Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Changes in Nicotinic Acid Content and Its Nucleotide Derivatives of Rice and Wheat Seeds during Germination

Relative distribution of bound and free forms of nicotinic acid in rice and wheat seeds and their metabolism during germination were the subject of the present investigation. Measurement of the levels of NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) formed another part of the work. Total nicotinic acid in both rice and wheat increased with germination and was maximum at 72 hours. From this time onwards, it began to decline rapidly and at the end of experiment, i.e., after 120 hours, it was lower than that for ungerminated seeds on per seedling basis, although it was slightly higher on per g dry weight basis. Ungerminated seeds of wheat and rice contained about 89 per cent and 80 per cent respectively of their total nicotinic acid in bound form which became partially free in course of germination. Total nucleotides (oxidised plus reduced forms) increased progressively up to 96 hours followed by a slight fall at 120 hours. NAD reached a maximum at 24 hours and fell gradually thereafter. The depletion of NAD was associated with a progressive accumulation of NADH. NADP decreased from the peak value at 72 hours. Formation of NADP and its maintenance at high level depend on NAD in the oxidised form and the content slowed down in tissues with higher NADU/NAD ratio. A relatively large amount of NADPH was present throughout the experiment.     

A RAPID METHOD FOR SELECTIVE SILVER STAINING OF NERVE FIBRES AND NERVE ENDINGS IN MOUNTED PARAFFIN SECTIONS

Abstract The staining described in this paper fundamentally is performed by two manipulations, namely (1) the treatment of deparaffinized sections for about fifteen minutes in a solution containing 15 per cent silver nitrate, 10 per cent potassium nitrate and 0.05 per cent glycine, and (2) the reduction for one minute in a solution of 1 per cent pyrogallol, 55 per cent ethyl alcohol and a trace (0.002 per cent) of nitric acid. After toning, dehydrating and mounting in the usual manner the sections generally are ready for examinations within less than an hour. The method is available for specimens fixed in the ordinary fixatives but those containing oxidizing metal compounds. The procedure is discussed from a theoretical point of view and some results are shown in photomicrographs.     

The growth of wheat plants in humic acid solutions under axenic conditions

Summary A technique is described for growing wheat plants in nutrient solutions containing C¹⁴-labelled humic acid under axenic conditions. The general appearance of axenic plants was indistinguishable from plants grown in association with microbes. C¹⁴-labelled humic acid enhanced the growth of both roots and shoots showing that by-products of microbial degradation of humic acid are unnecessary for this enhanced plant growth. Thus humic acid had a direct effect on the growth processes. The C¹⁴-labelled humic acid was taken up by the roots and virtually none was transported to the shoot. Only some 30 to 40 per cent of the incorporated radioactivity was associated with the root cell walls and thus more than 60 per cent was in the cytoplasm and may have influenced the biochemical processes involved in the regulation of plant growth.     

Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

Uric acid synthesis by rat liver supernatants from purine bases,nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0·5 mM and guanosine and guanine, 0·1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g^?1 of wet liver min^?1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylo-succinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min^?1 per mg^?1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0·1 and 0·05 mM concentrations was also determined. ATP (1 nM) strongly inhibited uric acid synthesis from 0·05 mM AMP (91 per cent) and from 0·05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM ) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0·05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0·012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.     

CRYSTALLIZATION OF PEPSIN FROM ALCOHOL

1. Pepsin is soluble in 65 per cent alcohol and may be readily crystallized from 20 per cent alcohol. The crystals appear as needles or plates which may be transformed into the usual hexagonal bipyramids by recrystallization from water. The different crystals are probably two crystalline forms of the same chemical substance. 2. The enzyme is quite stable in 20 per cent alcohol at pH 2.0 but is inactivated by high concentrations of alcohol. 3. The enzyme is stable for several hours in 65 per cent alcohol at pH 4.0 to 5.0 but is rapidly inactivated in more acid solution. 4. No increase in activity could be noted after treatment with hydrogen peroxide. 5. No proteolytic activity either before or after treatment with hydrogen peroxide could be found in trichloracetic acid filtrates, butyl alcohol extracts of pepsin preparations, or oxidized phenylhydrazine solutions.     

