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The effect of temperature upon the exchange rates between intra- and extracellular potassium in chick embryo muscle was determined by the use of radioactive potassium. The temperature coefficient of at least four-fifths of the cell potassium is large. At temperatures below 15°C., potassium is lost from the cell and is regained on warming. The results suggest the possibility that 20 per cent or less of the cell potassium may differ from the rest by being more rapidly exchangeable with the medium.

The objection by Bull to the estimation of bound water by ultrafiltration, because of an assumed adsorption of the reference substance, has been found invalid for glucose. No adsorption of glucose by the proteins, casein and gelatin, could be detected. The estimation of the bound water of proteins from the probable surface adsorption of water by proteins leads to only a small value.

Phenylhydrazine reacts with the periodate oxidation products of adenosine, guanosine, uridine, and cytidine to form bisphenylhy-drazones, which were isolated and characterized. These substances react further with phenylhydrazine to form adenine, guanine, uracil, and cytosine and 2- and 3- carbon fragments. Sodium borohydride reduces both aldehyde groups of the oxidized nucleosides when the reaction is carried out under alkaline conditions, but selective reduction of the aldehyde group distal to the purine or pyrimidine ring occurs under slightly acidic conditions. Ion exchange and paper chromatographic methods were developed for the examination of the dialdehydes and the corresponding reduction products were obtained from them. Evidence is presented indicating that the acid hydrolysis of the completely reduced products. the tri-alcohols, is probably initiated at the glycosidic linkage. A new micro method for measuring and removing periodate and iodate essentially in one step was developed. (auth)

The formation and metabolism of pseudouridine (5-ribosyluracil) in growing Tetrahymena pyriformis have been studied with the aid of labeled uracil, uridine, pseudouridine, and glucose as precursors. The pyrimidine rings of uracil, uridine, and pseudouridine serve equally well as precursors of the pyrimidine component of uridine and pseudouridine of ribonucleic acid. The ribosyl moiety of uridine labels the ribosyl portions of RNA uridine and RNA pseudouridine equally. Although the latter was not degraded to establish unequivocally that uridine ribosyl is present in pseudouridine ribosyl, the equivalence of specific activities and the evidence of the presence of precursor uridine ribosyl in the uridine of the RNA make it appear that the uridine-pseudouridine interconversion takes place by an intramolecular rearrangement and not principally through a free uracil or diribosyluracil intermediate. Although some transribosylation involving purine and pyrimidine ribosyl moieties and some formation of RNA pyrimidine ribosyl from glucose were observed, these appear to be of secondary importance. The data are consistent with, but do not prove, a rearrangement of pyrimidine nucleosides at the polynucleotide level.

The question of the permeability of mammalian erythrocytes to cations (with the exception of hydrogen ion) has long been a controversial one. The literature in the field has been reviewed by Ponder1 and Jacobs.2 Ponder states that, although there has been some evidence since 18913 that the red blood cell is permeable to cations, it has generally been assumed that it is not.4 Such an assumption requires awkward corrections to explain the experimental data. To avoid postulating a cation shift across the red cell membrane, Ponder1 states that Van Slyke and Cullen,5 Mellanby and Wood,6 and Doisy and Eaton7 have preferred to introduce corrections involving changes in red cell volume. However, Van Slyke and Cullen5 admit the possibility of a transfer of cations between plasma and corpuscles. For these volume changes, which are ascribed to water shifts, there is no good evidence, the most careful work giving results varying by as much as 100%8 or even varying in direction.9 Much of the interpretation is based on...

By using radioactive isotopes in tissue cultures, the rate of permeation of substances into cells can be measured independently of concurrent metabolic reactions of these substances. Techniques of obtaining and analyzing data are described. Examples are given using radioactive potassium and phosphorus. Using cultures of chick embryo muscle, turnover time for cell potassium is 6 hours, and for cell inorganic phosphate is 7 hours in the examples cited. Permeability rates, based on estimates of the cell surface involved and expressed as millimoles per cm.2 per hour, are of the order of magnitude of 10–6 for potassium and of 10–7 for phosphate.

