The hamster gene encoding the 78-kDa glucose-regulated protein (Grp78) was expressed in Escherchia coil as a fusion protein with glutathione S-transferase. After induction with isopropyl P-D-thiogalactopyranoside, the recombinant Grp78 was purified to homogeneity by affmiity column chromatography of the fusion protein followed by thrombin cleavage. The purified recombinant protein was compared with liver Grp78 for its ability to interact with ATP. Like liver Grp78, the recombinant protein contained a weak ATPase activity and a Ca2+-stimulated autophosphorylation activity. However, unlike liver Grp78, in which the autophosphorylation reaction is stimulated <50% by CaC12, the reaction with the recombinant Grp78 was stimulated about 15-fold in the presence of Ca2+. Although the liver protein showed at least four isoforms after two-dimensional gel electrophoresis, the recombinant Grp78 had one major species corresponding to the most basic form seen in liver. Both the liver Grp78 and the recombinant protein existed primarily as monomers and dimers. A small amount of oligomers was also present in the liver Grp78. When either protein was incubated with ATP, there was a conversion of the higher molecular weight species to the monomeric form.

The structural gene for l-lactamase II (EC 3.5.2.6), a metallothioenzyme, from Bacillus cereus 569/H (constitutive for high production of the enzyme) was cloned in Escherichia coli, and the nucleotide sequence was determined. This is the first class B ,l-lactamase whose primary structure has been reported. The amino acid sequence of the exoenzyme form, deduced from the DNA, indicates that P-lactamase II, like other secreted proteins, is synthesized as a precursor with a 30-amino acid N-terminal signal peptide. The pre-I8-lactamase II (Mr, 28,060) is processed in E. coli and in B. cereus to a single mature protein (Mr, 24,932) which is totally secreted by B. cereus but in E. coli remains intracellular, probably in the periplasm. The expression of the gene in E. coli RR1 on the multicopy plasmid pRWHO12 was comparable to that in B. cereus, where it is presumably present as a single copy. The three histidine residues that are involved (along with the sole cysteifte of the mature protein) in Zn(II) binding and hence in enzymatic activity against ,-lactams were identified. These findings will help to define the secondary structure, mechanism of action, and evolutionary lineage of B. cereus I8-lactamase II and other class B I8-lactamases. P-Lactamnases are widely distributed in bacteria (10). They differ in their enzymic and molecular properties and are thought to have a polyphyletic origin (2, 3). Some are chromosomally encoded (e.g., the ,-lactamase of Bacillus licheniformis), while others are plasmid borne (e.g., some P-lactamases of Staphylococcus aureus). P-Lactamases are grouped into three classes based on size and sequence homology (3, 19). Some of the secreted 3-lactamnases of Bacillus spp. and Staphylococcus spp. and the periplasmic R6K ,-lactamase carried on pBR322 in Escherichia coli (42) fall in class A. They are highly active on benzylpenicillin, have an Mr of-30,000, and show considerable homology with one another. They have a serine at the active site and are thought to have diverged from a single ancestral gene (3). Class B P-lactamases are Of Mr-23,000, are almost as active towards cephalosporins as penicillins, and are metallothioenzymes. This class is restricted to B. cereus and very closely related bacilli (21) and possibly Pseudomonas maltophilia P-lactamase Li (37). Primary structure has not been reported for any of the class B ,-lactamases. Class C was added by Jaurin and Grundstrom (19) to accommodate the ampC gene product, a chromosomally encoded cephalosporinase of E. coli. It is a mnolecule of Mr-39,000, with * Corresponding author. two Zn(II) ions per enzyme molecule (5, 13). Hydrolysis of

Recent studies have shown that ATP can dissociate dimers of the glucose-regulated protein Grp78 to monomers. In the present study, we have used purified recombinant Grp78 from Escherihia coli to investigate this reaction in more detail. During the course ofthe Grp78 dimer-monomer conversion, a stable Grp78 monomer-ATP complex is formed. Upon removal of the ATP, the Grp78 dimer is reformed. ADP, nonhydrolyzable ATP analogues, and GTP do not effect the dissociation of Grp78 dimers. A cell line that overproduces IgE Fc has been used to examine the nature of the Grp78-IgE Fc complexes present and the effect of ATP on them. Grp78-IgE Fc complexes ranging from 100 kDa to 300 kDa were observed by sucrose gradient analysis, suggesting that aggregate forms of Grp78 may be present in some of these complexes. Treatment of the extracts with ATP resulted in release of a Grp78 monomer from the complex. These results suggest that the dissociation of Grp78 oligomers by ATP may be involved in the function of Grp78 in protein translocation through the endoplasmic reticulum.

BiP is a member of the Hsp7O heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 pM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.Heat shock proteins (Hsps) or stress proteins, such as members of the Hsp7O family, interact with proteins to facilitate proper folding and assembly within the cell (1-3). These proteins are known to interact with ATP (4, 5) and possess a week ATPase activity (6-9) that can be stimulated by peptides and proteins that bind to the chaperone (7,10,11). ATP binding and/or hydrolysis is also believed to be involved in the dissociation of the Hsp70-protein complex (1)(2)(3)(10)(11)(12)(13)(14)(15), and recently, several studies have examined the interaction of ATP, ADP, and polypeptides with members of the Hsp7O family (4,(10)(11)(12)(13)(14).One member of this group called BiP (immunoglobulin heavy chain binding protein) or Grp78 (78-kDa glucoseregulated protein) is found in the lumen of the endoplasmic reticulum (ER), where it interacts transiently with some, but not all, nascent polypeptides destined to be secreted (3,(16)(17)(18)(19)(20). However, BiP also forms much tighter complexes with malfolded proteins (e.g., mutated and underglycosylated proteins), unassembled polypeptides, etc. (21)(22)(23)(24). In these cases the BiP-protein complexes are so tight that transport through the ER is prevented. Previously we initiated experiments to investigate the interaction of ATP with BiP, using either BiP isolated from liver (IBiP) or a recombinant form (rBiP) made in Escherichia coli (9, 13). We showed, using gel-filtration chromatography, that ATP could dissociate BiP dimers to monomers and that ATP bound to the monomer species (9, 13). In addition, in cell extracts a BiP-IgE Fc complex that accumulates in the ER was dissociated by ATP to yield a BiP monomer (13 (11) proposed a similar scheme for the interaction of BiP with substrates. In the present study we show that the 11-amino acid peptide substance P (SP), which was reported to bind to BiP (27), acts as a model substrate for BiP interactions on the basis of criteria first described by Flynn et al. (7). A rapid filter-binding assay has been used to examine the effect of SP on nucleotide binding to BiP, and our results are consistent with an initial interaction of SP with a BiP-ATP complex and a subsequent series...

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