Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

Hydrolysis of 3′,4′-dichloropropionanilide by an aryl acylamidase from Taraxacum officinale

An aryl acylamidase (aryl-acylamine amidohydrolase, E.C. 3.5. 1.a) which hydrolyses the herbicide propanil (3′,4′-dichloropropionanilide), was isolated from dandelion roots and partially purified and characterized. Specificity tests on the enzyme revealed that it could hydrolyse various chlorine ring-substituted propionanilides and 3,4-dichloroanilide alkyl compounds. The partially purified enzyme was inhibited by several sulfhydryl reagents and metal ions. The pH optimum was broad, between 7·4 and 7·8. The apparent activation energy, determined from an Arrhenius plot, was 9·0 kcal/mol (37 700 J/mol) for the hydrolysis of 3′,4′-dichloropropionanilide. The apparent K_m was 1·7 × 10⁻⁴ M with propanil as substrate.     

Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase

Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochrome c reductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochrome b-c₁ III complex (a highly purified ubiquinol-cytochrome c reductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of low K_m ferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochrome c reductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparent K_m for ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to the K_m when ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl^?. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.     

The effect of pH and ionic strength on the pre-steady-state reaction of cytochrome c and cytochrome aa3

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on cytochrome aa₃ increase by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa₃ did not cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H₂O and ²H₂O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa₃ requires a higher enthalpy of activation at temperatures below 20°C (15–16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.     

Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Non-linear arrhenius plots and the analysis of reaction and motional rates in biological membranes

We have systematically derived the rate-temperature relationships for a variety of models of membrane rate processes (particularly enzymic reactions) in order to predict the Arrhenius plot shape(s) appropriate to each model. We have explicitly considered the fact that most thermotropic changes in biological systems extend over finite and sometimes very broad temperature ranges. The rate-temperature relationships for most of the models considered can be expressed in a common, rather simple mathematical form suitable for application in computer data analysis. Only a few models predict Arrhenius plots with the “biphasic linear” form commonly reported in studies of membrane enzymes. However, many of the models yield plots which can be fitted to two intersecting lines within a quite modest experimental error, especially if the change in the slope of the plot around its “break” corresponds to a change in activation enthalpy of less than 15–20 kcal mol^?1. In general, Arrhenius-type plots of motional and reaction rates in membranes are found to be capable of indicating the midpoint but not the endpoints or the overall width of thermotropic transitions in the state of membrane components. Our findings clearly indicate a need for a more rigorous analysis of Arrhenius plot data in terms of graph shapes other than sets of intersecting lines and for more cautious interpretation of Arrhenius plot “breaks” with regard to their physical basis.     

Bovine adrenocortical microsomal hydroxylase and thermotropic transitions substrate-cytochrome P-450 binding reaction versus substrate hydroxylation

The effect of temperture on steroid C-21 hydroxylation and substrate-cytochrome P-450 binding reaction under turnover conditions (NADPH + O₂ are investigated. The Arrhenius activity plot exhibited a single break, while the van 't Hoff plot of the substrate dissociation constant (K_s) exhibited four breaks between 10 and 40°C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the K_s value, the detergent Triton X-114 was without effect on the Arrhenius activity plot. This indicates that the single break in the case of the enzyme activity is distinct from but not necessarily independent of the multiple breaks in the case of the K_s. At physiologic temperature and concentration of the substrate, the free energy (?9.5 kcal/mol) of the substrate-cytochrome binding reaction is more than sufficient to account for the apparent activation energy (6.6 kcal/mol) of the overall hydroxylation. This suggests that the substrate-cytochrome P-450 binding reaction has the potential of being a source of energy for the overall reaction.     

Thermodynamic analysis of purified human placental alpha-L-fucosidase

The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pK_m were for the 4-methylumbelliferyl-conjugate ΔF = ?6.6 kcal/mol, ΔH = ?8.5 kcal/mol, and ΔS = ?6.3 e.u. and for the p-nitrophenylconjugate ΔF = ?5.6 kcal/mol, ΔH = ?12.2 kcal/mol, and ΔS = ?21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = ?12.4 kcal/mol and ΔS = ?20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the “aglycone” moiety of the fluorogenic substrate to the enzyme.     

