Acid-soluble Phosphate Esters Research Articles

The Journal of Biological Chemistry, Volume 203, 1953: Enzyme secretion and the incorporation of P32 into phospholipides of pancreas slices.

1. When enzyme secretion was stimulated by carbamylcholine or acetylcholine (with eserine) in slices of pigeon pancreas, the incorporation of P32 into the phospholipide fraction of the stimulated slices was, after 2 hours, 4.8 to 8.7 (average, 7.0) times greater than the incorporation of P32 into the phospholipides of control slices. Neither respiration nor the incorporation of P32 into acid-soluble phosphate esters was increased. 2. Pilocarpine, which on a weight for weight basis was much less effective than carbamylcholine or acetylcholine in stimulating enzyme secretion in pancreas slices, was also much less effective in stimulating the uptake of P32 into phospholipides. 3. The stimulatory effects of carbamylcholine on both enzyme secretion and the incorporation of P32 into phospholipides were abolished by atropine. 4. The specific activity of the phospholipides from slices incubated anaerobically was less than 5 per cent of that observed aerobically. Anaerobically, carbamylcholine did not stimulate the incorporation of P32 into phospholipides to any significant extent. The specific activity of the acid-soluble phosphate esters after anaerobic incubation was 34 per cent of that found aerobically. 5. Cholinergic drugs had little or no effect on the incorporation of P32 into the phospholipides of the following tissue slices: pigeon and guinea pig liver, guinea pig heart ventricle, pigeon gizzard (smooth muscle), and guinea pig kidney cortex. A relatively slight stimulation of P32 uptake into phospholipides was observed in slices of pigeon brain (65 per cent) and guinea pig brain cortex (40 per cent). 6. Stimulation of amylase synthesis in slices of pigeon pancreas by the addition of a mixture of amino acids had no effect on the incorporation of P32 into phospholipides.

The bacterial wilt, uptake of phosphate, and phosphate ester levels in the resistant and susceptible alfalfa plants

7 days or 7 weeks old alfalfa plants (Medicago sativa L.), susceptible (S) and resistant (R) to bacterial wilt, were inoculated withCorynebacterium michiganense pv.insidiosum and on day 8 and 15 after inoculation the levels of acid-soluble phosphate esters (P-esters) were determinated by means of32P labelling in the shoots or roots. The most significant changes were recorded in the roots of the older R plants grown in full Knop nutrient solutions on day 8 after inoculation. The marked reduction of inorganic phosphate (P1) uptake by whole R plants is accompanied by a decrease in the levels of fructose-l, 6-bisphosphate (Fru-P2), glucose-6-phosphate (Glc-6-P), fructose-6-phosphate (Fru-6-P), adenosine mono-, and diphosphate (AMP and ADP), phosphorylcholine (P-choline) and a proportional increase in the level of P1. In the S plants, infection affected neither P1 uptake nor P1 proportions. In the plants grown after inoculation in diluted Knop’s solutions (0.147 mM KH2PO4), infection induced a reduction of the radial transport of P1 to the segments of R roots whereas a reduction of the levels was only recorded in some P-esters [AMP, ADP, dihydroxyacetone phosphate (DHAP), and P-choline, but no decrease of Fru-P2, Glc-6-P and Fru-6-P]. In the S plants, P1 transport and the levels of P-esters were increased by the infection. P1 transport exhibited considerable metabolic dependence (DNP, DCCD). Bacterial infection probably had no influence on the activity of the plasma membrane ATPases.

Characterization of phosphate esters in the shoots and roots of alfalfa plants susceptible and resistant to bacterial wilt

Acid-soluble phosphate esters were determined in extracts of plant material after a 24 h exposure of the roots of intact alfalfa plants to nutrient media labelled with32Pi. Similar phosphate ester patterns were found in 2-, 3-, 8-, and 9-week-old plants with the exception of Gra-P which could be detected only in shoot extracts. However, phosphate ester levels differed in the shoots and roots. Whereas Fru-P2, Glc-6-P, Fru-6-P, and adenine nucleotides were more abundant in the shoots, Grn-P and P-choline levels were higher in the roots. Certain differences in the levels of P-esters were also recorded between plants susceptible and resistant to bacterial wilt.

FRACTIONATION OF THE ACID-SOLUBLE PHOSPHATE ESTERS IN THE LESIONS OF ECZEMA AND PSORIASIS.

