Enzyme reactions at the surface of living cells. I. Electric repulsion of charged ligands and recognition of signals from the external milieu

The dynamic behaviour of a polyelectrolyte-bound enzyme is studied when diffusion of substrate or diffusion of product is coupled to electric repulsion and to Michaelis-Menten enzyme reaction. The definition of the classical concepts of electric partition coefficients and Donnan potential of a polyelectrolyte membrane has been extended under global non-equilibrium conditions. This extension is permissible when a strong repulsion exists of substrate and product by the fixed negative charges of the membrane. Coupling between product diffusion, electric repulsion and enzyme reaction at constant advancement may result in a hysteresis loop of the partition coefficient as the product concentration is increased in the reservoir. This hysteresis loop vanishes as the rate of product diffusion increases. No hysteresis loop may occur when electric repulsion effects are coupled to substrate diffusion and reaction. The existence of multiple values of the partition coefficient for a fixed concentration of product implies that the membrane may store short-term memory of the former product concentration present in the external milieu. The occurrence of hysteresis generated by coupling enzyme reaction, product diffusion, electric partition effects at constant advancement of the reaction may be viewed as a sensing device of product concentration in the external milieu. Surprisingly, non-linearities required to generate this sensing device come from electrostatic effects and not from enzyme kinetics.     

Cell membranes after malignant transformation. Part II: Dynamic stability with surface chemical reactions

The dynamic stability of cell membranes in presence of chemical reactions is analysed using the same hydrodynamic cell model as in Part I, with a spherical geometry. Chemical reactions give an additional contribution leading to instability even for positive total surface tension. The mechanical properties of the surface change drastically via the gradient of the surface tension (mechano-chemical coupling). An enzymatic regulation of cell division is proposed, via cAMP. Loss of contact-inhibition of division in cancer cells is interpreted as a lowering of the threshold for cell division, which is not modified at confluence. In that sense, failure of control mechanism in cancer cells is of more significance than rapid growth.     

Iodination of cells membranes: II. Characterization of HeLa cell membrane surface proteins

The molecular weights of the five iodinatable surface membrane proteins of HeLa cells were determined to be 170 000, 145 000, 130 000, 93 000 and 53 000. The proteolytic digestion of these proteins with pronase, trypsin and chymotrypsin was also studied.Metabolic studies showed that these iodinated surface proteins are released into the medium in both acid-soluble and acid-insoluble forms. Antibodies prepared towards these released membrane fragments as well as antibodies prepared towards whole membrane inhibit the growth of HeLa cells.     

Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor

Surface plasmon resonance (SPR)-based sensors have been used to detect the binding between interactive molecules. We applied the SPR technology to the analysis of interactions between living cells and molecules reactive to the cells, using mast cells and mast cell-reactive antigens. The exposure of dinitrophenol-human serum albumin (DNP-HSA), an antigen that stimulates mast cells, to IgE-sensitized mast cells induced a robust and long-lasting SPR signal in a dose-dependent manner. The maximal increase in SPR signal induced by 100 ng/ml DNP-HSA was 0.200 +/- 0.120 angle (mean +/- SD, n = 37), about 1000 times larger than the theoretically expected increase for the simple binding of DNP-HSA to Fc(epsilon)RI, the high-affinity IgE receptor. A small, but similarly prolonged signal was observed when the cells were stimulated by an agonist of the adenosine A3 receptor. The signal induced by DNP-HSA was abolished by genistein, and partially inhibited by phorbol 12-myristate 13-acetate and wortmannin. Interestingly, the signal induced by DNP-HSA was only weakly inhibited by DNP-lysine, suggesting that DNP-lysine manifests its action not by inhibiting, but by modulating the crosslinking of Fc(epsilon)RI. We concluded that SPR sensors can detect biologically significant signals in a real-time manner from the interactions between cells and molecules reactive to the cells.     

Alternative carboxylation reactions in type II methylotrophs and the localization of carboxylase activities in the intra-cytoplasmic membranes.

