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.     

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.     

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.     

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; )     

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.     

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.     

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.     

Importance of tyrosine for structure and function of mitochondrial malate dehydrogenases

Mitochondrial malate dehydrogenase from pig and chicken both contain one tyrosine/subunit with highly red-shifted spectrum, most probably involved in a hydrogen bond with a carboxylate group. The spectral changes of this tyrosine can be used as an indicator for alkaline denaturation, acid transition and coenzyme binding. Acid transition is coupled with breaking of this bond by protonation as monitored by loss of absorbance at 290 nm. Activity is lost and fluorescence intensity is increased at slightly higher pH, thus indicating increased mobility of the indicator and most probably of the whole protein prior to protonation of the indicator-tyrosine.     

A cytoplasmic inhibitor of DNA polymerase from the plasmodia of Physarum polycephalum.

A factor which inhibited DNA polymerase [EC 2.7.7.7] activity was isolated from the cytoplasm of plasmodia of true slime mold, Physarum polycephalum. This factor was purified by DEAE-Sephadex and CM-cellulose column chromatographies, heat treatment and gel filtration. This inhibitor was heat-stable, insensitive to trypsin [EC 3.4.21.4] and was not digested by RNase [EC 3.1.4.22] or DNase [EC 3.1.4.5]. The molecular weight was 16,000 as determined by gel filtration, and the isoelectric point was determined to be pH 10.1. In the presence of the inhibitor, Km for DNA in the DNA polymerizing reaction was markedly increased. The inhibitory effect was eliminated by addition of excess DNA, but the addition of excess enzyme or deoxyribonucleoside triphosphates had no effect on the inhibition.