MITOCHONDRIA AND THE CONTROL OF INTRACELLULAR CALCIUM

1.Because the calcium (Ca) ion is intimately associated with so many biochemical and physiological phenomena, it is fundamental to understand how intracellular Ca is maintained and controlled. This review draws attention to the vital role played by mitochondria in controlling intracellular Ca and describes how transport of the ion into and out of mitochondria may itself be controlled. 2.The heterogeneous distribution of Ca is a property of most, if not all cells. This arises because the ion binds strongly to a variety of biological compounds, especially those containing oxyanions, which themselves have a heterogeneous distribution in cells, but mostly because of the existence in the cell of specific Ca-ion transport systems. 3.Although the concentration of total Ca in the cell may be quite high, a very large proportion of it is bound and non-diffusible; a small fraction is diffusible but unionized. The proportion of Ca that is ionized is probably much less than I% of the total 4.The mechanisms by which Ca is transported into and out of the mitochondrial matrix are discussed. Inward movement of the ion occurs in response to the membrane potential (negative inside) generated by respiration. The process is carrier-mediated and exhibits characteristics such as substrate specificity, high affinity for Ca, satur-ability, cooperativity, stimulation by permeant anions and is specifically inhibited by low concentrations of Ruthenium Red and lanthanum. The properties of the Ca carrier are geared therefore to facilitate rapid inward movement of Ca into the mitochondria. Such a carrier system is found in mitochondria isolated from a wide variety of tissues and species. 5.Ionized Ca appears not to be distributed across the inner mitochondrial membrane according to the Nernst equation, so the possibility exists that the ion is transported as Ca/H⁺ antiport or as Ca/anion symport. Alternatively, an efflux system coupled to inward movement of a cation may serve to prevent the [Ca ion]_in/[Ca ion]_out from attaining equilibrium. These components together contribute to a Ca-translocation cycle that permits Considerable flexibility in the overall control of Ca flux. 6.Evidence for Ca cycling in mitochondria is presented and the influence of physiological agents such as Mg, phosphoenolpyruvate, inorganic phosphate and adenine nucleotides, on the influx and efflux components are discussed in some detail. Moreover, various hormones administered in vivo are able to induce changes in mitochondrial Ca cycling. One important feature that emerges from this collection of data is that the ability of mitochondria to retain Ca is associated with their ability to retain also their adenine-nucleotide complement. 7.Various lines of research provide convincing evidence in support of the view that mitochondria play a major role in controlling cell Ca in vivo. Especially significant are the observations that the ‘activity’ of mitochondrial Ca transport can change during development in both insect and mammalian tissue, can depend on the hormonal status of the tissue and undergoes a permanent change in certain tumour cells. 8.Finally, consideration is given as to how the mitochondrial Ca transport system is able to modify Ca-sensitive enzyme activities by regulating the Ca concentration in specific environments. Some biological activities that might be susceptible to such control are discussed.     

ELECTRON MICROSCOPE HISTOCHEMICAL EVIDENCE FOR A PARTIAL OR TOTAL BLOCK OF THE TRICARBOXYLIC ACID CYCLE IN THE MITOCHONDRIA OF PRESYNAPTIC AXON TERMINALS

Respiration-linked, massive accumulation of Sr²⁺ is used to reveal the coupled oxidation of pyruvate, α-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no α-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of α-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr²⁺ accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO₂ into some of the pyruvate.     

ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA

Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.     

THE INTRACELLULAR DISTRIBUTION AND HETEROGENEITY OF RIBONUCLEIC ACID IN STARFISH OOCYTES

A study has been made of the content and composition of RNA in cytoplasm, nucleoplasm, and nucleoli from growing oocytes of the starfish Asterias rubens. The determinations were carried out, using ultramicrochemical methods, on units isolated by microdissection from fixed sections. Macrochemical and interferometric control experiments show that RNA can be quantitatively evaluated in this way. The results show that the growing oocyte represents a system in which the relations between the quantities of nucleolar, nucleoplasmic, and cytoplasmic RNA undergo great changes. These changes are continuous for nucleolar and cytoplasmic RNA so that their amounts may be predicted from the size of the cell. Nucleoplasmic RNA, on the other hand, shows great variations among different cells, independent of cell size. Purine-pyrimidine analyses show that each cell component contains an RNA which differs significantly from that of the other two. Cytoplasmic and nucleolar RNA are closely related, the only difference being a slightly higher guanine/uracil quotient for the nucleolar RNA. They are both of the usual tissue RNA type, i.e., they show a preponderance of guanine and cytosine over adenine and uracil. Nucleoplasmic RNA deviates grossly from the RNA of the other two components. Here the concentrations of adenine and uracil are higher than those of guanine and cytosine, respectively. This RNA consequently shows some resemblance to the general type of animal DNA although the purine/pyrimidine ratio is far from unity. Our data favor a nucleolar origin for the stable part of the ribosomal RNA and a nucleoplasmic one for the unstable part (the messenger RNA).     

