Phosphate-free Medium Research Articles

Influence de l'acide glyoxylique sur les propriétés des mitochondries isolées

Glyoxalate is an effector of oxidative phosphorylation in isolated mitochondria : it slows down State 3 but does not affect State 4 respiration. This report presents the findings of our study on the mechanism of action of glyoxalate ; these findings are listed below. The inhibition of Stage 3 respiration by glyoxalate does not set in immediately, can be reversed in part by the addition of an uncoupling agent or a dithiol, is non-competitive against succinate and can be demonstrated with substrates requiring the involvement of other membrane transport systems. Glyoxalate prevents the increased oxygen uptake stimulated by 2,4-DNP or Sr++. Glyoxalate also inhibits phosphate transport and this inhibition can account for most of the effect observed. The inhibition of State 3 respiration is paralleled by a decrease in the mitochondrial accumulation of succinate : this decrease could arise from a direct effect of glyoxalate on dicarboxylic acid transport or could be the result of an inhibiton of the phosphate transport system, which is connected with the former. The decrease in the respiratory rate of uncoupled mitochondria placed in a phosphate free medium demonstrates that the effector acts directly at the substrate transport or/and electron transfer level. Phosphate, by delaying the respiratory inhibiton due to glyoxalate, has a protecting effect on mitochondrial functions. Glyoxalate is thus acting at several mitochondrial sites. It acts presumably by forming hemimercaptals, blocking sulfhydryl groups. Its effects can be accounted for by the unfolding of such (hemicercaptal) groups under the influence of ADP, Pi, uncoupling or others agents which bring about conformational changes in the internal mitochondrial membrane.

Rapid transient efflux of phosphate ions from pancreatic islets as an early action of insulin secretagogues.

Anionic fluxes during the membrane realignments of stimulated insulin release have not been characterized previously although cations have been implicated in stimulus-secretion coupling. We have shown that a limited packet pulse of phosphate release ("phosphate flush") begins at the same time that the first phase of insulin secretion may occur. To demonstrate this phenomenon, we have prelabeled islets, obtained from rat pancreas by collagenase digestions, by incubation with [(32)P]orthophosphate. When such prelabeled islets are perifused with Krebs-Ringer bicarbonate containing 0.5 mg/ml D-glucose, a basal rate of efflux of radioactivity is established; transfer to perifusates containing 3.0 mg/ml D-glucose elicits an increased (32)P efflux within 1-2 min to peak values which are 7- to 21-fold greater than basal. The total duration of this "phosphate flush" approximates 10 min and exceeds the duration of the first phase of stimulated insulin secretion. With lesser concentrations of glucose, the flush exhibits dose-response relationships, and with 3 mg/ml glucose, a second flush can be elicited by restoring basal conditions and stimulating anew with 3 mg/ml glucose. The phenomenon is highly specific and can be reduplicated by other secretagogues (L-leucine) or sugars (D-mannose) which are also known to elicit insulin release but not by sugars which fail to affect insulin secretion (D-galactose, D-fructose, i-inositol, L-glucose). The efflux of radioactivity consists entirely of [(32)P]orthophosphate. Phosphate flush persists in phosphate-free media, Ca(++)-free media, and when insulin release is obtunded by adding Ni(++) (2 mM) to the perifusates. Thus, efflux of [(32)P]orthophosphate can be dissociated from insulin extrusion, and from net influx of ionic phosphate or calcium. Membrane stabilization with D(2)O or 1.0 mM tetracaine reversibly inhibits phosphate flush. Although the mechanism by which this effect occurs has not yet been established, the phosphate flush appears to constitute one of the earliest and hitherto unknown indices of the excitatory state in pancreatic islets.

Differences in nuclear RNA labelling patterns of Burkitt lymphoma, leukaemic lymphosarcoma and various human leukaemias.

Nuclear RNA was isolated from citric acid nuclei derived from AML, CML, CLL, leukaemic lymphosarcoma and BURKITT lymphoma cells after 6 hours incubation with32P-orthophosphate in a phosphate-free medium. In fractionations on sucrose density gradients, marked differences were found in the distribution of the32P-radioactivity mainly in the 45S fraction containing the ribosomal precursor RNA. The lowest specific activities of nuclear 45S RNA were found in CML; very high labelling accurred in cells of AML, leukaemic lymphosarcoma and BURKITT lymphoma. In CLL cells which are known for lack in DNA synthesis, pre-ribosomal 45S and 35S RNA were labelled to a remarkable extent. Studies are in progress in order to define possible differences in nuclear RNA structures between lymphocytic and granulocytic cell lines.

