ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu 330 to Gln exchange (EQ-A8) in the Zn 2؉ binding motif (HE 330 LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substratebound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC 50 ϳ50 nM) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion. ADAM1 (a disintegrin and metalloprotease domain) proteins constitute a family of transmembrane glycoproteins and serve essential physiological roles in fertilization, myogenesis, and neurogenesis. These functions are due to distinct protein domains involved in cell-cell fusion, cell-cell interaction, or proteolysis of membrane proteins, a process termed ectodomain shedding (1). To date, the family of ADAM proteinases comprises more than 30 members in different species (2, 3), and 24 ADAM genes were found in the mouse genome. Fourteen of the murine ADAMs contain the catalytic consensus sequence HEXXHXXGXXHD in their metalloprotease domains and are therefore predicted to be proteolytically active (4). The cleavage of myelin basic protein (MBP) by ADAM10/MADM was the first demonstration of proteolysis by ADAMs (5). The tumor necrosis factor-␣ convertase (ADAM17) was purified on the basis of its ability to cleave tumor necrosis factor-␣ (6, 7) and a number of other peptide and protein substrates in vitro (1, 8). Proteolysis of membrane-bound surface molecules was also demonstrated for heparin-binding epidermal growth factor (9) and amyloid precursor protein (10,11), which are cleaved by ADAMs 9 and 10, respectively.Catalytically active ADAMs are usually activated by furincatalyzed removal of the prodomain or by other proprotein convertases. For cleavage by furin-like...

Alteration of the actin polypeptide chain within the DNnse I-binding loop by cleavage with E. coli A2 protease or suhtilisin was shown to increase the efficiency of energy transfer from tryptophan residues to AEDANS attached to Cys-374. Analysis of structural and fluorescence data suggested that only two of four actin tryptophan residues, namely, Trp-340 and/or Trp-356, can he energy transfer donors. It was also found that labelling with AEDANS induces perturbations in the environment of the tryptophan residues, these perturbations being smaller in the cleaved actin. These changes are consistent with a shift of the C-terminal segment of actin monomer upon cleavage and confirm the existence of high conformational coupling between subdomains 1 and 2 of actin monomer. We also suggest that tryptophan residues 340 and/or 356 are located in the focus of this coupling.

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Myotonias are muscle diseases in which the function of the muscular chloride channel ClC-1 is impaired. Null alleles of the corresponding Clc1 gene on mouse chromosome (Chr) 6 provide animal models for human myotonias. It was shown that the allele adr (Clc1adr) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1adr-mto) involves a stop codon near the N-terminus. We have determined the genomic organization of the mouse Clc1 gene and the sequence requirements for the transposon insertion in the Clc1adr allele. The mouse Clc1 gene is composed of 23 exons, ranging from 39 to 372 bp, and spans approximately 23 kb of genomic DNA. The exon/intron organization is highly homologous to that of the human CLCN1 gene; the homology of the coding sequence is 97% to rat and 89% to human. In the adr allele the ETn transposon is inserted into intron 12, the largest intron. Whereas the 5' and 3' LTR sequences of the ETn transposon are homologous to those reported for other insertional mutations of the mouse, no consensus motif for an insertion target site could be defined. On the basis of flanking sequences, we provide duplex PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1. Close to the 3' end of intron 12, a tetranucleotide repeat (AATC)n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies.

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