Effect of viscosity on enzyme-ligand dissociation. II. Role of the microenvironment

A theoretical treatment, describing a novel viscosity effect on decomposition of enzyme-ligand complexes, recently appeared (Somogyi et al., 1978). From this approach emerged a mechanistic picture of the manner in which increased viscosity lowers the value of the decomposition rate constant. A refined version of this model is presented herein. The analysis is extended to the molecular microenvironment ultimately responsible for mediating the "viscosity effect." Consideration is given to two major factors: (1) the role of viscosity in attenuating the excess chemical energy and (2) the statistical features of the microviscosity. In view of spatiotemporal inhomogeneity in the liquid structure, the concept of averaged microviscosity is introduced to parametrize the enzyme-ligand recombination probability. Quantitative predictions are consistent with models of liquid structure and with results from enzyme studies. The "viscosity effect" may contribute to substrate compartmentation in organized multi-enzyme systems in vivo.     

Microviscosity parameters and protein mobility in biological membranes.

A fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe were employed to determine the microviscosity, n, in liposomes and biological membranes of different cholesterol to phospholipid mol ratio. From the temperature profile of n the flow activation energy, deltaE, and the unit flow volume, V, were derived. The increase of cholesterol/phospholipid ratio in liposomes is followed by a marked increase in n and a decrease in both deltaE and V. Liposomes of the same phospholipid composition as human erythrocyte membranes display in the extreme cases of cholesterol/phospholipid ratios 0 and 1.4 the values of n(25 degrees C) = 1.8 and 9.1 P, and deltaE = 15.0 and 6.5 kcal/mol, respectively. For most membranes studied the fluorescence polarization characteristics and the corresponding n values are similar to those obtained with these liposomes when the cholesterol/phospholipid level of the liposomes and the membranes were the same. However, unlike in liposomes deltaE of all membranes is in the narrow range of 6.5-8.5 kcal/mol, regardless of its cholesterol/phospholipid level. It is plausible that this is a general characteristic of biological membranes which originates from the vertical movement of membrane proteins to an equilibrium position which maintains constant deltaE and V values. This type of movement should affect the interrelation between lipid fluidity and protein mobility. Lipid microviscosity and the degree of rotational mobility of concanavalin A receptor sites in cell membranes were therefore determined. The examined cells were normal and malignant fibroblasts, as an example of cells that form solid tumours in vivo, and normal and malignant lymphocytes, as an example of cells that form ascites tumours in vivo. In both cell systems, opposite correlations between the lipid fluidity and the mobility of concanavalin A receptors were observed. In the fibroblasts the malignant cells possess a lower lipid fluidity but a higher receptor mobility, whereas in the lymphocytes the malignant cells possess a higher lipid fluidity but a lower receptor mobility. Thus, in these cell systems the degree of rotational mobility of concanavalin A receptors increases upon decreasing the lipid fluidity and decreases upon increasing the fluidity of the lipid core. This dynamic feature is in line with the above proposal according to which the concanavalin A receptor sites become more exposed to the aqueous surrounding upon increasing the microviscosity of the lipid layer and vice versa.     

Microviscosity changes during differentiation of neuroblastoma cells

Microviscosity $\text{η}$ of the plasma-membrane lipid matrix was measured in exponentially growing and differentiating C1300 mouse neuroblastoma cells, attached to a glass substratum, by fluorescence polarisation of 1,6-diphenyl-1,3,5-hexatriene. Upon differentiation $\text{η}$ decreases progressively, reaching values below those observed in the growth phase. Treatment of the cells with dipalmitoyl phosphatidylcholine vesicles reversibly inhibits morphological differentiation. The results show that a high membrane fluidity is a prerequisite for differentiation.     

Transduction of enzyme-ligand binding energy into catalytic driving force.

We propose a testable general mechanism by which ligand binding energy can be used to drive a catalytic step in an enzyme catalyzed reaction or to do other forms of work involving protein molecules. This energy transduction theory is based on our finding of the widespread occurrence of ligand binding-induced protein macrostate interconversions each having a large invariant delta H0 accompanied by a small but highly variable delta G0. This phenomenon, which can be recognized by the large delta Cp0's it generates, can provide the necessary energy input step but is not in itself sufficient to constitute a workable transduction mechanism. A viable mechanism requires the additional presence of an 'energy transmission step' which is terminated to trigger the 'power' stroke at a precise location on the reaction coordinate, followed by an energetically inexpensive 'return' step to restore the machine to its initial conditions. In the model we propose here, these additional steps are provided by the existence of ligand inducible 2-state transitions in the free enzyme and in each of the enzyme complexes that occur along the reaction coordinate, and by the selective blocking of certain of these interconversions by high energetic barriers. We provide direct experimental evidence supporting the facts that these additional mechanistic components do exist and that the liver glutamate dehydrogenase reaction is indeed driven by just such machinery. We describe some aspects of the chemical nature of these transitions, and evidence for their occurrence in other systems.     