Phosphate as a control factor in cell division of Chlamydomonas reinhardti, studied in synchronous culture

Growth, cell division and time course of accumulation of macromolecules has been compared in Chlamydomonas reinhardti during synchronized vegetative growth in complete and phosphate free medium. Deprivation of P i from the growth medium at the beginning of the life cycle (zoospore stage) led to 1. 1. shortening of G1 phase with earlier start of DNA synthesis, karyokinesis, cytokinesis and sporulation (with reduction in spore number). 2. 2. Moderate variation in protein content and oscillation of RNA content. 3. 3. Decrease in degree of synchrony of nuclear division and sporulation. 4. 4. Derepression of phosphatases. Re-addition of P i at different times to cells grown in P-free medium from the beginning of the life cycle, demonstrated a transition period in the cells regarding the processes started by the removal of P i .

The influence of magnesium on calcium release from bone in vitro

1. 1. Bones from normal and magnesium-deficient rats were incubated in a calcium and phosphate-free medium which otherwise had an ionic composition similar to serum. 2. 2. Metabolically active bone released more calcium and magnesium to the incubation medium than inactive preparations of bone from the same animal. 3. 3. Magnesium increase the release of calcium from aged bone by physico-chemical processes which appeared to involve the exchange of magnesium for calcium on the bone surface. 4. 4. With metabolically active bone magnesium also increased calcium release by a mechanism which involved the breakdown of both the matrix and mineral phase, and it is suggested that magnesium stimulates the normal process of bone resoprtion.

Kinetic analyses of calcium movements in cell cultures. IV. Effects of phosphate and parathyroid hormone in kidney cells.

Several parameters of calcium and phosphate transport were measured in kidney cells at different medium phosphate concentrations and in the presence of PTH. Raising the medium phosphate from 0 to 10 mM increases calcium influx, the exchangeable calcium pool and the total cell calcium. The stimulation of calcium influx by PTH is obtained even in phosphatefree medium, whereas the expansion of the calcium pool cannot be obtained in the absence of external phosphate. PTH has no effect on the steady state phosphate influx and on the exchangeable phosphate pool. On the other hand, the total cell phosphate is increased by PTH but this effect is obtained only if calcium is present in the medium. Thus, the stimulation of calcium influx by PTH is independent of phosphate and is one of the primary effects of the hormone. (Endocrinology 86: 1389, 1970)

Untersuchungen zum Wirkungsmechanismus kurzfristiger Kälte auf den Intermediärstoffwechsel von Brassicaceen-Keimwurzeln: III. Mitteilung Phosphatstoffwechsel und Kältebehandlung

Summary 1. To investigate the mode of action of a short-term cold treatment, which caused a depression of respiration in the roots of Brassicaceae seedlings for several hours, the phosphate metabolism was examined. 2. The uptake of phosphate from phosphate buffer solution by isolated roots of rape was effected in dependence on the temperature and the concentration. Roots which were kept at 2°C for 3 and 5 hours showed a lower increase of phosphate than the 25°C controls. After exposition of only 1 hour no differences dependent on temperature were observed in the total phosphate content. 3. The phosphate increase took place in the ethanol soluble fraction. The determination of the 7-minute-phosphate showed, that a cold treatment of only one hour reduces the increase of the unstable organic phosphate molecules. 4. The use of phosphate free medium caused the same low temperature conditioned depression of the oxygen uptake in the roots. According to that a disturbance of the phosphate uptake cannot be the primary cause for the depression of the respiration. 5. A possible mechanism of the action of low temperature, which bases on changes in the membrane structures, is discussed.