The peripheral arrangements of the intra-cytoplasmic membranes (ICM) as well as the guanine + cytosine (G + C) content of 63% indicate that our obligate methanotrophic strain M 102 would belong to the type II Methylotrophs. There are no active transport mechanisms for some organic additives, some of which are known to stimulate CO2-fixation. The CO2-acceptor molecules, Pyr (pyruvate) and PEP (phospho-enolpyruvate), must be metabolically intracellularly furnished. More than one carboxylating system could be induced in cell fractions. The carboxylating activities were variably registered in cell-free extracts and fragments. Indications that the site of the carboxylating activities are the ICM are discussed. The fact that the CO2-fixation intensities decreased at about the middle of the active growing phase indicates that the formation of the intermediate C4-compound OAA (oxaloacetate) must be furnished through some reactions by-passing the PEP-carboxylation step. The common serine-pathway for C1-assimilation is rearranged so as to fit in the discussed findings.     

Cation-induced,inhibitor-resistant photosystem II reactions in cyanobacterial membranes

Ferricyanide-supported oxygen evolution in sonic vesicles from the cyanobacterium $\text{Spirulina}\text{platensis}$ is only partially sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and addition of cations to inhibited membranes stimulates the rate of oxygen evolution. The order of cation effectiveness (M³⁺ > M²⁺ > M⁺) suggests that this stimulation is due at least in part to surface charge screening effects which permit freer access of anionic ferricyanide to the vesicle membrane surface; La³⁺, Ca²⁺, and K⁺ are most effective in this regard. Ferricyanide photoreduction is completely sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and neither mono- nor divalent cations affect this inhibition. Addition of La³⁺, on the other hand, causes a nearly complete restoration of ferricyanide-supported oxygen evolution. We conclude that the membrane surfaces of these vesicles are uniquely different from those o higher plants; sites of ferricyanide reduction associated with the interphotosystem chain are surface localized, and the primary acceptor region of photosystem II is susceptible to a trivalent cation-specific reaction in which ferricyanide may directly oxidize the primary acceptor.     

Rapid microfluorimetry of enzyme reactions in single living cells

An optimized photon counting technique allows the microfluorimetric study of NAD⁺ (or NADP⁺) reduction-reoxidation transients in single living cells with a time resolution in the range of 1/50-1/100 sec. The transients resulting from the micro-electrophoretic addition of metabolites (e.g. Glc-6-P or Glc-1-P) can be analyzed in terms of early parameters (e.g. initial lag, rise half time or full rise time) and overall parameters (time of rise and half decay, amplitude, reoxidation time). Both the initial lag and rise half time are considerably longer with Glc-1-P than with Glc-6-P, possibly due to control at the phosphoglucomutase or compartmentation of glycolytic phosphate esters. While glycolytic NAD⁺ (or NADP⁺) reduction proceeds adequately in aerobic EL2 and EAT ascites cells (although ΔNADH/Δt is higher at anaerobiosis), it is critically dependent upon anaerobiosis in L and astrocytoma cells. Thus by rapid microfluorimetry it is possible to resolve the rising phase or other segments of the fluorescence transients into components each corresponding to a particular step in the sequence of intracellular events or control states.     

Destabilization of liposome membranes by the steroidal glycoalkaloid α-tomatine

The effect of the steroidal glycoalkaloid α-tomatine on the leakage of peroxidase from liposomes was studied. At pH 7.2, the optimum pH for disruption, tomatine had little effect at concentrations less than 10μM but was increasingly disruptive at concentrations of 10–100μM. Liposome destabilization was pH-dependent declining with decreasing pH until at pH 5 lysis was achieved only at tomatine concentrations of 500–1000μM. At all pH values tested (pH 5–8), tomatine caused significant disruption only if the membrane contained sterol. The extent of membrane damage was correlated with the concentration of sterol in the liposomes but not with the nature of the sterol or of the phospholipid. These findings are inconsistent with claims that surface glycosidases, which convert the glycoside to the aglycone, are prerequisites for tomatine action and that the aglycone is the active moiety.     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

