The sulphatase of ox liver. XXV. Sulphatase A as a hysteretic enzyme

The kinetic behaviour of the system native--substrate-modified sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) has been investigated and it has been shown that the progress curve of the complete reaction, including both the inactivation and reactivation stages, can be treated as that of a simple hysteretic system in which the substrate-modified enzyme is activated by a product of the reaction. It has been concluded that the early suggestions that the modification of sulphatase A was accompanied by the exposure of a second ligand-binding site are incorrect. It has been shown that, in the absence of sulphate, the rate of reversion of substrate-modified to native sulphatase A is increased by 4-nitrocatechol but not by the same concentration of 2-nitrophenol. A detailed reaction sequence is proposed. This explains the kinetic behaviour of sulphatase A with nitrocatechol sulphate or 2-nitrophenyl sulphate as substrate and in the presence or absence of sulphate.     

The effect of alkaloids on sugar receptors and the feeding behaviour of the blowfly

ABSTRACT. The deterrent effect of alkaloids on feeding by the black blowfly Phormia regina Meigen (Diptera: Calliphoridae) was tested by determining tarsal thresholds for mixtures of sucrose and alkaloids. The following alkaloids were used: atropine sulphate, berberine sulphate, quinine mono-hydrochloride, caffeine, yohimbine sulphonic ester, pilocarpine hydrochloride, coniine hydrochloride and codeine. The same alkaloids were tested electrophysiologically on tarsal chemoreceptors (D hairs). Both behaviourally and electrophysiologically alkaloids reduced response to sucrose. Deterrence and peripheral inhibition could be blocked by increasing the concentration of sucrose. Application of kinetic analyses to the electrophysiological data ruled out competitive, non-competitive, and uncompetitive inhibition at receptor sites. There is no correlation of thresholds with available data on lipid solubility or octanol/water partition coefficients. The diverse pharmacological properties of alkaloids suggest that there is no uniform limiting mechanism for this multiform array of compounds.     

Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport

Proton/sulphate co-transport in the plasma membrane of root cells is the first step for the uptake of sulphate from the environment by plants. Further intracellular, cell-to-cell and long-distance transport must fulfil the requirements for sulphate assimilation and source/sink demands within the plant. A gene family of sulphate transporters, which may be subdivided into five groups, has been identified with examples from many different plant species. For at least two groups, proton/sulphate co-transport activity has been confirmed. It appears that each group represents sulphate transporters with distinct kinetic properties, patterns of expression, and cell/tissue specificity related to specific roles in the uptake and allocation of sulphate. High-affinity sulphate uptake and low-affinity vascular transport, as well as vacuolar efflux, are controlled by the nutritional status of the plant. Most notably there is an apparent increase in capacity for cellular sulphate uptake and vacuolar efflux when sulphur supply is limiting. Within the groups, the individual sulphate transporters may be further subdivided by differences in temporal, cellular and tissue expression. Many of the transporters are regulated by the nutritional status of the individual tissues, to optimize sulphate movement within and between sink and source organs.     

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.     

A study on the kinetic mechanism of apoenzyme reconstitution from Aerococcus viridans lactate oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k3 and k-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

Validation of the catalytic mechanism of Escherichia coli purine nucleoside phosphorylase by structural and kinetic studies

The catalytic mechanism of Escherichia coli purine nucleoside phosphorylase (PNP) is revised using site-directed mutagenesis, kinetic studies and structure determinations.The experimental evidence on the role of the particular catalytic amino acid during catalysis has not been available. Therefore, the active site mutants Arg24Ala, Asp204Ala, Asp204Asn, Arg217Ala and Asp204Ala/Arg217Ala were prepared and their kinetics and thermodynamic studies were carried out. The activity tests with natural substrates and 7-methylguanosine confirmed the earlier hypothesis, that catalysis involves protonation of the purine base at position N7 by Asp204, which is triggered by Arg217.The crystal structures of the wild type in complexes with phosphate and sulphate, respectively, and of the Arg24Ala mutant in complex with phosphate/sulphate were determined. The structural data show that previously observed conformational change is a result of the phosphate binding and its interaction with Arg24.As E. coli PNP is a promising candidate for the tumour-directed gene therapy, our results may also help to design efficient mutants useful in gene therapy.     

