Inhibition of membrane transport ATPases by halenaquinol,a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata

Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na⁺, K⁺-ATPases and the rabbit muscle sarcoplasmic reticulum Ca²⁺-ATPase with I₅₀ values of 7.0×10⁻⁷, 1.3×10⁻⁶ and 2.5×10⁻⁶ M, respectively. Halenaquinol also inhibited K⁺-phosphatase activity of the rat brain cortex Na⁺, K⁺-ATPase with an I₅₀ value of 3×10⁻⁶ M. Ouabain-insensitive Mg²⁺-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect.     

In vitro transformation of dehydroepiandrosterone or its sulphate into androstenediol or its sulphate by rat brain and blood preparations

Abstract— –Enzymic transformation of [4-¹⁴C]dehydroepiandrosterone or [4-¹⁴C]dehydro-epiandrosterone sulphate to androstenediol or its sulphate occurred when incubated with a microsomal preparation of rat brain or a whole rat blood homogenate. The brain enzyme which appeared to cause this transformation had a pH optimum at 60, was NADPH₂-dependent, and had an apparent K_m of 4·6 × 10^?6m . When the subcellular fractions of rat brain were compared for transformation, microsomes had the highest specific activity, followed by the cytosol. The crude nuclear and mitochondrial fractions had no significant activity. The level of enzymic activity in the brain microsomes increased from that for rats sacrificed at 7 days of postnatal age to a maximum for rats sacrificed at 1 month of age; then the activity appeared to level off in rats older than 1 month. Microsomes obtained from the cerebellum had the highest specific activity in comparison to that obtained from the cerebral cortex, the diencephalon, and the brain stem. The incubated preparations of rat brain also converted dehydroepiandrosterone sulphate to androstenediol sulphate without hydrolysis. The enzyme in rat blood which was similar to that in the brain was also partially characterized. The blood enzyme had a pH optimum at 6–5, was nearly exclusively present in erythrocytes, was also NADPH₂-dependent, and had an apparent K_m of 2·7 × 10^?4m . The developmental pattern of the blood enzyme specific activity was similar to that of the rat brain enzyme. Upon haemolysis, most activity was recovered in the haemolysate.     

BIOGENIC AMINES AND THEIR METABOLITES AS SUBSTRATES FOR PHENOL SULPHOTRANSFERASE (EC 2.8.2.1) OF BRAIN AND LIVER

Phenol sulphotransferase activity in homogenates of rat liver and brain was determined spectrophotometrically. Rat liver had about 100-fold more phenol sulphotransferase activity than brain; however, both tissues showed about the same spectrum of activity towards the phenolic compounds tested. Dopamine and its acidic and neutral metabolites and the neutral metabolites of norepinephrine were the compounds most readily sulphury-lated in vitro. They were also the compounds most readily sulphurylated in vivo when they were injected intraventricularly together with labelled Na₂SO₄. When labelled Na₂SO₄ was injected alone, we detected conjugation of endogenous phenols. One of the compounds formed was identified by its chromatographic characteristics as 3-methoxy-4-hydroxy-phenylethyleneglycol sulphate. We detected other conjugates which appeared to be the sulphate esters of 3,4-dihydroxyphenylethyleneglycol; 3,4-dihydroxyphenylacetic acid; and homovanillic acid. In brain, sulphate conjugation may be a major route of metabolism for many of the phenolic compounds related to the biogenic amines and possibly for phenolic drugs which enter the brain.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

Apparent differences in tryptophan hydroxylation by cockroach (periplaneta americana) nervous tissue and rat brain