Publisher Summary This chapter describes the nomenclature and symbolism of α-amino acids. The nomenclature used for α-amino acids in any biochemical context, from proteins to metabolism to bacterial cell walls to antibiotics and hormones, utilizes almost exclusively the trivial names that are common in the pre-molecular-biology days, when the composition of proteins dominated biochemical research. The Commission on Biochemical Nomenclature (CBN), created by the International Union of Pure and Applied Chemistry (IUPAC) and the International Union of Biochemistry undertook the task of codifying abbreviations and symbols for chemical names of special interest in biological chemistry. The section on polypeptides and proteins was based on those traditional trivial names, which had been formally approved by the IUPAC Commission on the Nomenclature of Organic Chemistry in 1949, and the symbols derived from them in 1947 by Brand and Edsall. The only changes included to change Try to Trp, to prevent error by accidental inversion, Nval to Nvl, Ileu to Ile, CySH to Cys, Glu-NH2 and Asp-NH2 to Gln and Asn, and Hypro and Hylys to Hyp and Hyl. The three-letter system was considered, so that sequences could be aligned for comparison.

Publisher Summary The separation of AMP, ADP, and ATP by ion exchange chromatography is accomplished by selective elution from strong-base anion exchangers in either the chloride or formate form. A typical chloride separation is as follows. The mixture of AMP, ADP, and ATP, at a pH of6 to 9 and in the presence of less than 0.01 M salt, is absorbed on the column and washed through with 0.01 M NH 4 C1. A volume of 0.01 M NH 4 C1 is used which will reduce the pH to 7 or less and remove any undesirable anions which may have been absorbed previously. AMP is removed by 0.005 N HC1 (a volume of up to 50 C.V. is required). ADP is removed slowly (about 120 C.V.) by 0.01 N HC1 or more rapidly by 0.01 N HC1 plus 0.04 N NaC1. ATP is removed slowly by the last-named reagent or rapidly by 0.01 N HC1 plus 0.2 N NaC1. The removal of each substance may be followed by spectrophotometric observation of the eluates, and the most efficient conditions for each substance, in relation to possible contaminants, selected by varying the volumes and the chloride concentrations.

Publisher Summary The chapter discusses the principles, materials, and the procedure followed in the preparation of pseudouridylate and pseudouridine. Pseudouridine (5-β-D-ribosyluracil) is a constituent nucleoside of RNA, particularly in those RNA's known collectively as “sRNA” and “tRNA.” Pseudouridine appears as the free nucleoside in mammalian urine (man, rat, mouse) because of tRNA degradation. The most characteristic property of pseudouridine and its derivatives is that it allows detection, even in the presence of its usual contaminant, uridine, is the bathochromic shift in the ultraviolet-absorbance spectrum in alkaline media. To prepare pseudouridine from pseudouridylate, pseudouridylate is dephosphorylated and recovered by standard procedures from the enzyme solution.. Separations of pseudouridine from uridine, of pseudouridylate from uridylate, and of isomeric forms of pseudouridine itself, can be effected on paper in various solvent systems. The most useful and rapid one is isobutyrate buffer, in which the pseudo compounds have distinctly lower RI values than their uridine analogs. This is possibly the best way to separate uridine and pseudouridine in small quantities. Isopropanolacetic acid, butanol-H 2 0, and so on, are employed as well for analytical purposes.

