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The elongation of RNA by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide. Three systems were studied: a soluble chromatin, purified RNA polymerase II with DNA, and a HeLa cell extract with an adenovirus 2 promoter sequence.
Previously we reported that thiol depletion and lipid peroxidation were associated with the cytotoxicity of nephrotoxic cysteine S-conjugates, a group of toxins which kill LLC-PK1 cells after metabolic activation and covalent binding. To determine if this is a general mechanism of cytotoxicity in these cells, we compared the effect of antioxidants, an iron chelator, and a thiol reducing agent on the toxicity of an alkylating agent, iodoacetamide (IDAM), and an organic peroxidant, t-butylhydroperoxide (TBHP). IDAM or TBHP toxicity was concentration (0.01 to 1.0 mM) and time (1 to 6 h) dependent. Both toxins caused lipid peroxidation which occurred prior to cell death as determined by leakage of lactate dehydrogenase (LDH). The alkylating agent IDAM bound to cellular macromolecules and depleted cellular non-protein thiols almost completely by 1 h, while LDH release occurred first at 2 to 3 h. The toxicity of IDAM and TBHP was inhibited by the antioxidants DPPD, BHA, BHQ, PGA, and BHT and the iron chelator deferoxamine. However, DPPD blocked TBHP- and IDAM-induced lipid peroxidation and toxicity without affecting binding and depletion of cellular nonprotein thiols. Furthermore, the thiol reducing agent dithiothreitol was able to block lipid peroxidation and toxicity. Therefore it is possible that with an alkylating agent, depletion of cellular nonprotein thiols cooperates with covalent binding and contributes to lipid peroxidation and cell death. There appear to be common elements in the toxicity of alkylating agents and organic peroxidants in LLC-PK1 cells.
Treatment of BHK or HL60 cell lines with merocyanine 540 in the presence of the sulphydryl blocker iodoacetamide caused budding of the cell surface to release vesicles about 50-100 nm in diameter which accounted for up to 25% of the total surface membrane lipid. Smaller amounts of vesicular material were released in the presence of fluoride and merocyanine 540. The vesicles had a membrane lipid composition which was characteristic of other purified plasma membranes, with large amounts of sphingomyelin, phosphatidylserine and cholesterol and low proportions of phosphatidylinositol, phosphatidylcholine, triacylglycerol and cholesterol ester. This procedure for the isolation of vesicles should be a general method for the purification of plasma membrane components from a wide range of different cell types.
In the course of the polymerization of N-(1-pyrenyl) iodoacetamide-labelled actin the increase of the fluorescence is not directly proportional to the amount of the polymer mass formed. This precludes the use of the fluorescence signal as a tool for the quantitative description of the polymerization process.
Myosin and its subfragment 1 were labeled with the fluorescent probe N-(1-pyrenyl)iodoacetamide. Both of the labeled complexes exhibited the excimer band at 480 nm (pH 8.0, 25 degrees C). SH1 and SH2 are labeled with this probe as judged by Ca2+-ATPase of the labeled complex. Excimers arise both from the interaction of PIAAs in the two different heads within a single myosin molecule and also from the interaction of PIAAs in the same head. ATP affects these excimers depending on the concentration of Ca2+.
Rabbit polyclonal antibodies were raised against synthetic peptides of the bovine leukemia virus envelope glycoprotein gp51 and tested against the full size protein by the Western blotting technique. We show that acetylation of gp51 by iodoacetamide either maintains or significantly increases the antigen-antibody reaction and conclude therefrom that the reactive potential of an anti-peptide antibody may require acetylation of the sulfhydryl groups of the blotted protein.
