CHROMATOGRAPHIC ANALYSES OF ACID-SOLUBLE POLYPHOSPHATES IN CHLORELLA CELLS

Using uniformly ³²P-labeled Chlorella cells as material, compositionof acid-soluble inorganic polyphosphates was studied by paperchromatography and ion-exchange chromatography. 2.By the paper chromatographic analysis it was found that theacid-soluble polyphosphates consisted of highly condensed polyphosphates.Ring-forming tri- and tetrametaphosphates, pyrophosphate andtripolyphosphate were not detected in the acid-soluble fractionof the algal cells. 3.By an ion-exchange chromatography with the use of increasingconcentrations of KCl-solution as eluant, it was found thatthe acid-soluble polyphosphate was a mixture of polyphosphateswith a variety of condensation number (n-values). Polyphosphatesof the n-values between 3 and 15 were only 20% of the totalacid-soluble polyphosphate. The majority of the other polyphospateshad greater n-values which was eluted with 0.5–1.0 M KCl. (Received March 2, 1964; )     

EVIDENCE FOR THE OCCURRENCE AND DISTRIBUTION OF INORGANIC POLYPHOSPHATES IN VERTEBRATE TISSUES

Evidence for the occurrence of polyphosphates having apparent chain-lengths ranging from less than 10 to over 5000 orthophosphate units has been found in adult brain as well as in a number of other mammalian tissues which have been examined. There appears to be three times as much polyphosphate in rat brain as there is in rat liver. Adult rat brain appears to contain at least 15 μg of phosphorus as polyphosphate per g of fresh tissue. In addition, neural polyphosphate is extremely labile to catabolic degradation after death whereas hepatic polyphosphate is relatively more stable. The nature of this inorganic polymer was elucidated through use of the technique of ³¹P nuclear magnetic resonance spectroscopy. Further structural evidence was obtained by application of the isotopic dilution technique to ³²P-labelled neural polyphosphate mixed with an abiotically prepared inorganic polyphosphate. The conditions and rates of hydrolysis of the biological polyphosphate and a non-biological polyphosphate were comparable.     

31P-NMR STUDIES ON INORGANIC POLYPHOSPHATES IN MICROALGAE1

Using “P nuclear magnetic resonance analysis, total inorganic polyphosphate in algae could be quantitatively estimated, For this purpose the algal suspension, which had been kept in cold trichloroacetic acid, was further treated with 6 mM EDTA, or the cells were kept in 2 N KOH containing 100 mM EDTA for 18 h at 37°C. These simple methods avoid hydrolysis of cellular inorganic polyphosphate and, therefore, are useful for the study of phosphorus metabolism in algae. The effects of these treatments on visualization of the signal for inorganic polyphosphate in nuclear magnetic resonance spectra were discussed in comparison with in vivo, ‘P nuclear magnetic resonance spectra of algae.     

DISTRIBUTION AND TURNOVER OF PHOSPHATE COMPOUNDS IN GROWING CHLORELLA CELLS

1. Using the Chlorella cells which had been uniformly labeled with³²P, the distribution of phosphorus in various fractions ofcell material was investigated. Uniformly ³²P-labeled Chlorellawas further grown in a P-free medium or in a standard "cold"medium, and the change of distribution of ³²P (as well as theuptake of exogenous P) in various cell fractions was followed.
2. Analysis of the ³²P-labeled algal cells showed that the highestin P-content was the fraction of RNA followed by those of polyphosphates,lipid, nucleotidic labile phosphate compounds, DNA and protein(in decreasing order). ATP and ADP were found to be only minorfractions of the total labile phosphates.
3. On incubating the³P-labeled alga in a P-free medium, the P.contentsin the fractionsof DNA, protein, lipid and ATP increased, thosein polyphosphatesand ADP decreased, and that in RNA remainedalmost unchanged.When the ³²P-labeled alga was further grownin the normal "cold"medium, DNA and protein increased withthe expenditure of endogenous³²P, but with practically no incorporationof external P. Inthe meantime the P in polyphosphates decreasedconsiderably,and the RNA fraction incorporated a large amountof externalP but only a little of endogenous³²P.
4. It was inferred that,under the experimental conditions of thepresent study, thephosphorus used in the syntheses of DNA andprotein was primarilytaken from polyphosphates, while thatused in the synthesesof RNA, phospholipid and polyphosphateswas, for the most part,taken from the extracellular P-source.

