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1.
Exp Eye Res ; 79(6): 919-26, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15642330

ABSTRACT

Numerous animal studies indicate that aldose reductase inhibitors (ARIs) are beneficial for the prevention or amelioration of diabetic complications such as neuropathy, nephropathy and the ocular complications of cataract, retinopathy and keratopathy. To aid in the identification of novel potent ARIs, we have previously developed a screening method that is based on the formation of a non-covalent ternary tight-binding enzyme-inhibitor-nucleotide (AR-ARI-NADPH) complex that can be isolated using YM-10 filter units. Here, we report a modification of this method that permits us to rapidly identify tight binding ARIs that are isolated by denaturation from AR-ARI-NADPH complexes that are free of possible contamination resulting from the reaction of methanol with the YM-10 filter units. For the development of this procedure, nine structurally diverse ARIs were mixed with purified recombinant rat lens aldose reductase (RLAR) bound with NADPH to form tight-binding RLAR-ARI-NADPH complexes. These complexes were purified by high pressure Sephadex 75 size exclusion chromatography using ammonium acetate buffer and the formation of each complex was confirmed by electrospray ionisation mass spectrometry (ESI-MS). Each of the complexes was then denatured with methanol, rechromatographed on the size exclusion column, and the identity of the bound ARIs was confirmed by ESI-MS. The apparent ARI binding with aldose reductase to form a tight binding ARI complex appeared proportional to their IC50 values. This procedure allows for the rapid identification of tight binding ARIs with apparent IC50s<0.1 microm.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Enzyme Inhibitors/isolation & purification , Lens, Crystalline/enzymology , Animals , Chromatography, High Pressure Liquid/methods , Enzyme Inhibitors/chemistry , Molecular Weight , NADP/chemistry , Protein Denaturation , Rats , Spectrometry, Mass, Electrospray Ionization/methods
2.
J Biol Chem ; 276(51): 47763-6, 2001 Dec 21.
Article in English | MEDLINE | ID: mdl-11684673

ABSTRACT

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization. In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 microM comparable with its IC(50) in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation.


Subject(s)
Actins/metabolism , Biopolymers/metabolism , Glutathione/metabolism , Amino Acid Sequence , Cell Line , Cytoskeleton/metabolism , Epidermal Growth Factor/pharmacology , Humans , Molecular Sequence Data
3.
J Mass Spectrom ; 36(9): 1024-30, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11599080

ABSTRACT

Positionally isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione mono- and disulfonic acids give rise to similar electrospray ionization (ESI) and atmosphere pressure chemical ionization (APCI) mass spectra, which show very abundant MH(+) ions and negligible fragmentation. The MH(+) ions of these isomeric acids exhibit notably different behavior under collision-induced dissociation (CID) conditions. The acids with a sulfonic group at position 8' in the quinoline moiety, adjacent to the N-atom, exhibit highly abundant [MH - H(2)SO(3)](+) ions (m/z 272 for the mono- and m/z 352 for the disulfonic acids), which are of lower abundance in the CID spectra of isomers with the SO(3)H group at other positions, remote from the nitrogen atom. The latter isomers undergo efficient eliminations of SO(3) and HSO(3). The isomeric diacids with one SO(3)H group at position 4 of the indene-1,3(2H)-dione moiety, adjacent to one of the carbonyl groups, undergo highly efficient elimination of H(2)O. Mechanistic pathways, involving interactions between adjacent groups, are proposed for the above regiospecific fragmentations. Pronounced different behavior has been also observed in negative ion tandem mass spectrometric measurements of the sulfonic acids. The distinctive behavior of the isomeric acids was strongly pronounced when the measurements were performed with an ion trap mass spectrometer (LCQ), and much less so with a triple-stage quadrupole instrument (TSQ).


Subject(s)
Coloring Agents/analysis , Quinolines/chemistry , Sulfonic Acids/chemistry , Isomerism , Spectrometry, Mass, Electrospray Ionization
4.
Anal Chem ; 73(15): 3576-82, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11510821

ABSTRACT

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.


Subject(s)
Actins/chemistry , Iodoacetamide/chemistry , Iodoacetic Acid/chemistry , Peptides/analysis , Peptides/chemistry , Alkylation , Animals , Cattle , Chromatography, Liquid , Cysteine/chemistry , Hydrolysis , Mass Spectrometry , Oxidation-Reduction , Proteins/chemistry , Sulfhydryl Reagents/chemistry
5.
J Chem Ecol ; 27(6): 1219-28, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11504024

ABSTRACT

Abdominal defensive glands of both sexes of the Gulf fritillary butterfly, Agraulis vanillae (Linnaeus) (Nymphalidae:Heliconiinae) emit a pronounced odor when disturbed. We have identified 6-methyl-5-hepten-2-one; oleic, palmitic, and stearic esters of the corresponding alcohol 6-methyl-5-hepten-2-ol; hexadecyl acetate; 1,16-hexadecanediol diacetate; and 1,15-hexade-canediol diacetate in the glandular exudate. Since we have determined that free-flying birds or birds in a butterfly conservatory discriminate against A. vanillae as prey, we suggest that the constituents in the glands may play a defensive role against potential avian predators.