Nitrogen stress in wheat — Its measurement and relation to leaf nitrogen

Summary The intensity of nitrogen deficiency in the young wheat plant was measured over a range of 1 to 83 per cent of nitrogen stress. Within these limits of tress relative growth rate of tops fell from 11 to 2 per cent per day.Symptoms of nitrogen deficiency appeared when the stress was greater than 40 per cent.Nitrogen stress in the variety Gabo is calibrated in terms of the concentrations of total nitrogen, soluble nitrogen and free ninhydrin nitrogen of the youngest fully expanded leaf.     

The soluble components of chromaffin granules a carbon-13 NMR survey

Carbon-13 NMR spectra of the reconcentrated chromaffin granule lysate have been obtained at 50 MHz and 62.9 MHz. The spectrum contains a number of assignable resonances in addition to those of the main soluble components (catecholamines, adenine nucleotides and chromogranin). Guanine and uridine nucleotides are present at levels of 0.13 and 0.08 mol/mol adenine nucleotides, respectively. Concentrations of cytidine nucleotides and NAD⁺ are below the detection limit (0.02 mol/mol adenine nucleotides). An unidentified low molecular weight species, thought to be an adenine-containing oligonucleotide, is also present. Ascorbic acid was observed at a concentration of 0.14 mol/mol adenine nucleotides, but both dopamine and dehydroascorbic acid were below the detection limit. Protein resonances agree well with the reported amino acid composition of chromogranin A, with the exception of tryptophan and glutamine which have not previously been measured. The concentrations of these residues are estimated to be 12 ± 3 and 39 ± 5 residues per 77 000 dalton unit of chromogranin A. Substantial intensity due to unsaturated fatty acid side-chains in solubilized lipid is seen in the olefinic carbon region and in the methylene region, suggesting the presence of lipoprotein. Unassigned carbohydrate resonances are also present, but are largely obscured by sucrose in the isolation medium.     

THE EFFECT OF LOW OXYGEN TENSION ON TISSUE METABOLISM (RETINA)

Lactic acid production by rat retina in a medium containing phosphate was studied chemically. One half as much lactic acid was found as in a medium containing bicarbonate. In our experience the rate of respiration in a phosphate medium was sensitive to oxygen tension, for it was 38 per cent lower at 10 per cent and 51 per cent lower at 5 per cent oxygen than at 100 per cent oxygen. Previously Laser had reported no decrease in respiration at 5 per cent oxygen in phosphate medium. In phosphate medium, when the oxygen tension was varied, respiration and glycolysis bore a reciprocal relationship to each other. In bicarbonate medium, when the oxygen tension was lowered from 95 per cent to 5 per cent there was no significant change in the respiration, but glycolysis was increased nearly to the anaerobic level. This agrees with the earlier experiment of Laser in bicarbonate medium and adds support to his conclusion that the rate of glycolysis is controlled by oxygen tension rather than by the rate of respiration, under the conditions of the experiment.     

FRACTIONATION OF GELATIN

1. It is possible to fractionate gelatin by means of reprecipitation at 23°C. of a salt-free solution of pH 4.7 into two fractions, one of which is soluble in water at any temperature, and a second one which does not dissolve in water even when heated to 80°C. 2. The proportion of the soluble fraction in gelatin is much greater than of the insoluble one. 3. The insoluble fraction of gelatin does not swell when mixed with water, but it does swell in the presence of acid and alkali which finally dissolve it. 4. Blocks of concentrated gel made by dissolving various mixtures of the soluble and insoluble fractions of gelatin in dilute NaOH swell differently when placed in large volumes of dilute buffer solution pH 4.7 at 5°C. The gel consisting of the insoluble material shows only a trace of swelling, while those containing a mixture of soluble and insoluble swell considerably. The swelling increases rapidly as the proportion of the soluble fraction increases. 5. A 5 per cent gel made up by dissolving the insoluble fraction of gelatin in dilute NaOH loses about 70 per cent of its weight when placed in dilute buffer pH 4.7 at 5°C. A similar gel made up of ordinary gelatin loses only about 20 per cent of its weight under the same conditions. 6. It was not found possible to resynthesize isoelectric gelatin from its components. 7. An insoluble substance similar in many respects to the one obtained by reprecipitation of gelatin is produce on partial hydrolysis of gelatin in dilute hydrochloric acid at 90°C.     