Microviscosity changes during differentiation of neuroblastoma cells.

Microviscosity (eta) of the plasma-membrane lipid matrix was measured in exponentially growing and differentiating C1300 mouse neuroblastoma cells, attached to a glass substratum, by fluorescence polarisation of 1,6-diphenyl-1,3,5-hexatriene. Upon differentiation eta decreases progressively, reaching values below those observed in the growth phase. Treatment of the cells with dipalmitoyl phosphatidylcholine vesicles reversibly inhibits morphological differentiation. The results show that a high membrane fluidity is a prerequisite for differentiation.     

Microviscosity of human erythrocytes studied with hypophosphite and 31P-NMR

A 31P-NMR method, which complements earlier 13C-NMR procedures for probing the intra-erythrocyte microenvironment, is described. Hypophosphite is an almost unique probe of the erythrocyte microenvironment, since it is rapidly transported into the cell via the band 3 protein, and intra- and extracellular populations give rise to distinct resonances in the 31P-NMR spectrum. Relaxation mechanisms of the 31P nucleus in the hypophosphite ion were shown to be spin-rotation and dipole-dipole. Analysis of longitudinal relaxation rates in human erythrocytes, haemolysates and concentrated glycerol solutions allowed the determination of microviscosity using the Debye equation. Bulk viscosities of lysates and glycerol solutions were measured using Ostwald capillary viscometry. Translational diffusion coefficients were then calculated from the viscosity estimates using the Stokes-Einstein equation. The results with a range of solvent systems showed that 'viscosity' is a relative phenomenon and that bulk (i.e., macro-) viscosity is therefore not necessarily related to the NMR-determined viscosity. The intracellular NMR-determined viscosities from red cells, ranging in volume from 65.5 to 100.1 fl, varied from 2.10 to 2.67 mPa s. This is consistent with the translational diffusion coefficients of the hypophosphite ion altering by only 20%, whereas the values determined from bulk viscosity measurements conducted on lysates of these cells are consistent with a 230% change.     

New approach to heterogeneous enzyme immunoassays using tagged enzyme-ligand conjugates

A new approach to heterogeneous enzyme immunoassays has been developed that uses a tag molecule linked to an enzyme-ligand conjugate. The insoluble phase is an insolubilized receptor to that tag. The antibody to the ligand, in addition to complexing either the free ligand or the one covalently linked to the tagged enzyme, also serves to mask the tag on the tagged enzyme-ligand conjugate so that it can no longer bind to the insolubilized receptor. Accordingly, the proportion of enzyme conjugate associated with the insoluble fraction is proportional to the amount of analyte ligand being assayed. This heterogeneous EIA based on the “antibody masking the tag” is called AMETIA. In the model system we use DNP-lysine as the ligand, β-galactosidase as the enzyme, biotin as the tag, and avidin immobilized to Sepharose as the insoluble receptor.     

Water- and Air-Distributed Conidia Differ in Sterol Content and Cytoplasmic Microviscosity

Airborne and waterborne fungal spores were compared with respect to cytoplasmic viscosity and the presence of ergosterol. These parameters differed markedly between the two spore types and correlated with spore survival. This suggests that the mode of spore dispersal has a bearing on cellular composition, which is relevant for the eradication of industrially relevant fungal propagules.Contamination of food products by fungi often starts with dispersal vehicles that include air- and waterborne spores. The aim of this study was to assess whether air- and waterborne spores are not only different with respect to surface wettability but also have a distinct membrane and cytoplasmic composition. To this end, microviscosity and the presence of ergosterol in the plasma membrane were determined. Ergosterol is the target of many antifungals, and its presence or absence will affect sensitivity to such antifungals, including natamycin. Natamycin is considered a fungistatic antibiotic. It binds to ergosterol but is not able to disrupt the plasma membrane (9, 11). In this study, conidia of Penicillium discolor, Aspergillus niger (airborne), Fusarium oxysporum, and Verticillium fungicola (waterborne) were used. All of these species are relevant in applied situations ranging from postharvest diseases (Aspergillus and Fusarium) and food spoilage (Penicillium) to mycoparasitism of mushrooms (Verticillium). A. niger N402 and P. discolor CBS112557 were grown on malt extract agar (MEA; 7) at 25°C. F. oxysporum CBS116593 and V. fungicola MES12712 were grown on oatmeal agar (7) at 25°C. Low-temperature scanning electron microscopy of uncoated samples (8) clearly showed that the conidia of Verticillium and Fusarium were formed in large (spherical) clusters or on the surface of the colony amid the mycelium, while the other fungi showed clearly elevated spore-forming structures that formed chains of conidia (Fig. ​(Fig.1).1). Conidia of 10- to 12-day-old cultures were harvested in cold ACES buffer [10 mM N-(2-acetamido)-2-aminoethanesulfonic acid, 0.02% Tween 80, pH 6.8] and stored on ice before experimentation on the same day.Open in a separate windowFIG. 1.Formation of conidia by V. fungicola (Vf), F. oxysporum (Fo), P. discolor (Pd), and A. niger (An) observed by scanning cryoelectron microscopy. (A) Numerous conidia of A. niger are formed on erect conidiophores (B) Conidia of P. discolor are also formed on conidiophores, and the chains of the spores are notable. (C) Conidia of F. oxysporum are formed within the mycelium. (D) Conidia of V. fungicola are formed in large clusters that coalesce to form large aggregates of spores inside the mycelium. Bars, 10 μm.     

