Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.     

Ionic control of acid phosphatase activity in plant cell walls

Abstract. Purified acid phosphatase from sycamore cell walls is not activated by increasing the ionic strength of the reaction mixture. However activation occurs when the enzyme is bound to small cell wall fragments. The apparent activation of the bound enzyme by ions is paralleled by a decline of the substrate concentration C _1/2, that results in half of the maximum rate. Above ionic strengths of about 0.05 the bound and solubilized enzyme forms behave in the same manner. Titration of cell wall fragments at different ionic strengths show that the local pH, inside the cell wall fragments, is lower than the pH in bulk solution. These results are explained in the light of poly-electrolyte theory. The negative charges of the cell walls generate an electrostatic potential that results in the attraction or repulsion of ions. The local concentration of organic phosphate (the substrate of the enzyme) is then lower than its concentration in bulk solution. This concentration difference explains that the value of C _1/2, or of the apparent K_m of the bound enzyme, is greater than the true K_m of the solubilized enzyme. Increasing the ionic strength tends to equalize bulk and local ion concentrations, and therefore apparently activates the bound enzyme.     

Salt-induced Contraction of Bacterial Cell Walls

Intact Bacillus megaterium cells were found to contract as much as 26% in terms of dextran-impermeable volume when transferred from water to unbuffered, non-plasmolyzing NaCl solutions. This shrinkage appeared to be primarily due to electrostatic wall contraction rather than to any osmotic response of the cells. A variety of salts (but not sucrose) added to water suspensions of isolated cell walls caused protons to be released from the walls with resultant lowering of suspension pH and contraction of the structures. In effect, B. megaterium walls behaved as flexible, amphoteric polyelectrolytes, and their compactness in aqueous suspensions was affected by changes in environmental ionic strength and pH. Isolated walls were most compact in low ionic strength media with a pH of about 4, a value close to the apparent isoelectric pH of wall peptidoglycan. Electrostatic attractions appeared to play a major role in determining the compactness of highly contracted walls, and the walls responded to increased environmental ionic strength by expanding. In contrast, electrostatic repulsions were dominant in highly expanded walls, and increased environmental ionic strength induced wall contraction. Walls of whole bacteria also shrank when the cells were plasmolyzed. This second type of contraction seemed to result from relief of wall tension during plasmolysis, and it could be induced with nonionic solutes. Thus, cell wall tone in B. megaterium appeared to be set both by mechanical tension and by electrostatic interactions among wall ions.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic effects in soybean cell walls

The pectin methyl esterase from soybean cell walls has been isolated and purified to homogeneity. It is a protein with a relative molecular mass close to 33 000. The enzyme is maximally active at a pH close to 8 and its pH dependence may be explained by a classical Dixon model, where the two interconvertible enzyme ionization states coexist. The outflux of protons from cell walls, upon raising the ionic strength, may be taken as an indirect estimate of the fixed charge density. If the cell-wall fragments are pre-incubated at pH values between 5 and 9, the outflux of protons rises with the pH of pre-incubation. This implies, as postulated from the theory developed in the preceding paper, that alkaline pH favours the activity of pectin methyl esterase and that this enzyme effectively generates the fixed negative charges of the cell wall. Therefore the pectin methyl esterase reaction builds up the Donnan potential, delta psi, at the cell surface. The cell-wall charge density, estimated from the proton outflux, as well as from the titration of methyl groups on the cell wall, reaches a maximum between the third and the fourth day of growth. While the cell-wall volume increases and reaches a plateau, the fixed charge density increases at first and then declines. This is understandable if one assumes that the building up of a high charge density is a co-operative phenomenon and that the local pH inside the wall rises during cell growth. When both the cell-wall volume and the charge density increase together, this suggests that the local pH inside the wall lies within the critical pH range associated with the steep response of the system. When the cell-wall volume increases together with a decrease of the fixed charge density, the local pH should have dropped below this critical pH range. Under these conditions the pectin methyl esterase remains inactive, or poorly active. As the number of fixed negative charges increases, calcium becomes tightly bound to cell walls. This binding is so tight that the net charge density is minimum when the calcium concentration is maximum. The experimental results, presented above, offer experimental support to two important ideas discussed in the preceding paper, namely that pectin methyl esterase reaction builds up the Donnan potential at the cell surface, and that this response may be co-operative with respect to pH.     

