The tumor necrosis factor-alpha converting enzyme (TACE): a unique metalloproteinase with highly defined substrate selectivity

TNF alpha converting enzyme (TACE) processes precursor TNF alpha between Ala76 and Val77, yielding a correctly processed bioactive 17 kDa protein. Genetic evidence indicates that TACE may also be involved in the shedding of other ectodomains. Here we show that native and recombinant forms of TACE efficiently processed a synthetic substrate corresponding to the TNF alpha cleavage site only. For all other substrates, conversion occurred only at high enzyme concentrations and prolonged reaction times. Often, cleavage under those conditions was accompanied by nonspecific reactions. We also compared TNF alpha cleavage by TACE to cleavage by those members of the matrix metalloproteinase (MMP) family previously implied in TNF alpha release. The specificity constants for TNF alpha cleavage by the MMPs were approximately 100-1000-fold slower relative to TACE. MMP 7 also processed precursor TNF alpha at the correct cleavage site but did so with a 30-fold lower specificity constant relative to TACE. In contrast, MMP 1 processed precursor TNF alpha between Ala74 and Gln75, in addition to between Ala76 and Val77, while MMP 9 cleaved this natural substrate solely between Ala74 and Gln75. Additionally, the MMP substrate Dnp-PChaGC(Me)HK(NMA)-NH(2) was not cleaved at all by TACE, while collagenase (MMP 1), gelatinase (MMP 9), stromelysin 1 (MMP 3), and matrilysin (MMP 7) all processed this substrate efficiently. All of these results indicate that TACE is unique in terms of its specificity requirements for substrate cleavage.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

Shedding of interleukin-6 receptor and tumor necrosis factor alpha. Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins.

A functionally and structurally diverse group of transmembrane proteins including transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteolytic activity responsible for pro-tumor necrosis factor alpha (proTNFalpha) processing has been identified and named TACE (TNFalpha converting enzyme). In experiments with TACE deficient (TACE-/-) fibroblasts we found that 4beta-phorbol 12-myristate 13-acetate (PMA)-induced shedding of the interleukin-6 receptor (IL-6R) is strongly reduced. A basal hydroxamate sensitive release of IL-6R, however, could still be detected. This result demonstrates that TACE plays a role in IL-6R processing and that additional metalloproteases might be involved. PMA-induced shedding of IL-6R in TACE deficient mouse fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL-6R and proTNFalpha we generated chimeric IL-6R and proTNFalpha proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNFalpha and IL-6R, respectively. Interestingly, proTNFalpha chimeric proteins showed only minimal shedding. In contrast, IL-6R chimeras containing the proTNFalpha CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNFalpha CS is similar to that of proTNFalpha itself. We conclude that the amino-acid sequence at the proteolytic CS contributes to the cleavage characteristics of a protein. However, this information alone is not sufficient to transfer cleavability as seen with proTNFalpha chimeras containing the IL-6R CS and which were resistant to shedding.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha)

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.     

Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme

Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or "shedding" of several cell-surface proteins, including TNF and p75 TNFR. We established a TACE-reconstitution system using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, we identified two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, we studied the function of the different domains of TACE in three shedding activities. We found that TACE must be expressed with its membrane-anchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding required the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding required only the tethered TACE catalytic domain.     

TACE/ADAM-17 enzymatic activity is increased in response to cellular stimulation

Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease disintegrin that cleaves a variety of membrane proteins, releasing ("shedding") their extracellular domains from cells. Most TACE-mediated shedding events occur at low basal rates that are enhanced by treatment of cells with a variety of stimuli. To study the mechanism of induced shedding, we developed a peptide-cleavage assay that measures the cellular TACE activity. In unstimulated cells, cleavage of a TNFalpha processing-site peptide was mediated mainly by enzymes other than TACE. However, stimulation of cells with phorbol-12-myristate-13-acetate (PMA) increased peptide cleavage in a TACE-dependent manner. PMA treatment did not increase the amount of TACE on the cell surface. Moreover, the cytoplasmic domain of TACE was not required for the induced activity. Based on these observations, induction of TACE-mediated shedding events occurs at least in part via an increase in the enzymatic activity of cellular TACE, independent of its cytoplasmic domain.     