THE BIOSYNTHESIS OF C13 COMPOUNDS : I. THE BIOSYNTHESIS OF C13-LABELED STARCH

1. Starch, containing 7.05 atom per cent C¹³ excess has been produced in the mesophyll cells of bean leaves, starting with C¹³O₂ containing 7.26 atom per cent C¹³ excess. Approximately 67 per cent of the C¹³ taken up by the leaves was determined in the starch fraction. 2. The soluble carbohydrate, containing 6.72 atom per cent C¹³ excess, accounts for approximately 23 per cent of the C¹³ taken up by the leaves. The remainder was principally in the coarse tissue fragments fraction (9.73 per cent of the C¹³ utilized). 3. The apparatus and procedures used in this experiment are described.     

A comparative study of the effects of the diamine oxidase inhibitor aminoguanidine, with or without dietary restriction, on the nucleic acid and protein composition of cardiac and type I and type II skeletal muscles of the rat.

It has been proposed that the diamine oxidase inhibitor aminoguanidine may be a potential therapeutically important anabolic agent. An investigation was therefore made into the effects of aminoguanidine treatment with or without nutritional restriction, on cardiac and skeletal muscles containing mainly of either Type I (i.e. soleus) or Type II fibres (i.e. plantaris) or a mixture of Type I and II fibres (i.e. gastrocnemius). After 3 weeks, dietary restrictions reduced cardiac weight, protein, RNA and DNA contents by between 31 per cent and 36 per cent. Similar, but smaller, reductions were observed in the soleus (18-31 per cent), plantaris (22-34 per cent) and gastrocnemius (22-34 per cent). Aminoguanidine had no effect on the heart of the rats fed ad libitum, nor did it alter the response to dietary restriction. Treatment with aminoguanidine had no overt anabolic effect on skeletal muscle, but a reduction in DNA content was observed. It was concluded that cardiac protein and nucleic acid contents are more sensitive to dietary deprivation than either anaerobic or aerobic skeletal muscles. Furthermore, aminoguanidine does not appear to promote growth or reduce catabolism as previous studies have suggested.     

Studies on potassium release in an inceptisol soil (Holambi Series) at the minimum level of exchangeable potassium

Summary Ammonium acetate extractable potassium in the soil reached a minimum value of 6.8 mg K/100g soil after 14 crops of wheat and pearl millet in the field without applying any potassium fertilizer. At this level of ammonium acetate extractable K both wheat and pearl millet utilized about, 90 per cent of the total K from non-exchangeable sources. Wheat and pearl millet were grown in this soil in the greenhouse at different levels of K. At K₀ level wheat utilized 86 per cent of the total K uptake from the non-exchangeable source and pearl millet, 95 per cent. At K₁ level, wheat utilized only 19 per cent but at higher levels of K, there was build up in the K status of soils. In the case of pearl millet at K₁, K₂ and K₃ levels 59, 13 and 22 per cent of total uptake were contributed by non-exchangeable forms. The total K uptake by pearl millet was more than double that by wheat. Plant analysis showed that 83 per cent of the total K in wheat was contained in the shoot portion and the rest in the roots. The corresponding figures for pearl millet were 94 and 6 per cent.     

Ribonuclease in Bovine Milk

The RNase (EC 2.7.7.16) in bovine milk was partially purified from milk whey. The basic properties and the hydrolyzing specificity of this enzyme were studied. This enzyme was heat stable. When RNA was used as the substrate, the pH optimum was 7.5 and 3′- nucleotides were produced, but the extent of the hydrolysis stopped at 31 per cent and core was left. C! and U! were hydrolyzed but purine cyclic nucleotides were not. Poly U was digested to 3′-uridylic acid passing through U! but poly A was not. These properties are quite similar to pancreatic RNase.     