Tryptophan hydroxylation in cockroach (Periplaneta americana) nervous tissue was measured and compared to the hydroxylation of tryptophan in rat brain. Tryptophan hydroxylation in both tissues requires a pterine cofactor, and is inhibited by p-chlorophenylalanine. The molecular weight of the protein responsible for hydroxylation of tryptophan in cockroach nervous tissue obtained from gel filtration was estimated to be 54,000.The pH optima and enzyme kinetics differed greatly between the two hydroxylases. Hydroxylation of tryptophan by the enzyme obtained from cockroach tissues incubated with dimethyltetrahydropterine had a pH optimum of about 5.8–5.9 and a K_m in crude enzyme preparations of 2.6 × 10⁻⁶ M and is activity was substrate inhibited above 10⁻⁴ M tryptophan. Hydroxylation of tryptophan by the enzyme obtained from rat brain incubated with dimethyltetrahydropterine had a pH optimum of about 6.5–7.0, a K_m of about 6.7 × 10⁻⁴ M and exhibited no substrate inhibition at tryptophan concentrations up to 2 × 10⁻³ M.When incubated with biopterin, the presumed natural cofactor, the hydroxylase from cockroach tissues had a K_m of about 6.8 × 10⁻⁵ M and no substrate inhibition occurred at tryptophan concentrations up to 2 × 10⁻³ M. Under the same conditions rat hydroxylase had a K_m of 1.1 × 10⁻⁵M and substrate inhibition occurred above 10⁻⁴ M tryptophan.Unlike the mammalian situation, administration of tryptophan peripherally did not change the 5-hydroxytryptamine concentration in cockroach nervous tissue, but did increase tryptophan levels. The low V_max values of the cockroach hydroxylase and the inability of administered tryptophan to elevate 5-hydroxytryptamine levels suggest that in the cockroach hydroxylation of tryptophan itself may be the limiting factor in the biosynthesis of 5-hydroxytryptamine.     

Effect of stress on sulfated glycol, metabolites of brain norepinephrine.

The present study examined the effect of footshock stress on the formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihydroxyphenylglycol (DOPEG-SO₄). Rats receiving intraventricular injections of either ³HNE or Na₂³⁵SO₄ prior to 0.5 hour of footshock showed significant and comparable increases in both sulfated glycols labeled with ³H or ³⁵SO₄. Elevations were greatest in the hypothalumus using Na₂³⁵SO₄. In pheniprazine pretreated rats footshock did not increase the production of MOPEG-³⁵SO₄ from intraventricular labeled sulfate given alone or in combination with various doses of exogenous MOPEG. The results indicate that neuronally released brain NE is metabolized to form both MOPEG-SO₄ and DOPEG-SO₄. The increase in these metabolites results from an increased glycol production and not from a stress-induced activation of brain sulfation mechanisms.     

Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1. The metabolism of inorganic [³⁵S]sulphate (Na₂³⁵SO₄) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [³⁵S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high K_m of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.     

ATPases in rabbit erythrocytes: stimulation by HCO3-anion and by Na-cation-plus-K-cation.

A Mg²⁺Na⁺K⁺ATPase was found in a ghost preparation from rabbit erythrocytes, a finding in conflict with previous reports, but in agreement with the known kinetics of cation movements in these cells. However the Mg²⁺Na⁺K⁺ATPase was not inhibited by 10⁻⁴M ouabain, nor by 10⁻⁴M Ca²⁺. The physiological status of this enzyme is discussed. The basic Mg²⁺-ATPase activity in this preparation is also stimulated by HCO₃⁻; it is suggested that the HCO₃⁻-stimulated ATPases reported in a variety of other preparations are not necessarily due to mitochondrial contamination but could well originate from the plasma membrane.     

Determination and characterization of neuron specific protein (NSP) associated enolase activity.

Fractionation of the 40–80% (NH₄)₂SO₄ fraction of a soluble rat brain extract on DEAE cellulose resolves three species of enolase activity, two of which react with antiserum to neuron specific protein from rat (NSP-R) and one which does not react. Direct assay of pure neuron specific protein from rat, cat and human brain (NSP-R, NSP-C, NSP-H) as well as bovine brain 14-3-2, using 2 different assay systems demonstrate that all these preparations display enolase activity of between 40 and 70 units/mg. This activity is Mg⁺⁺ dependent and inhibited by fluorophosphate in all cases. Kinetic parameters such as Km for Mg⁺⁺, 2 PGA, and pH optimum were determined for the 2 different NSP preparations and also for bovine brain 14-3-2 protein.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

