Lethal factor active-site mutations affect catalytic activity in vitro.

The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.

Influence of chroma on spatial balance of complementary hues.

In 1905, Munsell proposed that if the relative areas of two colors are adjusted so that they mix to produce a neutral gray on a spinning Maxwell disk, these colors should appear balanced when juxtaposed with the same relative areas in a composition. This law of "inverse ratios of area" indicates that hues of low chroma should require larger relative areas to balance complementary hues of higher chroma. Several experiments have compared colors of varying chroma in terms of apparent weight, but none have assessed the validity of Munsell's law directly. A group of artists and a group of nonartists with no prior training in the use of color adjusted a panel until the relative areas of two complementary colors appeared balanced. Within-subject correlations between actual settings and theoretical predictions were predominantly high and positive. This was especially true of subjects with formal color training.