Surface charge measurements on Micrococcus lysodeikticus and the catalytic implications for lysozyme

Electrophoresis measurements on Micrococcus lysodeikticus have shown that the net surface charge density on the cell wall is constant at around -1.5 microC/cm2 for the pH range 4-8. This result has enabled a quantitative analysis to be made of how the electrostatic field associated with the negatively charged cell wall influences the ionic strength and pH dependency of the lytic activity of lysozyme towards M. lysodeikticus. A dominant effect is the creation of a local pH gradient at the cell wall, and at high ionic strengths the lytic activity is found to be controlled by an electrostatic force of attraction between the lysozyme molecule and the cell wall. As the ionic strength of the supporting electrolyte is decreased, however, an electrostatic force of repulsion becomes dominant and is associated with a negative charge carried by the lysozyme molecule, which could possibly be the ionized Asp-52 residue at the active site. This is considered to arise from the fact that at low ionic strengths the fine details of the heterogeneous charge distribution on the cell wall and lysozyme molecule are only partially screened by counter ions.     

Effect of Phosphorus Deficiency on Phosphatase Activity of Cell Walls from Roots of Subterranean Clover

Clover (Trifolium subterraneum L. cv. Mt. Barker) was grownin solution culture with adequate (+P) or no phosphate (–P).Cell walls were extracted from roots in such a way that theywere uncontaminated by other cellular materials. Phosphataseactivity was assayed using p-nitro-phenylphosphate (NPP). Phosphatasebound to cell walls had a pH optimum between 5.0 and 6.0, irrespectiveof the P supply to the plants. Activity of phosphatase boundto cell walls increased with electrolyte concentration of theassay medium at pH 6.5 but not at pH 5.5. This increase in activitywas probably due to a higher degree of ionization of the cellwall at pH 6.5 than at pH 5.5, and to effects of high ionicstrength in decreasing the mutual repulsion of negatively chargedNPP from negative charges on the cell walls. Cell wall-boundphosphatase did not exhibit Michaelis-Menten kinetics: the concentrationof NPP at which activity was half the maximum rate (S_0.5) was0.7 mM for cell walls extracted from roots of both +P and –Pplants. Up to 30% of the phosphatase activity bound to cellwalls could be removed using buffer solutions of high pH andhigh ionic strength which contained Triton X100. Both soluble and cell wall-bound phosphatase(s) of roots increasedin activity with P deficiency. The phosphatase activity of cellwalls increased 1.5 fold as the P concentration in the rootsfell from 0.4–0.2% dry weight. Experiments with sterileroots of clover showed that increases in cell wall-bound phosphataseactivity associated with P deficiency were not due to microbialcontamination. It is argued that phosphatase(s) in cell wallsof roots could make a substantial contribution to the P nutritionof clover in soils deficient in inorganic phosphate by hydrolysingorganic phosphate compounds in the soil. Key words: Phosphatase, Clover, Roots, Phosphorus deficiency, Cell walls     

Distribution of the anionic sites in the cell wall of apple fruit after calcium treatment

Summary The ripening and softening of fleshy fruits involves biochemical changes in the cell wall. These changes reduce cell wall strength and lead to cell separation and the formation of intercellular spaces. Calcium, a constituent of the cell wall, plays an important role in interacting with pectic acid polymers to form cross-bridges that influence cell wall strength. In the present study, cationic colloidal gold was used for light and electron microscopic examinations to determine whether the frequency and distribution of anionic binding sites in the walls of parenchyma cells in the apple were influenced by calcium, which was pressure infiltrated into mature fruits. Controls were designed to determine the specificity of this method for in muro labelling of the anionic sites on the pectin polymers. The results indicate that two areas of the cell wall were transformed by the calcium treatment: the primary cell walls on either side of the middle lamella and the middle lamella intersects that delineate the intercellular spaces. The data suggest that calcium ions reduce fruit softening by strengthening the cell walls, thereby preventing cell separation that results in formation of intercellular spaces.Abbreviations EDTA ethylenediaminotetraacetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate     

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.     

