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in baker‘s yeast (Saccharomyces cerevisiae), accumulation of the non-reducing disaccharide, trehalose, is triggered by stimuli that activate the heat-shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 179–186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose-6P-dehydrogenase, phosphoglucose-isomerase) as well as enzymes of non-yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat-induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose-6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells. [ABSTRACT FROM AUTHOR]

The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CM P-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialicacids to gain access to cellular organdies. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMPglycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N-fluoresceinylaminoacetylneuraminic acid (5-FTIUNeuAc; 5-N-fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N-acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminicacid analogues can be produced and used for the characterization of the Golgi CMP-sialic-acid carrier. [ABSTRACT FROM AUTHOR]

Translation of cellular and early vaccinia RNA in nuclease-treated lysates, derived from uninfected and vaccinia-virus-infected cells al the early stage, has been investigated. When using limiting amounts of RNA no discrimination of translation was observed in the infected cells lysates; This conclusion was confirmed by sensitive RNA competition experiments for translation in vitro and also when using two different fractionated systems for protein synthesis in vitro. This absence of detectable discrimination in vitro was established both by comparing incorporation of [35S]methionine into proteins and by analysis of the products thus synthesized by sodium dodecylsulfate gel electrophoresis. However, a modification of the translational machinery from vaccinia-virus-infected cells did occur since only the ribosomal salt wash derived from infected cells was able to reverse the inhibition of protein synthesis in vitro resulting from excess RNA (control or early). This property of vaccinia-virus-infected cell lysates may result from the synthesis of an early protein involved in translation or from a better recovery of translational factors from the infected cells., as suggested in the accompanying paper. [ABSTRACT FROM AUTHOR]

We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350-fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4-phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form. The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular-weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme. [ABSTRACT FROM AUTHOR]