Electro-osmosis at the surface of phospholipid bilayer membranes

The electro-osmotic velocity is the velocity of a fluid near an interface produced by an electric field parallel to a surface. The velocity adjacent to fixed phospholipid bilayer membranes was measured by observing the velocity of small vesicles suspended in the fluid. The charge densities of the bilayers ranged from 0 to 1 electronic charge per lipid and experiments were performed at temperatures above and below the transition temperature of the phospholipid bilayer in 1, 10 and 100 mM NaCl solutions. The Helmholtz-Smoluchowski equation correctly predicted the electro-osmotic velocity from the known value of zeta potential of the phospholipid bilayer.     

Destabilization of phosphatidylethanolamine-containing liposomes: hexagonal phase and asymmetric membranes

We have measured the temperature of the L alpha-HII phase transition, TH, for several types of phosphatidylethanolamine (PE), their binary mixtures, and several PE/cholesteryl hemisuccinate (CHEMS) mixtures. We have shown for liposomes composed of pure PE and in mixtures with CHEMS that there is an aggregation-mediated destabilization which is greatly enhanced at and above TH. We now ask the question: How well can a dioleoylphosphatidylethanolamine/CHEMS liposome, for example, destabilize TPE (transesterified from egg phosphatidylcholine)/CHEMS liposome and vice versa? We use Ca2+ and H+ to induce aggregation and to provide different values of TH: the TH of the PE/CHEMS mixture is much lower at low pH than with Ca2+. We find that if the temperature is above the TH of one lipid mixture, e.g., A, and below the TH of the other lipid mixture, e.g., B, then the destabilization sequence [measured by the fluorescent 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylylenebis(pyridinium bromide) leakage assay] is AA greater than AB much greater than BB. That is, the bilayer of the lipid A (which on its own would end up in the HII phase) destabilizes itself better than it destabilizes the bilayer of lipid B (which on its own would remain in the L alpha phase). The BB contact is the least unstable. From these experiments, we conclude that the enhanced destabilization of membranes provided by the polymorphism accessible to these lipids above TH is effective even if only one of the apposed outer monolayers is HII phase competent. The surprising result is that if the temperature is above the TH of both lipid mixtures, then the destabilization sequence is AB greater than AA, BB. That is, the mixed bilayers are destabilized more by contact than either of the pure pairs. We believe that this is due to specific differences in the kinetics of aggregation or close approach of the membranes. Similar results were obtained with pure PE liposomes induced to aggregate by Ca2+ at pH 9.5. We also found that the kinetics of low-pH-induced leakage from PE/CHEMS liposomes were initially faster when the CHEMS on both sides of the bilayer is fully protonated. However, in a citrate buffer, which cannot cross intact membranes, the leakage was eventually faster. Flip-flop of the protonated CHEMS to the inner monolayer can explain this observation.     

Interaction of biological membranes with the cytoskeletal framework of living cells

The review is focused on the molecular structure and function of the proteins composing the actin-based cytokeletal cortex, located at the cytoplasmic face of plasma membranes of eucaryotic cells, which stabilizes integral membrane proteins in separate domains of cell membranes. It includes a survey of the molecular properties of teh proteins of the erythrocyte membrane skeleton such as spectrin, ankyrin, protein 4.1, and adducin. The properties of the immunological counterparts of erythroid cortical proteins found in nonerythroid tissues and cells are compared. The structural organization and function of the newly discovered class of calcium-binding proteins, nonerythroid peripheral membrane proteins, calpactins, are also described. Finally, the discussion of some experimental models illustrates that the membrane skeleton of living cells is actively involved in a wide variety of essential biological functions ranging from differentiation, to maintenance of cell polarity and cell shape, and regulation of exocytotic processes.