Calcium and the cell wall

Abstract. From this brief review it appears that the interactions between calcium ions and cell walls play a key role in plant physiology. Calcium ions are involved in many mechnisms: for example, stabilization of cell wall structures, acidic growth, ion exchange properties, control of the activities of wall enzymes. All these properties originate from the tight binding of calcium ions to the pectins present in the cell walls. The factor most important for controlling wall behaviour is the density of non-diffusible charges and, due to its high affinity, calcium can significantly affect this factor. We also discuss the theoretical ion exchange models in relation to the specific role of calcium ions.     

Separation of beta-casein peptides through UF inorganic membranes.

Ultrafiltration of peptide mixtures is studied under various operating conditions (transmembrane pressure, tangential flow-rate) using two ultrafiltration inorganic membranes M5 and M1 with molecular weight cut-offs, MWCO 10 and 70 kD, respectively. It is shown that the separation of peptides is controlled by a dual mechanism: size exclusion and electrostatic repulsion. When the ionic strength is high enough to screen out the electrostatic interactions, experimental data are in good agreement with a sieving model developed to estimate the intrinsic transmission from the molecular weight of a component and from the MWCO of the membranes. Although the transmission so found is altered by concentration polarisation and pore blocking mechanisms, the results explain the apparent low transmission of peptides by ultrafiltration membranes. If the ionic strength of the fluid is low, electrostatic interactions can influence the transport phenomena, provided that the molecules are highly charged (at pHs away from the pI). For attractive interactions, an apparent partition coefficient larger than 1 is observed. Otherwise, the transmission is lower than predicted by the sieving theoretical equation, as if the partition coefficient were smaller than 1.     

The `compartmentation'' of choline acetyltransferase within the synaptosome

1. Choline acetyltransferase may be isolated in either a bound or soluble form after hypo-osmotic treatment of a crude synaptosome fraction, depending on the conditions. 2. In the bound form, the enzyme appears to be associated with the larger membrane fragments rather than with synaptic vesicles. 3. The bound form is predominant at slightly acid pH values and low ionic strength, the soluble form under more physiological conditions of pH and ionic strength. 4. Sodium chloride, potassium chloride, magnesium chloride and calcium chloride at similar ionic strengths solubilize the enzyme. 5. Choline acetyltransferase was found to be soluble under these conditions after release from synaptosomes from rat and pigeon cerebra, guinea-pig cortex and rabbit cortex, caudate nuclei, diencephalon and midbrain. 6. Certain isoenzymes of lactate dehydrogenase behaved similarly.     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

Yeast flocculation: competition between nonspecific repulsion and specific bonding in cell adhesion

Yeast flocculation is governed by the competition between electrostatic repulsion (nonspecific interaction) and polysaccharide-protein bonds (specific interaction). The electrical surface potential, which is mainly due to phosphodiester linkages (of the cell wall phosphomannan), maintains the cells dispersed. Polysaccharides and proteins of the cell surface can readily penetrate the potential barrier and may establish specific bonds. The specific inhibition of flocculation by various mannosyl derivatives suggested that the protein receptor binds to the group Man alpha----3 Man alpha----PO4- ----6 Man alpha----2 Man alpha... of phosphomannan. Calcium, which is required for flocculation, could act as a bridge between the negatively charged groups of phosphomannan and those of the protein receptor. The role of calcium, however, cannot be restricted only to charge neutralization because other divalent cations inhibit flocculation; our results show that cation binding is governed by strong stereochemical constraints. Studies on protein-polysaccharide interactions have shown that electrical charges may remain uncompensated at short distance, but can be stabilized by hydrogen bonds. Calcium could induce a "locked" conformation of the receptor; this conformation is the only one capable of binding phosphomannan strongly enough to make cell adhesion possible.     

