Search

The complexes of poly A and poly U are interesting models for studying the structure of nucleic acids. In the first part of this paper, the thermal stability of these complexes has been studied as a function of both pH and ionic strength in alkaline solution. The thermal stability is measured by the half transition temperatures, or Tm's. There are four different kinds of transitions: Tm2-1 refers to the dissociation of the two-stranded complex, poly (A + U); Tm2-1 to the dissociation of the three-stranded complex, poly (A + 2U) [4]. Tm8-2 is the temperature of half dissociation of poly (A + 2U) into poly (A + U) [1], and Tm2-3 is the temperature of rearrangement of 2 poly (A + U) into poly (A + 2U) [3] (Fig. 1). It is found that Tm2-1, and Tm8-1 decrease as pH increases above neutrality (Fig. 2 and 3), but that Tm2-8 does not vary (Fig. 3). This effect is readily explained if it is assumed that the ionisation of uracil brings a change in the state of the free poly U, but that the complexes themselves are not modified. In the second part of the paper a simple thermodynamic formulation is given which represents satisfactorily the relationship between the four equilibria, as observed at neutral pH (Fig. 1). A more complex treatment is then presented. It includes quantitation of electrostatic interactions, according to the theory of Kotin [18] and of the free enthalpy term corresponding to the organisation (stacking) of poly A. This leads to an excellent representation of the experimental data (Fig. 5). In alkaline solution, one can calculate the free enthalpy change associated with the ionisation of poly U at the Tm. According to our observations, it is likely that this is the only factor responsible for the decrease of Tm. Therefore, we can write that the sum of the free enthalpy of complex formation in the same conditions, but at pH 7, and of the free enthalpy of ionisation is zero. This leads to a determination of ΔG both as a function of temperature and ionic strength: the variation of ΔG with these factors is shown to be in good agreement with our detailed model. However, the absolute values are different, probably because of uncertainties concerning the data for poly A stacking enthalpy and entropy. Apart from providing evaluation of the thermodynamic parameters for double and triple helix formation, our results lead us to emphasize the importance of the thermodynamic state of the unassociated polynucleotides. This may be particularly relevant when one considers the partially complementary secondary structures of RNA's. [ABSTRACT FROM AUTHOR]

Presents an investigation of the sequencing of 65 amino acid residues at the carboxyl-terminus. Use of methods such as enzymic or chemical cleavages of the peptide chain; Fractionation of peptides by Sephadex or Dowex column chromatography, paper elctrophoresis or chromatography.

In the previous paper, a metabolic relationship between a cytochrome oxidase deficiency and threonine biosynthesis in Saccharomyces cerevisiae was proposed. It has been found that high concentrations of threonine or its immediate precursors L-aspartate- β-semi-aldehyde or homoserine, are able to restore cytochrome oxidase synthesis in threonine auxotrophs, grown in the presence of glucose as a carbon source. On the other hand, these strains are able to synthesize cytochrome oxidase when the carbon source is ethanol or glycerol even without an excess of threonine. The results show that this phenomenon is concerned with the regulation of cytochrome oxidase synthesis. Different hypotheses are presented to explain the possible role of threonine in this regulation. One of them stresses the possible role of a differential auxotrophy for the cytoplasmic and the mitochondrial protein synthesis systems. [ABSTRACT FROM AUTHOR]

