Binding of nitric oxide to thiols and hemes in hemoglobin H: implications for alpha-thalassemia and hypertension.

Our earlier studies suggested an association between alpha-thalassemia and hypertension. We postulated that this association might involve trapping of the vasodilator nitric oxide (NO) by hemoglobin (Hb). Hb A has recently been shown to carry NO on its sulfhydryl groups in addition to its hemes. In this report we studied the interaction of purified Hb H as well as Hb A with NO. The number of reactive sulfhydryls were determined spectrophotometrically with bis-dithionitrobenzoate. Spectral studies and nitrosothiol measurements after treatment with NO or nitrosothiols indicated that all eight reactive sulfhydryls of Hb H were capable of binding NO. Hb A, however, was only able to bind and transfer two molecules of NO per tetramer. These findings support the biochemical basis for the association between alpha-thalassemia and hypertension.

Mutational analysis and chemical modification of Cys24 of lactococcin B, a bacteriocin produced by Lactococcus lactis.

Using site-directed mutagenesis the single cysteine residue at position 24 of lactococcin B was replaced by all other possible amino acids. Most of these mutant molecules retained bacteriocin activity, with the exception of those in which cysteine was replaced by a positively charged amino acid. This would seem to be in agreement with the authors' earlier observation that treatment of the wild-type molecule with HgCl2 resulted in its inactivation. The factor that causes inactivation of lactococcin B seems to be the introduction of a positive charge at position 24 by HgCl2 rather than oxidation of this residue, as treatment of the bacteriocin with other oxidative chemicals did not interfere with the ability of lactococcin B to dissipate the membrane potential of sensitive cells. Results are also reported which imply that inactive lactococcin B can still bind to its receptor. It can be replaced by an active bacteriocin molecule, resulting in dissipation of the membrane potential.

Free fatty acids associated with the high molecular weight aminoacyl-tRNA synthetase complex influence its structure and function.

Aminoacyl-tRNA synthetases from higher eukaryotes often are isolated as high molecular weight complexes associated with other components such as lipids. Since hydrophobic interactions are involved in the organization of the complex, it has been suggested that interaction of synthetases with these lipids might be important for their structure and function. Delipidation is known to affect certain properties of synthetases within the complex including sensitivity to detergents plus salts, temperature inactivation, hydrophobicity, sensitivity to proteases, and, as shown here, sensitivity to p-mercuribenzoate and sites of papain cleavage. Of the lipids known to co-purify with the complex, cholesterol esters, phospholipids and free fatty acids, we show that the particular lipids responsible for many of these changes are the free fatty acids. Specific removal of fatty acids results in a complex with properties similar to one totally delipidated by detergent treatment, and readdition of the fatty acid fraction reverses the effects. The fatty acid fraction contains both saturated and unsaturated fatty acids, but unsaturated fatty acids are much more effective in reversing the properties of the delipidated complex that are saturated fatty acids. These results indicate that the free fatty acids co-purifying with the synthetase complex bind to the synthetases and affect their structure and function.

Topographical localization and characterization of microsomal glucose-6-phosphatase binding sites accessible to 4,4'-diazidostilbene 2,2'-disulfonic acid.

The effect of the photoactivated reagent 4,4'-diazidostilbene 2,2'-disulfonic acid (DASS) on rat liver microsomal glucose-6-phosphatase has been investigated in order to analyze the accessibility and the chemical nature of functional sites of the integral enzyme protein. The following results were obtained. (i) When native rat liver microsomes are irradiated with the photoactive reagent, the activity of glucose-6-phosphatase is progressively inhibited. However, complete reactivation is obtained by modification of the DASS-labeled microsomes with Triton X-114. (ii) Inhibition of glucose-6-phosphatase is also reversed when the DASS-labeled microsomes are treated with p-mercuribenzoate or dithiothreitol. (iii) When native microsomes are labeled with DASS an intensely fluorescent adduct is formed whose emission and excitation maximum corresponds with those obtained when cysteine or 3-mercaptopropionic acid are irradiated in the presence of the photolabile reagent. (iv) The data from fluorescence measurements show that p-mercuribenzoate and dithiothreitol reduce fluorescence labeling of the microsomes whereas Triton modification of the DASS-labeled membranes does not affect the DASS-induced fluorescence. (v) Glucose 6-phosphate hydrolysis of the partially purified glucose-6-phosphatase is also inhibited as observed with native microsomes. The DASS-induced inhibition is reversed and prevented by p-mercuribenzoate; however, the partially purified enzyme cannot be reactivated by Triton X-114. (vi) When glucose-6-phosphatase is partially purified from the DASS-labeled microsomes this enzyme preparation is fluorescence labeled and inhibited. From these results we conclude that DASS directly reacts with the integral phosphohydrolase mainly by chemical modification of essential sulfhydryl groups of the enzyme protein accessible from the cytoplasmic surface of the native microsomal membrane. The Triton-induced reactivation of the glucose-6-phosphatase of DASS-labeled microsomes is explained in terms of conformational changes of the integral protein elicited during modification of the surrounding membrane by detergent.

Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site.

Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl2. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by [3H] N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.

The effects of p-mercuribenzenesulfonate on purified spectrin and actin.

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.

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.

On the nature of the interaction between 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid and microsomal glucose-6-phosphatase. Evidence for the involvement of sulfhydryl groups of the phosphohydrolase.

The effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) on microsomal glucose 6-phosphate hydrolysis has been reinvestigated and characterized in order to elucidate the topological and functional properties of the interacting sites of the glucose-6-phosphatase. The studies were performed on microsomal membranes, partially purified and reconstituted glucose-6-phosphatase preparations and show the following. (a) DIDS inhibits activity of the glucose-6-phosphatase of native microsomes as well as the partially purified glucose-6-phosphatase. (b) Inhibition is reversed when the microsomes and the partially purified phosphohydrolase, incorporated into asolectin liposomes, are modified with Triton X-114. (c) Treatment of native microsomes with DIDS and the following purification of glucose-6-phosphatase from these labeled membranes leads to an enzyme preparation which is labeled and inhibited by DIDS. (d) Preincubation of native microsomes or partially purified glucose-6-phosphatase with a 3000-fold excess of glucose 6-phosphate cannot prevent the DIDS-induced inhibition. (e) Inhibition of glucose-6-phosphatase by DIDS is completely prevented when reactive sulfhydryl groups of the phosphohydrolase are blocked by p-mecuribenzoate. (f) Reactivation of enzyme activity is obtained when DIDS-labeled microsomes are incubated with 2-mercaptoethanol or dithiothreitol. Therefore, we conclude that inhibition of microsomal glucose 6-phosphate hydrolysis by DIDS cannot result from binding of this agent to a putative glucose-6-phosphate-carrier protein. Our results rather suggest that inhibition is caused by chemical modification of sulfhydryl groups of the integral phosphohydrolase accessible to DIDS attack itself. An easy interpretation of these results can be obtained on the basis of a modified conformational model representing the glucose-6-phosphatase as an integral channel-protein located within the hydrophobic interior of the microsomal membrane [Schulze et al. (1986) J. Biol. Chem. 261, 16,571-16,578].

Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase.

Plasma-membrane vesicles from rat corpus luteum showed an ATP-dependent uptake of Ca2+. Ca2+ was accumulated with a K1/2 (concn. giving half-maximal activity) of 0.2 microM and was released by the bivalent-cation ionophore A23187. A Ca2+-dependent phosphorylated intermediate (Mr 100,000) was detected which showed a low decomposition rate, consistent with it being the phosphorylated intermediate of the transport ATPase responsible for Ca2+ uptake. The Ca2+ uptake and the phosphorylated intermediate (E approximately P) displayed several properties that were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes. Both Ca2+ uptake and E approximately P discriminated against ribonucleoside triphosphates other than ATP, whereas the ATPase split all the ribonucleoside triphosphates equally. Both Ca2+ uptake and E approximately P were sensitive to three different Hg-containing inhibitors, whereas the ATPase was inhibited much less. Ca2+ uptake required added Mg2+ (Km = 2.2 mM), whereas the ATPase required no added Mg2+. The maximum rate of Ca2+ uptake was about 400-fold less than that of ATP splitting; under different conditions, the decomposition rate of E approximately P was 1,000 times too slow to account for the ATPase activity observed. All of these features suggested that Ca2+ uptake was due to an enzyme of low activity, whose ATPase activity was not detected in the presence of the higher-specific-activity Ca2+-dependent ATPase.

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.

In vitro studies of the mechanism of inhibition of rat liver uroporphyrinogen decarboxylase activity by ferrous iron under anaerobic conditions.