Transport phenomena associated with the deposition and disappearance of pyrophosphate granules in Tetrahymena pyriformis

1. 1. Tetrahymena pyriformis rapidly accumulate Mg 2+ and Ca 2+ when both cations, as well as phosphate, are present in the medium. The uptake of the ions is closely followed by the deposition of magnesium-calcium pyrophosphate granules. We believe that this process, through complexing the intracellular Mg 2+ and Ca 2+, facilitates their uptake by the cells. 2. 2. The process is reveresed as soon as the cells are placed in a phosphate-free medium. The granules disappear within 6–8 h, but there is a distinct difference in the transport of Ca 2+ and Mg 2+ out of the cells. Ca 2+ is removed from the cells by an active process (against a concentration gradient) from the moment the cells are placed in the phosphate-free medium. Ca 2+ efflux continues at a steady rate until the granules are depleted. No Mg 2+ leaves the cells until this time, and its efflux starts when all of the granules (and presumably Ca 2+) have been removed. 3. 3. The maintenance of the balance of intracellular Ca 2+ and Mg 2+ may be a part of the control mechanism governing the deposition and utilization of pyrophosphate. This may involve, on one hand, the ability of the two cations to complex pyrophosphate and, on the other hand, the activation by Mg 2+, and inhibition by Ca 2+, of inorganic pyrophosphatase.

Uncoupling of respiratory-chain phosphorylation by arsenate

1. The specific uncoupling by arsenate of oxidative phosphorylation in rat-liver mitochondria has been studied in EDTA-containing medium. The K m for arsenate for stimulation of the oxidation of succinate in phosphate-free medium is about 1 mM. 2. Arsenate-stimulated respiration is inhibited by low concentrations of phosphate, in the absence of ADP. The effects of arsenate and phosphate are competitive. A phosphate:arsenate ratio of 0.1 inhibits completely. In the presence of ADP, an arsenate:phosphate ratio of 5–10 is necessary for a decrease of the P:O ratio by 50 %. 3. In the absence of phosphate, ADP stimulates the arsenate-induced respiration by 60 %. Atractyloside has no effect on the ADP-independent respiration, but stops the stimulation by ADP, whether added before or after the ADP. 4. Low concentrations of aurovertin inhibit the arsenate-induced respiration to the same extent as oligomycin. However, with 9 μg aurovertin per mg protein, in the presence of arsenate, the respiration is greater than with low concentrations. This extra respiration is inhibited by oligomycin or by phosphate. 5. The arsenate-induced ATPase amounts to only about 15 nmoles phosphate per mg protein per min ( cf. 20 for endogenous ATPase, and 300 for 2,4-dinitrophenol-induced ATPase, in the same units). The arsenate-induced ATPase is inhibited by oligomycin, but not by azide. 6. It is concluded that stimulation by ADP of arsenate-induced respiration is not due to removal by ADP of endogenous phosphate. Nor is inhibition by phosphate due to removal of ADP. 7. It is concluded that the effects of arsenate provide strong evidence in favour of the existence of a ∼P intermediate of oxidative phosphorylation, either containing phosphate alone (X∼P) or containing ADP as well as phosphate (ADP-X∼P). 8. The different effects of oligomycin on dinitrophenol- and arsenate-induced respiration support the proposition that there are at least two non-phosphorylated high-energy intermediates (or one intermediate with two sites) in the sequence of energy-conserving reactions leading from the oxidoreduction reaction of the respiratory chain to ATP.

Oxidation of Long Chain Fatty Acids by Rat Liver Mitochondria

Abstract Long chain fatty acids are oxidized by rat liver mitochondria provided that endogenous adenosine triphosphate is not depleted. If endogenous ATP is depleted, e.g. by 2,4-dinitrophenol, external ATP as well as carnitine is required for fatty acid oxidation. Atractyloside does not block this reaction in media containing orthophosphate buffer. Oligomycin can substitute for carnitine, but the oligomycin-dependent route is inhibited by atractyloside. These experimental situations localize two ATP-dependent fatty acid-activating systems, one of which is linked to carnitine. The dinitrophenol-insensitive, GTP-specific fatty acyl coenzyme A synthetase is active only in the absence of orthophosphate. Inhibition of oxidation of fatty acids by atractylate in phosphate-free media is not relieved with oligomycin or carnitine.

