β-Poly(L-malate), a major constituent of nuclei in plasmodia of Physarum polycephalum, is enzymatically degraded to L-malate after secretion into the culture medium. This depolymerization is specifically catalysed by an endogenous polymalatase. The mode of action and the specificity criteria have been investigated by employing various chemical derivatives of β-poly (L-malate), including substitution at the hydroxy-terminus and carboxy-terminus of the polymer, esterification of the pending A-carboxylate, and β-poly (DL-malate). The results of the investigation were summarized in a specificity model that involved recognition of the hydroxy-terminus and of the A-carboxylate as substituents of the asymmetric carbon in the malic acid unit. Depolymerization proceeded from the hydroxy-terminus towards the carboxyterminus, thereby degrading the polymer to L-malate. When the terminal β-carboxylate had been amidated with the fluorescent N-(1-naphthyl)ethylenediamine, degradation was normal but was arrested at the level of the terminal β-carboxy-substituted dimer. It should be possible to employ polymalatase as a tool for the detection of branching and other modifications of β-poly(L-malate).Keywords : β-poly(L-malate); polymalatase; polymalate depolymerase; Physarum polycephalum ; specificity.Among eukaryotes, several forms of polynucleated cells are supply of nuclear proteins like histones and DNA polymerases (Angerer and Holler, 1995). The polymer is detectable only in known, such as in syncytia during an early stage of embryogenesis, or in developing sperm cells. Another multinucleated form the plasmodium and not in any of the other, mononucleate cell forms in the life cycle of P. polycephalum (spores, amoebae, and is found especially in lower eukaryotes, at best represented by the plasmodium of Physarum polycephalum (Holt, 1980; Sauer, certain durable cell forms for survival under nutrient and water shortages), in agreement with this assignment. The polymer con-1982). The plasmodium can become extremely large and contain several billions of nuclei. Typically, the plasmodium propagates sists of L-malate units, which are aligned via ester bonds between the hydroxy group and the β-carboxylic group. The carby synchronous nuclei division. At least two kinds of factors are required in order to generate a synchronous event of that type : boxylic groups in A-position of the L-malyl units (the pending group of the polymer chain) point away from the polymer backspecific kinases/phosphatases involved in cell-cycle regulation, and transporter (carrier) molecules that take care of a balanced bone. Under physiological condition, the carboxylic groups are ionized rendering the polymer a highly charged polyanion. It is supply of materials throughout the plasmodium. We have discovered the unusual polymer β-poly(L-malate) (Fischer et al., extremely concentrated in the nuclei of the plasmodium in terms of protein-polymer complexes. The level is homeostatically reg-1989) that can function as a molecular storage and carrier for the ulated...
Polymalatase from Physarum polycephalum calalysed the hydrolysis of b-poly [l-malate] and of the synthetic compounds b-di(l-malate), b-tetra(l-malate), b-tetra(l-malate) b-propylester, and l-malate b-methylester. Cyclic b-tri(l-malate), cyclic b-tetra(l-malate), and d-malate b-methylester were not cleaved, but were competitive inhibitors. The O-terminal acetate of b-tetra(l-malate) was neither a substrate nor an inhibitor. l-Malate was liberated; the K m, K i and V max values were measured. The appearance of comparable amounts of b-tri(l-malate), and b-di(l-malate) during the cleavage of b-tetra(l-malate) indicated a distributive mechanism for small substrates. The accumulation of a series of oligomers, peaking with the 11-mer and 12-mer in the absence of higher intermediates, indicated that the depolymerization of b-poly(l-malate) was processive. The results indicate that b-poly(l-malate) is anchored at its OH-terminus by the highly specific binding of the penultimate malyl residue. The malyl moieties beyond 12 residues downstream from the OH-terminus extend into a diffuse second, electrostatic binding site. The catalytic site joins the first binding site, accounting for the cleavage of the polymer into malate residues. It is proposed that the enzyme does not dissociate from b-poly(l-malate) during hydrolysis, when both sites are filled with the polymer. When only the first binding site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation.Keywords: polymalatase; polymalate; depolymerase; synthetic substrates; inhibitors.The plasmodium of Physarum polycephalum and of other myxomycetes produces b-poly(l-malate) (PMLA), an unbranched polyester of l-malic acid [1,2]. The highly charged polyanion binds the replicative DNA polymerases, histones, and other nuclear proteins forming higher-order protein complexes in the nuclei of only these types of cells [3,4±6]. Plasmodia are multinucleated cells typical for myxomycetes in the vegetative branch of their life cycle [7]. Because they develop into cells of extreme sizes and yet display a high degree of synchrony in the timing of cellular events, PMLA has been proposed to function as a storage and carrier matrix in the maintenance of an adequate supply of proteins for all nuclei. In high PMLA-producer strains, the polymer is secreted into the culture medium and depolymerized to l-malate. The hydrolase (polymalatase) has been identified and characterized [8]. The catalytic activity has its optimum at pH 3.5. The large quantity of polymalatase found in the extract of the plasmodium probably refers to the zymogen, which is spontaneously activated during cell lysis [9].Protein chemistry and inhibitor studies have suggested a catalytic mechanism that is unusual for esterases [8]. Structure±function correlation studies with PMLA-derivatives have indicated that the enzyme cleaves the ester bonds while moving in a downstream direction from the OH-terminus [10]. Because of their hydrolytic instability, the employed inhibitors and substrates were...
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