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1.
Biochim Biophys Acta ; 481(1): 212-21, 1977 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-557345

RESUMO

Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4-hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I-SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates. Titration of the enzyme with either Nbs2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or beta-decarboxylation.


Assuntos
Aldeído Liases/metabolismo , Fígado/enzimologia , Compostos de Sulfidrila , Aldeído Liases/antagonistas & inibidores , Animais , Bovinos , Ácido Ditionitrobenzoico/farmacologia , Glioxilatos/farmacologia , Ácidos Cetoglutáricos , Mercaptoetanol/farmacologia , Mercurobenzoatos/farmacologia , Conformação Molecular , Oxo-Ácido-Liases , Piruvatos/farmacologia , Relação Estrutura-Atividade
2.
Biochim Biophys Acta ; 445(2): 286-93, 1976 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-8142

RESUMO

Growth of Pullularia pullulans on L-rhamnose induces formation of L-rhamnofuranose dehydrogenase, whichreversibly converts L-rhamnofuranose to L-rhamnono-gamma-lactone with the concomitant reduction of NAD, but not of NADP. The dehydrogenase was purified 100-fold by MnCl(2) treatment...


Assuntos
Oxirredutases do Álcool/metabolismo , Fungos Mitospóricos/enzimologia , Oxirredutases do Álcool/isolamento & purificação , Desidrogenases de Carboidrato , Cátions Bivalentes , Ácido Edético/farmacologia , Concentração de Íons de Hidrogênio , Iodoacetatos/farmacologia , Cinética , Mercurobenzoatos/farmacologia , Ramnose/metabolismo
3.
Biochim Biophys Acta ; 482(1): 228-40, 1977 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-861231

RESUMO

Crystalline ribulose-1,5-bisphosphate carboxylase-oxygenase (3-phospho-D-glycerate carboxy-lyase (dimerising), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is partially inactivated by cold treatment and fully reactivated by simple heating in the absence of sulfhydryl reagents and effectors. Since the reversible cold inactivation of this bifunctional enzyme does not involve a gross change in the association state of subunits, a subtle conformational change induced by low temperatures was implicated (Chollet, R. and Anderson, L.L. (1976) Arch. Biochem. Biophys. 176, 344-351). Chemical modification of the cold-inactivated and heat-reactivated enzymes by 5,5'-dithiobis-(2-nitrobenzoate) and p-mercuribenzoate at 25 degrees C revealed no difference in the number of free -SH groups per mol protein. However, the reactivity of the sulfhydryl residues on the inactivated protein was considerably greater than that of the reactivated enzyme. Pretreatment of the two proteins with sodium dodecyl sulfate completely abolished the difference in -SH reactivity, indicating its dependence on protein conformation. Both the cold-inactivated and heat-reactivated enzymes enhanced the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) and caused a blue shift of the emission maximum from 510 to 472 nm. When the inactivated enzyme was reactivated by heating, the increase in catalytic activity was closely paralleled by a concomitant decrease in the fluorescence intensity of the ANS - protein complex at 25 degrees C. Fluorescence titration experiments revealed that the decrease in fluorescence intensity accompanying heat reactivation of the inactivated enzyme was due to a reduction in the number of hydrophobic sites available for ANS binding rather than to a change in the dissociation constant of the ANS - protein complex. These results indicate that the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase is associated with a reversible change in the conformation of the protein. This cold-induced conformational change resluts in a greater exposure of sulfhydryl groups and hydrophobic regions to the external environment and is closely paralleled by changes in the catalytic activity of the protein. By analogy to other oligomeric enzymes also subject to reversible cold inactivation, perhaps low temperatures induce a partial dissociation of the octameric structure of the hydrophobic catalytic subunits, but complete dissociation is arrested in some unknown manner by the small hydrophilic subunits.


