Human gastric lipase: a sulfhydryl enzyme

One sulfhydryl group was modified per mol of native human gastric lipase after incubation at pH 8.0 with 5,5'-dithiobis(2-nitrobenzoic acid) for 18 h or with 4,4'-dithiopyridine for 100 min. With both reagents a direct correlation was found between the modification of one sulfhydryl group and the loss of human gastric lipase activity. Incubation of human gastric lipase with a new hydrophobic sulfhydryl reagent dodecyldithio-5-(2-nitrobenzoic acid) in 30-fold molar excess, at pH 3.0, 5.0, and 8.0, induced immediate and complete human gastric lipase inactivation. Unlike 5,5'-dithiobis(2-nitrobenzoic acid) and 4,4'-dithiopyridine, dodecyldithio-5-(2-nitrobenzoic acid) almost instantaneously stopped the course of tributyrin hydrolysis by human gastric lipase. Human gastric lipase can thus be said to be a sulfhydryl enzyme.     

New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent.

A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.     

Re-evaluation of the role of thiol groups in rabbit muscle aldolase A

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.     

An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution

Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in the absence of Fru(1,6)P(2) under acidic conditions through the homotropic activation effect of substrate pyruvate. In this enzyme, a single amino acid replacement, i.e. that of His205 conserved in the Fru(1,6)P(2)-binding site of certain allosteric L-LDHs of lactic acid bacteria with Thr, did not induce a marked loss of the activation effect of Fru(1,6)P(2) or divalent metal ions, which are potent activators that improve the activation function of Fru(1,6)P(2) under neutral conditions. However, this replacement induced a great loss of the Fru(1,6)P(2)-independent activation effect of pyruvate or pyruvate analogs under acidic conditions, consequently indicating an absolute Fru(1,6)P(2) requirement for the enzyme activity. The replacement also induced a significant reduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P(2), through a slight decrease and increase of the Fru(1,6)P(2) sensitivity under acidic and neutral conditions, respectively, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L-LDH to Fru(1,6)P(2). The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs.     

Functional consequences of modifying highly reactive arginyl residues of fructose 1,6-bisphosphatase. Loss of monovalent cation activation.

Modification of pig kidney fructose 1,6-bisphosphatase with 2,3-butanedione (in the presence of AMP) results in the loss of activation of the enzyme by monovalent cations. Under these conditions about 8 arginyl residues per mole of enzyme were modified. No other residues were modified. No loss of monovalent cation activation occurs when modification with 2,3-butanedione is carried out in the presence of AMP plus the substrate fructose 1,6-bisphosphate and 3.2 less arginyl residues were modified. Since fructose 1,6-bisphosphatase contains 4 subunits, it is suggested that one arginyl residue per subunit plays an essential role in monovalent cation activation of the enzyme. Studies on sulfhydryl group reactivity toward 5,5'-dithiobis(2-nitrobenzoic acid) explain the protection exerted by fructose 1,6-bisphosphate against the loss of monovalent cation activation in terms of an enzyme conformational change induced by substrate, which makes unreactive the essential arginyl residue. The results of the present paper, as well as previous evidence, are discussed in terms of the mechanism of monovalent cation activation of fructose 1,6-biphosphatase.     

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver: some physicochemical properties.

A number of physiochemical properties of the cyclic AMP-adenosine binding protein of mouse liver (Ueland, P.M. and D?skeland, S.O. (1977) J. Biol. Chem. 252, 677--686) have been studied. 1. The specific extinction coefficient, E1%280nm, was estimated to 13.0. 2. Amino acid and amide group analyses confirmed the acidic properties of the protein as determined by electrofocusing (pI = 5.7). Based on the estimated partial specific volume (v = 0.74 cm3/g) the minimum molecular weight of the native, tetrameric protein was recalculated to be 185 000 (s20,w = 8.8 . 10(-13) s and Stokes radius = 48 A). 3. No NH2-terminal amino acid was found by the dansyl method using [14C]-dansyl chloride, indicating that the NH2-terminal groups are blocked. 4. Amino acid analyses gave 6 half-cystine residues per subunit, and the same number of free sulfhydryl groups was found by titration of the denatured protein with 5,5'-dithiobis (2-nitrobenzoic acid). 5. The reactivity of the SH groups in the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) revealed rapidly reacting (SHI), sluggishly reacting (SHII) and "masked" (SHIII) SH groups. ATP, adenosine, Mg2+ and KCl, factors known to affect the activation of cyclic AMP binding sites (Ueland, P.M. and D?skeland, S.O. (1978) Arch. Biochem. Biophys., in press) changed the reactivity of separate SH groups.     