BACKGROUND: To determine glycoserum, the nitroblue tetrazolium (NBT) assay is quick, economical, convenient and easily automated. This method, however, is vulnerable to interference by thiol group, which should not be ignored during the analysis. METHODS: Thiol group in glycoserum was blocked with iodoacetamide (IAM) before NBT was added. The reaction was carried out in a thermal bath and terminated on ice. The absorbance was measured at either 570 or 530 nm. RESULTS: IAM (3-10 mmol/l) did not give any detectable interference in the NBT assay. The absorbance at both 570 and 530 nm was linearly related to the concentration of either glycoserum or 1-deoxy-1-morpholino-D-fructose (DMF) that had been treated with IAM. The assay showed a good discrimination between diabetic and normal subjects (t-test, P < 0.001). Uric acid and lipemia under physiological conditions did not interfere with NBT reaction. The assay was affected by hyperlipemia and hematolysis. CONCLUSIONS: IAM-modified method prevented NBT assay from the interference by thiol group and uric acid.
The effect of iodoacetamide (IA), a sulphydryl blocking agent, on fertilisation in the mouse was examined by transferring zona-free oocytes into IA (500 microM)-containing medium at various times after insemination. When inseminated oocytes were transferred into IA medium 10 min after insemination, the oocyte chromosomes remained aggregated in one or two masses and the sperm nucleus failed to decondense. When oocytes were transferred during the second meiosis, oocyte meiosis was more or less arrested. The sperm nucleus decondensed but remained blocked at an early stage of decondensation. These observations suggest that thiol groups in the oocyte's cytoplasm and perhaps microtubules of the meiotic spindle play crucial roles in the completion of meiosis and the transformation of sperm nucleus into pronucleus.
Cystine linkages in proteins are often opened with reducing agents, sometimes to improve their digestion, often to eliminate disulfide linkages from complicating analysis of the digest. After reduction, the sulfhydryls are usually reacted with iodoacetamide (IAM), iodoacetic acid (IAA), or another electrophile to prevent reformation of disulfide linkages in a random manner. When the amount of protein may be reliably estimated, side reactions from excess IAM or IAA can be avoided. When this is not so, removal of excess iodoalkane can be accomplished by HPLC, by dialysis, or simply by allowing a reducing thiol to consume any excess. In mass spectrometric analysis of proteins isolated by 1D or 2D gels, removal of the excess iodoalkane is often accomplished simply by washing the gel prior to proteolytic digestion. During a recent study of the glutathionylation site mapping of actin, IAM was used to block any residual sulfhydryl groups remaining on the protein so that they would not displace glutathione from its initial site. In addition, to avoid losses due to actin polymerization during dialysis, the IAM was allowed to remain during the digestion. This further ensured that any sulfhydryl groups liberated during the digestion would be similarly blocked by the IAM. Under these conditions, we observed the peptides to undergo N- as well as S-carbamidomethylation. In examining a series of other peptides alkylated with IAM in this way, we have found N-alkylation to be the rule rather than the exception and even O-alkylation was detected. The main sites to which the carbamidomethyl group attaches to the peptides have been located with LC-MS2 using an ion trap mass spectrometer and found to be the N-terminal amino group. A simple expedient to prevent such reactions when an excess of reducing agent must be avoided is to run the alkylation in the presence of a thioether such as 2,2'-thiodiethanol rather than a thiol.
N-(1-Pyrenyl)iodoacetamide has been used to introduce fluorescent probes into Escherichia coli RNA polymerase. After an incubation time of 15 min, approximately 2 pyrene equiv was introduced per enzyme molecule. There was no further increase in modification after more extended periods of incubation. Neither calf thymus DNA nor nucleotides protected the holoenzyme from modification. Thus, the sites of modification do not appear to involve the binding sites for polynucleotides or the ribonucleoside triphosphates. From the isolation and analysis of the individual subunits, it was found that sigma contained approximately 1 pyrene equiv, beta contained 0.6, beta' contained 0.6, and alpha less than 0.1. Spectral and Stern-Volmer analyses indicate that the covalently attached pyrene molecules are in comparable apolar microenvironments. On the basis of CD analyses, the introduction of pyrene molecules into RNA polymerase alters its secondary structure. This alteration in secondary structure manifests itself by a reduction in overall enzymatic activity. Transcript analysis of the products obtained by using a linearized plasmid containing the A1 promoter and the Te terminator of bacteriophage T7 indicates that the pyrenyl derivative is capable of producing full-length transcripts and that it has an efficiency of chain termination comparable to the native enzyme. Analysis of tau plots for the interaction of the pyrenyl derivative and the native enzyme, respectively, with the A1 promoter yielded comparable values for the isomerization constant in the conversion of the closed complex to an open one. Comparable values were also obtained for the association constant. The rate of chain elongation for the pyrenyl derivative, however, is approximately 54% of that observed for the native enzyme. Thus, the decrease in overall transcriptional activity observed with the pyrenyl derivative is not due to a decrease in the efficiency of initiation or premature termination, but rather to a decrease in the rate of chain elongation.
Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.
The reaction of yeast hexokinase with iodoacetate or iodoacetamide has been investigated in detail, using pure hexodinase B. Of the four thiols in each subunit of the molecule, two (the "apparently essential thiols") are alkylated rapidly at 35 degrees, and the enzymic activity is lost in parallel with their reaction. The other two thiols react subsequently to completion, but at a very much slower rate. In the conditions use, no other uptake of the reagent occurs elsewhere during these thiol alkylations. Electrophoretically homogeneous kialkylated and tetraalkylated protein species are formed, in the two stages of the reaction. The inactivating reaction at 35 degrees with the apparently essential thiols is second order. The rate constant increases with increasing pH, in the range pH 7.0-8.5, in a manner consistent with control of the reaction by a group with pKa of approximately 10. The absolute (pH independent) rate constant is of the same order as that for a normal thiol in model compounds. The availability of the apparently essential thiols appears to be associated with some conformational change in the molecule in the monomer form: it declines at high ionic strengths, is maximal at intermediate values where the dimer first dissociates, but is lowered in the dimer at very low ionic strengths. The reaction also shows a sharp temperature dependence: the dimer at 30 degrees (in constrast to 35 degrees) shows no availability of the apparently essential thiols. A similar transition to a state permitting fast inactivation is found with pH, above pH 8.5. The reaction of the two apparently essential thiols is strongly inhibited by glucose. ATP and ADP, and their Mg complexes, protect significantly, but less effectively than does glucose. The affinities of these substrates at the active site of the enzyme are measured in this protection system. These various reactions appear to be of value for identifying the cysteine-containing regions that are involved in the active center or in its maintenance in the structure.
Environmental stress induces the synthesis of glucose-regulated proteins (Grps) in the endoplasmic reticulum (ER) and heat shock proteins (Hsps) in the cytoplasm. Iodoacetamide (IDAM), a prototypical alkyating agent, induces both Grp and Hsp synthesis in renal epithelial cells and causes necrosis which is prevented by prior activation of the ER stress response (pre-ER stress) [Liu, H., et al. (1997) J. Biol. Chem. 272, 21751-21759]. In this study, we examined the biochemical pathways leading to IDAM-induced apoptosis and investigated the role of the ER stress response in apoptotic cell death. The antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) prevented necrosis after IDAM treatment, but the cells went on to die with hallmarks of apoptosis, i.e., cell detachment, caspase-3 activation, cleavage of poly(ADP-ribose)polymerase (PARP), and DNA-ladder formation, all of which were blocked by the general caspase inhibitor zVAD. As with IDAM-induced necrosis, dithiothreitol protected against apoptosis, but cell permeable calcium chelators did not, suggesting that distinct biochemical pathways mediate these two forms of cell death. Pre-ER stress, but not heat shock, prevented IDAM-induced apoptosis. pkASgrp78 cells are deficient in Grp78 induction due to expression of a grp78 antisense RNA and are more sensitive to necrosis. However, these cells were resistant to IDAM-induced apoptosis and had increased basal levels of Grp94 and a KDEL-containing protein of about 50 kDa. Thus, the expression of grp78 antisense perturbs ER functions and activates expression of other ER stress genes accounting for the resistance to apoptosis. Taken together, the data describe functionally distinct signaling pathways through which the ER regulates apoptosis and necrosis caused by chemical toxicants.
