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At least 19 recordsLinked to original sources
PubMed1977

Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.

Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.

Alcohol Oxidoreductases↗
PubMed1986

Evidence for changes in the conformational status of rat liver microsomal glucose-6-phosphate:phosphohydrolase during detergent-dependent membrane modification. Effect of p-mercuribenzoate and organomercurial agarose gel on glucose-6-phosphatase of native and detergent-modified microsomes.

Comparative studies investigating influences of temperature and time of preincubation on the interactions of an organomercurial agarose gel and p-mercuribenzoate with glucose-6-phosphatase of native and Triton X-114-modified rat liver microsomes were carried out. The effect of p-mercuribenzoate on glucose 6-phosphate hydrolysis is a result of two processes, a moderate membrane perturbation connected with release of some latency and temperature- and time-dependent inhibition of the catalytic activity. Short-term preincubation with both organic mercurials at 37 degrees C is a necessary condition for the entire inhibition of the enzyme activity of native as well as of Triton X-114-modified microsomes. A binding site of the phosphohydrolase itself is accessible to p-mercuribenzoate and the phenyl mercury residue of the affinity gel from the cytoplasmic surface even in native microsomes. Kinetic analyses reveal a formally competitive mechanism of inhibition using native microsomes, but the kinetic picture changes to a noncompetitive pattern of Lineweaver-Burk plots when the inhibitor-loaded microsomes are modified optimally by Triton X-114. This behavior can be evaluated as the first convincing evidence for drastic changes of the conformational status of the phosphohydrolase during the membrane modification process. A combined conformational flexibility-substrate transport model characterizing the microsomal glucose-6-phosphatase as an integral channel-protein embedded within the hydrophobic interior of the membrane is proposed.

Animals↗
PubMed1978

Catalytic effects by thioltransferase on the transfer of methylmercury and p-mercuribenzoate from macromolecules to low molecular weight thiol compounds.

Thiol agarose and glyceraldehyde-3-phosphate dehydrogenase were blocked with methylmercury or p-mercuribenzoate. The exchange of mercurials between the thiol-containing polymers and glutathione or dithioerythritol was investigated. The activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited by blocking thiol-groups with the mercury compounds. Inhibition was reversible when a short period of inactivation was used. Inactivation for longer periods resulted in reduced regain of enzyme activity. The activity was in part regained when either of the 2 thiol compounds was added. Thioltransferase, known to catalyze thiol-disulfide exchange reactions, increased the regain of glyceraldehyde-3-phosphate dehydrogenase activity to nearly the original value. Here, thioltransferase is proposed to catalyze the transfer of organomercurial from one thiol complex to another. Some consequences of the observations in vivo are discussed.

Animals↗
PubMed1988

Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 2. Identification of a coenzyme A regulatory site by p-mercuribenzoate modification.

Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26, ACAT) is the major intracellular cholesterol-esterifying activity in vascular tissue and is potentially a key regulator of intracellular cholesterol homeostasis during atherogenesis. We have previously reported inhibition of microsomal ACAT by histidine and sulfhydryl-selective chemical modification reagents and present here a more detailed analysis of the effect of sulfhydryl modification on ACAT activity. This analysis indicated two effects of sulfhydryl modification on ACAT activity. Modification of aortic microsomes with relatively low concentrations of p-mercuribenzoate (PMB) (100-200 microM) identified an inhibitory coenzyme A binding site on ACAT which contains a modifiable sulfhydryl group. This site binds CoA tightly (Ki = 20 microM), and PMB modification prevented subsequent ACAT inhibition by CoA without itself inhibiting enzyme activity. At higher concentrations (1-2 mM), PMB inhibited ACAT activity, indicating the presence of a modifiable sulfhydryl group necessary for cholesterol esterification by ACAT. Modification of both sites by PMB was reversible by thiols, and protection against modification was afforded in both cases by oleoyl-CoA, indicating that these sites may also bind oleoyl-CoA. Thus, at least two sulfhydryl groups influence ACAT activity: one is necessary for cholesterol esterification by ACAT, and one is at or near an inhibitory CoA binding site, which may be occupied at intracellular concentrations of CoA.

Acyl Coenzyme A↗
PubMed1980

Proton magnetic resonance study of p-mercuribenzoate binding and structural changes in methemoglobin.

