Sulfoxide configuration in sparsomycin determines time-dependent and competitive inhibition of peptidyl transferase

Sparsomycin, S_cR_s configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation. In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay. The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin.     

Influence of 0.5 ppm nitrogen dioxide exposure of mice on macrophage congregation in the lungs

Summary The lungs of 12 mice, half of which were exposed to continuous 0.5 ppm nitrogen dioxide for 3 weeks, were explanted in culture, and the instances of macrophage congregation were quantitated according to numbers of target cells involved, categories of congregation from three to 11 or more, numbers of macrophages participating in each category for the total cultures, and the influence of delaying explantation for 24 and 96 hr. A total of 9042 macrophages and 2140 epithelial and spindle target cells were counted in the outgrowths from 306 explants. The incidence of macrophage congregation (or numbers of target cells) was greater for the cultures from the NO₂-exposed animals, both with respect to total incidences between groups (p→0), and the 0-hr (p<0.001) and 24-hr (p<0.01) culture subgroups. In addition, the values for T₃ to T₆ macrophage congregation were individually and consistently greater for the exposed animal group. Postmortem interval stress at 96 hr appeared to result in large colonies, but they were reduced greatly in number. Also the incidence of macrophage congregation fell by 28% as compared to 0-hr and 24-hr subgroups. Supported by Grants NHLI No. HL 17412 and EPA No. R. 800881.     

Significant changes in 16 S RNA conformation accompanying assembly of the 30 S ribosome in vitro

Our previous studies have shown that 16 S RNA can assume two different conformational forms as detected by agarose gel electrophoresis, and that these two forms vary in their ability to bind individual 30 S ribosomal proteins specifically. In this paper we show that the faster electrophoretic form can be converted to the slower electrophoretic form by the binding of either protein S4, S8, S7 or S15. The slower form can then be transformed into a fast form by heat-activating the reconstitution intermediate (RI) particle, which has been constructed under reconstitution conditions at 0 °C, to RI¹. We demonstrate that the transformation of the 16 S RNA conformation by binding of protein S7 permits the subsequent binding of protein S9 following deproteination. We propose that many of the classical assembly-dependent relationships are due to induced changes in the 16 S RNA conformation.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

Low hydration phase properties of phospholipid mixtures

An experimental investigation of the low hydration phase properties of phospholipid mixtures is described. ²H (D₂O) NMR, X-ray diffraction and differential scanning calorimetry have been used to elucidate the phase properties of mixtures of the mixed chain phospholipids palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylethanolamine (POPE). At 10% hydration pure POPE exhibited a H_II phase above 330 K, a fluid lamellar phase below 315 K, and a minimally hydrated crystalline phase below 300 K. For the 1:1 mixture, the samples exhibited only gel or fluid phases between 270 K and 360 K for hydrations in the range 15% to 30%. Below 15% hydration the mixture exhibited two fluid phases with different repeat spacings, as predicted previously.     

Primary cultures and the levels of cytochromeP450 in hepatocytes from mouse,rat, hamster,and rabbit liver

Summary Hepatocyte primary cultures (HPC) derived from rat, mouse, hamster, and rabbit liver were characterized for a variety of parameters. The conditions that maximized recovery, attachment, and survival varied between species. Hepatocytes from all four species were capable of attaching in serum-free Williams’ medium E (WME), but optimal attachment as monolayer cultures was achieved for mouse and hamster HPC in medium receiving 1% calf serum supplementation. Hamster hepatocytes required additional cations, whereas rabbit and rat hepatocytes displayed maximal attachment in medium supplemented with 10% calf serum. Survival of mouse and rabbit hepatocytes after 24 h in serum supplemented media was in the order of 90%. Rat and hamster hepatocyte 24 h survival was approximately 70 and 60%, respectively, and was not significantly affected by serum supplementation. Hepatocytes from each species varied in their content of cytochromeP450 at the time of isolation and in the rate of reduction during culture. Mouse and rat hepatocytes demonstrated the most rapid decline in content during the initial 24 h in culture, whereas concentrations in rabbit hepatocytes were virtually unchanged. The rate of decline inP450 concentrations in hamster hepatocytes was intermediate between those displayed by rat and rabbit hepatocytes. These studies have delineated conditions useful for the culture of hepatocytes from four species and have documented the status of an important parameter of their functional capability. This study was supported by EPA contract 68-01-6179. C. J. Maslansky was a recipient of a Monsanto Fund Fellowship in Toxicology.     

Isolation and properties of Tn10 insertions in the rac locus of Escherichia coli

Summary Two Tn10 insertions that are in the rac locus of the chromosome of Escherichia coli have been isolated and characterized. These insertions are located at min 29.7 and min 30.0. The insertions are stable when an F123 rac::Tn10 episome is transferred to an F^- rac ⁺ recipient, but they are lost at a high frequency when transferred to an F^- rac ^- recipient. This latter condition has been previously, demonstrated to cause the excision of the rac locus. The Tn10 insertions are also lost at a high frequency when strains containing them are lysogenized with reverse. If the lysogens that have lost the Tn10 insertion are subsequently cured of reverse, the cells no longer contain sequences homologous with rac locus DNA. These strains were rac ^- when tested for recombination activation (Low 1973), and this procedure consequently provides a simple means to make isogenic rac ^- and rac ^- strains.     

Stereochemical aspects of forcing special substrate hydrazides to behave either as electrophiles or as nucleophiles during catalysis by crude papain

Restriction of hydrazides of N-blocked amino acids mainly to electrophilic action, in acylating crude papain, has been achieved by means of a large amount of aniline, with formation of insoluble anilides of N-acylamino acids. Similarly, nucleophilic behavior, on the part of a hydrazide, has been promoted by introducing a large proportion of an N-acylamino acid to produce an insoluble N¹,N²-diacylhydrazine. Achiral, chiral and racemic hydrazides and their corresponding N-acylamino acids were utilized in the study. Among the more informative combinations of reactants were Z-dl-alanine hydrazide with aniline and then with Z-glycine. A stereospecific response in the former situation produced Z-l-alanine anilide. In the latter case, a stereoselective interaction produced Z-Gly-NHNH-lAla-Z more rapidly than Z-Gly-NHNH-d-Ala-Z. The final incubation period yielded an optically pure D product. Differences in stereochemical control have been delineated in terms of different spatial aspects for interactions at the S and S′ subsites of sulfhydryl proteolytic enzymes. A racemic reactant encountered firm stereospecificity as an electrophile at the S subsite but only modest stereoselectivity as a nucleophile at the S′ subsite. The ready availability of crude papain allows an effective procedure for the synthesis of substantial quantities of diacylhydrazines.     

Immobilization of proteins by reductive alkylation with hydrophobic aldehydes

A new method is described for the immobilization of enzymes and other proteins onto hydrophobic gels. Trypsin, for example, can be quantitatively immobilized by reaction with sodium cyanoborohydride and dodecyladehyde-coated Octyl-Sepharose. Noncovalent interactions between the hydrophobic gel and approximately 10 resulting dodecylamino groups in the modified enzyme bind it very strongly but do not affect its ability to hydrolyze benzolarginine ethyl ester. The same procedure can also be used to immobilize E. Coli asparaginase and yeast alcohol dehydrogenase in high yield.