Substrate inhibition of the mitochondrial and cytoplasmic malate dehydrogenases.

The mechanism that leads to an inhibition of enzyme activity in the presence of high concentrations of substrate was investigated with the two malate dehydrogenase isoenzymes obtained from pig heart. The inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of of oxalacelate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases from chicken hearts. The inhibition constants for oxalacetate are 2.0 mM with the mitochondrial enzyme and 4.5 mM with the cytoplasmic enzyme. Since the in vivo concentration of oxalacetate is reported to be about 10 micrometer, these data suggest that the substrate inhibition that is exhibited by the malate dehydrogenases may not be of any significance in vivo.     

Physarum polycephalum malate dehydrogenase: inhibitor analyses of the mitochondrial and supernatant isozymes

The effects of naturally occurring metabolites were tested on the malate dehydrogenase (L-malate: NAD+oxidoreductase, EC 1.1.1.37) isozymes from the eucaryotic protist Physarum polycephalum. Several of the Krebs cycle intermediates were inhibitors for each isozyme indicating that a similar catalytic process was involved for both forms. The metabolites ATP, ADP, and AMP were inhibitors competitive with NAD for the mitochondrial isozyme but not the supernatant form. Several other nucleoside phosphates had no effects. Tests of protein sulfhydryl, arginine- and tyrosine-modifying reagents revealed a similar functional sensitivity by both isozymes to these reagents. Those results are compared with data on isozymes from more complex tissue with comments on the physiological significance of those combined data.     

Calcium and malate are sporulation-promoting factors of Physarum polycephalum

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime mold Physarum polycephalum. The most important requirement for sporulation of Physarum is a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25 degrees C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state of Physarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.     

Kinetic characterization and thermostability of C. elegans cytoplasmic and mitochondrial malate dehydrogenases

Malate dehydrogenase (MDH) catalyzes the conversion of NAD⁺ and malate to NADH and oxaloacetate in the citric acid cycle. Eukaryotes have one MDH isozyme that is imported into the mitochondria and one in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 and mitochondrial MDH-2 in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we measured K_M values for oxaloacetate of 54 and 52 μM and K_M values for NADH of 61 and 107 μM for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH-1 and MDH-2 from a mixed population of worms and separated them using anion exchange chromatography. Both endogenous enzymes had a K_M for oxaloacetate similar to that of the corresponding recombinant enzyme. Recombinant MDH-1 and MDH-2 had maximum activity at 40 °C and 35 °C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Protein homology modeling predicted that MDH-1 had more intersubunit salt-bridges than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer intersubunit ionic interactions compared to mammalian MDH2 enzymes. These results suggest that the increased stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.     

Calcium wave for cytoplasmic streaming of Physarum polycephalum

The plasmodium Physarum polycepharum exhibits periodic cycles of cytoplasmic streaming in association with those of contraction and relaxation movement. In the present study, we injected Calcium Green dextran as a fluorescent Ca²⁺ indicator into the thin‐spread living plasmodium. We found changes in the [Ca²⁺]i (intracellular concentration of Ca²⁺), which propagated in a wave‐like form in its cytoplasm. The Ca²⁺ waves were also detected when we used Fura dextran which detected [Ca²⁺]i by the ratio of two wavelengths. We prepared the plasmodial fragment from the thin‐spread and found that the cycles of the contraction–relaxation movement was so synchronized that the measurement of its area provided an indication of the movement. We observed that [Ca²⁺]i also synchronized in the entire fragment and that the relaxation ensued upon the reduction in [Ca²⁺]i. We suggest that the Ca²⁺ wave generated periodically is one of the major factors playing a crucial role in the relaxation of P. polycepharum.     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白,并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ,１７．５ｋＤ）组成的大分子,其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性,Ｃａ~（２＋）离子也能提高其活性,Ｍｇ~（２＋）离子无明显影响。钒酸盐,碘乙酸,对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

Purification of an alkaline nuclease from Physarum polycephalum.