Unusual amino acid substitution in the anion-binding site of Lactobacillus plantarum non-allosteric L-lactate dehydrogenase.

In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L-LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G-->C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65 degrees C but more stable above 70 degrees C.     

Purification of the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus uberis and an investigation of its existence in different forms.

The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.     

Effect of oxidizing agents and sulfhydryl group reagents on beta toxin from Clostridium perfringens type C

Purified beta toxin from Clostridium perfringens type C was inactivated by the oxidizing agents o-iodosobenzoate (OIBA), oxidized glutathione, and ferricyanide, and by the sulfhydryl group regents 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, iodoacetamide, and iodoacetic acid, causing loss of activity in various degrees depending on the concentration used. The activity of the toxin was not influenced by exposure to 1.0 mM of p-chloromercuribenzoate. The toxin treated by OIBA or DTNB was reactivated by incubation with 2-mercaptoethanol and dithiothreitol. The data suggest that beta toxin contains thiol groups which are essential for the activity.     

Biochemical studies of taste sensation--VIII. Partial characterization of alanine-binding taste receptor sites of catfish Ictalurus punctatus using mercurials, sulfhydryl reagents, trypsin and phospholipase C.

1. Taste receptors for L-alanine in the channel catfish Ictalurus punctatus have been partially characterized. The binding activity, which is localized to a sedimentable fraction (Fraction P2), was assayed with L-[3H]alanine as the ligand. 2. Addition of HgCl2 or p-mercuribenzoate to the assay at 0.1-1 mM markedly inhibited binding. The effect was not reversible and was unaffected by increased L-alanine in the binding assay. 3. The sulfhydryl reagents iodoacetate, 5,5'-dithiobis(2-nitrobenzoic acid), arsenite, and N-ethylmaleimide did not show appreciable inhibition of binding. The results suggest that the inhibitory effect of mercurials is not on specific sulfhydryl groups at alanine-binding sites. 4. Treatment of Fraction P2 with phospholipase C decreased binding activity and treatment with trypsin led to increased binding activity.     

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups.

Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).     

Formiminotransferase-cyclodeaminase from porcine liver. A sulfhydryl essential for the deaminase activity of the bifunctional enzyme

Reaction of the bifunctional enzyme formiminoglutamate:tetrahydrofolate formiminotransferase (EC 2.1.2.5) - formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4) with the sulfhydryl reagent 5,5'-dithiobis (2-nitrobenzoic acid) selectively inactivates the cyclodeaminase. Loss of activity correlates with the modification of two sulfhydryl groups per subunit. The inhibitor folic acid reduces the rates of inactivation and sulfhydryl modification, and protection experiments demonstrate that only one of the two sulfhydryls modified is important for enzyme activity. The results indicate the presence of a cyclodeaminase site on each polypeptide, assuming one sulfhydryl per site, in agreement with a quaternary structure containing identical polypeptides. Modification does not cause dissociation of the enzyme and is reversible with dithiothreitol.     

Studies on aspartase. II. Role of sulfhydryl groups in aspartase from Escherichia coli.

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.     

Essential sulfhydryl groups of rat liver monoamine oxidase.