Interaction of human adult methemoglobin with p-mercuribenzoate (pMB) was examined at 21 degrees C by monitoring the hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of several high- and low-spin derivatives. The NMR spectra show that the heme methyl proton resonances from the beta subunits in methemoglobin were selectively affected by the binding of pMB regardless of whether the heme iron was saturated with high-spin or low-spin ligand. This observation suggests that the binding of pMB to methemoglobin induces a localized tertiary structural change around the beta heme, leaving the alpha heme unaffected. The structural change of the beta subunit was correlated with an increase in the high-spin character of the beta heme iron. A model study of the azide-methemoglobin complex suggested that the increase of the high-spin character of the beta heme iron is due to a conformational change of the proximal histidine which weakens the interaction between the heme iron and the proximal base. A similar and more pronounced spectral change due to binding of pMB was observed for the isolated beta subunit. The NMR spectral change in the isolated beta subunit also suggests that the binding of pMB to methemoglobin induces a localized conformational change within the beta subunit.

Adult↗
PubMed1989

Kinetics of p-mercuribenzoate binding to sulfhydryl groups on the isolated cytoplasmic fragment of band 3 protein. Effect of hemoglobin binding on the conformation.

Hemoglobin binds to the cytoplasmic domain of band 3 protein (CDB3) at physiologic pH and ionic strength in an oxygen-linked fashion, with deoxyhemoglobin having the higher affinity. The evidence in the literature suggests functional communication between the hemoglobin-binding site on CDB3 and the anion transport sites within the membrane-bound domain of band 3. Since the hemoglobin-binding site is estimated to be over 200 A from the transport domain, the functional communication hypothesis would require the existence of long-range, global changes in the CDB3 dimeric quaternary structure consequent to hemoglobin binding. In this report sulfhydryl reactivity toward p-mercuribenzoate is studied in an attempt to identify such long-range conformational changes. Formation of stoichiometric hemoglobin/CDB3 complexes is shown to produce major changes in sulfhydryl reactivity. Since the sulfhydryl pocket of CDB3 is known to lie at the dimeric interface over 100 A from the hemoglobin-binding site, the observed changes in reactivity suggest that hemoglobin complexation induces a global change in quaternary structure of the CDB3 dimer. This change offers a mechanism to explain functional connections between CDB3-binding sites and the anion transport sites on band 3. The existence of such long-range conformational changes would imply that the CDB3 dimer is poised to function as a cytosolic arm or lever in order to modulate the global structure of the porter.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1990

Effect of p-mercuribenzoate on the subestimation of angiotensin-converting enzyme measurement during chick retina development.

The time course of dipeptidase activity and the effect of p-mercuribenzoate (PCMB) on the subestimation of the fluorometric determination of angiotensin-converting enzyme (ACE, EC 3.4.15.1) during development was studied. ACE and dipeptidase activities were measured fluorometrically in homogenates of the developing chick retina using Hip-His-Leu and His-Leu as substrates, respectively, both either in the presence or in the absence of 1 mM PCMB. ACE activity was inhibited by captopril (IC50 1.7 nM), MK 422 (IC50 4.8 nM), BPP9a (IC50 0.25 microM) and BPP5a (IC50 1.2 microM), thus suggesting that avian retinal ACE catalytically resembles the mammalian enzyme. Dipeptidase activity varied 3.4-fold throughout development, leading to a large and variable (28-83%) subestimation of ACE activity during chick retina ontogenesis. PCMB (1 mM) inhibited 67-94% dipeptidase activity during development, thus greatly reducing any subestimation of ACE activity determination during the development of the chick retina.

Animals↗
PubMed1982

Myotonia in the rat diaphragm preparation caused by the sulfhydryl inhibiting para-substituted mercuribenzoates.

The sulfhydryl (SH) inhibiting para-substituted mercuribenzoates like pOHMB caused a myotonia which appeared as a smooth myotonic profile during recording of the response to twitch stimulation (0.1/s) of isolated rat diaphragm preparations. The maximum myotonic tension varied from about 10% to 200% of the twitch tension at pOHMB addition, and the myotonic repetitive action potential activity varied from a few to more than one hundred action potentials in different cells of the same preparation. The myotonia did not appear after pretreatment with the SH-reducing agent dithiothreitol, and both dithiothreitol and N-ethyl-maleimide inhibited the motonia. The myotonia increased with temperature, appearing at about 32 degrees C. Increased twitch frequency and tetanic stimulation decreased the myotonia. No change of threshold was observed in myotonic preparations. The myotonia was depressed in K+-free solution, and it was blocked by K+ concentrations exceeding 1.5 X normal. The myotonia was reduced when the NaCl was replaced by sucrose or choline chloride. In Ca2+-free solution the time to maximal myotonic tension and the variability of the maximal myotonia were reduced. CA2+ concentrations above normal inhibited the myotonia. No myotonia was observed in the slow twitch soleus muscle. pOHMB also caused a twitch depression during indirect and direct stimulation. The depression was observed in soleus muscle and in the diaphragm below 30 degrees C. The depression was thus independent of the myotonia.

Animals↗
PubMed1978

Increase in the susceptibility of hemoglobin to proteases upon treatment with p-mercuribenzoate.

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.

Caseins↗