We have assessed a previously proposed mechanism mediating 5'-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5'-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5'-deiodinase correlated with the amount of available (unalkylated) Fraction B.
1. In the present study we have examined the sensitivity of A and ASC amino-acid-carrier activities in rat liver plasma-membrane vesicles to the thiol-group modifying reagents N-ethylmaleimide (NEM) and iodoacetamide (IA). To this end, the different Na(+)-dependent entities involved in alanine transport were assessed. 2. NEM inactivated Na(+)-dependent alanine transport as a result of the inhibition of both system A and ASC transport activities. The functional sensitivity of system A to NEM was greater than that of system ASC. 3. The presence of L-alanine (10 mM) during the exposure of vesicles to NEM afforded partial protection to system A, but not to the ASC, carrier. This effect was specific, since the presence of L-phenylalanine (10 mM) did not cause any protection. 4. Na+ did not protect A or ASC carriers against NEM inactivation; however, the presence of Na+ (100 mM-NaCl) and L-alanine (10 mM) during the exposure of the vesicles to NEM protected against inactivation of system A and ASC transport activities. The extent of protection was greater in the case of the system ASC transport activity than in the case of the A carrier. 5. IA also diminished Na(+)-dependent alanine transport by inhibition of A and ASC transport activities. Sodium and L-alanine afforded protection to both A and ASC transport activities from the inhibitory action of IA. The extent of protection induced by substrates was similar for both carriers. 6. It is concluded that there is one, or several, free thiol groups in A and ASC carriers, the integrity of which is essential for transport activity. Sensitivity to thiol-group-specific reagents and the pattern of protection with substrates against inactivation is different in A and ASC carriers. That suggests the existence of topological dissimilarities regarding the thiol-group containing site(s) in A and ASC amino acid carriers.
Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.
1. RNase St was inactivated by iodoacetate. The inactivation was most rapid at pH 5.0-7.0. Competitive inhibitors protected RNase St from inactivation by iodoacetate. The protective effect of 2'-GMP was most effective among nucleotides tested. 2. RNase St was inactivated with the concomitant incorporation of one molar equivalent of carboxymethyl group. The carboxymethyl group incorporated into RNase St was liberated by treatment with 0.2 N NaOH or 1 M hydroxylamine. Thus the incorporation of a carboxymethyl group into a carboxyl group was demonstrated. 3. 14C-labeled CM-RNase St was digested successively with alkaline protease and aminopeptidase M. The 14C-labeled amino acid was identified as the carboxymethyl ester of glutamic acid by means of column chromatography. 4. By digestion of reduced carboxymethylated CM-RNase St with trypsin, a peptide containing a 14C-carboxymethyl group was isolated by Dowex AG-50W colum chromatography. alpha-Chymotryptic digestion of the radioactive tryptic peptide, Glu48-Lys65, produced a tetrapeptide containing a 14C-carboxymethyl group, that is, Tyr59-His60-Glu61-Tyr62. Therefore, it was concluded that Glu61 in RNase St was the site of carboxymethylation. 5. When RNase St was inactivated by iodoacetamide at pH 8.0, about 2 histidine residues were modified. The molar ratio of the products of carboxyamidomethylation were 52.3%, 21.7%, 21.0%, and 4.8% for 3-CAM-His, 1,3-di-CAM-His, 1-CAM-His, and di-CM-Lys, respectively. 6. CD spectra of CM-RNase St and CAM-RNase St were practically the same as that of the native RNase St indicating the maintenance of the native conformation during modification. 7. The binding constants of CM-RNase St and CAM-RNase St with 2'-GMP were about 1/150 and 1/38 of that of the native enzyme, respectively.
2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2-thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75. The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed.