The influence of spermine on the structural dynamics of yeast tRNAPhe

A tRNAPhe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588-4599) in the tRNA derivative the ethidium is present in three states (T1-T3) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10-100 ms) between T1 and T2, and a slow one (100-1000 ms) between T2 and T3. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T3, as does Mg2+, but the final ratio [T2]/[T1] obtained with spermine is higher than with Mg2+, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.     

Uptake and polar transport of indoleacetic acid in moss rhizoids

In the cucumber ( Cucumis sativus L. cv. Straight Eight) cotyledon expansion assay, cytokinin-stimulated ethylene production was separated from cytokinin-stimulated growth through the use of potassium and calcium salts. Low concentrations of KC1, which dramatically promoted growth induced by cytokinin, inhibited ethylene evolution, while CaCl₂ at a concentration that had no effect on growth, strongly promoted the cytokinin-induced ethylene evolution. In contrast to the growth response, stimulation of ethylene production was not directly related to the presence of potassium or calcium but to their relative concentrations. Concentrations of KCl and CaCl₂ which promoted ethylene evolution singly, strongly inhibited it when mixed together. Low rates of exogenous ethylene had no effect on the growth response. Both the growth and ethylene responses were found to be general cytokinin phenomena. Cotyledon respiration was promoted by KC1, CaCl₂ and cytokinin, but its stimulation was not correlated with either growth or ethylene production. In the presence of KClm cytokinin-induced respiration sharply lowered the content of certain sugars during the large growth response and followed KCl uptake. Analysis of KCl uptake showed that its growth promoting synergism with cytokinin was not due to osmotic effects.     

Biogenic Amine-Stimulated Cyclic Adenosine-3'',5''-Monophosphate Formation in the Rat Carotid Body

The subcutaneous injection of isoprenaline, salbutamol, histamine, and adrenaline to rats, which were subsequently killed by microwave irradiation, resulted in a rapid increase in the cyclic AMP content of the carotid body. On the other hand, noradrenaline, dopamine, adenosine, and 5-hydroxytryptamine, at doses at least 100 times greater than that of isoprenaline, did not significantly alter the cyclic nucleotide content in vivo. The response to isoprenaline was dose related, with an ED50 of 15 micrograms X kg-1, and reached a peak level 1-1.5 min after injection. Incubation of intact carotid bodies with isoprenaline (10(-5) M) in vitro also resulted in a 10-fold increase in cyclic AMP content. The in vivo response to isoprenaline could be blocked stereo-selectively by propranolol, and ICI 118.551, a beta 2-selective antagonist, blocks the isoprenaline-elicited increase in cyclic AMP completely at a dose of 30 micrograms X kg-1; whereas betaxolol, a beta 1-selective antagonist, was ineffective, even at a dose of 300 micrograms X kg-1. Hypoxia (5% oxygen in 95% N2) did not result in a significant increase in the cyclic AMP content, nor did it significantly alter the isoprenaline-stimulated increase in the cyclic AMP content of the rat carotid body. These results suggest that some catecholamines may stimulate cyclic AMP formation by interacting with a beta 2-adrenoceptor in the rat carotid body.     

A chemiluminescent method for measurement of activated oxygen forms in biological fluids and homogenates

A chemiluminescent method for measuring the concentration of activated oxygen species (O₂² and H₂O₂) is described. Its main features are: high sensitivity (10^?9 M H₂O₂), its applicability to systems with high optical absorbance in the visible spectral region, a wide linear dynamic range, and the possibility for recording the kinetics of the processes, in which activated oxygen species are involved.     