An alkaline nuclease was purified from microplasmodia of Physarum polycephalum. The nuclease, active on denatured DNA and RNA and free of contamination by other nucleolytic activities, appeared to be a zinc-metallo protein. The enzyme was only active under conditions, where Zn2+ were retained in the enzyme. Loss of zinc occurred by the chelating action of EDTA, EGTA or ampholines, by acid of highly alkaline pH conditions or by high ionic strength. The addition of ZnCl2 to compensate losses, restored all activity, while all other divalent cations caused inhibition. The nuclease, with a molecular weight of 32 000, was stable at neutral pH at high temperatures with a half-life of 20 min at 80 degrees C. It was inhibited by any salt of buffer concentration above the level of zero ionic strength and showed a special sensitivity towards phosphate ions. The possible similarity of this enzyme to nuclease S1 from Aspergillus oryzae is pointed out.     

Purification and partial characterization of transglutaminase from Physarum polycephalum.

An intracellular form of calcium ion-dependent transglutaminase (R-glutaminylpeptide:amine gamma-glutaminyltransferase, EC 2.3.2.13) was purified 818-fold to apparent homogeneity from acetone powder preparations of spherules of the acellular slime mold Physarum polycephalum. The enzyme was purified by combined methods of precipitation with 15% (wt/vol) polyethylene glycol, DEAE-cellulose chromatography, and isoelectric focusing in a pH 5 to 7 gradient. The isoelectric point of the enzyme was 6.1. The molecular mass of the denatured enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 39.6 kDa. A molecular weight of 77,000 was found by gel filtration of the native enzyme on a Superose 12 fast protein liquid chromatography column, indicating that the native functional protein is a dimer. The purified transglutaminase catalyzed the incorporation of [14C]putrescine into protein substrates including casein, N,N'-dimethylcasein, actin purified from P. polycephalum, and actin purified from bovine muscle. Actin was the preferred substrate for the enzyme, both as a purified protein and in crude extracts prepared from P. polycephalum. With N,N'-dimethylcasein as the amine acceptor substrate, [14C]putrescine, [14C]spermidine, and [14C]spermine were all effective amine donor substrates with Km values of 49, 21.4, and 31.7 microM, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200 microM. Upon starvation induced by depletion of a carbon source for growth, the specific activity of this enzyme increased sixfold during the differentiation of P. polycephalum microplasmodia to spherules. This suggests a role for transglutaminase in the construction of spherules, which have the capacity to survive starvation and dessication.     

Purification and properties of mitochondrial malate dehydrogenase of Toxocara canis muscle

Mitochondrial malate dehydrogenase was purified from muscle extracts of Toxocara canis by means of Sephadex G-100 gel filtration, DEAE-Sephadex ion-exchange chromatography and 5'AMP-Sepharose 4B affinity chromatography. The purified enzyme showed an optimum pH for the reduction of oxaloacetate of 7.3 in Tris-HCl buffer and of pH 7.5-7.8 in phosphate buffer. The m-MDH showed values of 3.2 kcal/mol and 10.5 kcal/mol for the energy of activation, calculated from the Arrhenius equation. The mitochondrial enzyme was found to be more susceptible to thermal inactivation as compared with the cytosolic isoenzyme. Kinetic experiments showed that the m-MDH of Toxocara canis is inhibited by excess oxaloacetate but not by excess NADH. The apparent Km for oxaloacetate reduction was 53 microM and 0.54 mM for L-malate oxidation.     

The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from Physarum polycephalum.

Cytoplasmic DNA polymerase (DNA deoxynucleotidyltransferase, EC 2.7.7.7) was partially purified from Physarum polycephalum. The first step of the purification procedure utilized the fact that the enzyme on gel filtration behaves in anomalous fashion. The second step was either ion-exchange chromatography or sucrose-density-gradient centrifugation. The partially purified DNA polymerase was heterogeneous and at least four species with different sedimentation coefficients (5.5S, 7.2S, 8.6S and 11.5S) were detected. Calculated molecular weights indicated a tendency for stoicheiometric polypeptide aggregation, accompanied by an alteration of the three-dimensional structure froma compact spheroid to a more open elliptical form. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and computed molecular weights suggest an active protomer in the range of 113000 daltons; all data pertain to I 0.045, which was maintained during the whole procedure.     

Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

Evolution of hierarchical cytoplasmic inheritance in the plasmodial slime mold Physarum polycephalum

A striking linear dominance relationship for uniparental mitochondrial transmission is known between many mating types of plasmodial slime mold Physarum polycephalum. We herein examine how such hierarchical cytoplasmic inheritance evolves in isogamous organisms with many self-incompatible mating types. We assume that a nuclear locus determines the mating type of gametes and that another nuclear locus controls the digestion of mitochondria DNAs (mtDNAs) of the recipient gamete after fusion. We then examine the coupled genetic dynamics for the evolution of self-incompatible mating types and biased mitochondrial transmission between them. In Physarum, a multiallelic nuclear locus matA controls both the mating type of the gametes and the selective elimination of the mtDNA in the zygotes. We theoretically examine two potential mechanisms that might be responsible for the preferential digestion of mitochondria in the zygote. In the first model, the preferential digestion of mitochondria is assumed to be the outcome of differential expression levels of a suppressor gene carried by each gamete (suppression-power model). In the second model (site-specific nuclease model), the digestion of mtDNAs is assumed to be due to their cleavage by a site-specific nuclease that cuts the mtDNA at unmethylated recognition sites. Also assumed is that the mtDNAs are methylated at the same recognition site prior to the fusion, thereby being protected against the nuclease of the same gamete, and that the suppressor alleles convey information for the recognition sequences of nuclease and methylase. In both models, we found that a linear dominance hierarchy evolves as a consequence of the buildup of a strong linkage disequilibrium between the mating-type locus and the suppressor locus, though it fails to evolve if the recombination rate between the two loci is larger than a threshold. This threshold recombination rate depends on the number of mating types and the degree of fitness reduction in the heteroplasmic zygotes. If the recombination rate is above the threshold, suppressor alleles are equally distributed in each mating type at evolutionary equilibrium. Based on the theoretical results of the site-specific nuclease model, we propose that a nested subsequence structure in the recognition sequence should underlie the linear dominance hierarchy of mitochondrial transmission.     

1,4-alpha-Glucan phosphorylase from the slime mold Physarum polycephalum. Purification, physico-chemical and kinetic properties

Glycogen phosphorylase from macroplasmodia of Physarum polycephalum was purified 76-fold to homogeneity. The native enzyme migrated as a single protein band on analytical disc gel electrophoresis coinciding with phosphorylase activity. After reduction in the presence of sodium dodecylsulfate one protein band was detectable which corresponded to an Mr of 93 000. The molecular weight of the native enzyme determined by gel sieving or gradient-polyacrylamide gel electrophoresis was 172000 and 186000, respectively. The enzyme contained about 1 mol pyridoxal 5'-phosphate and less than 0.1 mol covalently bound phosphate per mol subunit. The amino acid composition of the enzyme was determined. In the direction of phosphorolysis the kinetic data were determined by initial velocity studies, assuming a rapid equilibrium random mechanism. Glucose 1-phosphate and GDP-glucose were competitive inhibitors toward phosphate and noncompetitive to glycogen. 5'-AMP, a weak activator of the enzyme, counteracted the glucose-1-phosphate inhibition completely. Physarum phosphorylase was compared with phosphorylases from other sources on the basis of chemical and kinetic properties. No evidence for the presence of phosphorylated forms has yet been found.     

Calcium sensitivity of cytoplasmic actomyosin from Physarum polycephalum following different purification procedures

Cytoplasmic actomyosins purified from the acellular slime mold physarum polycephalum by application of two different procedures (Hatano and Tazawa, 1968; Kohama and Kendrick-Jones, 1986) were compared by SDS-PAGE and contraction experiments. In contrast to the 'Hatano actomyosin', 'Kohama actomyosin' contracts in a calcium sensitive manner, i.e., contraction occurs from zero calcium up to pCa4, and is inhibited at greater than or equal to pCa 3. Distinct differences in SDS gels are discussed.