超氧物歧化酶在植物细胞内的分布

大豆幼苗下胚轴的SOD活性主要存在于细胞溶质,约占细胞内总活性的87.3％,其次分布在线粒体,约占总活性的6.8～7.2％。细胞溶质的SOD以Cu-Zn-SOD(SODb_1b_2b_2)类型为主,它在细胞溶质中约占86％。线粒体的SOD主要是Mn-SOD(SOD_a)类型,它在线粒体中约占74～76％。大豆幼苗下胚轴的SOD同工酶活性,SOD_a(Mn-SOD)约占13％,SODb_1b_2b_2(Cu-Zn-SOD)约占77％,SODc_1c_2c_2(Cu-Sn-SOD)约占10％,表明大豆幼苗下胚轴的三组同工酶以SODb_1b_1b_2为最强。比较绿色与黄化花生幼苗子叶SODc_1c_2c_2的差异,证明SODc_1c_2c_2的形成与光照下叶绿体的正常发育有关。     

植物群落与异质性

异质性,对应于植物群落所要求的同质性模型,广泛地存在于植物群落中,但是对其生态学意义的解释却极为模糊。就植物群落中生物的化感作用和非生物的地形因子等两个方面的原因,揭示了植物群落异质性的潜在机制,并阐述了异质性对植物群落的物种组成、种群的分布格局、生态位和群落的动态等方面具有强烈的影响。其结果表明,植物群落组成、结构和动态,其实质就是异质性的趋同化和再异质化的过程。     

高等植物叶绿体和线粒体免疫亲近性的研究

以火箭免疫电泳分析表明:大豆叶绿体抗体与大豆线粒体有免疫交叉反应,同时大豆线粒体抗体与大豆叶绿体也有免疫交叉反应,但是大豆线粒体的抗体与鼠肝线粒体之间无免疫交叉反应。这说明高等植物线粒体对叶绿体比之对动物线粒体在免疫特性上有更大的亲近性,亦即高等植物线粒体和高等植物的叶绿体有更大的同源性。经火箭免疫电泳、交叉免疫电泳和线状免疫电泳进一步分析表明:菠菜偶联因子抗体(AbCF_1)和大豆线粒体、大豆叶绿体间,大豆线粒体抗体与CF_1和大豆叶绿体之间,以及大豆叶绿体的抗体(AbC)与CF_1和大豆线粒体间有免疫交叉反应,说明两种换能器之间有免疫亲近性,并分别与CF_1存在免疫亲近性。这揭示两种换能器免疫亲近性的表现是由于存在共同物质基础所致,这内在共同物质基础是偶联因子。这个结果有力地支持高等植物叶绿体和线粒体在结构和功能上以及发生上存在同源性的观点,在理论上也为两种换能器的起源和演化上存在同源性提供了一些依据。     

POSSIBILITIES FOR INTER- AND INTRACELLULAR TRANSLOCATION OF SOME ICOSAHEDRAL PLANT VIRUSES

Southern bean mosaic virus (SBMV) and Tomato ringspot virus (TomRV) were compared with regard to possible ways of inter- and intracellular translocation. The pore complexes in the nuclear membranes of nuclei in leaf palisade and mesophyll cells of several plant species commonly used in plant virus research were studied. The pore structure resembled that earlier described. The diameter of the pores was great enough to allow icosahedral plant viruses between 25 and 30 mµ wide to move through. SBMV occurred in noncrystalline form in nuclei of infected cells. Although this virus forms paracrystalline structures when partially purified, no virus crystals were seen in the cytoplasm of cells containing high concentrations of SBMV. It was established that this virus could move through nuclear pores. TomRV was found in infected leaf tissue in low concentrations. This virus showed a tendency to crystallize even when present in low concentrations. TomRV was observed only in the cytoplasm, not in nuclei. This virus was present in plasmodesmata, indicating the possibility of cell to cell translocation of whole particles through these structures.     

THE DEGENERATION AND REAPPEARANCE OF MITOCHONDRIA IN THE EGG CELLS OF A PLANT

Details are given of the elimination of mitochondria which occurs during the first 1 to 2 hours of the life of the egg of the fern Pteridium aquilinum (L.) Kuhn. During this phase the only mitochondria present are swollen and appear degenerate, but subsequently the cytoplasm of the egg becomes filled with large mitochondria containing numerous villi. Accompanying the appearance of these mitochondria, many, if not all of which have a peculiar umbo-like form, is the production by the nucleus of conspicuous and complex evaginations. The umbo-mitochondria are believed to be new, and a mechanism is suggested by which they may be generated from the complex evaginations of the nucleus.     