Electromechanical Interactions in Cell Walls of Gram-Positive Cocci

Isolated cell walls of Staphylococcus aureus and Micrococcus lysodeikticus were found to expand and contract in response to changes in environmental pH and ionic strength. These volume changes, which could amount to as much as a doubling of wall dextran-impermeable volume, were related to changes in electrostatic interactions among fixed, ionized groups in wall polymers, including peptidoglycans. S. aureus walls were structurally more compact in the hydrated state and had a higher maximum charge density than M. lysodeikticus walls. However, they were less responsive to changes in electrostatic interactions, apparently because of less mechanical compliance. In media of nearly neutral pH, S. aureus walls had a net positive charge whereas M. lysodeikticus walls had a net negative charge. These charge differences were reflected in Donnan distributions of mobile ions between wall phases and bulk medium phases. Cell walls of unfractionated cocci also could be made to swell and contract, and wall tonus in intact cells appeared to be set partly by electrostatic interactions and partly by mechanical tension in the elastic structures due to cell turgor pressure. The experimental results led to the conclusions that bacterial cell walls have many of the properties of polyelectrolyte gels and that peptidoglycans are flexible polymers. A reasonable mechanical model for peptidoglycan structure might be a sort of three-dimensional rope ladder with relatively rigid, polysaccharide rungs and relatively flexible polypeptide ropes. Thus, the peptidoglycan network surrounding cocci appeared to be predominantly an elastic restraining structure rather than a rigid shell.     

Electrostatic interactions during activation of coagulation factor IX via the tissue factor pathway: effect of univalent salts

Interaction between the Gla-domain of coagulation proteins and negatively charged phospholipid membranes is essential for blood coagulation reactions. The interaction is calcium-dependent and mediated both by electrostatic and hydrophobic forces. This report focuses on the electrostatic component of factor IX activation via the extrinsic pathway. Effective charges during the reaction are measured by ionic titration of activity, according to the Debye-Huckel and Gouy-Chapman models. Rates of activation decrease with ionic strength independently of the type of monovalent salt used to control ionic strength. Moreover, the effect of ionic strength decreases at concentrations of charged phospholipid approaching saturation levels, indicating that membrane charges participate directly in the ionic interaction measured. The effective charge on calcium-bound factor IX during activation on phospholipid membranes is 0.95+/-0.1. Possible sites mediating contacts between the Gla-domain and membranes are selected by geometrical criteria in several metal-bound Gla-domain structures. A pocket with a solvent opening-pore of area 24-38 A2 is found in the Gla-domain of factors IX, VII, and prothrombin. The pocket contains atoms with negative partial charges, including carboxylate oxygens from Gla residues, and has a volume of 57-114 A3, sufficient to accommodate additional calcium atoms. These studies demonstrate that electrostatic forces modify the activity coefficient of factor IX during functional interactions and suggest a conserved pocket motif as the contact site between the calcium-bound Gla-domain and charged membranes.     

Engineering of human lysozyme as a polyelectrolyte by the alteration of molecular surface charge

The surface positive charges of human lysozyme were either increased or decreased to alter the electrostatic interaction between enzyme and substrate in the lytic action of human lysozyme using site-directed mutagenesis. The amino acid substitutions accompanying either the addition or the removal of two units of positive charge have shifted the optimal ionic strength (NaCl concentration in 10 mM Mes buffer, pH 6.2) for the lysis of Micrococcus lysodeikticus cell from 0.04 M to 0.1 M and from 0.04 M to 0.02 M respectively. In addition to the change in ionic strength-activity profile, the pH-activity profile and the effect of a polycationic electrolyte, poly-L-Lys-HCl, on the lytic activity were significantly changed. Owing to the shifts in both ionic strength profiles and pH profiles the Arg74/Arg126 mutant has become a better catalyst than wild-type enzyme under the conditions of high ionic strength and high pH, and the Gln41/Ser101 mutant has become a better catalyst under the conditions of low ionic strength and low pH.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

Carbamoylation of Cu,Zn-superoxide dismutase by cyanate: Role of lysines in the enzyme action

Reaction with cyanate leads to a reversible change of the EPR spectrum of Cu,Zn-superoxide dismutase and to time-dependent carbamoylation of the lysine residues of the enzyme, producing a stable covalent derivative with more negative charge. The carbamoylated enzyme is less active than the native enzyme in spite of unaltered EPR spectra. The extent of this inactivation is much less when the enzyme activity is measured at low ionic strength. These results show that integrity of the active site is not the sole factor playing a role in the enzyme mechanism and that the ionic strength effect is related to electrostatic interactions between O⁻₂ and surface charges of the protein.     

Invertase activity associated with the walls of Solanum tuberosum tubers

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.