High and Low Energy States of Cytochromes: III. IN REACTIONS WITH CATIONS

Upon addition of low concentrations of calcium or manganese to pigeon heart mitochondria in a phosphate-free medium, a characteristic pattern of steady state changes ensues which leads to a high absorbance at 560 mµ (at 300° K) characteristic of cytochrome b. Other components of the respiratory chain are highly oxidized; electron transport from succinate and glutamate is inhibited, as is energy transfer into the pathway for diphosphopyridine nucleotide reduction. The spectroscopic changes and the inhibition of electron transfer represent State 6. Low concentrations of cations are required for half-maximal change in cytochrome b; about 15 µ m calcium gives half-maximal effect. The calcium concentration required is a function of pH; the response of cytochrome b of the largest magnitude is observed at pH 8. High concentrations of calcium (50 to 150 µ m ) decrease this effect. The inhibited state is readily reversed by phosphate, leading to reduction of all the components of the respiratory chain except cytochrome b, which proceeds to a less absorbing state. Under these conditions, the functions of electron transport, energy-linked pyridine nucleotide reduction, and response to adenosine diphosphate are largely restored. The inhibited state of electron transfer is readily reactivated by uncoupling agents, but energy-linked pyridine nucleotide reduction is not reactivated. Examination of cytochrome b spectra at low temperature (77° K) indicates the accumulation of a compound absorbing at 555 mµ in the presence of low concentrations of cations and its transformation to a compound absorbing at 565 mµ upon addition of phosphate. Cytochrome c responds most rapidly to cations; cytochrome b and reduced pyridine nucleotide respond less rapidly, but at low concentrations of cations. The alkalinization of the cristal membrane (29) is a slower reaction that requires higher cation concentrations. Both the reaction of cytochromes with cations and the alkalinization of the cristal membrane are considered as factors in causing the inhibited state of electron transfer, State 6.

Metabolic roles of inorganic polyphosphates in chlorella cells

1. 1. A new type of inorganic polyphosphate (poly-P i) was detected in Chlorella cells and named Poly-P i “D”. It is extractable with 2 N KOH at 37° together with Poly-P i “C” which has been reported earlier. Upon neutralization of the extract with perchloric acid, Poly-P i “C” is co-precipitated with perchloric acid while Poly-Pi “D” remains dissolved. 2. 2. Using uniformly 32P-labeled Chlorella cells which are subjected to various environmental conditions, the metabolic role of Poly-P 1 “D”, as well as those referred to earlier as Poly-P i's “A”, “B” and “C” were investigated. 3. 3. On incubating the 32P-labeled algal cells in a phosphate-free medium under photosynthetic conditions, ribonucleic acid increased and Poly-P i “D” decreased. When the 32P-labeled algal cells were subcultured in the normal “cold” medium under photosynthetic conditions, a large amount of external phosphate but only a small amount of endogenous 32P was incorporated into the ribonucleic acid fraction. In the meantime, the 32P in Poly-P i's “C” and “A” decreased considerably while that in Poly-P i's “B” and “D” decreased slightly or remained constant. It has been reported that deoxyribonucleic acid and phosphoprotein increased with contamitant decrease of Poly-P 1's “A”, “B” and “C” in a phosphate-free medium under photosynthetic condition, and that 32P in the fractions of deoxyribonucleic acid and protein continued to increase when the 32P-labeled algal cells were subcultured in the normal “cold” medium 1. It was therefore, inferred that, under photosynthetic conditions, Poly-P i's “C” and “A” serve as intermediates in the transfer of phosphate to the phosphorus compounds synthesized such as deoxyribonucleic acid and phosphoprotein, while Poly-P i's “B” and “D” function as reservoirs of phosphate which are utilized only in the absence of exogenous phosphate source. 4. 4. In the dark the 32P in Poly-P i “A” continued to increase slowly both in the phosphate-free medium and in the normal cold medium, while no significant change was observed in the levels of 32P or total phosphate of the other phosphorus compounds. This suggests that Poly-P i “A” accepts phosphate—independently of light— from some intracellular phosphate source.

Derepression of alkaline phosphatase synthesis by chloramphenicol and canavanine inhibition

Derepression of alkaline phosphatase synthesis in Escherichia coli strain PR1 is produced by growth of the cells in excess phosphate medium containing the protein synthesis inhibitors chloramphenicol or canavanine. Upon reversal of protein synthesis inhibition, repression is restored only very gradually at 37°C, the more rapidly the lower the temperature. This slow re-establishment of repression is similar to the repression lag observed after derepressed growth in phosphate-free medium ( Gallant & Stapleton, 1964 ). This effect of protein synthesis inhibition is consistent with the notion that the repressor of alkaline phosphatase is a protein which is metabolically unstable. Inhibition of its continued synthesis accordingly leads to a drop in repressor level and consequent derepression in excess phosphate. Canavanine appears to be at least 3 times as effective as chloramphenicol in inhibiting repressor synthesis, which suggests that the repressor protein is rich in arginine.