Extracellular phosphorylation of collagen XVII by ecto-casein kinase 2 inhibits ectodomain shedding

Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-alpha converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [gamma-(32)P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser(542) and Ser(544) in the ectodomain as phosphate group acceptors. Phosphorylation of Ser(544) in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitope-specific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser(544) to Ala significantly increased ectodomain shedding, whereas overexpression of CK2alpha inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch

TNF-alpha-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18-54 (N-TACE(18-54)) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE(18-54) attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE(18-54) inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30-48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.     

Aminoalkylcarbamoylphosphonates reduce TNFα release from activated immune cells

Some carbamoylphosphonates (CPOs) inhibit matrix metalloproteinases (MMPs). Although MMPs are involved in inflammatory processes, the anti-inflammatory activity of CPOs has not been reported. In this context we compared the biological activity of the three aminoCPOs, PYR-CPO, PIP-CPO and cis-ACCP. We were particularly interested in their capability to modulate the secretion of tumor necrosis factor alpha (TNFα). LPS-activated mouse peritoneal macrophages and LPS-activated mouse splenocytes were used to explore this question. It was found that the aminoCPOs were able to reduce TNFα secretion to a level equivalent to the reduction caused by the steroid drug budesonide (BUD). The reduction in TNFα levels was neither accompanied by cytotoxicity, nor did it inhibit cell proliferation. To explicate whether the aminoCPOs affect TNFα processing by TNFα-converting enzyme (TACE), TACE inhibitory properties of the three molecules was tested in vitro. Only PIP-CPO exerted TACE inhibitory activity at therapeutic (non-cytotoxic) concentrations, indicating on its potential to serve as an anti-inflammatory agent by reducing TNFα secretion.     

Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice

Mammalian angiotensin-converting enzyme (ACE) is one of several biologically important ectoproteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic cleavage. It has been suggested that a common proteolytic system is responsible for the cleavage of a diverse group of membrane ectoproteins, and tumor necrosis factor-alpha-converting enzyme (TACE), a recently purified disintegrin-metalloprotease, has been implicated in the proteolytic cleavage of several cell surface proteins. Mice devoid of TACE have been developed by gene targeting. Such mice could provide a useful system to determine if TACE is responsible for the cleavage of other ectoproteins. Cultured fibroblasts without TACE activity, when transfected with cDNA encoding for the testicular isozyme of ACE (ACET), synthesized and secreted ACET normally after a proteolytic cleavage near the C terminus. In addition, similar quantities of the soluble, C-terminally truncated somatic isozyme of ACE (ACEP) were present in the serum of wild-type and TACE-deficient mice. These results demonstrate that TACE is not essential in the generation of soluble ACE under physiological conditions. Finally, we also report solubilization of ACE-secretase, the enzyme that cleaves ACE, from mouse ACE89 cells and from rabbit lung. We demonstrate that soluble ACE-secretase from both sources failed to cleave its substrate in solution, suggesting a requirement for anchoring to the membrane.     

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.     

Evidence for a role of a tumor necrosis factor-alpha (TNF-alpha)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a member of the TNF family, is a dendritic cell survival factor and is essential for osteoclastogenesis and osteoclast activation. In this report we demonstrate (i) that TRANCE, like TNF-alpha, is made as a membrane-anchored precursor, which is released from the plasma membrane by a metalloprotease; (ii) that soluble TRANCE has potent dendritic cell survival and osteoclastogenic activity; (iii) that the metalloprotease-disintegrin TNF-alpha convertase (TACE) can cleave immunoprecipitated TRANCE in vitro in a fashion that mimics the cleavage observed in tissue culture cells; and (iv) that in vitro cleavage of a TRANCE ectodomain/CD8 fusion protein and of a peptide corresponding to the TRANCE cleavage site by TACE occurs at the same site that is used when TRANCE is shed from cells into the supernatant. We propose that the TRANCE ectodomain is released from cells by TACE or a related metalloprotease-disintegrin, and that this release is an important component of the function of TRANCE in bone and immune homeostasis.     

Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells

Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.     

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.     

Substitution of methionine 435 with leucine, isoleucine, and serine in tumor necrosis factor alpha converting enzyme inactivates ectodomain shedding activity.

Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is a zinc metalloprotease that has emerged as a general sheddase, which is responsible for ectodomain release of numerous membrane proteins, including the proinflammatory cytokine TNF-alpha, the leukocyte adhesin L-selectin and epidermal growth factor receptor ligand-transforming growth factor alpha (TGF-alpha), and related family members. Structurally, TACE belongs to a large clan of proteases, designated the metzincins, because TACE possesses a conserved methionine (Met435), frequently referred to as the met-turn residue, in its active site. A vital role of this residue in the function of TACE is supported by the fact that cells expressing the M435I TACE variant are defective in ectodomain shedding. However, the importance of Met435 in TACE appears to be uncertain, since another metzincin, matrix metalloprotease-2, has been found to be enzymatically fully active with either leucine or serine in place of its met-turn residue. We constructed TACE mutants with leucine or serine in place of Met435 to further examine the role of the met-turn residue in TACE-mediated ectodomain cleavage. Similar to the M435I TACE mutant, both the M435L and M435S constructs are defective in cleaving transmembrane TNF-alpha, TGF-alpha, and L-selectin. Comparative modeling and dynamic computation detected structural perturbations, which resulted in higher energy, in the catalytic zinc complexes of the Met435 TACE mutants compared with that in the wild-type enzyme. Thus, Met435 serves to maintain the stability of the catalytic center of TACE for the hydrolysis of peptide bonds in substrates.     

A continuous fluorimetric assay for tumor necrosis factor-alpha converting enzyme

Fluorogenic peptide substrates with fluorophore/quencher-capped ends have found extensive use in monitoring protease activity in the screening of small-molecule libraries for protease inhibitors. We report here the identification and characterization of a fluorogenic substrate for tumor necrosis factor-alpha converting enzyme (TACE). This substrate is a 10-amino-acid peptide (LAQAVRSSSR) capped with an o-aminobenzoyl group on the N-terminal end and with a 3-(2,4-dinitrophenyl)-L-2,3-diaminopropionic amide group on the C-terminal end. Exhaustive enzymatic conversion of the substrate to products resulted in a fluorescence enhancement of -11-fold. A single cleavage occurred at the A-V scissile bond of the peptide. The validity of this fluorimetric assay for TACE was corroborated by an independent HPLC method. Interestingly, the hydrolysis of the substrate displayed positive cooperativity with a Hill coefficient of 1.5, while the hydrolysis of the corresponding uncapped peptide displayed Michaelis-Menten kinetics. A k(cat) value of 21.6 s(-1) and an S(0.5) value of 342 microM were obtained for the fluorogenic substrate. The addition of the two capping groups on the two ends of the peptide enhanced the k(cat) value by 64-fold. Nine additional decapeptides that contained the same capping groups on the two ends and substitutions at the P1 and P1' sites were also tested. TACE appears to slightly prefer the A-V scissile bond. The enzyme also cleaves scissile bonds such as F-V, A-I, and A-L efficiently.     

Characterization and purification of a mammalian endoribonuclease specific for the alpha -globin mRNA.

The alpha-globin mRNA has previously been shown to be the target of an erythroid-enriched endoribonuclease (ErEN) activity which cleaves the mRNA within the 3'-untranslated region. We have currently undertaken a biochemical approach to purify this enzyme and have begun characterization of the enzyme to determine requirements for substrate recognition as well as optimal cleavage conditions. Through mutational analysis and truncations we show that a 26-nucleotide region of the alpha-globin 3'-untranslated region is an autonomous element that is both necessary and sufficient for cleavage by ErEN. Mutations throughout this region abolish cleavage activity by ErEN suggesting that the entire sequence is important for recognition and cleavage. ErEN is most active under biological salt concentrations and temperature and activity of the enzyme does not require cations. The size for ErEN was estimated by denaturing gel filtration analysis and is approximately 40 kDa. Interestingly, the exquisite specificity of ErEN cleavage became compromised with increased purity of the enzyme suggesting the involvement of other proteins in specificity of ErEN cleavage. Nondenaturing gel filtration of MEL extract demonstrated that ErEN is a component of an approximately 160 kDa complex implying that additional proteins may regulate ErEN activity and provide increased cleavage specificity.