EXPERIMENTS BEARING ON THE NATURE OF INTRACELLULAR INCLUSIONS IN PLANT VIRUS DISEASES

The intracellular changes resultant on infection with aucuba mosaic and Hy. III diseases are described and are compared with the cytological effects of tobacco mosaic virus. With the two former viruses, inclusion bodies are formed by the aggregation and fusion of minute particles which appear in the cytoplasmic stream. With tobacco mosaic disease an amoeba-like body is produced and this persists for some weeks before suddenly disappearing again. It is accompanied by striate material all of which ultimately fuses into one large body.
Attempts have been made to parallel these conditions in healthy cells of Solanaceous plants by treatment with substances known to coagulate protoplasm. Almost all the reagents used induced stimulation of the cytoplasmic stream similar to the initial sign of virus infection. With salts of molybdic acid, all the cytological abnormalities due to aucuba mosaic or Hy. III disease have been imitated. Treatment with lactic acid induces the formation of amoeboid bodies like the X-bodies of tobacco mosaic, but these bodies persist for only a few hours.
Attempts have also been made to inhibit the formation of inclusion bodies induced by several different diseases in a number of hosts but no success was obtained.
The experiments support the view that the intracellular inclusions of plant virus diseases are essentially products of the host cell.     

EVIDENCE FOR THE HETEROGENEITY OF L-2,4-DIAMINOBUTYRIC ACID UPTAKE IN RAT CORTEX

The uptake of L-[³H]DABA by rat cerebral cortex slices was studied. Analysis of the kinetic data obtained provides evidence that DABA entry is mediated by both high and low affinity carriers. When cortical slices were incubated in the presence of equimolar [³H]DABA and [¹⁴C]GABA the ratio of entry of the two radionuclides was found to depend upon the loading concentration. The specificity of the uptake of 1 μM and 1 mM-L-DABA was examined: GABA and DABA were relatively potent inhibitors of 1 μM-DABA uptake whereas an equal concentration of histidine did not produce significant inhibition. In contrast, DABA and histidine were markedly more potent as inhibitors of 1 mM-DABA uptake than was GABA. It is concluded from these experiments that L-DABA is transported into cortical slices by a carrier which has high affinities for both DABA and GABA and by a second lower affinity carrier which prefers DABA as a substrate to GABA. On the basis of a comparison of the effects of inhibitors on [³H]DABA and [³H]GABA uptake it is estimated that approx 26% of DABA uptake at 1 μM does not occur by the high affinity carrier whereas at 1 mM-DABA this proportion rises to 62–67%.     

ULTRASTRUCTURAL AND ENZYMATIC EVIDENCE FOR THE PRESENCE OF MICROBODIES IN THE FUNGUS ACHLYA

Ultrastructural evidence for structures resembling microbodies is presented for the fungus Achlya ambisexualis Raper. These structures are DAB positive and thus presumably contain the enzyme catalase. Activities from mycelial homogenates for. the following enzymes are given: catalase, glycolate oxidase, uricase, isocitrate lyase, malate dehydrogenase, citrate synthetase, malate synthetase and glutamate: oxaloacetate transaminase. These results suggest that Achlya contains microbodies and that they may be of the glyoxysome type. The specific activity of catalase increases substantially following initiation of antheridial hyphae by the hormone antheridiol.     

THE ENZYMATIC MACERATION OF PLANT TISSUES: OBSERVATIONS USING A NEW METHOD OF MEASUREMENT

Mc Clendon , J. H., and G. F. Somers . (U. Delaware, Newark.) The enzymatic maceration of plant tissues: observations using a new method of measurement. Amer. Jour. Bot. 47(1) :1-7. Illus. 1960.—An apparatus is described for measuring the breaking strength of tissue slices. The apparatus was used in the measurement of maceration of potato tuber slices by fungal and tomato enzymes. During the enzymatic maceration of the slices, the strength fell in an approximately logarithmic manner to a stable value less than 5% of the initial strength. Calcium ion did not prevent enzymatic maceration, although it increased the strength of the tissue. Chelating agents used alone did not macerate but facilitated the enzymatic maceration. There was a pH optimum at 3.0—3.5 with a commercial “Pectinase” and enzymes from Botryosphaeria ribis but near 4.7 for a preparation from tomato fruit. The reciprocal of the time for a set strength reduction was proportional to the square root of the enzyme concentration. The relative strength remaining [(initial strength/final strength)–1] after an arbitrary reaction time was proportional to the enzyme concentration raised to the 0.8 power. The temperature coefficient was about 2.5, but other evidence indicated some limitation by diffusion. Non-enzymatic maceration increased rapidly below pH 3 and was especially prominent after subsequent neutralization.