Human porphyria cutanea tarda (PCT) is an unusual consequence of common hepatic disorders such as alcoholic liver disease and iron overload, where hepatic iron plays a key role in the expression of the metabolic lesion, i.e., defective hepatic decarboxylation of porphyrinogens. In this investigation, kinetic studies on a partially purified rat liver uroporphyrinogen decarboxylase have been conducted under controlled conditions to determine how iron perturbs porphyrinogen decarboxylation in vitro. The enzyme, assayed strictly under anaerobic conditions in the dark, was inhibited progressively by ferrous iron. Approximately 0.45 mM ferrous ammonium sulfate was required to observe about 50% inhibition of enzyme activity measured with uroporphyrinogen I as substrate. We showed that (a) all the steps of enzymatic decarboxylation (octa-, hepta-, hexa-, and pentacarboxylic porphyrinogen of isomer I series) were inhibited by ferrous iron. The inhibition was competitive with respect to uroporphyrinogen I and III substrates; (b) the cations, e.g., Fe3+ and Mg2+, had no effect, whereas sulfhydryl group specific cations and compounds such as Hg2+, Zn2+, p-mercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoate) all inhibited the enzyme; (c) the enzyme could be protected from inhibition by Fe2+ and p-mercuribenzoate by preincubation with pentacarboxylic porphyrinogen, a natural substrate and competitive inhibitor. These data suggest for the first time a direct interaction of ferrous iron with cysteinyl residue(s) located at the active site(s) of the enzyme.

Effect of sulfhydryl modification on the activities of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Alkylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with p-mercuribenzoate caused a rapid stimulation of the kinase and an inhibition of the bisphosphatase. At later times, the kinase activity also became inhibited. In contrast, treatment with N-ethylmaleimide abolished kinase activity but had no effect on the bisphosphatase. Selective modification of residues involved in the kinase reaction was also seen with iodoacetamide, which caused a 10-fold stimulation of the kinase Vmax without affecting the bisphosphatase. The stimulatory effect of carboxyamidomethylation was seen when the kinase was assayed in the presence of inorganic phosphate, an allosteric activator of the enzyme. The iodoacetamide-treated enzyme had a 10-20-fold higher Km for fructose 6-phosphate than the native enzyme and the Ki for fructose 2,6-bisphosphate was also increased. However, the adenine-nucleotide site did not seem to be affected since there was no change in the Km for ATP, the Ki for ADP, or the adenine-nucleotide exchange. There was also a direct correlation between the incorporation of [14C]acetamide into the enzyme and activation of the kinase. The residues modified by iodoacetamide were shown to be cysteines by the exclusive appearance of carboxymethylcysteine in protein hydrolysates. Activation was associated with alkylation of 2 cysteines/subunit, of the 12 which could be alkylated after denaturation/reduction. Iodoacetamide-activated kinase was inhibited by ascorbate/Fe3+, which has been shown to modify sulfhydryl groups in the native enzyme, with concomitant loss of kinase activity.

The action of organic mercurials on the erythrocyte membrane.

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.

Indirect inactivation of eukaryotic initiation factor 2 in reticulocyte lysate by selenite.

Addition of selenite to rabbit reticulocyte lysate produces a biphasic pattern of translational inhibition. Sucrose density gradient shows that the onset of translational inhibition is accompanied by decreased Met-tRNAf binding to 43 SN ribosomal subunits and loss of polysomes. Control rates of translation are restored by the addition of exogenous eukaryotic initiation factor 2 (eIF-2). Selenite also directly inhibits Met-tRNAf binding activity of eIF-2. While selenite could react directly with unpaired cysteine residues of eIF-2 to inhibit protein synthesis initiation, a more complex mechanism than a direct inactivation of eIF-2 is suggested by the following observations: 1) translational inhibition produced by selenite is accompanied by an apparent increase in the phosphorylation state of eIF-2alpha; and 2) the extent of translational inhibiton is not proportional to steady-state level of phosphorylation. Rather, the time required for the onset of translational inhibition decreases as the level of eIF-2alpha phosphorylation is increased. This suggests a multistep sequence for eIF-2 inactivation, dependent upon an initial activation of eIF-2alpha kinase and followed by additional eIF-2 modification(s).

The effect of N-(methoxy polyethylene glycol) rho-hydroxymercuribenzamide, a polymeric sulfhydryl group reagent, on spermatozoan motility.

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.

The interaction of L-ascorbic acid with the active center of myrosinase.

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.

The binding and catalytic activities of forms of ligandin after modification of its thiol groups.

Ligandin (glutathione S-transferase B, EC 2.5.1.18)was treated with p-mercuribenzoate, N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide, 5,5,-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetamide or iodoacetate. Although performic acid oxidation revealed the presence of four cysteines, p-mercuribenzoate and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, the most effective of the reagents studied, reacted with only three residues. N-Ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) each reacted with two cysteines: iodoacetamide reacted with only one cysteine and iodoacetate was essentially unreactive. Modification of three thiol groups decreased both the enzymic and binding activities of ligandin although the number of binding sites was unaffected. Modification of only one or two of the thiol groups had little effect on the ligandin activities. It therefore appears that there is a thiol group in the common hydrophobic-ligand- and substrate-binding site of ligandin. Ligandin was separated into two fractions on CM-cellulose. Both fractions gave the same results with p-mercuribenzoate and iodoacetamide.