Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90␣ and HSP90␤ isoforms. Among them, ten and three mAbs reacted specifically with HSP90␣ and HSP90␤, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90␣, i.e. amino acids 227-310 (designated Region I) and 702-716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90␣ at low concentrations revealed the existence of major susceptible sites at Arg 400 -Glu 401 , Lys 615 -Ala 616 , and Arg 620 -Asp 621 . Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1-400, 401-615, and 621-732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90.The 90-kDa heat-shock protein (HSP90) 1 is one of the major stress proteins in eukaryotic cells. There are at least two HSP90 genes, and two HSP90 isoform proteins, ␣ and ␤, are HSP90 is believed to have a chaperone-like activity for particular molecules that are involved in signal transduction, such as steroid receptors (6), casein kinase II (7), pp60 v-src (8), elF2␣ kinase (9), and aryl hydrocarbon receptor (dioxin receptor) (10). HSP90 specifically binds to these proteins; and, in most cases, this interaction is essential for the function of the proteins (9,11,12). However, a variety of evidence, i.e. the abundance of HSP90 in cells even under nonstressed conditions, the conserved amino acid sequences from prokaryotic to eukaryotic cells (13), and the indispensability in yeast (3), strongly suggests that HSP90 is involved in more fundamental functions of cells. In fact, several studies have recently shown that HSP90 functions as a general chaperone. That is, it interacts with various proteins less specifically and modulates their conformation. For instance, the refolding of citrate synthase is significantly enhanced by the co-presence of HSP90 (14). The spontaneous refolding of denatured dihydrofolate reductase and irreversible denaturation of firefly luciferase are prevented b...

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