Comparative study of solubilized and membrane-bound acetylcholinesterase of sarcolemma]

Comparative kinetic studies of membrane-bound and solubilized sarcolemmal acetylcholinesterase reveal some difference in concentration-activity curves. A deviation from normal Michaelis-Menten kinetics is found in case of membrane-bound acetylcholinesterase. The solubilization of sarcolemma by a solution of high ionic strength or by sonication normalizes the reaction kinetics. It is shown that the amount of SH-groups in intact sarcolemma available for DTNB in the presence of sodium dodecyl sulphate, is about twice of that in the absence of sodium dodecyl sulphate. In case of the solubilized fraction of sarcolemma (by a solution of high ionic strength or by sonication), the amount of SH-groups available for DTNB in the presence and in the absence of sodium dodecyl sulphate is similar.     

Partial purification and properties of mevalonate kinase from neonatal chick liver

Mevalonate kinase from neonatal chick liver has been partially purified by ammonium sulphate precipitation and Sephadex G100 and DEAE-cellulose fractionation. The kinetic characteristics agreed with the sequential mechanism suggested for the enzyme and provided apparent Km values of 0.01 mM for mevalonic acid and 0.25 mM for ATP. Partially purified mevalonate kinase from neonatal chick liver showed an absolute specificity for ATP. Mn2+ was a better activator than Mg2+ at low concentrations (0.1-1.0 mM). Higher Mn2+ concentrations produced a clear inhibition of mevalonate kinase. Likewise, addition of EDTA, with or without metal ions, clearly inhibited the enzymatic reaction.     

The determination of kinetic parameters for carrier-mediated transport of non-labelled substrate analogues: a general method applied to the study of divalent anion transport in placental membrane vesicles

A method is described that allows the determination of kinetic parameters for carrier-mediated transport of unlabelled compounds. The ability of these compounds to relieve the inhibition of labelled substrate efflux produced by addition of an external competitive inhibitor is studied. The method is of general applicability and does not depend upon any intrinsic asymmetry in the ratio of the rates of translocation of the loaded and unloaded carrier. In this paper it is used in a study of the human placental sulphate transporter. Experiments on brush-border membrane vesicles show the method to predict quantitatively kinetic parameters for unlabelled sulphate entry. Further analysis shows this carrier to have a broad specificity for other oxyanions (selenate, tung-state, molybdate and chromate) with the following selectivity sequence: for rate of translocation, SO4, SeO4 greater than WO4 greater than MoO4 much greater than CrO4; for binding affinity, CrO4 greater than MoO4, WO4 greater than SO4, SeO4.     

Anaerobic treatment of sulphate-containing waste streams

Sulphate-containing wastewaters from the paper and board industry, molasses-based fermentation industries and edible oil refineries present difficulties during anaerobic treatment, leading to problems of toxicity, reduction in methane yield, odour and corrosion. The microbiology and biochemistry of dissimilatory sulphate reduction are reviewed in order to illustrate the potential competition between sulphate reducers and other anaerobes involved in the sequential anaerobic mineralisation process. The theoretical considerations which influence the outcome of competition between sulphate reducers and fermentative, syntrophic, homoacetogenic and methanogenic bacteria are discussed. The actual outcome, under the varying influent organic composition and strength and sulfate concentrations which prevail during digestion of industrial wastewaters, may be quite different to that predicted by thermodynamic or kinetic considerations. The factors governing competitive interactions between SRB and other anaerobes involved in methanogenesis is discussed in the context of literature data on sulphate wastewater treatment and with particular reference to laboratory and full-scale digestion of citric acid production wastewater.     

A Study on the Kinetic Mechanism of Apoenzyme Reconstitution from Aerococcus viridans Lactate Oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k₃ and k_-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

Properties of steroid sulphatase and arylsulphatase activities of human placenta

1. The properties of enzyme activities hydrolysing the sulphate esters of dehydroepiandrosterone, oestrone and p-nitrophenol are reported. The preparation studied was obtained from the microsomal fraction of human placenta by ultrasonic treatment and addition of Triton X-100. 2. The behaviour of the preparation during sedimentation at 105000g and attempts at purification indicated that the activities were particulate. Electron microscopy demonstrated the rupture of vesicular structures approx. 0.5mu in diameter concurrent with the release of activity. 3. The three activities were always associated throughout repeated attempts at separation by sucrose-density-gradient centrifugation and Sephadex-gel filtration. On the basis of kinetic studies, stability studies and treatment with butanol and ribonuclease it was concluded that a separate enzyme is responsible for each of the three activities. Widely varying plots of activity as a function of pH were consistent with this conclusion. 4. On the basis of sensitivity of the enzymes hydrolysing dehydroepiandrosterone sulphate and oestrone sulphate to ribonuclease and sensitivity of all three enzymes to lipase, it was concluded that the three enzymes are bound to a particle containing lipid and RNA. Enzymic activity is dependent on structural integrity of the particle. 5. A spectrophotometric method for the assay of oestrone sulphate hydrolysis is described. 6. Hydrolysis of nitrocatechol sulphate by human placenta under conditions described for arylsulphatases A and B is reported.     