Subject(s)
Acetates/isolation & purification , Exocrine Glands/chemistry , Lepidoptera/physiology , Abdomen , Acetates/chemistry , Animals , Birds , Female , Male , Odorants , Predatory Behavior
6.
Anal Biochem ; 285(2): 205-10, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11017703

ABSTRACT

We have determined the major ingredients of the commercially available reagents M-PER, Y-PER, and B-PER from Pierce Chemical Co. using electrospray mass spectrometry. These three proprietary reagents have been widely used in the biochemical community as cell membrane dissolving tools during the initial step of protein purification. However, the identity and mechanism of these reagents remained unknown. In this paper, we identified these reagents as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-octyl-beta-d-thioglucopyranoside, respectively. In addition, we wish to stress here the increasing importance of the role of electrospray mass spectrometry in the analysis of such proprietary biological preparations which are increasingly finding their way into the biochemical literature.


Subject(s)
Cholic Acids/chemistry , Detergents/chemistry , Quaternary Ammonium Compounds/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Thioglucosides/chemistry
7.
Biochemistry ; 39(27): 8007-16, 2000 Jul 11.
Article in English | MEDLINE | ID: mdl-10891082

ABSTRACT

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14.17. 3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.


Subject(s)
Fatty Acids, Monounsaturated/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes , Catalytic Domain , Hydroxylation , Mass Spectrometry , Mixed Function Oxygenases/antagonists & inhibitors , Recombinant Proteins/metabolism , Substrate Specificity
8.
Biochem J ; 346 Pt 2: 305-11, 2000 Mar 01.
Article in English | MEDLINE | ID: mdl-10677347

ABSTRACT

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , Oxidoreductases/metabolism , Amino Acid Sequence , Aspartic Acid Endopeptidases/chemistry , Dimerization , Enzyme Activation , HIV Protease , HIV-2/physiology , Humans , Methionine Sulfoxide Reductases , Molecular Sequence Data , Oxidation-Reduction , Oxidoreductases/chemistry , Sequence Alignment , Virus Replication
9.
Biochemistry ; 38(20): 6699-705, 1999 May 18.
Article in English | MEDLINE | ID: mdl-10350489

ABSTRACT

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.


Subject(s)
Cysteine/metabolism , Glutathione/metabolism , Protein Tyrosine Phosphatases/metabolism , Amino Acid Sequence , Binding Sites , Cysteine/chemistry , Dithionitrobenzoic Acid/chemistry , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Glutathione/chemistry , Glutathione/physiology , Glutathione Disulfide/chemistry , Glutathione Disulfide/metabolism , Glutathione Disulfide/physiology , Humans , Kinetics , Mass Spectrometry , Molecular Sequence Data , Oxidation-Reduction , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/chemistry , Titrimetry
10.
Anal Biochem ; 270(1): 159-66, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10328778

ABSTRACT

Oleamide is a putative endogenous sleep-inducing lipid which potently enhances currents mediated by GABAA and serotonin receptors. While a quantitative assay would aid in determining the role of oleamide in physiological processes, most of the available assays are lacking in sensitivity. We now describe a quantitative assay for measuring low nanogram amounts of oleamide in biological fluids using GC/MS in the selective ion-monitoring mode. The internal standard (13C18 oleamide) was added to known concentrations of oleamide, which were converted to the N-trimethylsilyl or N-tert-butyldimethylsilyl derivatives before analysis by GC/MS, yielding linear calibration curves over the range of 1-25 ng of oleamide when monitoring the m/z 338/356 fragments. Using this technique, oleamide levels were determined following solvent extraction of normal rat cerebrospinal fluid and plasma to be 44 and 9.9 ng/ml, respectively. This technique constitutes a sensitive and reliable method for determining low nanogram quantities of oleamide in biological fluids.


Subject(s)
Gas Chromatography-Mass Spectrometry/methods , Oleic Acids/analysis , Animals , Male , Oleic Acids/blood , Oleic Acids/cerebrospinal fluid , Rats , Rats, Sprague-Dawley , Sensitivity and Specificity
11.
Anal Chem ; 70(20): 4453-5, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9796428

ABSTRACT

Wang and Hackett have recently proposed (Anal. Chem. 1998, 70, 205-212) a new electrospray interface described as a "concentric cylindrical capacitor". Excellent spectra were obtained, especially in the negative ion mode, with lipid A, a 15-mer oligonucleotide, and a series of proteins. We find that similar results can be obtained merely by ensuring a direct electrical contact between the analyte inside a fused-silica capillary and the high-voltage supply. No capacitor effect need be invoked.