The major nucleosides obtained by hydrolysis of RNA are quantitatively assayed in less than 1 hr at the level of 1 or more nmoles by cation-exchange chromatography and a sensitive, continuous ultraviolet-absorbance monitoring device. A uniform cation-exchanger bead preparation (17.5 ± 2 μm diameter) permits operation at low (6–16 psi) pressures and rapid flow rates (1 ml/cm2 min) without loss of resolution. Analytical sensitivity is enhanced by improved flow cell and electronic circuit designs. A multi wavelength monitoring unit incorporated into the monitoring system aids in assaying substances of differing ultraviolet-absorption characteristics. Minor or altered nucleosides (20) and purine and pyrimidine bases (8) are separable, with few exceptions, by means of the single solvent chosen for the elution. The parameters determining the effectiveness of the separations have been explored and are expressed in terms permitting design modifications to improve the separation of refractory pairs or shorten the analysis time or both.

Ion-exclusion chromatography has been applied to separate nucleosides for analytical and preparative purposes. The ionized substances are variably repulsed or excluded from a negatively charged cation exchanger according to the degree to which they are negatively ionized by the pH of the eluting solution. The method reported successfully resolves a mixture of nanomole amounts of 4-thiouridine, pseudouridine, uridine, ribothymidine, guanosine, cytidine and adenosine in 1 h or less, and utilizes dilute, volatile eluants (0.02 M (NH 4 ) 2 CO 3 , pH 9.3 or 9.8).

The acid hydrolysis of calf thymus DNA by normal HCl at 100° results primarily in nucleotides containing one more phosphate than pyrimidine, a number of which have been isolated and identified by ion-exchange chromatography. This is consistent with an elimination mechanism that leaves on the pyrimidine residue those phosphates formerly lying between purine and pyrimidine residues. It also indicates that the loss of purine residues is a necessary prerequisite to the rupture of the polynucleotide chain by mild acidic hydrolysis. The presence of nucleotides containing equal amounts of pyrimidine and phosphate may be caused by the hydrolysis of phosphate from the initial products. About 33% of the phosphate appears as inorganic phosphate after 1 hour of hydrolysis; the bulk of this appears to be phosphate originally lying between purine residues in the original DNA. There is a slow rise to a new plateau of 50% at 3 to 4 hours. The sum of inorganic plus monoesterified phosphate remains constant from 1 hour to 4 hours, indicating a slow hydrolysis of monoester phosphate, presumably from the 3′ positions. The presence of all varieties of mono-, di-, and trinucleotide sequences that are possible from cytidylic and thymidylic acids indicates that all possible sequences of pyrimidine and of purine nucleotides exist in thymus DNA.

This particular symposium on tracer methodology coincides almost exactly with the twentieth anniversary of the first shipment of radioactive material by the Oak Ridge National Laboratory to the general scientific public. It might be appropiate, therefore, to pause for a glance back to the years when the radioisotope production facilities at Oak Ridge, which underlie so much of biological research in the past two decades, were being developed [1]. Since my concern with radioisotopes, no more than average since then, played a central role in that development, such a glance may give your chairman a bit of “status” in this company [2].

THOUGH the manner in which the four nucleotides of ribonucleic acid are combined in the intact molecule is not completely settled, the data obtained from analyses of the chemical degradation products are most compatible with phosphoryl-group attachments through the C2 and C3 positions of the ribose group1. The exclusion of C5 phosphoryl linkages rests principally upon the failure to isolate 5′ nucleotides or the acid-stable ribose-5-phosphate from acid hydrolysates of ribonucleic acid. However, Gulland and Jackson2, hydrolysing yeast ribonucleic acid with a snake venom containing a phosphodiesterase and a specific 5′ nucleotidase, found 35 per cent of the total phosphate liberated as inorganic phosphate, suggesting that a large number of the phosphoryl groups were attached to the ribose C5 position. Recently, Schmidt3, utilizing periodate titrations of ribonuclease-hydrolysed yeast ribonucleic acid, concluded that some nucleotide groups had been linked to positions other than ribose C2 or C3 in the original ribonucleic acid. The experiments described below indicate that intact 5′ nucleotides can be isolated from enzymatically hydrolysed calf liver ribonucleic acid.