mediated by stable NO-carriers such as S-nitrosoglutathione and related S-nitrosothiols. In this report we show that irradiation of S-nitrosoglutathione at either absorption band (lambda max = 340 nm or 545 nm) results in the release of nitric oxide. Photolysis of S-nitrosoglutathione at 545 nm exhibited a quantum yield of 0.056 +/- 0.002 and was best approximated by a first-order process with kobs = 4.9 x 10(-7) +/- 0.3 x 10(-7) s-1. The photolytic release of NO from S-nitrosoglutathione resulted in an enhanced cytotoxic effect of S-nitrosoglutathione on HL-60 leukemia cells. That the cytotoxic effect of S-nitrosoglutathione was diminished by the addition of oxyhemoglobin strongly suggests that NO is the cytotoxic species. The finding that NO can be readily liberated from S-nitrosoglutathione by visible radiation indicates that the photochemical properties of this compound in the visible spectrum must be considered in order to obtain meaningful data as to its physiological role and the S-nitrosoglutathione and related compounds may find use as photochemotherapeutic agents.

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Protein disulfide isomerase (PDI), is a member of the thioredoxin superfamily of redox proteins. PDI has three catalytic activities including, thiol-disulfide oxireductase, disulfide isomerase and redox-dependent chaperone. Originally, PDI was identified in the lumen of the endoplasmic reticulum and subsequently detected at additional locations, such as cell surfaces and the cytosol. This review will provide an overview of the recent advances in relating the structural features of PDI to its multiple catalytic roles as well as its physiological and pathophysiological functions related to redox regulation and protein folding.

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