¹A part of this paper was read at the Vth International Congressof Biochemistry, Moscow, August 10–16, 1961. (Received June 4, 1961; )     

TURNOVER OF PHOSPHATE COMPOUNDS IN CHLORELLA CELLS UNDER PHOSPHATE EFICIENCY IN DARKNESS

1. With the view to get information as to the mode of phosphatetransfer among the intracellular phosphorous compounds undernonphotosynthesizing conditions, investigation was made on thechanges of P-distribution occurring when the algal cells wereincubated in a P-deficient medium in the dark. 2. During the incubation, appreciable decrease of P-contentwas observed only in the fraction of polyphosphate "B". In parallelwith this decrease, an increase of P occurred only in the RNAfraction,indicating that, under non-photosynthesizing conditions, RNAis synthesized with the expenditure of phosphorus of polyphosphate"B". 3. By comparing the present results with those obtained earlierunder photosynthesizing conditions, the effect of light on themode of intracellular mobilization of phosphorous compoundswas discussed. (Received June 8, 1961; )     

CHANGES IN LEVELS OF PYRIDINE NUCLEOTIDES IN CHLORELLA CELLS DURING THE COURSE OF PHOTOSYNTHESIS

1. Using intact cells of Chlorella, the effects of CO₂ on thelevelsof oxidized and reduced forms of DPN and TPN in the lightandin the dark were investigated.
2. It was found that the light-inducedchanges of the DPNH-levelwere not affected by the presenceor absence of CO₂. On theother hand, the light-induced increaseof TPNH was suppressedin the presence of CO₂ and the levelof TPNH which was raisedon illumination in the absence of CO₂was lowered by the provisionof CO₂.
3. On the basis of thesefindings, it was concluded that TPNH,but not DPNH, is participating,in some way, in the mechanismof photosynthesis.
4. Discussionswere made on the difference in the sites of participationofTPNH and of the photogenic reducing agent (R) in the pathofcarbon in photosynthesis.

(Received February 28, 1960; )     

CHANGE IN GLUCOSAMINE CONTENT OF CHLORELLA CELLS DURING THE COURSE OF THEIR LIFE CYCLE

1. By the VAN WISSELINGH color reaction and the chitosan sulfatetest it was revealed that Chlorella cells contain chitosan probablyin their cell walls. 2. By fractionating the cell material into several fractionsfollowed by their hydrolysis, it was revealed that the majorityof glucosamine was present in the residue material remaininginsoluble in ethanol-ether and perchloric acid (PCA) solution.Conceivably, this glucosamine has derived, for the most part,from the chitosan contained in the cell wall material. 3. During the course of life cycle of the algal cells, the increasein content of glucosamine occurred in three steps: first, inproportion to the growth of smaller (young) cells into largercells; second, corresponding to the formation of autosporeswithin ripened cells; and third, in parallel with the growthof newly born daughter cells. 4. Between the first and second phase mentioned above, thereoccurred an abrupt breach in the increase of glucosamine. Thisphenomenon was presumed to be closely related to the profoundchange in the permeability of cell walls occuring at this transitionalstage of cell development. (Received September 5, 1960; )     

AN EXPERIMENTAL SEPARATION OF OXYGEN LIBERATION FROM CARBON DIOXIDE FIXATION IN PHOTOSYNTHESIS BY CHLORELLA

Using intact cells of Chlorella pyrenoidosa it is possible to obtain oxygen by the reduction of certain reducible materials other than carbon dioxide. Of these, benzaldehyde was studied in some detail. This reduction does not involve the production of carbon dioxide from the benzaldehyde. Stoichiometrical relationships as expressed by the following equation: 2C₆H₅CHO + 2H₂O → 2C₆H₅CH₂OH + O₂ are somewhat difficult to obtain because the benzaldehyde can disappear from the reaction mixtures by dark reactions. The technique is now available which permits detailed studies of the oxygen-liberating mechanisms in photosynthesis.     