Microviscosity in lecithin liposomes: effect of nicotinic acid

We have studied the effect of nicotinic acid, a drug commonly used as a vasodilatory agent and also for the treatment of hypercholesterolemia, on the fluidity profile of liposomes of egg lecithin and dipalmitoyl lecithin, using a fluorescent polarization probe. In both cases the drug decreases the membrane fluidity and for cholesterol-probed liposomes, it disrupts the "intermediate fluid condition" induced by cholesterol. The drug also affects the activation energy for diffusion in the hydrophobic region of the liposomes.     

Microviscosity and calcium exchange in yeast cells and effects of phenethyl alcohol

The microviscosity of the yeast cell interior determined by a fluorimetric method is 13.6 cp. Treatment with 0.5% phenethyl alcohol increases viscosity to 15 cp and increases calcium content fivefold. Phenethyl alcohol at low concentrations causes reversible increase in cell membrane permeability to fluorescein.     

Microviscosity of mouse splenocyte cytoplasmic membrane modified by some antibiotics

This study has assessed the changes in the polarization fluorescence of 1,6-diphenyl-1,3,5-hexatriene in the splenic membrane of mice treated with clindamycin, penicillin, cefoxitin, cefamandole, cephaloridine, tetracycline, and erythromycin. The data show that most of the antibiotics tested modified membrane activity, as was shown by the changes in membrane microviscosity parameters.     

Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies

Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane Microviscosity Modulates μ-Opioid Receptor Conformational Transitions and Agonist Efficacy

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.     

Microviscosity of plasmalemmas in rose petals as affected by age and environmental factors

The microviscosity of the plasmalemma of protoplasts isolated from rose (Rosa hyb. cv. Golden Wave) petals was measured by fluorescence depolarization. The plasmalemma's microviscosity was found to increase in petals which were allowed to age on cut flowers or after isolation as well as in isolated protoplasts aged in an aqueous medium. Increasing the temperature of the cut flowers or the isolated protoplasts enhanced the increase of the microviscosity of the protoplast plasmalemma. The mole ratio of free sterol to phospholipid was greater in protoplasts isolated from old flowers or in protoplasts aged after isolation than in protoplasts isolated from younger flowers. Microviscosity was greatest when protoplasts were aged at pH 4.4 and in the presence of Ca²⁺. Artificial alterations of the sterol to phospholipid ratio in the protoplasts, induced by treatment with liposomes, caused similar changes in their measured microviscosity.

These findings strongly suggest that the increase in the petal plasmalemma microviscosity with age is associated with an increase in the sterol to phospholipid ratio which results, at least partially, from the activity of endogenous phospholipases.

     

Thermodynamic quantitative structure-activity relationship analysis for enzyme-ligand interactions in aqueous phosphate buffer and organic solvent

Thermodynamic quantitative structure-activity relationships (QSAR) for chymotrypsin-ligand binding is developed, and the results are compared for the effects of organic solvent on the substrate specificity of the enzymes to those in aqueous phosphate buffer. This is the first of such analysis utilizing thermodynamic QSAR. A possible explanation for the difference describing the effects of organic solvent for the binding of substituted phenyl esters of N-benzoyl L-alanine analogues [PhCONHCH(Me)COOC(6)H(4)-p-X, I] observed in both the classical and the thermodynamic QSAR is presented.     

Inactivation of human gamma-glutamylcysteine synthetase by cystamine. Demonstration and quantification of enzyme-ligand complexes.

Human erythrocyte gamma-glutamylcysteine synthetase is inactivated by the disulfide cystamine (2,2'-dithiobis-(ethylamine)) at pH 8.2 with a rate constant of 1020 min-1 mM-1. Magnesium ion and various combinations of substrates and products confer differing degrees of protection against cystamine inactivation, thus allowing the detection and quantification of certain enzyme-ligand interactions. By measuring inactivation rates as a function of ligand concentrations in incomplete reaction mixtures, we have obtained evidence for the following complexes: enzyme . Mg2+; enzyme . Mg2+ . MgATP2-; enzyme . Mg2+ . L-glutamate; enzyme . Mg2+ . MgATP2- . L-glutamate; enzyme . Mg2+ . L-gamma-glutamyl-L-alpha-aminobutyrate. The data also imply the existence of enzyme . (Mg2+)2 . MgATP2- . L-glutamate and several enzyme forms resulting from the weak binding to L-alpha-aminobutyrate. The methods used permit the calculation of cystamine inactivation rates for most of these enzyme forms and also give values for the equilibrium constants describing their formation.