Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme

Uridine diphosphoglucose dehydrogenase (EC 1.1.1.22: UDPglucose dehydrogenase) at pH 5.5-7.8 is a stable homohexamer of 305 +/- 7 kDa that does not undergo concentration-dependent dissociation at enzyme concentrations greater than 5 micrograms/mL. Chemical cross-linking of the native enzyme at varying glutaraldehyde concentrations yields dimers, tetramers, and hexamers; at greater than 2% (w/v) glutaraldehyde, plateau values of 21% monomers, 16% dimers, 5% tetramers, and 58% hexamers are obtained. Dissociation at acid pH (pH 2.3) or in 4-6 M guanidine hydrochloride leads to inactive monomers (Mr 52,000). Denaturation at increasing guanidine hydrochloride concentration reveals separable unfolding steps suggesting the typical domain structure of dehydrogenases holds for the present enzyme. At greater than 4 M guanidine hydrochloride complete randomization of the polypeptide chains is observed after 10-min denaturation. Reconstitution of the native hexamer after dissociation/denaturation has been monitored by reactivation and glutaraldehyde fixation. The kinetics may be described in terms of a sequential uni-bimolecular model, governed by rate-determining folding and association steps at the monomer level. Trimeric intermediates do not appear in significant amounts. Reactivation is found to parallel hexamer formation. Structural changes during reconstitution (monitored by circular dichroism) are characterized by complex kinetics, indicating the rapid formation of "structured monomers" (with most of the native secondary structure) followed by slow "reshuffling" prior to subunit association. The final product of reconstitution is indistinguishable from the initial native enzyme.     

Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima.

The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem. The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources. In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system. In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions. D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110 degrees C. The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts. The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme. At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable. Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism. An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature. Under destabilizing conditions, e.g. moderate concentrations of chaotropic agents, low temperature favors denaturation. The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80 degrees C reactivation exceeds 80%. Shifting the temperature from approximately 0 degrees C to greater than or equal to 30 degrees C releases a trapped tetrameric intermediate in a fast reaction. Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps. Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60 degrees C.     

Chicken Intestinal Alkaline Phosphatase : I. The kinetics and thermodynamics of reversible inactivation II. Reactivation by zinc ions

Purified chicken intestinal alkaline phosphatase is active at pH 8 to 9, but becomes rapidly inactivated with change of pH to 6 or less. Also, a solution of the inactivated enzyme at pH 4.5 rapidly regains its activity at pH 8. In the range of pH 6 to 8 a solution of purified alkaline phosphatase consists of a mixture of active and inactive enzyme in equilibrium with each other. The rate of inactivation at lower pH and of reactivation at higher pH increases with increase in temperature. Also, the activity at equilibrium in the range of pH 6 to 8 increases with temperature so that a solution equilibrated at higher temperature loses part of its activity on cooling, and vice versa, a rise in temperature shifts the equilibrium toward higher activity. The kinetics of inactivation of the enzyme at lower pH and the reactivation at higher pH is that of a unimolecular reaction. The thermodynamic values for the heat and entropy of the reversible inactivation and reactivation of the enzyme are considerably lower than those observed for the reversible denaturation of proteins. The inactivated enzyme at pH 4 to 6 is rapidly reactivated on addition of Zn ions even at pH 4 to 6. However, zinc ions are unable to replace magnesium ions as cocatalysts for the enzymatic hydrolysis of organic phosphates by alkaline phosphatase.     

Authenticity and reconstitution of immobilized enzymes: characterization and denaturation/renaturation of glucoamylase II.

Glucoamylase II (GA II) immobilized to Eupergit C and CIZ as a porous and nonporous matrix shows enzymatic characteristics indistinguishable from those of the free enzyme, except for reduced specific activity. Since this decrease is equally observed for both matrices, it has to be ascribed to nonproductive fixation of the enzyme or steric hindrance rather that perturbations caused by "inner diffusion" effects. Authenticity refers to the optimum pH for catalytic activity, Michaelis constants for starch and maltoheptaose, as well as identical stability toward temperature, pH, and guanidinium chloride (GdmCl). On the basis of these data, the two-state mechanism observed for the equilibrium transitions of the free enzyme may be assumed to hold also for the immobilized enzyme. Renaturation after preceding denaturation in 6.4 and 7 M GdmCl leads to widely differing yields depending on the conditions. Shifting the denaturant concentration stepwise back to nondenaturing GdmCl concentrations leads to a broad range of "hysteresis" accompanied by aggregation. Rapid dilution of the free and immobilized enzymes at pH greater than 6 and sufficiently low protein concentration leads to reactivation yields of 80 and 45%, respectively. For the free enzyme, reconstitution at lower pH is determined by the kinetic competition of folding and aggregation. In the case of the immobilized enzyme, "entangling" of the matrix with the unfolded polypeptide chain competes with renaturation.     