Purification and properties of arylsulphatase from the brain of the silkworm Bombyx mori

(1) Arylsulphatase of the silkworm Bombyx mori was partially purified using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and Con-A Sepharose chromatography. (2) The purified enzyme preparation was not homogeneous but showed no beta-glucuronidase or beta-galactosidase activities. (3) The kinetic properties of the enzyme indicated that it could be classified under type-2 arylsulphatases of vertebrates. (4) The purified enzyme shows very little activity towards p-nitrophenyl sulphate and none towards cerebroside 3-sulphate.     

Binding of substrates and other anions to yeast phosphoglycerate kinase.

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.     

The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

A study of sulphate transport by lactating rat mammary tissue slices: evidence for anion exchange

1. The efflux of radiolabelled sulphate from lactating rat mammary tissue slices has been studied. Sulphate efflux was found to be time- and temperature-dependent. 2. 4,4'-Diisothiocyanostilbene-2,2'-disulphonate (DIDS) inhibited a portion of sulphate release, whereas bumetanide was without effect. 3. The anions chloride, iodide and sulphate trans-stimulated sulphate efflux when added to the incubation medium. The increase in the efflux rate of sulphate found with chloride could be markedly inhibited by DIDS. Thiocyanate, unlike the other anions tested, only had a small effect. 4. The results strongly suggest that there is an anion exchange mechanism in the mammary gland which can mediate the transport of sulphate. This transporter may be important for the metabolism of sulphate by the mammary gland and may also help determine milk anion concentrations.     

Kinetic study of ferrous sulphate oxidation of Acidithiobacillus ferrooxidans in the presence of heavy metal ions

Acidophilic microorganisms such as Acidithiobacillus ferrooxidans have the capability to carry out processes of bioleaching, biosorption and bioprecipitation of heavy metal ions, which have important environmental applications. At. ferrooxidans derives the energy for their metabolism from ferrous iron oxidation, process, which can be affected by the presence of heavy metals in the medium. Moreover, organic matter produces an inhibitory effect over the ferrous iron oxidation of At. ferrooxidans. In this work, heterotrophic bacterium Acidiphilium sp. was added when the medium is supplemented with organic matter to reduce this negative effect. The purpose of this work is the kinetic study of ferrous sulphate oxidation by At. ferrooxidans in the presence of different concentrations of several heavy metal ions (Cr(III), Cu(II), Cd(II), Zn(II) and Ni(II)) and compare this kinetic behaviour with a mixed culture with Acidiphilium sp.The obtained results show a non-competitive inhibition of heavy metals over bacterial oxidation of ferrous sulphate. In accordance with this kind of inhibition, a kinetic equation has been proposed to predict the behaviour of At. ferrooxidans in the presence of heavy metals in the range of concentrations studied.     

Method for isolation of aminoacyl-tRNA synthetases from plants: purification and some properties of methionyl,phenylalanyl and arginyl tRNA synthetases from yellow lupin seeds

A method for the simultaneous purification of methionyl-, phenylalanyl- and arginyl-tRNA synthetases from yellow lupin seeds (Lupinus luteus) is described. The method uses ammonium sulphate fractionation, and DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. Molecular weight and kinetic parameters of the pure enzymes are reported.     

Purification and properties of aspartate transcarbamylase from Mycobacterium smegmatis

Aspartate transcarbamylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) has been purified from Mycobacterium smegmatis TMC 1546 using streptomycin sulphate precipitation, ammonium sulphate precipitation, DE-52 chromatography, second ammonium sulphate precipitation, Sephadex G-200 gel filtration, and aspartate-linked CNBr-activated Sepharose 4B affinity chromatography in successive order. The enzyme was purified 231.6-fold, and the preparation was found to be homogeneous on column chromatography and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 246,000 and was composed of two asymmetrical subunits. The kinetic and regulatory properties of aspartate transcarbamylase from M. smegmatis were also studied. The enzyme was found to be an allosteric in nature with carbamyl phosphate showing positive cooperativity and UMP exhibiting a negative cooperativity. CTP was found to be the most potent inhibitor among nucleotides. Phosphate acted as a non-competitive product inhibitor with respect to aspartate. Succinate and maleate exerted a competitive inhibition when aspartate was the variable substrate.