Structure of homo- and hetero-oligomeric meprin metalloproteases. Dimers,tetramers, and high molecular mass multimers

Meprin A and B, metalloproteases consisting of evolutionarily related alpha and/or beta subunits, are membrane-bound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each approximately 80 kDa) form disulfide-bridged homo- and heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin beta subunits only, was dimeric under a wide range of conditions. By contrast, meprin alpha homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from approximately 1.0 to 6.0 MDa. The size of the meprin alpha homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin alphabeta heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin beta, which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin alpha homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.     

Developmental expression of meprin metalloprotease subunits in ICR and C3H/He mouse kidney and intestine in the embryo, postnatally and after weaning

Meprins are secreted and membrane-bound metalloendopeptidases highly expressed in kidney and intestinal epithelial cells. They are oligomeric glycoproteins composed of evolutionarily related alpha and/or beta subunits. The present work revealed that the messages for both meprin subunits were expressed in intestine and kidney in ICR and C3H/He mouse embryos (as early as day 11), indicating developmental functions for both subunits. During the first 2 weeks after birth, the mRNA levels for both subunits increased in ICR mice, but between 10 days and 3 weeks (time of weaning) the alpha subunit level in the intestine fell markedly. In adult ICR mice, meprin beta mRNA was consistently expressed in both kidney and intestine, whereas meprin alpha mRNA was highly expressed in kidney but only present at low levels in intestine. In C3H/He mice, the pattern of meprin alpha and beta subunit mRNA expression was similar to that of ICR mice, except that meprin alpha was barely detectable in kidney after birth. The results of postnatal studies indicate that the meprin alpha subunit has a role in the intestine during suckling but is not essential after weaning, and that the beta homooligomer is the major meprin form after weaning in both kidney and intestine.     

Critical amino acids in the active site of meprin metalloproteinases for substrate and peptide bond specificity

The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.     

Intersubunit interactions in human X,K-ATPases: role of membrane domains M9 and M10 in the assembly process and association efficiency of human, nongastric H,K-ATPase alpha subunits (ATP1al1) with known beta subunits

Na,K- and H,K-ATPase (X,K-ATPase) alpha subunits need association with a beta subunit for their maturation, but the authentic beta subunit of nongastric H,K-ATPase alpha subunits has not been identified. To better define alpha-beta interactions in these ATPases, we coexpressed human, nongastric H,K-ATPase alpha (AL1) and Na,K-ATPase alpha1 (alpha1NK) as well as AL1-alpha1 and alpha1-AL1 chimeras, which contain exchanged M9 and M10 membrane domains, together with each of the known beta subunits in Xenopus oocytes and followed their resistance to cellular and proteolytic degradation and their ER exit. We show that all beta subunits (gastric betaHK, beta1NK, beta2NK, beta3NK, or Bufo bladder beta) can associate efficiently with alpha1NK, but only gastric betaHK, beta2NK, and Bufo bladder beta can form stably expressed AL1-beta complexes that can leave the ER. The trypsin resistance and the forces of subunit interaction, probed by detergent resistance, are lower for AL1-beta complexes than for alpha1NK-beta complexes. Furthermore, chimeric alpha1-AL1 can be stabilized by beta subunits, but alpha1-AL1-gastric betaHK complexes are retained in the ER. On the other hand, chimeric AL1-alpha1 cannot be stabilized by any beta subunit. In conclusion, these results indicate that (1) none of the known beta subunits is the real partner subunit of AL1 but an as yet unidentified, authentic beta should have structural features resembling gastric betaHK, beta2NK, or Bufo bladder beta and (2) beta-mediated maturation of alpha subunits is a multistep process which depends on the membrane insertion properties of alpha subunits as well as on several discrete events of intersubunit interactions.     

Meprin proteolytic complexes at the cell surface and in extracellular spaces

Meprins are metalloproteinases of the astacin family and metzincin superfamily that are composed of evolutionarily related alpha and beta subunits, which exist as homo- and hetero-oligomeric complexes. These complexes are abundant at the brush border membranes of kidney proximal tubule cells and epithelial cells of the intestine, and are also expressed in certain leucocytes and cancer cells. Meprins cleave bioactive peptides such as gastrin, cholecystokinin and parathyroid hormone, cytokines such as osteopontin and monocyte chemotactic peptide-1, as well as proteins such as gelatin, collagen IV, fibronectin and casein. Database predictions and initial data indicate that meprins are also capable of shedding proteins, including itself, from the cell surface. Membrane-bound meprin subunits are composed of dimeric meprin beta subunits or tetrameric hetero-oligomeric alpha beta complexes of approx. 200-400 kDa, and can be activated at the cell surface; secreted forms of homo-oligomeric meprin alpha are zymogens that form high-molecular-mass complexes of 1-6 MDa. These are among the largest extracellular proteases identified thus far. The latent (self-associating) homo-oligomeric complexes can move through extracellular spaces in a non-destructive manner, and deliver a concentrated form of the metalloproteinase to sites that have activating proteases, such as sites of inflammation, infection or cancerous growth. Meprins provide examples of novel ways of concentrating proteolytic activity at the cell surface and in the extracellular milieu, which may be critical to proteolytic function.     

