Immunochemical characterization of the adenine nucleotide translocator. Organ specificity and conformation specificity

Antibodies have been prepared against purified preparations of the heart and kidney nucleotide translocator in the 'c'-conformation. The results show organ-specific antigenic determinants on the translocator proteins isolated from heart, kidney and liver although a partial cross-reactivity between these three proteins was demonstrable. The organ specificity was observed both with the solubilized and with the membrane-bound translocator protein indicating organ-specific determinants on exposed regions of the carrier. An organ-specific inhibition of the nucleotide transport in heart mitochondria by the heart carboxyatractylate-protein antiserum leads to the conclusion that the organ specificity is at least partially conditioned by the binding site for the substrate and/or the closely linked gate of the carrier protein. Apart from the organ specificity the results also demonstrate a specificity of the antibodies for the translocational conformations of the carrier: the 'c'-conformation stabilized in the carboxyatractylate-protein complex and the 'm'-conformation present in the bongkrekate-protein complex. However, after denaturation of the carboxytraktylate-protein and bongkrekate-protein complexes the binding of the anti-(carboxyatractylate-protein) antiserum to both inhibitor-protein complexes was nearly identical. The conformation specificity was also expressed by the inhibition of the conformation transition from the 'c'- to the 'm'- state. This side-specific inhibition of the nucleotide transport and the identical binding activity of the carboxyatractylate-protein antiserum against the denatured carboxyatractylate-protein and bongkrekate-protein complexes suggested that the conformation-specific antigenic determinants are topographic surface regions which are determined by the chain folding.     

On the specificity of caffeine effects

Summary Caffeine in 0.1% or higher concentration reversibly inhibits E. coli and yeast growth. It inhibits RNA and protein synthesis (Tables 1+3) within a few minutes after being added to the incubation medium (Fig. 2). The suggestion is made that these effects of caffeine, as well as its synergism with some mutagens, are due to its ability to costack with free purines and to form complexes with single-stranded nucleic acids.     

Features of the intrageneric Alnus-Frankia specificity

Host specificity between local Frankia strains and native alders [Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn.] was evaluated in inoculation experiments. Pure cultures of Frankia , whether originating from A. incana or A. glutinosa , were infective and effective on both host species. These pure cultures were isolated from spore-negative (Sp⁻) nodules. From spore-positive (Sp⁺) nodules no Frankia isolates were obtained. This strain type resisted all our isolation attempts and therefore crushed nodules had to be used for Sp⁺ type inoculations.
The Sp⁺ type Frankia populations differed in their host specificity. Sp⁺ nodules from A. glutinosa were effective on both alder species, but Sp⁺ nodules from A. incana induced effective nodules only on the original host; on A. glutinosa only small (1-3mm) prenodule-like structures were found. Such A. glutinosa plants died on N-free medium, thus showing that these nodules were ineffective. In the effective nodules the middle cortex was dominated by infected cells filled with vesicle clusters. In the ineffective nodules only a few cortical cells were infected and sporangia predominated in these cells. Surprisingly enough they also contained vesicle-like structures as demonstrated in electron micrographs.     

Substrate specificity of the SecB chaperone

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is approximately 9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20-30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.     

Host-Frankia specificity within the Casuarinaceae

Summary Fifteen species from three genera of the Casuarinaceae were inoculated with suspensions ofFrankia prepared from single nodule-lobes collected from different species and genera within the Casuarinaceae. Host-endophyte specificity was expressed mainly at the generic level. There was marked cross-inoculation within Casuarina and little nodulation ofCasuarina species from Allocasuarina sources with the exception of 3 sources ofFrankia fromA. torulosa which showed a high tendency to nodulateCasuarina species. Few sources from Casuarina nodulated species of Allocasuarina and while cross-inoculation within Allocasuarina was frequent it was less marked than within Casuarina. SomeFrankia inocula had wider host ranges than others, nodulating outside the genus or series of origin. It was not possible to determine if these apparent wider ranges in host spectra reflected genotypic differences betweenFrankia or were associated with the presence of more than oneFrankia strain in some inocula.     

Assessment of the specificity of norethisterone radioimmunoassays

Specifities of 4 different norethisterone (Nor) antisera (coded A,B,C, and D) were evaluated and compared by cross-reaction studies to relate the antiserum specificity to the overall specificity of the radioimmunoassay (RIA), as established by plasma levels measured in women regularly taking the microdose of Nor (300 mcg/day). Using any of the 4 antisera, no significant deviation from parallelism were found among graded doses of authentic Nor and increasing volumes of plasma from women taking Nor for contraception. Cross-reaction studies preceded by chromatography to decrease plasma blanks are described, with each antiserum compared to the others for its efficacy in estimating plasma Nor values. It was concluded that 1) the significance of cross-reaction studies as well as that of a parallelism test for assessing overall specifity of the RIA is limited; 2) a single chromatography before RIA improves assay specificity but may not be sufficient to remove all interfering compounds; and 3) a comparison of direct and chromatographic procedures using several different antisera is useful for selection of the relatively most specific RIA procedure. These study results indicated that either antiserum C or D (preceded by chromatography) will yield better results than A or B.