Assuntos
Carboxiliases , Oxigenases , Ribulose-Bifosfato Carboxilase , Naftalenossulfonato de Anilina , Sítios de Ligação , Carboxiliases/metabolismo , Temperatura Baixa , Ácido Ditionitrobenzoico/farmacologia , Cinética , Mercurobenzoatos/farmacologia , Oxigenases/metabolismo , Plantas/enzimologia , Plantas Tóxicas , Ligação Proteica , Ribulose-Bifosfato Carboxilase/metabolismo , Espectrometria de Fluorescência , Espectrofotometria , Nicotiana/enzimologia
4.
Biochim Biophys Acta ; 403(1): 221-31, 1975 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-240429

RESUMO

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli W contains 38 half-cystine residues per tetrameric enzyme molecule. Two sulfhydryl groups were modified with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) per subunit, while 8.3 sulfhydryl groups were titrated with p-mercuribenzoic acid. In the presence of 4 M guanidine - HCl, 8.6 sulfhydryl groups reacted with DTNB per subunit. Aspartase was inactivated by various sulfhydryl reagents following pseudo-first-order kinetics. Upon modification of one sulfhydryl group per subunit with N-Ethylmaleimide, 85% of the original activity was lost; a complete inactivation was attained concomitant with the modification of two sulfhydryl groups. These results indicate that one or two sulfhydryl groups are essential for enzyme activity. L-Aspartate and DL-erythro-beta-hydroxyaspartate markedly protected the enzyme against N-ethylmaleimide-inactivation. Only the compounds having an amino group at the alpha-position exhibited protection, indicating that the amino group of the substrate contributes to the protection of sulfhydryl groups of the enzyme. Examination of enzymatic properties after N-ethylmaleimide modification revealed that 5-fold increase in the Km value for L-aspartate and a shift of the optimum pH for the activity towards acidic pH were brought about by the modification, while neither dissociation into subunits nor aggregation occurred. These results indicate that the influence of the sulfhydryl group modification is restricted to the active site or its vicinity of the enzyme.


Assuntos
Amônia-Liases/metabolismo , Aspartato Amônia-Liase/metabolismo , Cistina/análise , Aspartato Amônia-Liase/análise , Ácido Aspártico/análogos & derivados , Sítios de Ligação , Ácido Ditionitrobenzoico/farmacologia , Escherichia coli/enzimologia , Etilmaleimida/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Mercurobenzoatos/farmacologia , Ligação Proteica , Conformação Proteica
5.
Biochim Biophys Acta ; 567(2): 384-91, 1979 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-109123

RESUMO

Only L-ascorbic acid activated plant myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), whereas ascorbic acid analogs did not. The enzyme protein was conformationally changed by the addition of L-ascorbic acid to the spectrophotometric analysis, approx. 1.5 amino residues appeared on the surface of the enzyme and about 2.3 tryptophan residues were buried in the molecule when 1 mM L-ascorbic acid was added. Optimum temperature for the myrosinase activity was approx. 55 degrees C without L-ascorbic acid, but with L-ascorbic acid it was about 35 degrees C; that for beta-glucosidase activity was the same (55 degrees C) with or without L-ascorbic acid. The effect of chemical modification of the functional groups of myrosinase on the interaction of L-ascorbic acid was investigated and the interaction of L-ascorbic acid with the active center of the enzyme is proposed.


Assuntos
Ácido Ascórbico/farmacologia , Glicosídeo Hidrolases/metabolismo , 2-Hidroxi-5-nitrobenzil Brometo/farmacologia , Ácido Ascórbico/análogos & derivados , Sítios de Ligação , Mercurobenzoatos/farmacologia , Plantas/enzimologia , Conformação Proteica , Temperatura , Tioglucosídeos , Tropanos/farmacologia , beta-Glucosidase/metabolismo
6.
Biochim Biophys Acta ; 957(2): 243-53, 1988 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-3191142