Oxygen-dependent desulphation of monomethyl sulphate by Agrobacterium sp. M3C

Agrobacterium sp. M3C, previously isolated from canal-water for its ability to grow on monomethyl sulphate, degraded this ester with stoichiometric liberation of inorganic sulphate. In contrast with the biodegradation of monomethyl sulphate in Hyphomicrobium sp., and of other longer-chain alkyl sulphates in Pseudomonas spp., the pathway in Agrobacterium appeared not to involve a sulphatase enzyme capable of catalysing ester-bond hydrolysis. No such sulphatase was detectable under a range of conditions of bacterial culture, or using various methods for preparing cell-extracts, or different assay conditions. There was no incorporation of ¹⁸O-label from H₂ ¹⁸O into the liberated inorganic sulphate. No methanol was detectable during biodegradation, and the organism was incapable of growth on methanol, and did not produce methanol dehydrogenase activity when grown on monomethyl sulphate. Tracer studies using mono[¹⁴C]-methyl sulphate indicated that formate serine and glycine were produced during the biodegradation. The presence of these amino acids, together with high activity of hydroxypyruvate reductase, indicated the operation of the serine pathway common in methylotrophs. Use of an oxygen electrode in conjunction with monomethyl[³⁵S]sulphate showed that release of ³⁵SO₄ ^2- was dependent on availability of O₂, and that there was equimolar stoichiometry among monomethyl sulphate degraded, O₂ consumed and ³⁵SO₄ ^2- released. A proposed pathway for the degradation involved an initial mono-oxygenation to methanediol monosulphate with subsequent elimination of SO₄ ^2- and concomitant formation of formaldehyde. The pathway was compared with degradation mechanisms for other C₁ compounds and for other sulphate esters.     

Two different modes of inhibition of the rat brain Na+,K+-ATPase by triterpene glycosides,psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na⁺,K⁺-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na⁺,K⁺-ATPase with I₅₀ values of 1×10⁻⁴ M and 3×10⁻⁴ M, respectively. PsA significantly stimulated [³H]ATP binding to Na⁺,K⁺-ATPase, weakly increased [³H]ouabain binding to the enzyme, and inhibited K⁺-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [³H]ATP binding to Na⁺,K⁺-ATPase, and had no effect on [³H]ouabain binding to the enzyme. K⁺-Phosphatase activity was inhibited by PsB in parallel with Na⁺,K⁺-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na⁺,K⁺-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

Sulphate uptake and metabolism in the chrysomonad,Monochrysis lutheri

The intracellular concentration of inorganic ³⁵SO₄ in Monochrysis lutheri cells exposed to 0.513 mM Na₂ ³⁵SO₄ for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this ³⁵SO₄ with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 μmoles/g cells/hr). The flux of free ³⁵SO₄ to organic ³⁵S was 0.029 μmoles/g cells/hr. Assuming an internal electrical potential in the cells of-70 mV, this intracellular concentration of inorganic ³⁵SO₄ was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a K _m of 3.2×10^-5 M and a V _max of 7.9×10^-5 μmoles sulphate/hr/10⁵ cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the proteins(s) associated with sulphate transport.     

On the presence of sulphate in pituitary lutropin

The presence of sulphate in the carbohydrate of pituitary lutropin from different species has been investigated using a biosynthetic approach. Pituitaries from rats, rabbits, goats, and buffaloes were incubated in the presence of³⁵SO ₄ ^- and the³⁵SO ₄ ^- -labelled proteins in the tissue immunoprecipitated with a well characterized anti-sheep lutropin serum. The incorporation into immunoreactive lutropin was low in the case of rat, rabbit and goat pituitaries while, it was considerable in the case of buffalo pituitaries. Hence further characterization studies were carried out on³⁵SO ₄ ^- -labelled proteins of buffaloes. The physico-chemical, immunological and biological properties of radio-labelled buffalo pituitary material were shown to be similar to those of standard lutropin. Inin vitro conditions of incubations, most of the incorporation of³⁵SO ₄ ^- was observed into tissue lutropin while under similar conditions of incubation, [¹⁴C]-amino acids were found to get incorporated mostly into medium lutropin. The physiologically specific releasing hormone, lutropin-releasing hormone was found to stimulate the release of³⁵SO ₄ ^- -labelled lutropin from the rabbit pituitaries into the medium. These results give indirect evidence that sulphate could be present in pituitary lutropin.     

Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid.

The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals.     

Effects of acutely and chronically administered antidepressants on the clonidine-induced decrease in rat brain 3-methoxy-4-hydroxyphenylethylene-glycol sulphate content.

