Characterization of sulfhydryl groups of actin

Lusty, Carol J.; Fasold, H.

doi:10.1021/bi00835a036

Cited by 96 publications

(50 citation statements)

References 26 publications

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“…Perhaps the reciprocal action between sites is why labeling "SH1" activates the calcium activated ATPase of S-1. Lusty and Fasold reported that binding of myosin to actin inhibits the reaction of actin Cys-373 with 2,2'-dicarboxy-4'-iodoacetamidoazobenzene (18). The present work shows that binding of myosin S-1 to actin activates actin Cys-10 (and, reciprocally, inhibits myosin "SH1") towards reaction with [3H]1,5-IAEDANS.…”

Section: Methodssupporting

confidence: 60%

Reciprocal reactivities of specific thiols when actin binds to myosin.

Duke¹,

Takashi²,

Ue³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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We report measurements of the reactivity (degree of labeling, as mole of ligand per mole of protein, at constant exsure time) of the reactive thiol, "SHI", of a subfragment of myosin (S-i), and of Cys-10 of F-actin under various conditions, using N-iodo{3Hjacetyl-N(1-sulfo-5-naphthyl)ethylenediamine, a fluorescent radioactive iodoacetamide analog. When either ADP or adenyloyl imidodiphosphate (simulating unhydrolyzed ATP) is bound to the enzymatic site of S-i, the reactivity of "SHI" is slightly enhanced, but when active ATPase is going on, reactivity is reduced by about a third, presumably due to the species, (S.1)**ADPPi. The reactivity of Cys-10 alone is very low. When the complex, (S-I)F-actin, is formed, the reactivity of SHI is strongly decreased, and the reactivity of Cys-10 is strongly increased. The foregoing results explain our further observation (on glycerol-treated rabbit psoas fibers) that when fibers labeled in relaxation solution are compared with fibers labeled in rigor solution, myosin is more reactive and actin is less reactive, in the former case; a-actinin and C-protein are also less reactive in the former case.Our laboratory has used the compound N-iodo-[3H]-acetyl-N'-(l-sulfo-5-naphthyl)ethylenediamine ([3H]1,5-IAEDANS) extensively to fluorescence-label myosin and muscle fibers because this compound has a strong specificity for the reactive thiol (SHI) in the S-1 moiety of myosin.While searching for conditions in which the proteins of glycerinated psoas fibers are selectively labeled, we noted that the "relaxation" state tended to favor myosin labeling and to suppress actin labeling, while the "tension-generating" and "rigor" states tended to favor the converse. In order to clarify this reciprocity between myosin and actin, and to investigate the chemical changes that occur during myosin-actin binding, we forsook fibers and experimented with purified proteins and various nucleotides. We have thus been led into work along the lines pioneered by Barany et al. (1). We have learned that the reactivity of "SH1" with [3H]1,5-IA-EDANS depends upon the occupancy of the ATPase site; it is slightly enhanced by either ADP or adenyloyl imidodiphosphate (AMPPNP), and is significantly depressed during hydrolysis. It also depends on the occupancy of the actin-5-naphthyl)ethylenediamine; SH1, the "fast reacting" and calcium ATPase activating thiol located on the heavy chain of S-1; 5-1, subfragment resulting from papain hydrolysis of myosin; AMPPNP, adenyloyl imidodiphosphate; Cys-10 andCys-373, cysteinyl residue in position 10 (NH2-terminal peptide) and 373 (COOH-terminal peptide), respectively, of the amino-acid sequence of actin; TPCK, L-1-tosylamide-2-phenylethylchloromethyl ketone (chymotrypsin inhibitor); TLCK, N-a-p-tosyl-l-lysine chloromethyl ketone.HCI (papain inhibitor); EGTA, ethylene-glycol-bis(,3-aminoethyl ether) N,N'-tetraacetic acid; NaDodSO4, sodium dodecyl sulfate; Tes, Ntris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. binding site and is very strongly depressed when actin i...

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Section: Methodssupporting

confidence: 60%

Reciprocal reactivities of specific thiols when actin binds to myosin.

Duke¹,

Takashi²,

Ue³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

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“…The dependence of thioester accumulation on glutamine concentration (Fig. 1) is also consistent with the K,, for glutamine hydrolysis [12]. Taken together this evidence supports the notion that the thioester is an obligate intermediate in the hydrolytic reaction.…”

Section: Febs Letterssupporting

confidence: 84%

“…This data lends further support for the idea that the stably bound glutamine counts are present as an acyl ester of glutamate. The additional observation that the acyl ester is not formed with the Cys269Ser mutant enzyme argues that glutamine hydrolysis procoeds through an enzyme.bound thioester, as proposed in prior studies [6] and in more recent schemes [7,12,17,18] for the reaction mechanism of glutaminedependent carbamyl pho.~phate synthetase. Even though the Cys:~Ser enzyme fails to form the covalent aeyl ester, previous data obtained with gephadex-spun columns indicated substantial binding of glulamine under non.equilibrium conditions iS].…”