Equilibrium studies on the refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH4-5tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process. At pH approximately 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 degrees C, enzyme concentration 0.4-59 mug/ml) up to 95% enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.     

The reversible inactivation of pig kidney alkaline phosphatase at low pH

1. Pig kidney alkaline phosphatase is inactivated by treatment with acid at 0°. 2. Inactivated enzyme can be partially reactivated by incubation at 30° in neutral or alkaline buffer. The amount of reactivation that occurs depends on the degree of acid treatment; enzyme that has been inactivated below pH3·3 shows very little reactivation. 3. Studies of the kinetics of reactivation indicate that the process is greatly accelerated by increasing temperature and proceeds by a unimolecular mechanism. The reactivated enzyme has electrophoretic and gel-filtration properties identical with those of non-treated enzyme. 4. The results can be best explained by assuming that a lowering of the pH causes a reversible conformational change of the alkaline phosphatase molecule to a form that is no longer enzymically active but is very susceptible to permanent denaturation by prolonged acid treatment. A reactivation mechanism involving sub-unit recombination seems unlikely.     

Effects of low concentrations of guanidine . HCl on the reconstitution of lactic dehydrogenase from pig muscle in vitro. Evidence for guanidine binding to the native enzyme

The presence of low concentrations of guanidine . HCl has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscles after preceding dissociation and deactivation in various denaturants. The obseverd attenuation is a function of the amount of guanidine . HCl present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCl on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.     

Renaturation and urea-induced denaturation of 20 beta-hydroxysteroid dehydrogenase studied in solution and in the immobilized state

Tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) from Streptomyces hydrogenans was reactivated after inactivation, dissociation and denaturation with urea. The effect of several factors such as NAD+, NADH, substrate, sulphydryl reducing agents, extraneous proteins, pH and enzyme concentration on reactivation was investigated. The coenzymes were found to be essential for obtaining a high reactivation yield (about 90%), since in their absence the reactivation was less than 10%. NADH was effective at lower concentrations than NAD+. The reactivated enzyme, after the removal of inactive aggregates, showed physical and catalytic properties coincident with those of the native enzyme. The mechanism by which NADH affects the reconstitution of 20 beta-hydroxysteroid dehydrogenase was investigated using both soluble enzyme and enzyme immobilized on Sepharose 4B. The immobilization demonstrates that isolated subunits are inactive and incapable of binding NADH and suggests that subunit association to the tetramer is essential for enzymatic activity. NADH appears to act, after subunit assembly has taken place, by stabilizing tetramers and preventing their aggregation with monomers that would give rise to inactive polymers.     

Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates

Noncovalent aggregation is a side reaction in the process of reconstitution of oligomeric enzymes (e.g., lactic dehydrogenase) after preceding dissociation, denaturation, and deactivation. The aggregation product is of high molecular weight and composed of monomers which are trapped in a minium of conformational energy different from the one characterizing the native enzyme. This energy minimum is protected by a high activation energy of dissociation such that the aggregates are perfectly stable under nondenaturing conditions, and their degradation is provided only by applying strong denaturants, e.g., 6 M guanidine hydrochloride at neutral or acidic pH. The product of the slow redissolution process is the monomeric enzyme in its random configuration, which may be reactivated by diluting the denaturant under optimum conditions of reconstitution. The yield and the kinetics of reactivation of lactic dehydrogenase from pig skeletal muscle are not affected by the preceding aggregation-degradation cycle and are independent of different modes of aggregate formation (e.g., by renaturation at high enzyme concentration or heat aggregation). The kinetics of reactivation may be described by one single rate-determining bimolecular step with k2 = 3.9 x 10(4) M-1 s-1 at zero guanidine concentration. The reactivated enzyme consists of the native tetramer, characterized by enzymatic and physical properties identical with those observed for the enzyme in its initial native state.     

Reactivation of denatured citrate synthase.

1. The imported mitochondrial enzyme citrate synthase can be partially (less than or equal to 45%) reactivated after denaturation in guanidinium chloride, if the concentration of the denaturing agent is lowered by dialysis, rather than by dilution, when essentially no reactivation is observed. 2. The presence of a reducing agent (dithiothreitol) is necessary for regain of activity. 3. Optimum regain of activity occurs at enzyme concentrations of about 10-20 micrograms/ml; at higher concentrations there is significant formation of aggregates.     

Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism and specificity of reconstitution of dimeric lactate dehydrogenase from Limulus polyphemus

D-Lactate dehydrogenase (EC 1.1.1.28) from Limulus polyphemus is a homodimer which is composed of identical subunits of Mr = 35 000. The enzyme may be reversibly denatured and dissociated at acid pH or in 6M guanidine X HCl. The sigmoidal time course of reactivation obeys a consecutive uni-bimolecular mechanism with k1 = 6 X 10(-4) S-1 and k2 = 1.3 X 10(-4) M-1 S-1 (20 degrees C) as first- and second-order rate constants. Cross-linking experiments with glutaraldehyde prove that reactivation and dimer formation run parallel. Joint "synchronous" reconstitution of the enzyme with dimeric porcine mitochondrial malate dehydrogenase (after denaturation in 6M guanidine X HCl) does not yield active hybrids. The unchanged kinetics of reactivation in the absence and presence of the prospective partner of hybridization prove that inactive hybrid intermediates may also be excluded. The absence of hybrids upon synchronous reconstitution of the two closely related dimeric NAD-dependent dehydrogenases clearly suggests that the assembly of nascent oligomeric proteins must be highly specific.     

Renaturation studies of free and immobilized D-amino-acid oxidase

The renaturation of free and Sepharose-immobilized D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (less than or equal to 4+), was obtained with the free enzyme, is spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of D-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate congruent to iodide greater than chloride greater than phosphate congruent to sulphate congruent to citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of D-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.     

Effect of sheep haptoglobin on the hemoglobin molecule in the Hp-Hb complex

Sheep haptoglobin (HpC) binding hemoglobin increases the stability of the latter to acid denaturation and oxidation by atmospheric O2. HpC is also capable of binding methemoglobin (MetHb) denaturated at pH 3.5 to form a stable complex. This process is accompanied by partial reconstitution of the structural integrity and peroxidase activity of MetHb. Consequently, the formation of a HpC-MetHb complex leads to changes in the tertiary structure of the MetHb molecule. The increase in the peroxidase activity of MetHb at pH less than or equal to 4.0 after its binding to HpC is due to the stabilizing and stimulating activity of HpC.     

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.     

Isolation and characterization of the cytosolic and chloroplast forms of spinach leaf fructose diphosphate aldolase

Two different isoenzymes of fructose-P2 aldolase can be resolved by chromatography of crude spinach leaf extracts on DEAE-cellulose columns. The acidic isoenzyme comprises about 85% of the total leaf aldolase activity. The two forms differ in primary structure as judged by their distinctive amino acid compositions, tryptic peptide patterns, and immunological properties. Only the acidic isoenzyme was detected in extracts of isolated chloroplasts, suggesting that this molecule represents the chloroplast form of spinach leaf aldolase while the basic isoenzyme is of cytosolic origin. The cytosolic (basic) isoenzyme and chicken aldolase A4 are similar in the following respects. 1) They have similar specific catalytic activity (10-15 units/mg); 2) they are both highly sensitive to inactivation by very limited digestion with bovine pancreatic carboxypeptidase A; 3) they both have subunit molecular weights of 40,000; 4) they both have derivatized (blocked) NH2-terminal structures; 5) they are both resistant to thermal denaturation at 50 degrees C; and 6) they both regain catalytic activity following reversible denaturation at pH 2.3 or in 5.8 M urea. Also, the cytosolic aldolase cross-reacted immunologically with the single aldolases present in spinach seeds and in wheat germ. Further, this isoenzyme readily "hybridized" with chicken aldolase A4 in vitro. These observations demonstrate the close homology between the cytosolic aldolases derived from plant and animal origins. The chloroplast aldolase had a specific catalytic activity of about 8 units/mg and, like its cytosolic counterpart, was severely inactivated by limited digestion with carboxypeptidase A. However, this isoenzyme was distinct from the cytosolic aldolase in the following characteristics: 1) its "small" subunit size (Mr congruent to 38,000); 2) its underivatized NH2-terminal structure; 3) its high sensitivity to thermal denaturation at 50 degrees C; and 4) its inability to refold into an enzymatically active conformation following denaturation at pH 2.3 or in 5.8 M urea. The distinctive properties of the chloroplast aldolase may be expected for an enzyme which is synthesized as a higher molecular weight precursor on cytosolic polysomes and is then proteolytically processed to the "mature" form during its migration into the chloroplast organelle.     

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].     

Substrate specifity in Amoeba proteus

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.     

Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase

Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.     

On the denaturation of enzymes in the process of foam fractionation

Experimental study on the denaturation of enzyme during the separation by foaming was conducted with trypsin and catalase in aqueous medium as model system respectively. The effects of operating pH and sparging gas composition on the denaturation of an enzyme were examined respectively. The oxidative deactivation of enzyme at the gas-liquid interface was identified, which could be reduced by applying nitrogen or carbon dioxide as sparging gas. At suitable conditions, the loss of enzyme activity can be reduced to less than 10% in case of trypsin and to zero in case of catalase. With its proven mildness and effectiveness, foam fractionation in a loop bubble column is applicable for recovery and concentration of enzymes from aqueous solutions.