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The glutathione transferase from squid digestive gland is unique in its very high catalytic activity toward 1-chloro-2,4-dinitrobenzene and in its ancestral relationship to the genes encoding the S-crystallins of the lens of cephalopod eye. The three-dimensional structure of this glutathione transferase in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene (GSDNB) has been solved by multiple isomorphous replacement techniques at a resolution of 2.4 A. Like the cytosolic enzymes from vertebrates, the squid protein is a dimer. The structure is similar in overall topology to the vertebrate enzymes but has a dimer interface that is unique when compared to all of the vertebrate and invertebrate structures thus far reported. The active site of the enzyme is very open, a fact that appears to correlate with the high turnover number (800 s-1 at pH 6.5) toward 1-chloro-2,4-dinitrobenzene. Both kcat and kcat/KmCDNB exhibit pH dependencies consistent with a pKa for the thiol of enzyme-bound GSH of 6.3. The enzyme is not very efficient at catalyzing the addition of GSH to enones and epoxides. This particular characteristic appears to be due to the lack of an electrophilic residue at position 106, which is often found in other GSH transferases. The F106Y mutant enzyme is much improved in catalyzing these reactions. Comparisons of the primary structure, gene structure, and three-dimensional structure with class alpha, mu, and pi enzymes support placing the squid protein in a separate enzyme class, sigma. The unique dimer interface suggests that the class sigma enzyme diverged from the ancestral precursor prior to the divergence of the precursor gene for the alpha, mu, and pi classes.

The murine aB-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of aB-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse aB-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the aB-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of aB-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.The crystallins compose over 90% of the water-soluble proteins in the vertebrate eye lens and are encoded by four major gene families (for a review, see reference 28). The high-molecular-weight a-crystallin is composed of protein aggregates derived from the products of two genes, axA and aB, which are located on different chromosomes (2,20,31,36). aA-crystallin and aB-crystallin are evolutionarily related, exhibiting 56% homology at the amino acid level in the bovine lens (37); in addition, both crystallins are partially homologous to small heat shock proteins (19,36,41) and to an egg antigen (p40) of the blood fluke Schistosoma mansoni (24).The mouse axA-crystallin promoter has been studied extensively. A gene fragment from positions -366 to +46 base pairs (bp) directed expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically in lens both in culture (5) and in transgenic mice (27). Lens-specific expression was also observed with an aA-crystallin promoter fragment as small as from positions -111 to +46 bp (6), and this DNA fragment can bind lens nuclear proteins from embryonic chicks (33a).To extend our understanding of the a-crystallin genes, we isolated the mouse aB-crystallin gene and began to characterize its expression. We show here, unexpectedly, that the mouse aB-crystallin gene is expressed not only in lens but also in a variety of other tissues, particularly heart, kidney, skeletal muscle, lung, and brain. It was recently reported that aB-crystallin RNA is present in hamster brain (12), and results of a preliminary report by Bhat and co-wo...

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