Kinetic characteristics of bicarbonate-chloride exchange across the neonatal human red cell membrane

The kinetics of HCO₃^?/Cl^? exchange across red cell membrane of newborn infants was studied using a stopped-flow rapid reaction apparatus with a glass pH electrode attached. The measured apparent permeability P is (1.35±0.08 (S.E.)) · 10^?4 cm/s (n=30) for newborns, compared with (3.1 ± 0.4) · 10^?4 cm/s (n=15) for adults. These correspond to half-times of 0.2 s for newborns and 0.1 s for adults indicating that neonatal red cells exchange Cl^? for HCO₃^? only half as fast as do adult cells. The temperature dependence of the exchange rate was studied from 2 to 42°C. From the Arrhenius plot the activation energy of the exchange process in neonatal red cells changes from 22.9 kcal/mol (low temperature) to 4.8 kcal/mol (physiological temperature) at a transition temperature of 17°C. These values are lower than the corresponding values for adult red cells, 34.7 and 10.2 kcal/mol. HCO₃^?/Cl^? exchanges in both adult and neonatal red cells are inhibited by phlorizin. Inhibition constants K_i are 0.8 mM and 2.5 mM for adults and newborns, respectively. The differences in the values of the HCO₃^?/Cl^? exchange rate constant and the activation energy of the exchange process between neonatal and adult red cells indicate that there is a modification of HCO₃^?/Cl^? transport system in the neonatal red cell membranes.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

The allantoinase of Lathyrus sativus

The presence of a stable allantoinase in Lathyrus sativas and its de novo synthesis at a maximal rate in the first 48 hr of germination have been demonstrated. The plumule and radicle together exhibited highest enzyme activity. L. sativas allantoinase has been purified nearly 35-fold. The purified enzyme was optically active around pH 7.5, did not require any metal ion for activity and exhibited a K_m of 2·56 mM for (±)-allantoin, and an activation energy of 5·6 kcal/mol. Unlike other plant allantoinases, the L. sativus enzyme is highly specific for (±)-allantoin and is shown to be a sulfhydryl enzyme which apparently exists in a stable form in vivo obviating the need for added sulfhydryl compounds for maximal activity.     

Molecular Asymmetry in Urease of Water Melon (Citrullus vulgaris)

Kinetics of urease-catalysed urea hydrolysis follows Arrhenius equation in the temperature range 10-50°C and shows an energy of activation equal to 7.14 kcal/mol. The kinetics of thermal inactivation of the enzyme is biphasic, In that half of the initial activity is destroyed more rapidly than the remaining half. The data are consistent with the rate equation: A_t = A_fast·e^-k _fast ^-t + A_slow ·e^-K _slow ^-t where A_t is the residual activity at time t, A_fast and A_slow, k_fast and k_slow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. A similar activity decay (namely blphaslc) is also observed on storing the enzyme at +4 and ?4OC. The data suggest the existence of half-and-half distribution of sites which is a manifestation of a pre-exlstent site heterogeneity in the oligomeric protein molecule.     

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

Kinetic manifestations of allosteric interactions in models of regulatory enzymes with "indirect" co-operativity

The shape of the plots of initial reaction rate (ν) versus initial substrate concentration ([S]₀) and versus initial concentration of allosteric effector ([F]₀) for the model of allosteric enzyme of Monod, Wyman &; Changeux (1965) and for the model of dissociating regulatory enzyme has been analysed by means of the inconstant exponent (q) for substrate or effector concentration, respectively. It has been shown that allosteric interactions in above-mentioned models with “indirect” co-operativity may be manifested not only by the sigmoidal shape of the plot of ν versus [S]₀ or ν versus [F]₀ (with one point of inflexion) but also by the increase in the magnitude of exponent q in progress of saturation process of the enzyme by the substrate or by the effector in the absence of the sigmoidal shape of these plots. It has been shown also that the plot of ν versus [S]₀ has two inflexion points when the parameters have certain definite values. One of these inflexion points (or even both at definite values of the parameters) is hardly discernible. At certain definite values of the parameters two inflexion points may be kinetically manifested by such phenomenon as “negative” co-operativity (q < 1). This is possible if one of the interconvertable enzyme forms exceeds another not only in the affinity to the substrate but also in the value of the rate constant for catalytic breakdown of the enzyme-substrate complex.     

Breaks in arrhenius plots of reactions involving membrane-bound and solubilized sialyltransferases,due to temperature dependence of kinetic parameters

Temperature dependence of asialomucin-sialyltransferase ($\text{CMP}\text{-N-}\text{acetylneuraminate:}\text{D}\text{-galactosyl-glyco-protein}$) N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in K_m and V_max values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities.     

Evidence for a glucosyl-enzyme intermediate in the beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside

The reaction of almond β-glucosidase with $\text{p}$-nitrophenyl-β-$\text{D}$-glucoside has been investigated over the temperature range +25° to ?45° using 50% aqueous dimethyl sulfoxide (DMSO) as solvent. At temperatures below those at which turnover occurs a “burst” of $\text{p}$-nitrophenol proportional to the enzyme concentration is observed. Such a “burst” suggests the existence of a glucosyl-enzyme intermediate whose breakdown is rate-limiting, and provides a method for measuring the active-site normality. At pH 5.9, 25°, the presence of 50% DMSO causes an increase in K_m from 1.7×10^?3M (0%) to 1.7×10^?2M, whereas V_max is unchanged. The DMSO thus apparently acts as a competitive inhibitor with K_i = 0.7M. The Arrhenius plot for turnover is linear over the accessible temperature range with E_a = 23.0 ± 2.0 kcal/mole.