ANALYSIS OF STARCH CONTENT IN CHLORELLA PYRENOIDOSA (CHLOROPHYTA) BY MORPHOLOGICAL AND QUANTITATIVE TECHNIQUES

Chlorella pyrenoidosa (Chick) was grown heterotrophically in batch culture on defined medium with glucose. Morphometric analysis of cells in the exponential growth phase showed that starch accounted for 57% of the volume of the chloroplast and 36% of the total cell volume. During the stationary growth phase, the amount of starch accounted for only 36% of the chloroplast volume and 13% of the total cell volume. This represented a 36% decrease in the amount of starch/cell between the exponential and stationary phase. Determination of starch as grams/cell using quantitative techniques on cell extracts showed a comparable decrease in the amount of starch during this same transition. Based on these results, morphometric techniques provided an accurate assay of starch and have the added advantage of visualization of cellular structures not available when quantitative techniques are used.     

动情周期中大鼠子宫和输卵管壁肥大细胞数量变化的研究

用放射免疫分析法对动情周期中大鼠血清雌二醇浓度进行检测;取子宫、输卵管常规石蜡切片、H－E染色,并用甲苯胺蓝染色显示肥大细胞,于光镜低倍视野下计数。结果显示：动物血清雌二醇浓度依次为：动情期（E）组＞动情前期（PE）组＞动情后期（ME）组＞动情间(DE)且,各组间差异均有显性;在子宫,肥大细胞分布于宫壁肌怪平滑肌束间的结缔组织内、近小血管处,以微血管周居多,常见单个散在,于ME子宫内膜尚偶见肥大细胞;输卵管肥大细胞局限于其外膜层内、近小血管周围,亦多散在。子宫、输卵管壁内的肥大细胞镜下呈圆形、椭圆形或略不规则形,胞浆内充满紫红色粗大颗粒,子宫肥大细胞数量依次为：ME＞DE＞PE＞E,各组间差异有生（DE与PE、PE与E,P＜0.05,余组间P＜0.01）;输卵管壁内肥大细胞数量各组间差异无显性（P＞0.05）。本尚对大鼠血清雌二醇水平波动与子宫、输卵管壁内肥大细胞数量变化的关系及其生理意义进行了讨论。     

ROLE OF PROLINE ACCUMULATION IN RESPONSE TO TOXIC COPPER IN CHLORELLA SP. (CHLOROPHYCEAE) CELLS

The green alga Chlorella sp. (strain 2350) was found to accumulate proline, up to a maximum of 0.027 fmol·cell⁻¹, under stressful concentrations of cupric ions. The function of the accumulated proline was studied with respect to its effect on copper (Cu) uptake. By induction with salt stress, cells with various levels of intracellular proline were used for this study. It is shown that the amount of Cu taken up by the cells was reduced when the intracellular proline levels were enhanced. When proline was exogenously supplied prior to Cu treatment, the adsorption of Cu was markedly reduced. When exogenous proline was supplied after Cu treatment, it resulted in a remarkable desorption of the adsorbed Cu immediately after the addition of proline. The results of the present study indicate that proline may exert some action on the cell surface and suggest that one function of accumulated proline is to reduce the uptake of metal ions. The accumulation of proline may be related to a tolerance mechanism for dealing with Cu stress.     

LIGHT-INDUCED OXYGEN EVOLUTION CAUSED BY HYDROGEN PEROXIDE IN CATALASE-INHIBITED CHLORELLA CELLS

1. Chlorella cells and spinach chioroplasts, whose catalase activityhad been more than 90% inhibited by 10^–5 M azide, werefound to decompose H₂O₂ photochemically to liberate oxygen,indicating that H₂O₂ was used as an oxidant of the HILL reaction.
2. That, however, the observed phenomena cannot be fully accountedfor in terms of the HILL reaction with H₂O₂ was revealed bythe observation that an extract of Chiorella cells, which hadbeen completely freed from chlorophyll, also showed a light-acceleratedO2 evolution from H₂O₂ in the presence of 10^–5 M azide.This extract contained a large quantity of catalase, which seemedto have been, in some way, involved in the reaction in question.
3. The catalatic H₂O₂ decomposition caused by crystalline catalaseof mammalian liver (in the presence of 10^–5 M azide) wasnot accelerated by the effect of light.

¹ Present address: Department of Biology, Faculty of Science,Niigata University, Niigata. (Received June 4, 1961; )     

ENZYMIC DETERMINATION OF NITRATE BY USE OF FROZEN/THAWED CHLORELLA CELLS

Frozen/thawed cells of Chlorella reduce nitrate quantitatively to nitrite which can be determined colorimetrically. 14–420 ng nitrate-N can be determined. The method is suitable for the determination of nitrate in sea water.