THE CONDITIONS OF RECOVERY OF TRANSMISSIVITY OF NEWLY REPASSIVATED IRON WIRES IN NITRIC ACID

1. Passive steel wires were activated in a bath (Bath A) containing 70 v. per cent HNO₃ (in which they undergo prompt repassivation), and immediately transferred to a second bath (Bath B) containing HNO₃ of a concentration varying in different experiments. After varying intervals in this bath they were transferred while still passive to a third bath (Bath C) containing strong HNO₃ (70 or 100 v. per cent) and there immediately activated. 2. During the immersion in Bath B the wires progressively recover their ability to transmit activation waves in strong HNO₃. The measure of this recovery is the distance travelled by the activation waves in Bath C after the varying times of exposure in Bath B. Transmissivity as thus measured is at first incomplete (decremental) and later becomes complete. The minimal exposures in Bath B required to render wires completely transmissive in the strong acid of Bath C were determined for concentrations of HNO₃ between 10 and 100 v. per cent. With 100 v. per cent HNO₃ in Bath C, these exposures range from 40 minutes or more in 15 v. per cent to 10 minutes in 100 v. per cent HNO₃ (temperature 19–20° in all baths). 3. The time required for complete recovery varies inversely with the concentration of the acid in the recovery bath (Bath B), but increases rapidly with the concentration of the acid in the testing bath (Bath C). Hence at a time when a wire has recovered just sufficiently to transmit non-decrementally in a given strong acid (e.g., 70 v. per cent) it still transmits decrementally in a stronger acid. Complete recovery for transmission in 100 v. per cent HNO₃ requires about twice as long as for 70 v. per cent HNO₃. In HNO₃ of 50 v. per cent and less decremental transmission does not occur. 4. The indications are that recovery is an effect of the progressive solvent action of the external acid on the passivating oxide film, which at its first deposition appears to be relatively thick and hence resistant to electrochemical reduction. The final stage of recovery, when electrical sensitivity and speed of transmission are maximal, would on this hypothesis correspond to minimal thickness, possibly monomolecular. 5. The rate of recovery in Bath B is not far from proportional to the concentration of HNO₃ in the more dilute solutions, but in the higher, especially the strongly passivating, concentrations (70 to 100 v. per cent) the rate becomes appreciably slower than proportional, apparently because of the intense oxidizing action of these solutions, which reinforces the oxide sheet and retards the thinning process. 6. The bearing of these observations on the problem of the conditions of recovery in irritable living tissues (such as nerve) during the absolute and relative refractory periods is briefly discussed.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

NADPH-cytochrome P-450 reductase. Circular dichroism and physical studies.

NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis after mineral or organic acid hydrolysis. The free mobility of cytochrome P-450 reductase in sodium dodecyl sulfate was identical to that of several soluble proteins used as standards (i.e. ovalbumin, bovin serum albumin, erythrocuprein, beta-galactosidase). Molecular weight estimates from sedimentation equilibrium studies in the presence of guanidine hydrochloride (76,500) are consistent with those determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at various per cent gel concentrations (79,000 to 80,000). Computer analysis of circular dichroism spectra of cytochrome P-450 reductase in the far ultraviolet region indicated the presence of 34 per cent alpha helical and 16 per cent beta structure. The amount of random structure was calculated to be 50 per cent.     

The composition of soluble nucleotides in the developing wheat grain

A method is described for the extraction and measurement of soluble nucleotides from wheat grain. Nucleotides were separated (80-90% recovery) by paper chromatography followed by electrophoresis. The nucleotides extracted were ADP-glucose, ATP, ADP, AMP, and NAD; UDP-glucose, UTP, UDP, and UMP with smaller quantities of cytidine nucleotides.     

Free Amino Acids in the Leaves of Salvinia natans and Azolla filiculoides Grown in Light and Dark

Free amino acids were determined quantitatively by thin-layer chromatography and identified by various chromatographic methods in the leaves of two floating water ferns. Special interest was paid to the amount of γ-amino butyric acid. Water soluble nitrogen compounds increase in the leaves of both plants during the growth of three to four weeks in darkness. Compounds especially accumulating are ammonia and the amides asparagine and glutamine. The amount of most proper amino acids is reduced in the dark, except γ-amino butyric acid, which, in addition to amides, is the main amino compound increasing in darkness. 0.1 M HCl extracts the greatest amount of nitrogen compounds of low molecular weight from the dried leaves of the experimental plants. If we denote the amount of the dissolved amino compounds as 100 per cent, we can say that water extracts 96 per cent of the amino compounds, and that an 80 per cent ethanol solution extracts 79 per cent of these compounds.