The inhibition by some thiol reagents of partly purified mitochondrial monoamine oxidase (MAO) (EC 1.4.3.4) from rat liver was studied, and the molar content of sulfhydryl groups in the enzyme determined. Sodium nitroprusside and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) inhibited the enzyme, apparently reversibly, while sodium arsenite was not inhibitory. Concentrations of the respective inhibitors causing 50% inhibition after 15 min of preincubation with the enzyme at pH 7.0 and 37 degrees C are 5.80 times 10(-4) M and 4.35 times 10(-5) M. The thiol compounds cysteine, dithiothreitol, and 2-mercaptoethanol did not inhibit MAO. The average number of sulfhydryl groups per mole of enzyme, determined by reaction with DTNB, increased from 3.6 +/- 0.2 freely reacting sulfhydryl groups (n = 4) to 18.4 to total sulfhydryl groups (n = 2) on denaturation with 8 M urea.     

Spin-labelling of phosphorylase b using a paramagnetic 1-fluoro-2,4-dinitrobenzene derivative

Phosphorylase b (1,4-alpha-D-glucan:1,6-alpha-D-glucan 6-alpha-glucosyltransferase, EC 2.4.1.1) can be specifically spin-labelled at a site essential for the catalytic action of the enzyme. A paramagnetic analogue of 1-fluoro-2,4-dinitrobenzene was synthesized and used as a dinitrophenylating agent. Reaction of phosphorylase b with the paramagnetic probe combined with the thiolysis method, leads to spin-labelling of a single -NH2 group (0.75 groups per subunit) with concomitant loss of 50% of the catalytic activity. Dinitrophenylation does not change the sedimentation profile of the enzyme. The ESR spectrum of modified phosphorylase b indicates that the attached label has rather limited segmental mobility and its environment is slightly hydrophobic. Small but subtle conformational changes induced by ligands in this critical site of the macromolecule can be directly detected by the spin-label. Also, sulfhydryl group modification of the spin-labelled enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) has a pronounced effect on the resonance spectrum.     

Chemical modifications of the crystalline quinolinate phosphoribosyltransferase from hog liver.

Amino acid analysis and chemical modification of the crystalline quinolinate phosphoribosyltransferase (EC 2.4.2.19) from hog liver were performed. The enzyme contained 29 residues of half cystine per mol. The enzyme activity was strongly inhibited by sulfhydryl reagents. The number of reactive (exposed) sulfhydryl group was determined to be 10.2 and total sulfhydryl group was to be 25.2 per mol by using 5,5'-dithiobis(2-nitrobenzoic acid). The enzyme activity was also inhibited by lysine residue-, histidine residue-, and arginine residue-modifying reagents. These results and the effect of preincubation with the substrates on chemical modifications suggest that the lysine residue, histidine residue and sulfhydryl group may be closely related to the binding site of quinolinic acid.     

Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.

The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).     

Inactivation of Salmonella phosphoribosylpyrophosphate synthetase by oxidation of a specific sulfhydryl group with potassium permanganate.

Phosphoribosylpyrophosphate synthetase from Salmonella typhimurium contains four cysteine residues per subunit. Three of these react readily with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB), forming an active derivative with kinetic and physical properties similar to the native enzyme, but one reacts only under denaturing conditions. Stoichiometric amounts of KMnO4 inactivate the DTNB-treated enzyme. The loss of activity is correlated with the oxidation of the remaining cysteinyl group to cysteic acid by KMnO4. Amino acid analysis indicates that no other residues are altered. The rate of inactivation of the enzyme is decreased 30-fold by saturatin g concentrations of the substrate ATP. Inorganic phosphate also protects substantially against KMnO4. Titration of the native enzyme with limiting amounts of KMnO4 shows that the sulfhydryl group essential for activity competes effectively with the other sulfhydryl groups for KMnO4. These results suggest that the essential sulfhydryl group is near the active site, and that KMnO4, a phosphate analogue, can act as an active site-directed reagent at the ATP binding site of the enzyme. The KMnO4-oxidized enzyme is more highly aggregated than untreated enzyme and fails to bind ATP appreciably.