The capacity to potentiate the sedative action of pentobarbitone by the volatile oil of Indian Acorus calamus has been used to screen various fractions of the oil for the presence of the principle responsible. The methods used for the removal of the oxygenated components of the oil did not remove the active material. The results show that the active principle resides in the hydrocarbon fraction of the oil or in an oxygenated component not removed by the methods employed. The volatile oil from the European Acorus calamus showed activity similar to the oil from the Indian drug.

Ethoxyformic anhydride abolishes the peptidyl transferase activity of 50-S ribosomal subunits, LiCl split proteins and L16. Hydroxylamine treatment results in reactivation. Erythromycin exhibits significant protection with 50-S ribosomal subunits. With LiCl split proteins and L16 significant protection was exhibited only after reconstitution.The results indicate that the ethoxyformic anhydride is reacting with approximately six histidines in LiCl split proteins and one in L16. Since L16 has been reported to contain a single histidine, the results presented indicate the involvement of this histidine in peptidyl transferase activity.Peptide bond formation or peptidyl transferase activity of the 50-S ribosomal subunits may be determined using the fragment assay [I]. Under the conditions of this assay peptide bond formation is not dependent on the presence of the 30-S ribosomal subunits, mRNA, intact tRNA, supernatant protein factors or GTP [2].The involvement of certain 50-S ribosomal subunit proteins in peptidyl transferase activity has been indicated by partial reconstitution experiments in which L11 has been suggested to be the peptidyl transferase protein [3,4] and in another report L16 [5]. Although the nature and degree of the involvement of L11 in peptidyl transferase activity remains to be clarified, the essential nature of L16 appears clear.The molecular weight of L16 has been reported as 17 400 and 17 900 by sodium dodecylsulphate gel electrophoresis [6,7]. Other methods of molecular weight determination, chemical and sedimentation equilibrium, reveal higher molecular weights [7]. The complete sequence of this protein has also been reportedThe involvement of a single amino acid residue, namely histidine, was suggested by our earlier report [9] in which photochemical oxidation of 50-S ribosomal subunits resulted in a rapid and irreversible loss of peptidyl transferase activity. The pH profile of inactivation exhibited a maximum at pH 7.5. Fahne- PI.Dejinition. A260 unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a l-cm pathlength cell. stock [lo] also suggests the involvement of a.histidine residue in peptidyl transferase activity. We report the effect on peptidyl transferase activity of the chemical modification of 50-S ribosomal subunits, LiCl split proteins and L16 by ethoxyformic anhydride, a relatively specific histidine reagent.

The requirements for peptide-bond synthesis and transesterification reactions of Escherichia coli 70s ribosomes, 50s native or reconstructed 50s subunits were examined using Met-tRNA as donor substrate and puromycin or a-hydroxypuromycin as acceptors.We report that the soluble protein EF-P, purified to apparent homogeneity, stimulates the synthesis of N-formylmethionylpuromycin or N-formylmethionylhydroxypuromycin by 70 S ribosomes or reassociated 30s and 50s subunits.In the presence of EF-P, 70s ribosomes are significantly more efficient than 50s particles in catalysing either peptide-bond synthesis or transesterification. The involvement of 50s subunit proteins in EF-P-stimulated peptide-bond formation and transesterification was studied. 50s subunits were dissociated by 2.0 M LiCl into core particles and 'split' proteins, several of which were purified to homogeneity. When added to 30s . A-U-G . f[35S]Met-tRNA, 50s cores or 50s cores reconstituted with L6 or L11 promoted peptide-bond synthesis or transesterification poorly. EF-P stimulated peptide-bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF-P stimulated a low level of transesterification by cores reconstituted with L6 and L1 1 . In contrast, core particles reconstituted with L16 exhibited both peptide-bond-forming and transesterification activities and EF-P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF-P-stimulated peptide-bond and transesterification reactions of the peptidyl transferase.Ethoxyformylation of either 50s subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide-bond formation and transesterification. Similarly ethoxyformylation of EF-P resulted in a 25 -50% loss of its ability to stimulate both reactions. 30s subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF-P in the catalytic mechanism of peptidyltransferase is discussed.Much of our understanding of the mechanism of protein synthesis and of the topology of ribosomes derives from studies on how these particles catalyze peptide-bond synthesis Several approaches have been used to define the proteins required for the peptidyltransferase activity of ribosomes. These include attempts to localize the individual proteins by affinity labeling of the 3' portion of tRNAs or specific reagents This paper is dedicated to Dr Fritz Lipmann on the occasion of his 85th birthday.Definitions. 1 A230 unit of a protein solution is equivalent to 250 pg [32]. 1.0 AZG0 unit is the quantity ofmaterial contained in 1 ml of a solution which has an absorbance of 1 at 260 mm, when measured in a 1-cm-path-length cell. The concentrations of puromycin and cr-hydroxypuromycin were determined spectrophotometrically in 0.1 M HCl at .2 , , , = 267.5 using E = 2.0 x lo4 M-' cm-' for both compounds.Abbreviations. Abbreviations for nuclei...

The photochemical oxidation of the 5 0 3 ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first-order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivated. The pH profile of inactivation exhibits a maximum at pH 7.5.Erythromycin at a low concentration (0.04 pmol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect.The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.Photo-oxidation in the presence of the anionic dye Rose Bengal effectively modifies proteins under very mild conditions and exhibits under these conditions a high degree of specificity for histidine and guanine [I]. Garvin et al. [2] have reported inactivation of Escherichiu coli 70-S ribosomes by photo-oxidation in the presence of Rose Bengal and other sensitizing dyes. Noller ef al. [3] have shown that photo-oxidation of 30-S ribosomal subunits in the presence of the anionic dye Rose Bengal leads to a rapid loss of activity in protein synthesis and that three proteins and about three histidine residues are protected from photooxidation by the presence of poly(U) and tRNA.Affinity analogues of peptidyl-tRNA [4] and chloramphenicol [5,6] have provided evidence that the proteins L16, L2, L26-27 are located at the peptidyl transferase center and are probably closely associated with each other. We report here the dye-sensitized photoinactivation of peptidyl transferase activity, some characteristics of the inactivation and the protection of peptidyl transferase activity by erythromycin and puromycin but not by sparsomycin, lincomycin or chloramphenicol. MATERIALS AND METHODS Muter ials

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