The study of pectin methylesterase and wall-loosening enzyme activities in situ, as well as the estimation of the electrostatic potential of the cell wall, suggest a coherent picture of the role played by metal ions and pH in cell-wall extension. Cell-wall growth brings about a decrease of local proton concentration because the electrostatic potential difference (delta psi) of the wall decreases. This in turn activates pectin methylesterase, which restores the initial delta psi value. This process is amplified by the attraction of metal ions in the polyanionic cell-wall matrix. The amplification process is basically due to the release of enzyme molecules that were initially bound to 'blocks' of carboxy groups. This increase of metal-ion concentration also results in the activation of wall-loosening enzymes. Moreover, the apparent 'inhibition' of pectin methylesterase by high salt concentrations may be considered as a device which prevents the electrostatic potential from becoming too high.
The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur. The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme. With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase. This is explained by the existence of 'blocks' of carboxy groups on pectin that may trap enzyme molecules and thus prevent the enzyme reaction occurring. Metal ions may interact with these negatively charged groups, thus allowing the enzyme to interact with the ester bonds to be cleaved. At high concentrations, however, metal ions inhibit the enzyme reaction. This is again understandable on the basis of the view that some carboxy groups must be adjacent to the ester bond to be cleaved in order to allow the reaction to proceed. Indeed, if these groups are blocked by metal ions, the enzyme reaction cannot occur, and this is the reason for the apparent inhibition of the reaction by high concentrations of metal ions. Methylene Blue, which may be bound to pectin, may replace metal ions in the 'activation' and 'inhibition' of the enzyme reaction. A kinetic model based on these results has been proposed and fits the kinetic data very well. All the available results favour the view that metal ions do not affect the reaction through a direct interaction with enzyme, but rather with pectin.
The kinetic study of the de-esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell-shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell-wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell-wall enzymes are involved. . Although their pathways of biosynthesis are well characterized, their role in the physiology of the plant cell remains uncertain at this time [7]. Three polyamines (putrescine, spermidine and spermine) have been localized in the cell walls of mung bean hypocotyls [S] and in the primary cell walls of carrot cells grown in vitro [9]. The amount of polyamines found in primary cell walls depends on their physiological state [S, 91 and the polygalacturonic acid content [5, 101. It was demonstrated that the carrot cell walls preferentially adsorb large polyamines like spermidine 191 and that this interaction leads to the release of calcium which may act as a second messenger [ I l l . As the negative charges of polygalacturonic acids are responsible for the wall electrostatic potential [12], the results of the binding of polyamines is to lower the electrostatic potential. Since the cell-wall potential plays a central role in the regulation of the plant cell-wall growth and is generated by the catalytic activity of the cellwall pectin methyl esterase [13], one may expect the activity of this enzyme to be modulated by the presence of polyamines. If such is the case, polyamines could be involved in regulation of the growth process. The aim of this paper is to answer this important question. MATERIALS AND METHODSSoybean pectin methyl esterase was isolated and purified to homogeneity from cell-wall fragments obtained from in Enzyme. Pectin methyl esterase (EC 3.1.1.1 1). ~-vitro cultured cells as previously described [14]. Orange pectin methyl esterase was obtained from Sigma Chemicals Company and was dialysed before use against bidistilled water. It was used without further purification.Pectin hydrolysis was followed on a commercial pectin preparation extracted from apple (Fluka) or orange (Serva). Before use, pectic solutions were chromatographed through Sephadex G-100 (Pharmacia) and the main peak of 40 kDa was collected and utilized in the experiments. The mean degree of methylation of the pectin utilized was 70%. As already mention...
The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergendphospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D, the maximum hydrolysis rate V , and the kinetic constant K:, were determined with the micellar substrate.The specificity of various substrates was examined by comparing the V,,,IK,B, values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present in mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme.Keywords: phospholipase D ; phospholipid; mixed micelles; sunflower oil bodies.Phospholipase D (PLD) catalyses the hydrolysis of the ester linkage between phosphatidic acid and various alcohol moieties of several phospholipid species [I-31. This enzyme is widespread in the plant and animal kingdoms. It has been reported to play an important role in signal-transduction cascades in a variety of mammalian cells [4], where it appears to be activated by various hormones and neurotransmitters and some growth factors. In plants PLD is known to be involved in several cellular processes but its role has not been established clearly. The enzyme is present in the protein bodies of germinating mung bean cotyledons [5] and is associated with plasma and intracellular membranes in seedling tissues of castor bean [6]. Activity changes have been observed in conjunction with water stress [7], senescence [S] and pathogenic infection [9]. During mung bean seed germination and seedling growth, the decrease in the phospholipid content has been correlated with an increase in PLD activity [S]. In castor bean endosperm, immunoblot analyses have shown that the amount of PLD increases during the first five days of germination. It has therefore been suggested that these activity changes may be due to metabolic reactions involving membrane phospholipids, which are essential to plant growth and development [lo]. In rice bran, crude PLD prepara- (EC 3.1.4.4). tions were found to be able to react ...
A new model which provides an explanation for pH-induced co-operativity of hysteretic enzymes is proposed. The essence of the model is that a region, or a domain, of the enzyme undergoes a spontaneous 'slow' conformationa1 change which does not affect the geometry of the active site. The region which undergoes this spontaneous conformational transition bears an ionizable group. Kinetic co-operativity occurs if the pK of this ionizable group changes upon this conformational transition. Thus co-operativity does not arise from a distortion of the active site.An interesting prediction of the model is that at 'extreme' pH values co-operativity must be suppressed. Although the kinetic equation pertaining to the model is of the 2 : 2 type, co-operativity is not maximum or minimum at half-saturation of the enzyme by the substrate, as occurs with 2: 2 binding isotherms. A new index of maximum or minimum kinetic co-operativity, whether this extreme occurs at half-saturation or not, has been proposed which allows the change of kinetic co-operativity to be followed as a function of pH.It is believed that this model will be useful in explaining the behaviour of enzymes attached to biological polyelectrolytes, such as membranes or cell envelopes.It has been shown in recent years that some enzymes located on cell envelopes display a quite unexpected pH response [l-31. Owing to the polyanionic nature of these envelopes, the local pH inside the polyanionic matrix may be quite different from the one prevailing in free solution. It is therefore tentatively believed that this change of kinetic behaviour originating from pH changes represents a regulatory device that is operative in vivo [I, 3,4].In addition to the classical enzyme co-operativity, generated by site-site interactions, many enzymes have been shown to display a special type of kinetic co-operativity which results from 'slow' conformational transitions of the active site occurring far from pseudo-equilibrium conditions. These enzymes, which may be polymeric or monomeric, were given the name of 'hysteretic enzymes' [5-71. If some of these hysteretic enzymes are located on cell envelopes, one may wonder whether pH changes may not affect enzyme conformational transitions, thus inducing or affecting the kinetic co-operativity of the enzyme.As outlined above, kinetic co-operativity of hysteretic enzymes results from the existence of two conformations of the active site borne by the free enzyme. Since the enzyme exists under two different ionization states, however, one may wonder whether these two conformations of the active site are effectively required to generate co-operativity.The aim of this paper is to work out precisely a theoretical model where co-operativity of a hysteretic enzyme does not originate from the existence of two conformations of the active site borne by the free enzyme, but rather from the ionization, or the protonation, of a group located outside the active centre, in a different region of the protein. In the following paper [8] we have shown this mode...
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