S J Singer

On the translocation of proteins across membranes

Many proteins of intracellular organelles are first synthesized in the cytoplasm and are then specifically transferred across the membranes of the organelles. On the assumption that these transfers all occur by the same basic mechanism, we enumerate the rather stringent requirements that the mechanism must satisfy. A unitary molecular mechanism is then proposed that meets these requirements.

Kinetic evidence for a conformational change by an anti-hapten antibody upon binding multivalent hapten.

Abstract Previous ultracentrifugal studies (Warner & Schumaker, 1970 a ) have shown that equimolar amounts of rabbit anti-2,4-dinitrophenyl (Dnp)† antibodies and either di-Dnp—lysine or di-Dnp—cystine react to form monomeric and dimeric complexes only. The sedimentation data indicate, at least for the monomer, a change in antibody conformation upon binding hapten. A stopped-flow kinetic study of binding in these systems shows the presence of a relatively very slow step with a concentration-independent half-time of about 5 sec. The concentration dependencies of the fraction of the total fluorescence quenching associated with this slow step show it to be the formation of the fourth antibody-hapten bond in the bridged dimer. We conclude that this rate reflects the necessity for a slow antibody conformational change to precede the formation of this last bond. In other words, an energetically unfavorable, and thus infrequently attained, state of the antibody molecules is necessary for closure of the bridged dimeric structure.

Specificity of affinity labeling of anti-DNP antibodies☆

Abstract A method is described for quantitatively evaluating the specificity of affinity labeling of antibody molecules. This method involves the reaction of a mixture of two non-cross-reacting antibodies with a reagent which will undergo affinity labeling with only one of the antibodies, followed by specific precipitation of the antibody which has not been specifically labeled. Data for the specificity of the reaction of anti-(2–4, dinitrophenyl) antibody with two different affinity labeling reagents is presented. The specificity observed for these reagents is shown to be sufficient for use in fragmentation studies to identify peptides derived from the active site without ambiquity arising from non-specific modification.