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.     

Transport of meprin subunits through the secretory pathway: role of the transmembrane and cytoplasmic domains and oligomerization

The meprin alpha subunit, a multidomain metalloproteinase, is synthesized as a type I membrane protein and proteolytically cleaved during biosynthesis in the endoplasmic reticulum (ER), consequently losing its membrane attachment and COOH-terminal domains. The meprin alpha subunit is secreted as a disulfide-linked dimer that forms higher oligomers. By contrast, the evolutionarily related meprin beta subunit retains the COOH-terminal domains during biosynthesis and travels to the plasma membrane as a disulfide-linked integral membrane dimer. Deletion of a unique 56-amino acid inserted domain (the I domain) of meprin alpha prevents COOH-terminal proteolytic processing and results in the retention of this subunit within the ER. To determine elements responsible for this retention versus transport to the cell surface, mutagenesis experiments were performed. Replacement of the meprin alpha transmembrane (alphaT) and cytoplasmic (alphaC) domains with their beta counterparts allowed rapid movement of the alpha subunit to the cell surface. The meprin alphaT and alphaC domains substituted into meprin beta delayed movement of this chimera through the secretory pathway. Replacement of glycines in the meprin alphaT domain GXXXG motif with leucine residues, alanine insertions in the meprin alphaT domain, and mutagenesis of basic residues within the meprin alphaC domain did not enhance the movement of the alpha subunit through the secretory pathway. By contrast, a mutant of meprin alpha (C320AalphaDeltaI) that did not form disulfide-linked dimers or higher order oligomers was transported through the secretory pathway, although more slowly than meprin beta. Taken together, the data indicate that the meprin alphaT and alphaC domains together contain a weak signal for retention within the ER/cis-Golgi compartments that is strengthened by oligomerization.     

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.     

Probing the active sites and mechanisms of rat metalloproteases meprin A and B

Meprin A and B are highly regulated, secreted and cell-surface homo- and hetero-oligomeric enzymes. Meprins are abundantly expressed in kidney and intestine. The multidomain alpha and beta subunits have high sequence identity, however they have very different substrate specificities, oligomerization potentials and are differentially regulated. Here we describe that meprin subunit activities are modulated differently by physico-chemical factors. Homo-oligomeric meprin B had an acidic pH optimum. The low pH protonation indicated the existence of at least two ionizable groups. An additional ionizable group generated a shoulder in the basic pH range. Homo-oligomeric meprin A had a neutral pH optimum and the activity curve revealed that two ionizable groups might be protonated at acidic pH similar to meprin B. Increasing the concentration of salt generally inhibited meprin B activity. Meprin A was inhibited at low salt concentrations but activated as salt was increased. This work has important implications in the elucidation of the catalytic mechanisms of meprins and other metalloproteases. In addition, the activity of meprin oligomers that arise in tissues will be affected by variations in pH and NaCl. This could have profound implications because meprins are exposed to a range of conditions in the extracellular milieu of renal and intestinal tissues and in inflammation and cancer.     

Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers.

We have expressed glycine receptor (GlyR) alpha and beta subunit cDNAs in HEK-293 cells to study the functional properties of homo- versus hetero-oligomeric GlyR channels. Dose-response curves of whole-cell currents in cells expressing alpha 1 subunits revealed an average Hill coefficient of h = 4.2. Co-expression with the beta subunit markedly increased glycine-gated whole-cell currents, which now exhibited a mean Hill coefficient of only h = 2.5. For alpha 1, alpha 2 and alpha 3 homo-oligomers, the main-state single-channel conductances were 86, 111 and 105 pS, respectively, recorded at symmetrical Cl- concentrations of 145 mM. The mutant alpha 1 G221A gave rise to a main-state of 107 pS. This indicates that the main-state of alpha homo-oligomers depends on residue 221 which is located within transmembrane segment M2. Importantly, the main-state conductances of alpha 1/beta, alpha 2/beta and alpha 3/beta hetero-oligomers were only 44, 54 and 48 pS, respectively. The latter values are similar to those found in spinal neurons, suggesting that native GlyRs are predominantly alpha/beta hetero-oligomers. Co-expression of alpha 1 with mutant beta subunits revealed that residues within and close to segment M2 of the beta subunit determine the conductance differences between homo- and hetero-oligomers.     

