Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

Structure-Function Studies with the Unique Hexameric Form II Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) from Rhodopseudomonas palustris

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a “closed” conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile¹⁶⁵ and Met³³¹) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala⁴⁷) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme''s k_cat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.     

The Roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel

The human and rat forms of the Kv2.1 channel have identical amino acids over the membrane-spanning regions and differ only in the N- and C-terminal intracellular regions. Rat Kv2.1 activates much faster than human Kv2.1. Here we have studied the role of the N- and C-terminal residues that determine this difference in activation kinetics between the two channels. For this, we constructed mutants and chimeras between the two channels, expressed them in oocytes, and recorded currents by two-electrode voltage clamping. In the N-terminal region, mutation Q67E in the rat channel displayed a slowing of activation relative to rat wild type, whereas mutation D75E in the human channel showed faster activation than human wild type. In the C-terminal region, we found that some residues within the region of amino acids 740-853 ("CTA" domain) were also involved in determining activation kinetics. The electrophysiological data also suggested interactions between the N and C termini. Such an interaction was confirmed directly by using a glutathione S-transferase (GST) fusion protein with the N terminus of Kv2.1, which we showed to bind to the C terminus of Kv2.1. Taken together, these data suggest that exposed residues in the T1 domain of the N terminus, as well as the CTA domain in the C terminus, are important in determining channel activation kinetics and that these N- and C-terminal regions interact.     

The C terminus of mammalian phospholipase D is required for catalytic activity

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the -2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal alpha carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.     

Determination of amino acid sequences of two subunits in sarcosine oxidase from Corynebacterium sp. U-96

The primary structures of the C and D subunits of sarcosine oxidase from Corynebacterium sp. U-96 were determined by sequencing the peptide fragments derived from their enzymatic digestions. The C and D subunits were shown to be composed of 199 and 92 residues, respectively. Each amino acid sequence showed a high homology with the sequence of the corresponding subunit from Corynebacterium sp. P-1. However, there were some differences between these two species, that is, four N-terminal residues were truncated in the C subunit, but six C-terminal residues were truncated in the D subunit. The D subunit contained three cysteine residues, but no disulfide bonds are in the subunit. Overall sequences of both subunit showed no homology with any other protein in the data base.     

Carboxy-terminal processing of the large subunit of [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757

hydA and hydB, the genes encoding the large (46-kDa) and small (13. 5-kDa) subunits of the periplasmic [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757, have been cloned and sequenced. The deduced amino acid sequence of the genes product showed complete identity to the sequence of the well-characterized [Fe] hydrogenase from the closely related species Desulfovibrio vulgaris Hildenborough (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). The data show that in addition to the well-known signal peptide preceding the NH2 terminus of the mature small subunit, the large subunit undergoes a carboxy-terminal processing involving the cleavage of a peptide of 24 residues, in agreement with the recently reported data on the three-dimensional structure of the enzyme (Y. Nicolet, C. Piras, P. Legrand, E. C. Hatchikian, and J. C. Fontecilla-Camps, Structure 7:13-23, 1999). We suggest that this C-terminal processing is involved in the export of the protein to the periplasm.     

The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin.     

Identification of the Shiga toxin A-subunit residues required for holotoxin assembly.

Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.     

Primary structure of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.     

Cloning and sequencing of the leucine dehydrogenase gene from Bacillus sphaericus IFO 3525 and importance of the C-terminal region for the enzyme activity

The structural gene (leudh) coding for leucine dehydrogenase from Bacillus sphaericus IFO 3525 was cloned into Escherichia coli cells and sequenced. The open reading frame coded for a protein of 39.8 kDa. The deduced amino acid sequence of the leucine dehydrogenase from B. sphaericus showed 76–79% identity with those of leucine dehydrogenases from other sources. About 16% of the amino acid residues of the deduced amino acid sequence were different from the sequence obtained by X-ray analysis of the B. sphaericus enzyme. The recombinant enzyme was purified to homogeneity with a 79% yield. The enzyme was a homooctamer (340 kDa) and showed the activity of 71.7 μmol·min⁻¹·mg⁻¹) of protein. The mutant enzymes, in which more than six amino acid residues were deleted from the C-terminal of the enzyme, showed no activity. The mutant enzyme with deletion of four amino acid residues from the C-terminal of the enzyme was a dimer and showed 4.5% of the activity of the native enzyme. The dimeric enzyme was more unstable than the native enzyme, and the K_m values for -leucine and NAD⁺ increased. These results suggest that the Asn-Ile-Leu-Asn residues of the C-terminal region of the enzyme play an important role in the subunit interaction of the enzyme.     