The influence of spermine on the structural dynamics of yeast tRNA

A tRNA^Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T₁–T₃) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T₁ and T₂, and a slow one (100–1000 ms) between T₂ and T₃. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T₃, as does Mg²⁺, but the final ratio obtained with spermine is higher than with Mg²⁺, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

The influence of spermine on the structural dynamics of yeast tRNAPhe

A tRNA^Phe derivative carrying ethidium at position 37 in the anticodon loop has been used to study the effect of spermine on conformational transitions of the tRNA. As previously reported (Ehrenberg, M., Rigler, R. and Wintermeyer, W. (1979) Biochemistry 18, 4588–4599) in the tRNA derivative the ethidium is present in three states (T₁–T₃) characterized by different fluorescence decay rates. T-jump experiments show two transitions between the states, a fast one (relaxation time 10–100 ms) between T₁ and T₂, and a slow one (100–1000 ms) between T₂ and T₃. In the presence of spermine the fast transition shows a negative temperature coefficient indicating the existence of a preequilibrium with a negative reaction enthalpy. Spermine shifts the distribution of states towards T₃, as does Mg²⁺, but the final ratio $\text{[}\text{T}{}_2\text{]}\text{[}\text{T}{}_1\text{]}$ obtained with spermine is higher than with Mg²⁺, which we tentatively interpret to mean that spermine stabilizes one particular conformation of the anticodon loop.     

T-antigen expression by polyoma mutants with modified RNA splicing

Polyoma virus mutants were constructed that could not express all the three T-antigens. The mutagenesis was directed to the two 5' splice sites utilized in the maturation of early RNA. The mutant bc1051 had a base change at the splice site of large T-antigen mRNA, and the mutants dl1061 and dl1062 had deletions at the corresponding splice point of small and middle T-antigen mRNA. The site was removed in mutant dl1061 and altered by fusion to upstream sequences in mutant dl1062. Analysis of viral RNA showed that dl1061 and dl1062 formed only large T-antigen mRNA, whereas bc1051 did not produce this RNA-species. However, the biological properties of dl1062 suggested that it also produced mRNA directing the synthesis of a small T-antigen-related polypeptide, at least in low amounts. Only mutant bc1051 could induce transformation of rat cells. In mouse 3T3 cells dl1062 multiplied to a limited extent, while bc1051 and dl1061 failed to produce virus. However, dl1061 DNA was synthesized at a low rate which could be increased to normal levels by co-transfection with mutant bc1051. This result suggests that polyoma small and middle T-antigen have a previously unrecognized function in the early phase of the infection process, or in viral DNA-synthesis.     

Immunoglobulin synthesis and glucose-6-phosphate dehydrogenase as cell markers in human lymphoblastoid cell lines

Multiple lymphoblastoid cell lines were established from each of seven Burkitt lymphoma biopsies and from tonsils, removed from four patients with chronic tonsillitis. The cellular origin of the lines was studied using as markers the pattern of immunoglobulins secreted into the medium and the cells' glucose-6-phosphate dehydrogenase (G-6-PD) phenotypes.Lines from the same tonsil biopsy differed from each other by their patterns of immunoglobulin synthesis and G-6-PD phenotypes. All tonsil-derived lines secreted complete immunoglobulins. Newly established lines usually produced several heavy and light chain types, indicating multicellular origin, but the number of components produced decreased during the course of long-term cultivation. G-6-PD phenotypes of lines established from the same tonsil removed from a G-6-PD heterozygote differed—B, A and B/A phenotypes were found. The B/A lines rapidly changed to a single enzyme phenotype (B or A) when maintained in culture.The immunoglobulin and G-6-PD phenotypes in lines derived from Burkitt lymphomas differed from those of tonsil lines in several respects: (1) Some lines produced no immunoglobulins; (2) in immunoglobulin-synthesizing lines, the patterns of heavy and light chain production were more restricted than in tonsil lines; (3) after some months in culture, a uniform pattern of immunoglobulin synthesis was found in all lines derived from the same tumour; (4) lines from G-6-PD heterozygotes had the same single enzyme phenotypes as were found in the tumours.The data strongly suggest that most lines from Burkitt lymphomas are derived from the tumour clones and that most tonsil-derived lines have multicellular origin.