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Translocation of the zinc-dependent metalloendopeptidase anthrax lethal factor (LF) from the endosome to the cytosol requires an acidic endosomal milieu. In the current study, we utilized immobilized (to prevent protein aggregation below pH 5.5) and native LF to assess the effect of pH on the function and metal content of LF. Our results reveal the diminution of LF's catalytic competence under moderately acidic conditions (pH ∼6) to be uncorrelated to the metal content of the protein. However, a significant degree of demetallation of LF (∼30%) was observed at pH values close to those found in late endosomes (pH ∼5), thus raising the possibility that a substantial proportion of LF molecules may not be in their zinc-bound state prior to translocation., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Prostaglandin H synthase (PGHS) as apo-PGHS, holo-PGHS, and holo-PGHS, inactivated in the course of catalysis was studied using chemical modification with diethyl pyrocarbonate (DEPC). The exhausted reaction with DEPC corresponded to the modification of 7 histidine residues in apo-PGHS and 4 in holo-PGHS. All 18 histidine residues became accessible for modification with DEPC in the enzyme, inactivated in the course of catalysis. The velocities of tryptic cleavage of all the three forms into two fragments were fairly different but independent of modification. Based on the results we hypothesize fast and dramatic changes in the protein structure in the course of the substrate conversion.

A simple procedure was devised for isolating from homogenates of mitotic cells the human homolog to the fission yeast cdc2 gene product. The identity of the purified protein was established with anti-p34cdc2 antibodies and p13suc 1, both specific ligands for p34cdc2. Active-site labeling with oxidized [alpha 32P]ATP showed the purified molecule to be an ATP-binding protein. Its ability to phosphorylate casein but not histone, and its phosphorylation on tyrosine, detected by anti-phosphotyrosine antibodies, indicates the form of p34cdc2 purified is the inactive or apoenzyme form. Purified quantities of human p34cdc2 should be of considerable value in establishing the mechanism of its activation at mitosis by phosphatases.

Limited proteolysis was used to probe the structure of the apo- and holoenzyme of rat liver tyrosine aminotransferase. Both were subjected to trypsinolysis and the major fragments were isolated and characterized. Trypsin cleaves the apoenzyme after residues Arg57, Lys64, and Lys71 and the holoenzyme after Arg37 and Lys38. The difference in the accessibility of the enzyme deprived or associated with pyridoxal 5'-phosphate reflects two distinct conformations. The activity, the affinity for the ligands and the thermostability of the purified truncated enzyme forms are similar to those of the native apo- and holoenzyme. A model for the domain structure of mammalian tyrosine aminotransferase and a mechanism for its rapid turnover are proposed.