Characterization of alpha 2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.     

Detection of a glycosylated, incompletely folded form of chorionic gonadotropin beta subunit that is a precursor of hormone assembly in trophoblastic cells

The alpha and beta subunits of human chorionic gonadotropin are secreted both as a combined, noncovalently linked dimer form as well as uncombined, free forms by human trophoblastic cells. We have utilized the cultured choriocarcinoma cell line JAR to determine what regulates the combination of the two subunits. The human chorionic gonadotropin subunits produced by JAR cells were biosynthetically labeled with [35S] cysteine or [3H]mannose by a pulse-chase protocol, purified by immunoprecipitation with specific antisera that recognize free or combined subunits, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. Radioactively labeled bands were eluted from the gels and analyzed for total counts/minute incorporated, the ratio of free thiols to intramolecular cystine disulfides, and oligosaccharide composition. In some experiments, labeled gel bands were eluted with trypsin under nonreducing conditions, and the trypsin-released peptides were analyzed by high performance liquid chromatography. Using these procedures, the following results were obtained. The earliest, biosynthetically labeled form of the beta subunit detected in JAR cells contains high mannose N-linked oligosaccharides and has one-half of its incorporated cysteines present as free thiols. This form, termed pre-beta 1, has not yet combined with the alpha subunit even though the biosynthetically labeled alpha subunit is present in the cells at the same time. The pre-beta 1 form has a t1/2 of about 4 min and has a precursor-product relationship with a more completely disulfide-bonded form, termed pre-beta 2, which does combine with the alpha subunit to form a dimer. A subset of beta molecules produced in JAR cells does not attain the same disulfide bonding pattern as the pre-beta 2 form, does not combine with the alpha subunit, and is secreted as a free beta subunit into the culture medium. On the other hand, the earliest detectable form of the alpha subunit in JAR cells has all its thiols present as cystine disulfides, at a time when dimerization with the beta subunit has not yet taken place. These results strongly suggest that intramolecular disulfide bond formation in the beta subunit is the crucial and rate-limiting event in alpha beta dimer formation. The subset of beta molecules that remain free do not appear to form the appropriate intramolecular disulfides and thus do not achieve the correct conformation to combine with the alpha subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the cell surface heterodimer VLA-4 and related peptides

A monoclonal antibody (B-5G10) was produced which specifically recognizes the Mr 150,000/130,000 VLA-4 complex on the surface of human cells. Cross-linking studies indicated that the Mr 150,000 alpha 4 subunit of VLA-4 is in noncovalent 1:1 association with the Mr 130,000 VLA beta subunit. In the absence of cross-linking, the VLA-4 alpha 4 beta subunit complex was easily dissociated, especially in Nonidet P-40 detergent, or at elevated pH (above 8.0). Studies of dissociated subunits showed that B-5G10 recognizes an epitope on the Mr 150,000 alpha 4 subunit of VLA-4, whereas the beta subunit is immunologically identical to the Mr 130,000 beta subunit common to all VLA heterodimers. VLA-4 is widely distributed on hematopoietic cells, including thymocytes, peripheral blood lymphocytes, monocytes, activated T cells, T and B lymphoblastoid cell lines, and myeloid cell lines. However, VLA-4 is only weakly expressed on most adherent cell lines tested. Immunoprecipitates of VLA-4 often contain additional proteins of Mr 80,000 and Mr 70,000. These are probably derived from the Mr 150,000 alpha 4 subunit because: 1) they are both recognized by anti-alpha 4 sera, but not anti-beta sera; 2) the sum of their sizes is equal to the size of alpha 4; 3) they are selectively coexpressed with alpha 4 and not other VLA alpha subunits; 4) the Mr 80,000 protein has an identical NH2-terminal sequence to alpha 4; 5) like alpha 4, the Mr 70,000 and 80,000 peptides can variably associate with the VLA beta subunit; and 6) trypsin appears to cleave the Mr 150,000 alpha 4 subunit into products of Mr 70,000 and 80,000.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Activation of human meprin-alpha in a cell culture model of colorectal cancer is triggered by the plasminogen-activating system