RESUMO

The compound p-mercuribenzenefulfonate was found to affect the self-association behavior of both spectrin and actin. The reagent brings about the depolymerization of F-actin, as judged from the decrease in the fluorescence of an attached pyrene label, with a second-order rate constant an order of magnitude less than that for the disruption of isolated erythrocyte cytoskeletons. Therefore, it is unlikely that the depolymerization of actin is the rate-determining step in the mercurial-dependent disruption of the erythrocyte cytoskeleton. Low reagent concentrations caused an initial rapid dissociation of spectrin tetramers at a rate comparable with that of cytoskeleton disruption. Prolonged incubation, or higher reagent concentrations, resulted in subsequent aggregation of spectrin. The reagent also prevented the interaction between spectrin and actin, presumably through its depolymerization of actin and its effects on spectrin. The early event in the disruption of isolated erythrocyte cytoskeletons by p-mercuribenzenesulfonate thus appears to be the dissociation of spectrin oligomers. Subsequent depolymerization of actin brought about by the reagent then results in total disruption of the cytoskeleton.


Assuntos
Actinas , Membrana Eritrocítica/efeitos dos fármacos , Mercurobenzoatos/farmacologia , Espectrina/efeitos dos fármacos , Actinas/sangue , Eletroforese em Gel de Poliacrilamida , Membrana Eritrocítica/ultraestrutura , Etilmaleimida/farmacologia , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Polímeros , Ligação Proteica/efeitos dos fármacos
7.
Biochim Biophys Acta ; 649(1): 98-104, 1981 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-7306547

RESUMO

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although N-ethylmaleimide and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.


Assuntos
Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Mercurobenzoatos/farmacologia , Compostos de Fenilmercúrio/farmacologia , Animais , Bovinos , Cães , Membrana Eritrocítica/metabolismo , Etilmaleimida/farmacologia , Humanos , Iodoacetamida/farmacologia , Marsupiais , Proteínas de Membrana/metabolismo , Mercúrio/metabolismo , Acetato de Fenilmercúrio/farmacologia , Ratos , Solubilidade , Especificidade da Espécie , Espectrina/metabolismo
8.
Endocrinology ; 96(5): 1201-9, 1975 May.
Artigo em Inglês | MEDLINE | ID: mdl-235420

RESUMO

Nuclear proteins extracted from purified nuclei with 0.4M KCl at pH 7.4 OR 8.5 are able to bind L-triiodothyronine (T3) giving rise to nuclear thyroid hormone binding protein-T3 (NTBP-T3) complexes. Binding is maximum in 3 h at 20 C. It is thermolabile even at 36 C, inhibited by p-hydroxymercuribenzoate and markedly enhanced by dithiothreitol. Optimum pH is between 7.8 and 8.5. Divalent cations are not necessary. The NTBP-T3 complex exhibits similar anodal electrophoretic migration in polyacrylamide gel at pH 8.5, whether formed in vivo or in vitro. Scatchard plots obtained with various amounts of T3 from 0.15 nM TO 0.15 MUM and either unlabeled nuclear proteins or in vivo formed NTBP-[125I]-T3 complexes, give apparent association constants K-a of 0.2 X 10-10 M minus at pH 7.4 and 0.8 X 10-10 M minus 1 at pH 8.5. Capacity is about 0.5 pmol T3 per mg protein or 800 pg/g liver. The presence of dithiothreitol markedly enhances the Ka. The nuclear binding sites are not highly specific for L-T3 since they are able to bind D-T3 with almost equal affinity and triiodothyroacetic acid with a higher affinity. L-thyroxine (T4) can also displace L-T3 but with about 10-fold lesser effectiveness. Nuclear binding proteins of low capacity and high affinity have been demonstrated in vitro. The NTBP-T3 complexes formed in vivo, with whole nuclei, or in vitro are indistinguishable.