The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain.     

Thermal aggregation of ribonuclease A. A contribution to the understanding of the role of 3D domain swapping in protein aggregation

By lyophilizing RNase A from 40% acetic acid solutions, two dimeric aggregates, the "minor" and "major" dimers (named here N-dimer and C-dimer, respectively), form by 3D domain swapping at a ratio of 1:4. Trimeric and tetrameric aggregates are also obtained. The two dimers and the higher oligomers also form without a lyophilization step. By keeping RNase A dissolved at a high concentration (generally 200 mg/ml) in various media at temperatures ranging from 23 to 70 degrees C for times varying from a few minutes to 2 h, various oligomers, in particular the two dimeric conformers, formed in quite different amounts, often inverting their relative quantities depending on the more or less severe unfolding conditions. When unfolding mainly concerned the N terminus of the protein, richer in hydrophilic residues, the N-dimer, formed by 3D domain swapping of the N-terminal alpha-helix of each monomer, prevailed over the C-dimer. Under more vigorous denaturing conditions, where also the C terminus of RNase A, richer in hydrophobic amino acids, unfolded, the C-dimer, formed by 3D domain swapping of the C-terminal beta-strand, prevailed over the other, possibly because of the induction to aggregation promoted by the hydrophobic residues present in the C termini of the two monomers.     

Structural characterization of Escherichia coli phosphatidylserine decarboxylase

Phosphatidylserine decarboxylase of Escherichia coli is one of a small group of pyruvoyl-dependent enzymes (Satre, M., and Kennedy, E.P. (1978) J. Biol. Chem. 253, 479-483). The DNA sequence of the structural gene (psd) and partial protein sequence studies demonstrate that the enzyme contains two nonidentical subunits, alpha (Mr = 7,332) and beta (Mr = 28,579), which are derived from a single proenzyme. These two subunits are blocked at their respective amino termini. Reduction of the enzyme with NaCNBH3 in the presence of radiolabeled phosphatidylserine resulted in association of the label with the alpha subunit. Similar reduction in the presence of ammonium ions exposed a new amino terminus for the alpha subunit beginning with alanine. Therefore, the pyruvate prosthetic group is in amide linkage to the amino terminus of the alpha subunit. The amino terminus of the beta subunit was determined to be formylmethionine. The carboxyl terminus of the beta subunit was determined to be glycine as predicted by the DNA sequence. Comparison of the DNA sequence and protein sequence information revealed that the decarboxylase is made as a proenzyme (Mr = 35,893), and the predicted amino acid at the position of the pyruvate within the open reading frame of the proenzyme is serine. Therefore, as with other pyruvoyl-dependent decarboxylases, the prosthetic group is derived from serine through a post-translational cleavage of a proenzyme.     

Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.     

COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini

From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus.     

Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein

Bacterial outer membrane proteins are supposed to span the membrane repeatedly, mostly in the form of amphipathic beta-sheets. The last ten C-terminal amino acid residues of PhoE protein are supposed to form such a membrane-spanning segment. Deletion of this segment completely prevents incorporation into the outer membrane. Comparison of the last ten amino acid residues of other outer membrane proteins from different Gram-negative bacteria revealed the presence of a potential amphipathic beta-sheet with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7 and 9 from the C terminus, in the vast majority of these proteins. Since such sequences were not detected at the C termini of periplasmic proteins, it appears to be possible to discriminate between the majority of outer membrane proteins and periplasmic proteins on the basis of sequence data. The highly conserved phenylalanine at the C termini of outer membrane proteins suggests an important function for this amino acid in assembly into the outer membrane. Site-directed mutagenesis was applied to study the role of the C-terminal Phe in PhoE protein assembly. All mutant proteins were correctly incorporated into the outer membrane to some extent, but the efficiency of the process was severely affected. It appears that both the hydrophobicity and the aromatic nature of Phe are of importance.     

Isolation and analysis of the gene encoding the pyruvate-ferredoxin oxidoreductase of Desulfovibrio africanus, production of the recombinant enzyme in Escherichia coli, and effect of carboxy-terminal deletions on its stability.

Previous studies have shown that the pyruvate-ferredoxin oxidoreductase (POR) of the sulfate-reducing bacterium Desulfovibrio africanus is a homodimer that contains one thiamine pyrophosphate and three [4Fe-4S]2+/1+ centers/subunit. Interestingly, the enzyme isolated from a strictly anaerobic bacterium is highly stable in the presence of oxygen, in contrast to the other PORs characterized in anaerobic organisms (L. Pieulle, B. Guigliarelli, M. Asso, F. Dole, A. Bernadac, and E. C. Hatchikian, Biochim. Biophys. Acta 1250:49-59, 1995). We report here the determination of the nucleotide sequence of the por gene encoding the D. africanus POR. The amino acid sequence deduced from this nucleotide sequence corresponds to the first primary structure of a homodimeric POR from strictly anaerobic bacteria. The subunit of the D. africanus POR contains two ferredoxin-type [4Fe-4S] cluster binding motifs (CX2CX2CX3CP) and four additional highly conserved cysteines belonging to a nontypical motif. These 12 cysteine residues may coordinate the three Fe-S centers present in D. africanus POR. The thiamine pyrophosphate binding domain is located in the C-terminal part of the protein close to the four conserved cysteine residues. The D. africanus enzyme sequence appears homologous to the other POR sequences. However, the enzyme differs from all other PORs by a C-terminal extension of about 60 residues of its polypeptide chain. The two cysteine residues located in this additional region may be involved in the formation of a disulfide bridge associated with the activation process of the catalytic activity. The por gene has been expressed, for the first time, in anaerobically grown Escherichia coli behind the isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter, resulting in the production of POR in its active form. The recombinant enzyme is stable toward oxygen during several days, and initial characterization of the recombinant POR showed that its activity increased in the presence of dithioerythritol. These properties indicate that the recombinant POR behaves like the native D. africanus enzyme. The study of carboxy-terminal deletion mutants strongly suggests that deletions in the C-terminal region of D. africanus enzyme can have dramatic effects on the stability of the enzyme toward oxygen.     

The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.     

The alpha subunit of meprin A. Molecular cloning and sequencing, differential expression in inbred mouse strains, and evidence for divergent evolution of the alpha and beta subunits.

Meprin A, a membrane-bound oligomeric metalloendopeptidase, contains two different subunits, alpha and beta. We report here the cloning and sequencing of the alpha subunit cDNA. The translated polypeptide consists of 760 amino acids, including a preprosequence (77 amino acids) that precedes the NH2 terminus of the purified enzyme. The next 198 amino acids constitute the "astacin family" protease domain, which includes the astacin family signature sequence, HE(L,I)XHXXGFXHE(Q,H)XRXDRDX(Y,H)(V,I)X(I,V). An immunoglobulin/major histocompatibility complex protein signature was found at the end of the protease domain. At the COOH terminus of the alpha subunit, there is an epidermal growth factor-like domain, followed by a transmembrane domain, and six additional amino acids. Ten potential glycosylation sites have been identified, and at least three of those sites are glycosylated. Northern blot analyses of kidney tissue from C57BL/6 and C3H/He mice indicate that variations in meprin A activity in these strains reflect differences in the levels of the alpha subunit mRNA. Several internal peptide sequences obtained from the beta subunit indicate that it is approximately 50% identical to the alpha subunit. Furthermore, NH2-terminal sequence analyses (39 residues) indicate that rat and mouse alpha are 79% identical, rat and mouse beta are 74% identical, and that alpha and beta subunits for both species are 47% identical. These data indicate that alpha and beta are closely related products of divergent evolution.