EFFECT OF RADIOISOTOPE DISINTEGRATION ON CELL CULTURE CELL VIABILITY.

Stent and Fuerst(1) have reviewed the applications of the decay of biologically incorporated radioisotopes to genetic and physiological studies in phages and bacteria, By controlled incorporation of a radioisotope into a particular biochemical compound and by using suitable experimental manipulations one may obtain transmutation of one element into another in situ and determine the biological consequences of transmutation under varying physiological conditions. If applicable to mammalian cells in culture, studies similar to those reviewed by Stent and Fuerst(1) could be made with animal cells. The functioning of the blueprint, DNA, for phage synthesis has been examined in detail by Stent and his co-workers (2,3) using 32P decay. They found that the information was transferred to a structure not inactivated by 32P decay after 9 minutes under growing conditions and that protein synthesis was required for this transfer. These studies have rarely been extended to higher organisms except for the observations that growth in a solution containing a high level of 32P was abnormal and that the 32P disintegrations were mutagenic in Droso-philia and Habrobracon (4,5,6). A priori mammalian cells would presumably be more sensitive to decay of 32P than microbial cells since the β particle emitted has a longer track inside the mammalian cells, thus superimposing ionizing effects onto that of nuclear recoil and transmutation. The wider function of phosphorus-containing compounds in mammalian cells might be expected either to increase or decrease their sensitivity to this radiation depending upon the essentiality and number of replicas of the incorporating material. Carrier free 32P as phosphate was obtained from the Oak Ridge National Laboratory and was sterilized by autoclaving. Phosphate-free medium 199 was obtained from Microbiological Associates, Bethesda, Md., and for the purposes of this experiment it was supplemented with 1 mμmole per ml of PO4.

THE SUCCINIC OXIDASE ACTIVITY OF RAT LIVER MITOCHONDRIA

Rat liver mitochondria diluted into a high-potassium phosphate-free medium containing 2 × 10−5 M DNP take up oxygen readily if succinate is added at the same time as the mitochondria. If the addition of succinate is delayed, the respiration elicited is markedly reduced, falling to about 5% of the zero-time control at 2 minutes. Succinate-linked respiration can be restored by the addition of ATP or of cysteine sulphinic acid. A spontaneous recovery occurs on prolonged incubation (> 20 minutes). The inactivation, at least at such short incubation times, is dependent on the presence of DNP, and partial protection is provided by inorganic phosphate and the substitution of sucrose for KCl. Considerable protection is also provided by the presence of 1.5 mM Amytal. These results are all consistent with the hypothesis that oxaloacetate is produced from endogenous substrate during the first 1 or 2 minutes after dilution of the mitochondria, and that the failure to demonstrate succinic oxidase activity after this time is due to the inhibition of succinic dehydrogenase by oxaloacetate.

Über die Wirkung von Arsenat auf Nitratreduktion, Atmung und Photosynthese von Grünalgen

The effect of sodium arsenate on the metabolism of the green alga,Ankistrodesmus braunii (strain Marburg) increases with increasing arsenate concentration and with decreasing phosphate concentration. Above 10−4 M arsenate, in a phosphate-free medium of pH 4.1, an increase in respiratory oxygen uptake and inhibition of photosynthesis were observed. At the highest concentration used (5·10−2 M arsenate), the rate of respiration was about 250% of that of the controls, whereas photosynthesis had dropped to about 20% of the normal value. In addition, arsenate was found to be an inhibitor of the reduction of nitrite in the dark, whereas it does not inhibit the first step of nitrate reduction,i.e. the reaction nitrate → nitrite. Therefore, in a solution containing nitrate, addition of arsenate leads to accumulation of nitrite. These results further support the assumption, derived previously from experiments with 2,4-dinitrophenol, that the further reduction of nitrite requires high-energy phosphate, in contrast to the first step of nitrate reduction.