Assuntos
Fígado/metabolismo , Nucleoproteínas/metabolismo , Tri-Iodotironina/metabolismo , Animais , Sítios de Ligação , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Masculino , Mercurobenzoatos/farmacologia , Monoiodotirosina/análogos & derivados , Ratos , Tiroxina/metabolismo , Fatores de Tempo
9.
J Biochem ; 84(1): 205-11, 1978 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-690099

RESUMO

Native human oxyhemoglobin, which has a rigid conformation resistant to proteases such as trypsin and subtilisin, could be hydrolyzed by these proteases at pH 7.0 after treatment with p-chloromercuribenzoate. The digestion curve of hemoglobin as a function of concentration of the mercurial was essentially parallel to the titration curve of hemoglobin with the mercurial, indicating that a relationship exists between susceptibility to proteases and modification of thiol groups of the protein. On the other hand, when myoglobin was used as a substrate, the degree of proteolysis did not increase after treatment with the mercurial. Circular dichroism measurements and gel-filtration experiments showed that the observed increase in susceptibility of hemoglobin to proteases was not due to a conformational change involving unfolding of alpha-helical structure, but was due to the dissociation of the tetrameric hemoglobin molecule into dimer and monomer after treatment with the mercurial.


Assuntos
Mercurobenzoatos/farmacologia , Oxiemoglobinas/metabolismo , Subtilisinas/metabolismo , Tripsina/metabolismo , Caseínas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Metemoglobina/metabolismo , Peso Molecular , Mioglobina/metabolismo , Conformação Proteica , Compostos de Sulfidrila/análise , Inibidor da Tripsina de Soja de Kunitz/farmacologia
10.
Naunyn Schmiedebergs Arch Pharmacol ; 294(2): 179-85, 1976 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-13312

RESUMO

The reaction of tris(2-chloroethyl)amine (TCEA) with purified hemoglobin and its effect on properties of hemoglobin was studied using 14C-labeled TCEA. Hemoglobin remained soluble after binding as much as 4 TCEA per heme. In concentrations which did not denature hemoglobin TCEA reacted only with a small proportion of the free SH groups; blockade of the SH groups with PMB did not noticeably affect the binding of TCEA to hemoglobin. Hydrolysis by trypsin or chymotrypsin of hemoglobin which had reacted with TCEA yielded radioactive peptides besides not radioactive peptides and radioactive compounds not reacting with ninhydrin. The reaction with TCEA caused a change in electrophoretic mobility of hemoglobin and prevented its complete disintegration by PMB into subunits. After reaction with TCEA the affinity of hemoglobin for oxygen was strongly increased and the heme-heme interaction strongly diminished. The Bohr effect and the effect of 2,3-diphosphoglycerate on oxygen affinity remained unchanged. The effect of TCEA on the properties of hemoglobin points to specificity in its reaction with functional groups of hemoglobin.


Assuntos
Hemoglobinas/metabolismo , Compostos de Mostarda Nitrogenada/farmacologia , Sítios de Ligação , Ácidos Difosfoglicéricos/sangue , Eletroforese , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Mercurobenzoatos/farmacologia , Compostos de Mostarda Nitrogenada/metabolismo , Oxiemoglobinas/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos
11.
Contraception ; 20(2): 159-65, 1979 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-226329

RESUMO

The water-soluble, polymeric sulfhydryl group reagent N-(methoxypolyethylene glycol) rho-hydroxymercuribenzamide (Mw 5000) inhibited the motility of human spermatozoa. Its activity profile was very similar to that of rho-hydroxymercuribenzene sulfonate, a charged sulfhydryl group reagent that is a very poor membrane penetrant. These results suggest that functional sulfhydryl groups of the spermatozoan membrane are easily accessible and externally oriented on the membrane surface.


Assuntos
4-Cloromercuriobenzenossulfonato/farmacologia , Mercurobenzoatos/farmacologia , Compostos de Fenilmercúrio/farmacologia , Polietilenoglicóis/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Reagentes de Sulfidrila/farmacologia , Transporte Biológico , Membrana Celular/fisiologia , Humanos , Hidrólise , Masculino , Mercaptoetanol/farmacologia
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