British Journal of DermatologyVolume 75, Issue 8-9 p. 358-362 FRACTIONATION OF THE ACID-SOLUBLE PHOSPHATE ESTERS IN THE LESIONS OF ECZEMA AND PSORIASIS. M.G.P. McCABE B.Sc, M.G.P. McCABE B.Sc Research Assistant. The Institute of Dermatology, St. John's Hospital for Diseases of the Skin, Lisle Street, London, W.C.2. The Institute of Diseases of the chest, Brompton Hospital, Fulham Road, London, S.W.3.Search for more papers by this authorP. D. MIER B.Sc, P. D. MIER B.Sc Lecturer. The Institute of Dermatology, St. John's Hospital for Diseases of the Skin, Lisle Street, London, W.C.2.Search for more papers by this author M.G.P. McCABE B.Sc, M.G.P. McCABE B.Sc Research Assistant. The Institute of Dermatology, St. John's Hospital for Diseases of the Skin, Lisle Street, London, W.C.2. The Institute of Diseases of the chest, Brompton Hospital, Fulham Road, London, S.W.3.Search for more papers by this authorP. D. MIER B.Sc, P. D. MIER B.Sc Lecturer. The Institute of Dermatology, St. John's Hospital for Diseases of the Skin, Lisle Street, London, W.C.2.Search for more papers by this author First published: August 1963 https://doi.org/10.1111/j.1365-2133.1963.tb13574.xCitations: 7 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume75, Issue8-9August 1963Pages 358-362 RelatedInformation

A Positive Feedback Mechanism of Adenosine Triphosphate Synthesis in Erythrocytes

IN previous papers, Nakao et al.1,2 reported that adenosine triphosphate (ATP) is regenerated in erythrocytes stored for more than several weeks or treated with fluoride3, when the cells are incubated with nucleoside and adenine at 37° C. In these aged or fluoride-treated cells, most of the nucleotides and other acid-soluble phosphate esters were decomposed almost completely and energy metabolism almost entirely stopped. To elucidate the mechanism of adenosine triphosphate synthesis in such erythrocytes, the following observations were carried out :

Glycolysis and respiration in Ehrlich ascites tumour cells. IV. Pasteur effect, malonate Pasteur effect and Crabtree effect.

Summary.1. The aerobic inhibition of glucose utilization i. e. the Pasteur effect in Ehrlich ascites tumour cells decreased when the concentration of inorganic phosphate in the medium and the level of intracellular inorganic phosphate increased. It was accompanied by a rise in acid soluble phosphate esters. A higher aerobic than anaerobic glucose uptake was observed in IAA inhibited cells, or by incubation in a strong phosphate buffer (negative Pasteur effect).2. In ascites tumour cells, partially blocked with malonate, fumarate in contrast to citrate, stimulated oxygen uptake and inhibited glycolysis (the malonate Pasteur effect). This effect also decreased when the concentration of inorganic phosphate in the medium and the level of intracellular, inorganic phosphate increased. It was inhibited by DNP and anaerobiosis.3. In glycolyzing cells, blocked with malonate, fumarate further enhanced the acid labile phosphate and ATP, in spite of the accompanying rise in oxygen uptake. Thus the Crabtree effect could not be explained by lack of total cellular inorganic phosphate or phosphate acceptors.4. The Pasteur and Crabtree effects in Ehrlich ascites tumour cells are explained by the preferential channeling of limited a‐mounts of cellular inorganic phosphate which is to some extent carried by adenine nucleotides into certain reactions at the expense of others.

Glycolysis and respiration in Ehrlich ascites tumour cells. I. Phosphate metabolism in relation to glycolysis and the Crabtree effect.

Summary.1. Between 0—40 μmoles of inorganic phosphate per ml of extracellular fluid was rate determining for aerobic glycolysis in Ehrlich ascites tumour cells. The oxygen uptake during aerobic glycolysis was either unaffected or slightly inhibited by phosphate.2. Aerobic glycolysis was accompanied by a reduction in cellular inorganic phosphate and an accumulation of total acid soluble organic phosphate, acid labile phosphate, hexosephosphates and ATP. The ATP increased at the expense of ADP and AMP. In IAA inhibited cells, acid soluble phosphate esters accumulated during aerobic glycolysis, but the total adenine nucleotides decreased markedly.3. Glycolysis as well as respiration greatly stimulated the uptake of P32 into organic phosphate fractions, while the concentration of inorganic phosphate in the medium seemed to be a major factor determining the total uptake of P32 by the ascites tumour cells.