Subject(s)
Mass Spectrometry/instrumentation , Lipid A/chemistry , Oligonucleotides/chemistry , Proteins/chemistry , Silicon Dioxide
12.
Rapid Commun Mass Spectrom ; 12(20): 1457-62, 1998.
Article in English | MEDLINE | ID: mdl-9796533

ABSTRACT

The oxidation state of the cysteine residue at the active site of human protein tyrosine phosphatase (PTP-1B) greatly affects its enzymatic activity. We wished to examine peroxide-treated preparations for modifications of this enzyme with electrospray mass spectrometry in order to determine the locations and oxidation states of the cysteines or other residues involved in the process. Since these reaction products contained large amounts of salts and buffers, they required desalting prior to analysis. Existing on- and off-line methods presented certain difficulties in handling and sample usage. Based on recent experience with direct syringe admission of sample, we developed a procedure as a simple, inexpensive alternative to full high-performance liquid chromatography systems that provides on-line desalting using only a few microL of sample. The method was applied to the analysis of oxidized PTP-1B preparations where conversion of cysteine 215 to both sulfinic and sulfonic acid residues was demonstrated.


Subject(s)
Cysteine/chemistry , Protein Tyrosine Phosphatases/chemistry , Amino Acid Sequence , Binding Sites , Humans , Mass Spectrometry , Molecular Sequence Data , Oxidation-Reduction , Recombinant Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
13.
Pflugers Arch ; 436(4): 545-52, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9683727

ABSTRACT

A peptide with neurotoxic effect on mammals, purified from the venom of the spider Phoneutria nigriventer, was studied regarding its primary structure and its effects on voltage-gated calcium channels. The peptide, named ω-phonetoxin-IIA, has 76 amino acids residues, with 14 Cys forming 7 disulphide bonds, and a molecular weight of 8362.7 Da. The neurotoxicity is a consequence of the peptide's blocking effects on high-voltage-activated (HVA) calcium channels. N-type HVA calcium channels of rat dorsal root ganglion neurons are blocked with affinity in the sub-nanomolar concentration range. The toxin also blocks L-type channels of rat ß pancreatic cells, with an affinity 40 times lower. Although not studied in detail, evidence indicates that the toxin also blocks other types of HVA calcium channels, such as P and Q. No effect was observed on low-voltage-activated, T-type calcium channels. The significant homologies between ω-phonetoxin-IIA and the peptides of the ω-agatoxin-III family, and the overlapping inhibitory effects on calcium channels are discussed in terms of the structure-activity relationship.


Subject(s)
Calcium Channel Blockers/chemistry , Calcium Channels/metabolism , Peptides/chemistry , Spider Venoms/chemistry , Spiders/chemistry , Amino Acid Sequence , Animals , Calcium/metabolism , Cells, Cultured , Electrophysiology , Female , Ganglia, Spinal/cytology , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Male , Molecular Sequence Data , Neurons/cytology , Neurons/metabolism , Rats , Rats, Wistar , Sequence Alignment , Spider Venoms/genetics
14.
J Am Soc Mass Spectrom ; 9(1): 15-20, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9679592

ABSTRACT

The hexakis (4-diemthylaminopyridyl) benzene hexacation of 1 is investigated as an extreme example of the ability of electrospray ionization to allow transfer of small multivalent ions to the gas phase. The hexacationized benzene ring ions are stabilized by forming gas phase complexes with two to five trifluoromethanesulfonate counterions. MS/MS analysis reveals that their fragmentation takes place by loss of neutrals such as trifluoromethanesulfonic acid and 4-dimethylaminopyridine; no rupture of the benzene or pyridine rings was observed in spite of accumulation of positive charge in a restricted geometry.


Subject(s)
Benzene Derivatives/chemistry , 4-Aminopyridine/analogs & derivatives , 4-Aminopyridine/chemistry , Crystallization , Electrochemistry , Indicators and Reagents , Mass Spectrometry , Mesylates
16.
Biochemistry ; 36(8): 2221-6, 1997 Feb 25.
Article in English | MEDLINE | ID: mdl-9047323

ABSTRACT

Myelin-associated glycoprotein (MAG) is a transmembrane structural protein that is thought to be involved in the formation and/or maintenance of the myelin sheath. MAG is proteolyzed at a discrete location near its transmembrane domain by a calcium activated myelin-associated cysteine protease in the central nervous system. The soluble proteolysis product, dMAG, can be found in the cerebrospinal fluid. The proteolysis of MAG may be involved in the molecular mechanism of demyelination, as the proteolytic degradation of myelin proteins has been observed in disease states. The site for the proteolysis of MAG to dMAG was identified. This was accomplished by developing a protocol for the purification of soluble dMAG and by protein sequencing of short peptides containing the carboxy-terminus of dMAG. The results from these experiments indicated that the native proteolytic site in MAG was located extracellularly and occurred between residues 512 (Ala) and 513 (Lys), with a large hydrophobic residue at the P2 position (Trp-511). This finding in turn indicated that the protease for which MAG was a substrate had cathepsin L-like activity. Cathepsin L-like activity in myelin was confirmed by peptidolysis experiments using known cathepsin L substrates. Additional experiments are in progress to determine the identity of this protease.