Previous work of several laboratories has shown that the two carbon atoms C-28 and C-29 of the ethyl or ethylidene side chains of phytosterols are introduced on a C-24 unsaturated side chain by two successive C-methylation steps. A scheme, based on previous work of Nes et al. and Goodwin et al., gives the various intermediates which can be formed by stabilisation of the intermediate carbonium ion (II). It was usually thought that ethylidene compounds such as (III) were the precursors of ethyl compounds such as (VIII) and this has been proved recently in the case of poriferasterol (IX) which contained four deuterium atoms when Ochromonas malhamensis was grown in presence of [Me-²H3] methionine [8]. In the present paper it is reported that the sterol of the slime mold Dictyostelium discoideum [Δ 22-stigmasten-3-Β-ol (Xa)] contains five deuterium atoms, when extracted from D. discoideum fed on cells of a methionine-less strain of E. coli grown in presence of [Me-²H3] methionine. The unsaturated sterol (Xa) is accompanied by the saturated sterol (XIa) which was not previously found in this organism. Mass spectrometry of the pentadeuterated sterols (Xa) and (XIa) seemed to show conclusively that all five deuterium atoms are located in the ethyl side chain [11], but it was thought necessary to confirm this conclusion by a detailed analysis of the side chain; thus, the sterol (Xa) was ozonised and part of the side chain isolated as a crystalline p-phenylphenacyl ester (XIIIa). Mass spectrometry of this compound and of a synthetic non-labelled (XIllb) and the corresponding monodeuterated derivative (XTIJc) confirms that all five deuterium atoms of the Dictyostelium sterol (Xa) are in the ethyl side chain. These results exclude an ethylidene compound (such as III) as a precursor of the sterol (Xa); they do not allow us to choose between other possible routes illustrated th the scheme. Our experiments also show that there is no hydrogen shift during the second methylation step. [ABSTRACT FROM AUTHOR]

In the previous paper [1] we proposed a mechanism for the reaction of pancreatic ribonuclease with nucleotide diesters. in which the kinetic constants k+1 and k-1 + k+2 are assigned to a base catalysed step for the formation, and a proton catalysed step by the conjugate acid for the breakdown, of an enzyme stabilized intermediate. In the hydrolysis reaction of the 2′,3′- cyclic diesters the rates depend on the structure of the catalysing base, and in particular on its polarisability. in addition to this effect, we found that, in the dinucleoside phosphate series, the rates increased up to three orders of magnitude, depending on the nature of the base of the second nucleoside [4]. According to our suggested mechanism I-VI the accelerated rates could be due to (a) the second base influencing the polarisability of the catalysing system by π-electron interactions between the stacked bases. This effect should be reflected in the enthalpy term and should parallel the hypochromicity of the dinucleoside-phosphates exactly: (b) pre-ordering of the reacting atoms (C-2-oxygen, 2′-OH-group and phosphorus) by the base stacking, which would be reflected in a lowering of the activation energy in the entropy term and should only roughly parallel the hypochromicity. To differentiate between these two possibilities we prepared the diesters IX -XXXIV, determined the hypochromic effect and also the values of km and k+2 at pH 7. There is only a partial correlation between k+3 values and the hypochromic effect. The k+2 values vary with the structure of the second nucleoside and show a particular dependence upon the nature of the link between base and phosphate group. Highest values are found with adenosine and deoxyadenosine. Elongating the ribose with mercaptoethanol (XIX) or substituting the relatively rigid ribofuranose ring with flexible n-hexyl-, pentyl- or butyl-chains (XX-XXIII appreciably reduces the hypochromicities and also the k+2, values, in the case of a propyl link (XXIII, XXIV( or in the analogously structured 3′-3′-dinucleosidephosphate XXV no hypochromic effect is found and the k+2 values are as low as that of 3′-cytidylic acid benzyl ester. A second nucleoside attached to the 5′ end of a substrate (XXXIII. XXXIV) does not result in an increase of the rate of diester hydrolysis at the 3′ end though there is a normal hypochromicity. Therefore a pre-ordering of the reacting atoms (demonstrable on models) appears to be the accelerating factor. A specific interaction between the second nucleoside and the enzyme being responsible for the pre-ordering effect can be excluded by either blocking or varying all the potential interaction sites. The concept of a pre-ordering in the substrate due to bass stacking agrees with the results from measurements of the temperature dependence of k+2 and calculations of the enthalpy (ΔH≠) and entropy (Δ,S≠) term in the activation energy of this step. When diesters with an exactly analogous structure of the intermediate II such as CpA (IX) and Cp-butyladenine (XXII) are compared, the differences are found in the entropy term only. Between the first and second step substrates such as CpA and 2′3′-Cp the differences are also found in the entropy term with a small factor in the enthalpy term. From a comparison of Up- and Cp-diesters it can be seen that due to the differences in the enthalpy term the Up-diesters should react 5–10 times faster than Cp-diesters, but that due to the differences in the entropy term Cp-diesters react 20 times faster than Up-diesters.… [ABSTRACT FROM AUTHOR]