Interrelations in respiratory, phosphorylative and mitotic activities of Ehrlich ascites tumor cells: Influence of dinitrophenol

The effect of graded concentrations of 2,4-dinitrophenol (DNP) on interrelationships between respiration and phosphorylations of Ehrlich ascites tumor cells was studied in vitro. DNP influence seemed to be mediated through two distinct mechanisms. One, coming into play at low DNP concentrations, effected the breakdown of labile and other acid-soluble phosphate esters in the cell, leading to increased concentrations of inorganic phosphorus in cells and medium. No pronounced impairment of inorganic phosphate uptake from medium into cells or of labile phosphorus formation within cells occurred, as evidenced by the continued incorporation of P 32. Associated with this phase of DNP action, there was a rise in the rate of cellular respiration. As the concentration of DNP passed a certain threshold level, a second mode of action became apparent. Incorporation of P 32 into cellular phosphate fractions, as well as respiration, became increasingly inhibited. Because of increased permeability of cells to inorganic and ester phosphorus at higher DNP concentrations and because of the inability of cells to regenerate labile phosphorus, it was suggested that the second phase of DNP action was associated with changes in the cell membrane. Incorporation of P 32 into DNA-P was enhanced by a low concentration of DNP. There appeared to be some indication that cellular synthesis of DNA might be independently related to both the rate of O 2 uptake and the availability of high-energy phosphorus.

The incorporation of 32P into the nucleotides of ribonucleic acid in pancreas slices during enzyme synthesis and secretion

The incorporation of 32P into the nucleotides of RNA has been followed in pigeon pancreas slices under a variety of conditions. This incorporation is inhibited 90–95% anaerobically. Stimulation of enzyme synthesis about 100% by the addition of a complete amino acid mixture is accompanied by 20–40% increases in respiration and the specific activities of RNA and acid-soluble phosphate esters. Omission of tryptophan abolishes the stimulatory effect of the complete amino acid mixture of amylase synthesis but has no effect on respiration or the turnover of RNA or acid-soluble phosphate esters. These observations suggest that the slight stimulation of 32P incorporation into RNA by the complete amino acid mixture is due to the stimulation of general metabolism by the amino acid mixture, rather than to a link between enzyme synthesis and RNA synthesis. Stimulation of enzyme secretion in vitro with carbamylcholine or acetylcholine is not accompanied by any increase in the rate of incorporation of 32P into RNA. Stimulation of enzyme secretion in mouse pancreas in vivo is accompanied by an approximate 50% decrease in the specific activity of RNA relative to that of the acid-soluble phosphate esters. A possible mechanism whereby nucleic acids may function in the synthesis of proteins and other specific ( e.g. nucleic acids and type-specific polysaccharides) is discussed.

ENZYME SECRETION AND THE INCORPORATION OF P32 INTO PHOSPHOLIPIDES OF PANCREAS SLICES

1. When enzyme secretion was stimulated by carbamylcholine or acetylcholine (with eserine) in slices of pigeon pancreas, the incorporation of P32 into the phospholipide fraction of the stimulated slices was, after 2 hours, 4.8 to 8.7 (average, 7.0) times greater than the incorporation of P32 into the phospholipides of control slices. Neither respiration nor the incorporation of P32 into acid-soluble phosphate esters was increased. 2. Pilocarpine, which on a weight for weight basis was much less effective than carbamylcholine or acetylcholine in stimulating enzyme secretion in pancreas slices, was also much less effective in stimulating the uptake of P32 into phospholipides. 3. The stimulatory effects of carbamylcholine on both enzyme secretion and the incorporation of P32 into phospholipides were abolished by atropine. 4. The specific activity of the phospholipides from slices incubated anaerobically was less than 5 per cent of that observed aerobically. Anaerobically, carbamylcholine did not stimulate the incorporation of P32 into phospholipides to any significant extent. The specific activity of the acid-soluble phosphate esters after anaerobic incubation was 34 per cent of that found aerobically. 5. Cholinergic drugs had little or no effect on the incorporation of P32 into the phospholipides of the following tissue slices: pigeon and guinea pig liver, guinea pig heart ventricle, pigeon gizzard (smooth muscle), and guinea pig kidney cortex. A relatively slight stimulation of P32 uptake into phospholipides was observed in slices of pigeon brain (65 per cent) and guinea pig brain cortex (40 per cent). 6. Stimulation of amylase synthesis in slices of pigeon pancreas by the addition of a mixture of amino acids had no effect on the incorporation of P32 into phospholipides.