A study was undertaken of the effects of acutely and chronically administered agents on rat brain MHPGSO₄ content and on the ability of a low dose of clonidine (25 μg/kg) to lower brain MHPGSO₄ levels. This effect of clonidine is attributed to an activation of presynaptic α-adrenoceptors. The agents studied were desipramine and nisoxetine, both inhibitors of NA uptake, Org 6582, a specific inhibitor of 5-HT uptake, and iprindole and mianserin, two atypical antidepressants. In all studies, a dose of 10 mg/kg was used. Acutely administered mianserin increased rat brain MHPGSO₄ levels and prevented the clonidine-induced reduction. The clonidine-induced fall in MHPGSO₄ levels was also absent following the acute administration of desipramine or nisoxetine. However, the observed effect cannot be unequivocally attributed to a blockade of presynaptic α-adrenoceptors since both drugs decreased basal levels of MHPGSO₄. For chronic studies drugs, other than Org 6582, were injected every 12 h for 14 days and experiments were undertaken 12 h after the last injection. Org 6582 was injected once daily for 14 days and experiments undertaken 24 h after cessation of administration. Chronic mianserin and nisoxetine increased MHPGSO₄ levels. Only chronic desipramine blocked the clonidine-induced fall, the phenomenon developing between 5 and 9 days of chronic desipramine administration. This study indicates that, under the experimental conditions employed, the ability of chronic desipramine to elicit subsensitivity of presynaptic α-adrenoceptors does not extend to the other four agents studied.     

Central noradrenergic activity and the formation of glycol sulfate metabolites of brain norepinephrine

The present study utilized intraventricular injection of Na₂³⁵SO₄ to detect drug induced changes in the $\text{in vivo}$ formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihyd (DOPEG-SO₄). Assays involved the hypothalamus only. Rats pretreated with clonidine showed a reduced formation of both MOPEG-³⁵SO₄ and DOPEG-³⁵SO₄ after intraventricular Na₂³⁵SO₄ as well as reduced synthesis of ³H-NE from intraventricular ³H-tyrosine. Phenoxybenzamine (POB) produced increases in the synthesis of both ³⁵S-labeled conjugates and ³H-NE. Neither drug altered the loss of exogenous ³H-MOPEG-SO₄ but clonidine increased both the accumulation of labeled sulfate and the sulfation of exogenous MOPEG in pheniprazine treated rats. These results show that the rates of formation of the labeled glycol sulfates are sensitive indicators of changes in brain NE turnover but can also be influenced by factors involved in sulfation that are unrelated to NE turnover. Blockade of NE synthesis with alpha methyltyrosine did not affect resting or POB-elevated levels of the labeled conjugates until stores of NE were reduced by 40%. The latter findings suggest that central noradrenergic neurons can release and metabolize NE at a normal rate despite synthesis blockade so long as adequate stores of NE are available.     

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Highly purified preparations of Na⁺+K⁺-dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I₅₀ values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10^?4M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.     

Effect of sulfated polysaccharides and sulfate anions on the AMP-dependent activity of phosphorylase b

The effect of sulfated polysaccharides on the AMP-dependent activity of rabbit muscle phosphorylase b as compared with that of Na₂SO₄ has been studied. It has been shown that sulfated polysaccharides and Na₂SO₄ greatly stimulated AMP-activation of the enzyme at low AMP concentrations. Dextran sulfate and Na₂SO₄ desensitized the allosteric interactions of the enzyme towards the nucleotide activator and reversed the enzyme inhibition caused by glucose-6-phosphate and glucose. Furthermore, it was found that while dextran sulfate decreased the K_m value for both substrates, glucose-1-phosphate and glycogen, sulfate anions decreased only the K_m value for glycogen.     

Partitioning of amylase produced by Aspergillus niger in solid state fermentation using aqueous two-phase systems

Equilibrium data of aqueous two-phase systems composed of polyethylene glycol (4000 g mol⁻¹ or 6000 g mol⁻¹) and Li₂SO_4, (NH₄)₂SO₄ or Na₂SO₄ at pH 6.5 and 25 °C were obtained. The efficiency of these in the partition of amylases derived from Aspergillus niger was determined. The experimental data of binodal curves and tie lines were used to estimate the group interaction parameters using the UNIFAC model. Additionally, the influence of phases on the activity of the enzymes was investigated. The results indicate that the polymer molar mass did not influence the biphasic region size. However, the cations under study presented differences in induction to phase formation. It was verified that the systems formed with the Na⁺ presented a larger biphasic region. The increase in the molar mass of the polymer caused the increase in the exclusion volume from 3970.732 g mol⁻¹ to 5700.873 g mol⁻¹. The transfer Gibbs free energy of enzymes presented values between −1296.30 kJ mol⁻¹ and −2867.70 kJ mol⁻¹, that is, the process was spontaneous for all systems studied. The systems formed by (NH₄)₂SO₄ and PEG 4000 g mol⁻¹ presented the best K_e result (3.421) and theoretical recovery of 80.35 %.