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Semliki-Forest-virus 42-S virion RNA is translated in a mouse ascites cell-free system. One of the major polypeptides made in vitro has been identified as Semliki-Forest-virus capsid protein by co-electrophoresis on dodecylsulfate -- polyacrylamide gels and by tryptic peptide analysis on paper. Another major product of the cell-free system has been characterised as envelope protein by peptide analysis but it does not co-electrophorese on gels with envelope proteins from purified virions. It is possible that the nascent, non-glycosylated polypeptide is partially degraded by proleolytic enzymes present in the cell-free extract, or alternatively that fragments of envelope protein are produced by premature termination on the viral RNA. Several other polypeptides are synthesised in the cell-free system in variable amounts but these have not been identified. The genetic content and the mechanism of translation of Semliki-Forest-virus 42-S RNA are discussed. [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

The analytical active-enzyme-centrifugation method permits the determination of the hydrodynamic properties of enzymes while they are fully active on their substrates: the centrifugation can be performed at the very dilute concentrations used in kinetic studies (&aysmp; 1 µg/ml): furthermore a purified enzyme preparation is not necessary and in many cases a crude extract is sufficient. This method is beginning to be used in several laboratories even though few of its experimental aspects have been published. This paper reports the details for carrying out, an activeenzyme-centrifugation run, discusses the experimental conditions anti details with artifacts and ways, which are easy, to overcome them. [ABSTRACT FROM AUTHOR]

The Cbl-1 mutant of Bacillus subtilis 168 WT is the result of a spontaneous mutation presenting a pleiotropic character. In this paper, the results of the morphological and chemical analyses of the cell walls isolated from both strains are compared. When either the whole cells or the isolated cell walls of both strains were fixed with osmium tetroxide, the examination by electron microscopy showed extensive morphological differences in structure due to the partial destruction of the mutant cell walls. The chemical investigations performed on the isolated cell walls revealed surprising differences in chemical structure. In the cell walls of the mutant, teichoic acid was completely missing and was replaced by protein. A major protein-containing fraction accounting for about 50% of the material solubilized by autolysis of the mutant cell walls was isolated. Polyacrylamide gel electrophoresis showed that this fraction was composed mainly of a single protein of high molecular weight When denatured by sodium dodecyl sulfate treatment in the presence of β-mercaptoethanol, the protein yielded by gel electrophoresis two major bands with molecular weights of approximately 105000 and 155000, respectively. The molecular weight of the Cbl-1 cell wall protein determined by high speed sedimentation equilibrium was 255 600. [ABSTRACT FROM AUTHOR]

Semihemoglobins α and β are derivatives of human hemoglobin which possess the usual chain composition (α2β2 or αβ) but which carry the heine prosthetic group on only one kind of chain (α or β), the complementary chain (β° or α°) being heme-free. In this paper, new results on two compounds of this type, namely, semiHbαD (αβ°) and semilHbβ (α°β) have been presented; existing data on other earlier preparations have also been reviewed. Methods for preparation of semiHbαD and semiHbβ have been described-. Some of their properties including optical absorption, circular dichroic spectra, oxygen equilibrium and kinetics of carbon monoxide binding have been measured; these properties are then compared with those possessed by intact hemoglobin (β2β2), isolated chains (α,β) and the berne-free apoprotein (α°β°). 1. The contribution of the heine moiety to the optical absorption and circular dichroic spectra of the semihemoglobins are found to be intermediate between those of hemoglobin and those of the corresponding isolated chain. This result indicates that the effect of interaction between the unlike chains of these molecules are transmitted as far as the heine prosthetic group. The structure of the heme-free chain is also modified as the result of this interaction as revealed by titration of sulfhydryl groups, although circular dichroic spectra in the far ultraviolet are not conclusive. 2. The oxygen affinity and the Bohr effect of the semihemoglobins are again intermediate between those of hemoglobin and isolated chain, the semiHbβ being closer to hemoglobin than semiHbαD. The same is true concerning the velocity of carbon monoxide binding. 3. The kinetics of fixation of carbon monoxide to semihemoglobins is strongly biphasic. It is suggested that this property could reflect the existence of semihemoglobins as two types of filmers (αβ) but differing among themselves on account of different contact regions brought into play. (α1β1 and α1β1 in the nomenclature of Perutz.) 4. The significance of the fact that semiHbα is obtained in multiple forms is considered. 5. The ensemble of the results are discussed in the context of chain-chain interactions. [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]

Binding of tryptophan, tryptamine and ATP to tryptophanyl-tRNA synthetase was studied by equilibrium dialysis experiments. There arc two binding sites per mole of enzyme both for tryptophan and for tryptamine. Ks for tryptophan is 0.95 μM and for tryptamine is 1.8 μM. In the case of ATP no binding could he measured over the range of concentrations examined. The Scatchard plots of tryptophan and tryptamine binding do not shown any cooperativity between the subunits. Dissociation of the dimeric enzyme was studied at very low protein concentration. Upon dilution of the enzyme both the [32P]PPi-ATP isotope exchange activity and the tRNA charging activity are lost simultaneously. Kinetic evidence demonstrates that the inactivation is primarily due to the dissociation of the active direct into inactive monomers. The dissociation is strongly promoted by alkaline pH and is inhibited by the presence of tryptophan or tryptophanyladenylate but not by that of tRNA. The dissociation constant of the dimer-monomer equilibrium at pH 8.5 is 15 nM at 25 °C. The dissociation form is more susceptible to denaturation than the dimeric species. The results reported in the paper suggest that even if there are not binding interactions between the subunits, the active conformation of the enzyme depends on their associated state. [ABSTRACT FROM AUTHOR]

With the help of an automatic protein sequenator, we have verified a prediction made by us in a recent paper, namely that residue 16 in Candida krusei cytochrome c is a glutamine and not a glutamic acid. Neurospora crassa cytochrome c now remains the only mitochondrial cytochrome c which apparently does not have a glutamine at this position. In the course of this investigation, from two strains of Candida krusei we isolated two cytochromes which differ in amino acid composition. One of them seems to correspond to that described in the literature. Two of the differences have been located. [ABSTRACT FROM AUTHOR]