Section: Febs Lettersmentioning

confidence: 67%

Detection of an enzyme bound γ‐glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli

Lusty¹

1992

FEBS Letters

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E. colt carbamyl phosphate synthetase binds 0.2-0.4 mol equivalents ofglutamine m an acid resmtant form. The bound material is quantitativdy released as glutamate by weak base hydroly~ib and as a mixture of 12% glutamate, 10% 7-glutamylhydroxamate, and 70% pyrrollidoneearboxylie acid by hydrolysis with hydroxylamilxe. These results provide direct evidence Ibr a 7-8lutamyl aeyl ester on the enzyme The absence of the aeyl ester in a mutant ¢arbamyl phosphate sYnthetase with a Cys ~-~ .-o Ser subshtut~on in the glutaminase subumt further suggests that the covalent intermediate is a thioestei of Cy~ -'t~, Under equihbrium conditions, the Cys"~Ser mutant enzyme brads glutamme with a Ka of 7 +_. 1 ,aM, indicating tlmt Cys ~ is essential for acyl ester formation but not for binding of ~utamme.Carbamyl phosphate synthetase; Thioester intermediate; Glutamine anaidotransferase; Glulamine hydrolysis INTR.ODUCT1ONThe synthesis of carbamyl phosphate by glutaminedependent carbamyl phosphate synthetase of E. co,',: de;-pearls on an initial hydrolysis of glutamine by the glutaminase snbunit [11. The release of ammonia from glutaraine has been shown to depend on an active site cysteine, as judged by inhibition of the reaction by 2-amino-4-oxo-5-chloropentanoic acid [2,3] or cyanate [4] and lack of enzyme activity in a mutant with a substitution of a serine for a cysteine residue in the glutarninase domain of the protein [5]. Kinetic evidence and partitioning experiments showing that the enzyme catalyzes formation of 7-glutamylhydroxamate from glutamine and hydroxylamine also suggest the formation of a 7-glutamyl thioester in the reaction mechanism [6,7]. Despite much evidence favoring the participation of a thioester intermediate similar to that shown to occur m the hydrolytic reaction catalyzed by papain [8,9], its existence in carbamyl phosphate synthetase has not been directly demonsuated and as a result has been a subject of ~ome controversy [6,10].In the present study the question of whether a 7-glutamyl thioester is an intermediate in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase has been re-examined. Evidence is presented that the • ~.,,,, CAD, ~hc .,u,,,,ane,~,,a, p~nm,..,ne-speeu,c enzyme comprising glutamine-dependent earbamyl phosphate synthetase, asparrate transearbamylase and dihydroorotase.acid-stable form of glutamine bound to the wild-type holoenzyme and previously inferred to be a thioester adduct of glutamate [1,6], is nearly quantitatively released by reaction with hydroxylamine as 7-glutamyl hydroxamate and its cyclic derivative° pyrollidonecarboxylic acid. This demonstration indicates that glutamine can be converted to an enzyme-bound acyl ester. The absence of the ester in a mutant enzyme with a Cys "*69 ~ Set substitution in the glutaminase component further argues that the covalent compound is a thioester Of Cys ~'°). EXPERIMENTAL Reagentse-[U-'4ClGlutamine (270 mCi/mmol) and L-lG-~H]~lutamine (56 Ct/ retool) were purcl~ased from Amersham. Other reagents were of the high~st quality ava...

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“…In G-actin in the presence of ATP, only the thiol group in position 374 is exposed [14]. Alkylation of this SH group with Thiolyte MQ yielded a conjugate containing 0.7 moles of label per mol of actin.…”

Section: Resultsmentioning

confidence: 99%

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

et al. 1993

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By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosinp4 (T/94), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation, Based on this effect, we determined the Kn value of the thymosin @l complex as 0.8 PM, a value that is in agreement with previous determinations. In addition to the main compound thymosin/?4, bovine tissue contains a related peptide, thymosin p (Tp9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T/$?, similar to Tp4, forms a 1:l complex with monomeric actin, and hereby inhibits actin polymerization. With a K, value of 1.1 FM the affinity of TjT9 is in the same range as that of Tp4, suggesting that T@9, like T/?4, contributes to maintaining the pool of monomeric. actin in bovine non-muscle cells. Further proof of the interaction of T/I9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslink~g experiments. Using different crosslinking reagents, like water-soluble ~~iimide (EDC), ~-maleimido~~oyl-~-hydroxys~nimi~te (MBS), and disu~inimidylsu~rate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tfi9 could be identified by imm~oblot~ng. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T/39 must be located close to the carboxy-terminus.

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Characterization of sulfhydryl groups of actin

Cited by 96 publications

References 26 publications

Reciprocal reactivities of specific thiols when actin binds to myosin.

Reciprocal reactivities of specific thiols when actin binds to myosin.

Detection of an enzyme bound γ‐glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli

Use of bimanyl actin derivative (TMB‐actin) for studying complexation of β‐thymosins

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