The incorporation of radioactive phosphate into feline pneumonitis virus in the chick embryo yolk sac.

that the energy for the synthesis of feline pneumonitis virus in chick embryo yolk sac is supplied by the aerobic oxidation of endogenous substrates and the concomitant production of organic acidsoluble phosphate esters. Infected yolk sacs, during the 24 hours just preceding the death of the embryos, incorporate phosphorus into their desoxypentose nucleic acid (DNA) fraction much more rapidly than do uninfected yolk sacs. The infection may also cause an increase in the rate of incorporation of phosphorus into pentose nucleic acid (PNA). This investigation was made to clarify the mode of feline pneumonitis virus reproduction. Virus purified from infected yolk sacs labeled with radioactive phosphorus (P32) was fractionated and compared with the yolk sac pool from which it was prepared. Both DNA and PNA appear to be integral components of the virus, and the nucleic acids of the purified virus have higher specific activities than any chemical fraction of the yolk sac pool from which the virus is isolated.

An autoradiographic study with P32 of the rabbit uterus.

In recent years the biochemistry of the endometrium has been extensively studied, especially with regard to the manner in which oestrogens and progesterone regulate the phosphorus metabolism. As early as 1930 Okey, Bloor & Corner reported increased amounts of phospholipids in the pig endometrium during the luteal phase and later Walaas & Walaas (1950 a and b) observed that oestradiol benzoate increased the turnover of acid-soluble phosphate esters in the rat myometrium.

Failure of phosphorus-32 to exchange with organic phosphorus compounds.

WHEN radioactive phosphate is used in biological experiments, the interpretation of the results depends upon the assumption that the labelled inorganic phosphate does not exchange with the phosphate of organic esters except via metabolic reactions. Evidence to support this assumption is limited. Hevesy and Aten1 were satisfied that phosphorus-32 did not exchange with the Embden ester (hexose-6-phosphate), and apparently inorganic phosphate does not exchange with glycerophosphoric acid2. Studies on human erythrocytes in this laboratory3 have made it desirable to determine whether phosphorus-32 is able to enter certain other acid-soluble phosphate esters by simple exchange.

Metabolism in Different Parts of the Brain, Especially in the Epiphysis, Measured with Radioactive Phosphorus

This investigation constitutes an attempt to divide the brain into anatomically well defined regions and to compare the phosphate metabolisms of the latter by means of radioactive P. The highest phosphate content expressed in per cent of wet weight was found in the epiphysis, the lobus anterior and posterior of the hypophysis, the plexus chorioideus and the substantia perforata. Measurements with radioactive phosphorus suggested that a division of the parts of the brain into two groups was possible, viz. an active group with a lively phosphate metabolism and a less active group with a slow phosphate metabolism. The former group includes the epiphysis, the lobus anterior and posterior of the hypophysis and the plexus chorioideus, whereas the latter consists of the lobi olfactorius, parietalis and occipitalis, the thalamus, the anterior part of tuber cinereum, the posterior part of tuber cinereum, the corpus mamillare, the cerebellum, the pons, the corpora quadrigemina and the medulla oblongata. The substantia perforata has an intermediate position, but seems in the majority of cases to belong to the group with a lower phosphate metabolism. The activities were measured in relation to that of the cerebellum, which was taken as 100. The radioactive phosphorus accumulates 40 minutes after injection in the epiphysis to about 65 % in form of acidsoluble phosphate esters, in about 25 % in form of free phosphate and in about 10 % in compounds insoluble in trichloracetic acid. The activity of the epiphysis is generally higher than that of the hypophysis. The phosphate metabolism in this part was often twice or three times as vigorous as that of the lobus anterior hypophysis. It is therefore most probable that the epiphysis has an important function. Experiments are being carried out to investigate the function of this organ by the method described in this paper. The most active brain components, following immediately after the epiphysis, are the plexus chorioideus and the lobus anterior and posterior of the hypophysis. Owing to the considerable quantities of blood contained in the plexus chorioideus, particular conditions prevail in that organ. Those conditions are discussed in some detail. The average phosphate metabolism of these parts of the brain is four to five times that of the cerebellum. Other parts of the brain tested exhibit, generally speaking, the same metabolism as the cerebellum. It seems to be significant that the anterior part of the tuber cinereum exhibits a somewhat livelier activity than the majority of the brain components belonging to this group. We owe thanks to Docent J. MÖLLERSTRÖM for having placed his laboratory facilities at our disposal and for the interest he payed to our investigations and to Prof. MANNE SIEGBAHN for having provided us with the radioactive phosphorus necessary for this work.