Subject(s)
Cathepsins/metabolism , Endopeptidases , Myelin-Associated Glycoprotein/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cathepsin L , Cattle , Cysteine Endopeptidases , Molecular Sequence Data , Myelin-Associated Glycoprotein/metabolism , Peptide Fragments/analysis , Peptide Fragments/isolation & purification , Sequence Analysis
18.
Anal Chem ; 68(18): 3288-9, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8797388

ABSTRACT

Using MS/MS, we have identified ions at m/z 399, 421, 609.4, and 819.9 as those of tris (2-butoxyethyl) phosphate, originating in the gray rubber stoppers commonly used as closures in commercial sources of peptides and proteins. The compound is readily extracted by contact with organic solvents such as alcohol or acetic acid and even by water alone. Zinc salts of proteins are also observed, presumably from zinc oxide fillers present in the stoppers.


Subject(s)
Peptides/analysis , Proteins/analysis , Rubber/analysis , Drug Packaging , Mass Spectrometry
19.
J Chromatogr A ; 724(1-2): 179-84, 1996 Feb 16.
Article in English | MEDLINE | ID: mdl-8819795

ABSTRACT

The present studies have been conducted to investigate the cause of an unusually broad peak of ovalbumin obtained by countercurrent chromatography (CCC) reported earlier [K. Shinomija et al., J. Chromatogr., 644 (1993) 215]. A series of CCC experiments using our prototyte of the cross-axis coil planet centrifuge revealed that commercial ovalbumin products were classified into two groups: group A formed two peaks of ovalbumin at pH 7.0 and 5.8, while group B showed a relatively sharp single peak in a broad range of pH. Electrophoresis indicated that the group A ovalbumin consisted of both natural and denatured products: the natural ovalbumin is a monomer (Mr 45 000) whereas the denatured products form dimers (Mr 90 000). The abnormally broad peak obtained from the group A ovalbumin at pH 9 is apparently caused by the heterogeneity of the sample protein.


Subject(s)
Chromatography, Liquid/methods , Ovalbumin/chemistry , Animals , Chick Embryo , Countercurrent Distribution , Egg White , Electrophoresis, Polyacrylamide Gel
20.
Biochemistry ; 35(7): 2482-8, 1996 Feb 20.
Article in English | MEDLINE | ID: mdl-8652592

ABSTRACT

The homodimeric protease of the human immunodeficiency virus 1 contains two cysteine residues per monomer which are highly conserved among viral isolates. However, these cysteine residues are not essential for catalytic activity which raises the question of why they are conserved. We have found previously that these cysteine residues are unusually susceptible to oxidation by metal ions, and this results in inhibition of protease activity. Recombinant protease mutants (C67A, C95A, and the double mutant C67A,C95A) were prepared to assess the possible role of these cysteines in redox regulation of the enzyme. Mixed disulfides were formed between the cysteine residues of the enzymes and low molecular weight thiols. Enzyme activity was lost when a mixed disulfide was formed between 5,5'-dithiobis(2-nitrobenzoic acid) and cysteine 95, while the same mixed disulfide at cysteine 67 reduced activity by 50%. This effect was reversible as normal activity could be restored when the enzyme was treated with dithiothreitol. The cysteines could also be modified with the common cellular thiol glutathione. Modification with glutathione was verified by mass spectrometry of the protein peaks obtained from HPLC separation. Glutathiolation of cysteine 95 abolished activity whereas modification at cysteine 67 increased the k(cat) by more than 2-fold with no effect on K(m). In addition, glutathiolation at cysteine 67 markedly stabilized the enzyme activity presumably by reducing autoproteolysis. These results demonstrate one possible mechanism for regulation of the HIV-1 protease through cysteine modification and identify additional targets for affecting protease activity other than the active site.


Subject(s)
Cysteine/genetics , HIV Protease/metabolism , Catalysis , Chromatography, High Pressure Liquid , Dithionitrobenzoic Acid/chemistry , Escherichia coli/genetics , Glutathione/metabolism , HIV Protease/genetics , HIV Protease/isolation & purification , Hydrolysis , Oxidation-Reduction
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