The activation of latent proenzymes is an important mechanism for the regulation of localized proteolytic activity. Human meprin-alpha, an astacin-like zinc metalloprotease expressed in normal colon epithelial cells, is secreted as a zymogen into the intestinal lumen. Here, meprin is activated after propeptide cleavage by trypsin. In contrast, colorectal cancer cells secrete meprin-alpha in a non-polarized way, leading to accumulation and increased activity of meprin-alpha in the tumor stroma. We have analyzed the activation mechanism of promeprin-alpha in colorectal cancer using a co-culture model of the intestinal mucosa composed of colorectal adenocarcinoma cells (Caco-2) cultivated on filter supports and intestinal fibroblasts grown in the companion dish. We provide evidence that meprin-alpha is activated by plasmin and show that the presence of plasminogen in the basolateral compartment of the co-cultures is sufficient for promeprin-alpha activation. Analysis of the plasminogen-activating system in the co-cultures revealed that plasminogen activators produced and secreted by fibroblasts converted plasminogen to active plasmin, which in turn generated active meprin-alpha. This activation mechanism offers an explanation for the observed meprin-alpha activity in the tumor stroma, a prerequisite for a potential role of this protease in colorectal cancer.     

Heterologously overexpressed, affinity-purified human meprin alpha is functionally active and cleaves components of the basement membrane in vitro

Meprins are astacin-like metalloproteases of renal and intestinal epithelia and embryonic neuroepithelial cells. The full length cDNA of the human meprin alpha subunit has been overexpressed in baculovirus-infected insect cells yielding the tetrameric proprotein which could be proteolytically activated and affinity-purified to homogeneity. Recombinant meprin alpha hydrolyzes the synthetic substrate N-benzoyl-tyrosyl-p-aminobenzoic acid (PABA-peptide) and cleaves by limited proteolysis the basement membrane constituents laminin 1 and laminin 5. This supports a concept that meprin alpha, when basolaterally secreted by human colon carcinoma epithelial cells, increases the proteolytic capacity for tumor progression in the stroma.     

Targeted disruption of the meprin beta gene in mice leads to underrepresentation of knockout mice and changes in renal gene expression profiles

Meprins are multidomain zinc metalloproteases that are highly expressed in mammalian kidney and intestinal brush border membranes and in leukocytes and certain cancer cells. Mature meprins are oligomers of evolutionarily related, separately encoded alpha and/or beta subunits. Homooligomers of meprin alpha are secreted; oligomers containing meprin beta are plasma membrane associated. Meprin substrates include bioactive peptides and extracellular matrix proteins. Meprins have been implicated in cancer and intestinal inflammation. Additionally, meprin beta is a candidate gene for diabetic nephropathy. To elucidate in vivo functions of these metalloproteases, meprin beta null mice were generated by targeted disruption of the meprin beta gene on mouse chromosome 18q12. Analyses of meprin beta knockout mice indicated that (i) 50% fewer null mice are born than the Mendelian distribution predicts, (ii) null mice that survive develop normally and are viable and fertile, (iii) meprin beta knockout mice lack membrane-associated meprin alpha in kidney and intestine, and (iv) null mice have changes in renal gene expression profiles compared to wild-type mice as assessed by microarray analyses. Thus, disruption of the meprin beta allele in mice affects embryonic viability, birth weight, renal gene expression profiles, and the distribution of meprin alpha in kidney and intestine.     

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.     

Characterization of the lateral interaction between human erythrocyte spectrin subunits.

A technique in which the subunits of human erythrocyte spectrin were immobilized on a nitrocellulose membrane was developed to study which domains of the subunit are able to bind to the counterpart subunit. The limited tryptic digestion of the isolated alpha and beta subunits of human erythrocyte spectrin produced eight fragments in the alpha subunits and nine fragments in the beta subunit. Four fragments of the beta (80, 60, 44, and 18 kDa) and two of the alpha (82 and 33 kDa) bound to alpha and beta subunits which were immobilized on nitrocellulose membrane strips, respectively. The binding affinities of all the fragments to the subunits, however, were remarkably lower than that of the mother proteins. The titration of fluorescence anisotropy of N-(1-anilinonaphthyl-4)maleimide which was covalently attached to the subunit by the trypsin-digested fraction of the counterpart subunit also indicate weak binding of the fragments even in solution. These findings suggest that the high-affinity binding of the alpha subunit to the beta subunit to form spectrin alpha beta dimer occurs only when the binding domains are arrayed along the polypeptide chains at the appropriate positions on the subunits.