Therapeutic drug monitoring of amlodipine and the Z-FHL/HHL ratio: Adherence tools in patients referred for apparent treatment-resistant hypertension.

BACKGROUND: Non-adherence to antihypertensives is a cause of 'pseudo-treatment-resistant' hypertension. OBJECTIVE: To determine whether monitoring plasma amlodipine concentrations and inhibition of angiotensin-converting enzyme (ACE) can be adjunct adherence tools. METHODS: Patients with hypertension who were prescribed enalapril and amlodipine were enrolled. Blood pressures (BPs) were monitored and an adherence questionnaire was completed. Steady-state amlodipine was assayed using liquid chromatography-mass spectrometry and degree of ACE inhibition using the Z-FHL/HHL (z-phenylalanine-histidine-leucine/hippuryl-histidine-leucine) ratio. RESULTS: One hundred patients (mean (standard deviation) age 50.5 (12) years, 46% male) were enrolled. Based on plasma assays, 26/97 patients (26.8%) were unsuppressed by enalapril and 20/100 (20%) were sub-therapeutic for amlodipine. There were significant BP differences based on plasma levels of the medication: 21/20 mmHg lower in the group with suppressed ACE and 26/20 mmHg in the group with steady-state amlodipine concentrations. CONCLUSIONS: Monitoring antihypertensive adherence by assaying plasma medication concentrations is a feasible option for evaluating true v. pseudo-resistant hypertension.

Therapeutic drug monitoring of amlodipine and the Z-FHL/HHL ratio: Adherence tools in patients referred for apparent treatment-resistant hypertension

Background. Non-adherence to antihypertensives is a cause of 'pseudo-treatment-resistant' hypertension.Objective. To determine whether monitoring plasma amlodipine concentrations and inhibition of angiotensin-converting enzyme (ACE) can be adjunct adherence tools.Methods. Patients with hypertension who were prescribed enalapril and amlodipine were enrolled. Blood pressures (BPs) were monitored and an adherence questionnaire was completed. Steady-state amlodipine was assayed using liquid chromatography-mass spectrometry and degree of ACE inhibition using the Z-FHL/HHL (z-phenylalanine-histidine-leucine/hippuryl-histidine-leucine) ratio.Results. One hundred patients (mean (standard deviation) age 50.5 (12) years, 46% male) were enrolled. Based on plasma assays, 26/97 patients (26.8%) were unsuppressed by enalapril and 20/100 (20%) were sub-therapeutic for amlodipine. There were significant BP differences based on plasma levels of the medication: 21/20 mmHg lower in the group with suppressed ACE and 26/20 mmHg in the group with steady-state amlodipine concentrations.Conclusions. Monitoring antihypertensive adherence by assaying plasma medication concentrations is a feasible option for evaluating true v. pseudo-resistant hypertension

ACE activity and endurance performance during the South African Ironman triathlons.

The insertion allele of the angiotensin-converting enzyme (ACE) gene has been associated with endurance performance. Since a large portion of the variance seen in circulatory ACE levels is unaccounted for by the insertion/deletion polymorphism it is likely that the ACE phenotype would serve as a more informative marker in assessing elite endurance performance. The aim of this study was to correlate plasma ACE activity with performance of a homogenous population of South African-born Caucasian male triathletes. Plasma ACE activity was determined in 145 triathletes, representing the fastest and slowest subgroups who completed either the 2000 or 2001 South African Ironman Triathlon. There was a trend for lower mean plasma ACE activity in the fastest (28.85±8.60 mU/ml) when compared to the slowest finisher subgroup (31.65±8.75 mU/ml, P=0.055). There was a significant positive correlation between plasma ACE activity and overall finishing time within the participants who completed the event in under 15 h (r=0.192, P=0.029). There was also a positive correlation with cycle (r=0.195, P=0.034) and run (r=0.184, P=0.040) stages but not the swim stage (r=0.084, P=0.353). In conclusion, this is the first study to report a relationship between plasma ACE activity and endurance performance in humans.

Structure based drug design of angiotensin-I converting enzyme inhibitors.

Cardiovascular disease (CVD) is responsible for ∼27% of deaths worldwide, with 80% of these occuring in developing countries. Hypertension is one of the most important treatable factors in the prevention of CVD. Angiotensin-I converting enzyme (ACE) is a two-domain dipeptidylcarboxypeptidase that is a key regulator of blood pressure as a result of its critical role in the reninangiotensin- aldosterone and kallikrien-kinin systems. Consequently, ACE is an important drug target in the treatment of CVD. ACE is primarily known for its ability to cleave angiotensin-I to the vasoactive octapeptide angiotensin-II, but is also able to cleave a number of other substrates including the vasodilator bradykinin and N-acetyl-seryl-aspartyl-lysyl-proline (acetyl-SDKP), a physiological modulator of hematopoiesis. Numerous ACE inhibiors are available clinically, and these are generally effective in treating hypertension. However some adverse effects are associated with ACE inhibition, such as the persistent dry cough and the potentially fatal angioedema. The solution of ACE crystal structures over the last decade has facilitated rational drug design which has contributed to the development of domain-selective ACE inhibitors, the most notable of which include RXP407 (N-domain) and RXPA380 (C-domain), which in principle may herald new therapeutic approaches for ACE inhibition. Additionally, dual inhibitors to ACE and other targets such as neprilysin, endothelin converting enzyme and chymase have been developed. The success of ACE inhibitors has also led to the search for novel inhibitors in food and natural products and the structure guided screening of such libraries may well reveal a number of new ACE inhibitors.

Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding.

A panel of monoclonal antibodies (mAbs) raised against both the N and C domains of angiotensin-I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) have been extensively mapped and have facilitated the study of various aspects of ACE structure and biology. In this study, we characterize two mAbs, 9B9 and 3G8, that recognize the N domain of ACE and that influence shedding and dimerization. Fine epitope mapping was performed, which mapped the epitopes for these mAbs to the N terminal region of the N domain where they overlap to a large extent, despite having different effects on ACE processing. The mAb 3G8 epitope appears to be shielded by the C domain and to be carbohydrate dependent as binding increased significantly as a result of underglycosylation, whereas these factors did not influence mAb 9B9 recognition. Three mutations within the overlapping region of these two epitopes, Q18H, L19E, and Q22A, which decreased mAb 3G8 binding to the soluble N domain, were introduced into full-length somatic ACE (sACE) to determine their influence on ACE expression and processing. Increased ACE expression, cell surface expression, and basal shedding were observed with all three mutations. Furthermore, cross-linking and western blotting of Chinese hamster ovary (CHO) cell lysates detected two distinct ACE dimers, a native and cross-linked dimer. Increasing amounts of the cross-linked dimer were observed for the mutant sACEQ22A, further implicating the overlapping region of the mAb 9B9 and 3G8 epitopes in ACE processing.

Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

Structure of angiotensin I-converting enzyme.

Angiotensin-converting enzyme (ACE) is a zinc- and chloride-dependent metallopeptidase that plays a vital role in the metabolism of biologically active peptides. Until recently, much of the inhibitor design and mechanism of action of this ubiquitous enzyme was based on the structures of carboxypeptidase A and thermolysin. When compared to the recently solved structures of the testis isoform of ACE (tACE) and its Drosophila homologue (AnCE), carboxypeptidase A showed little structural homology outside of the active site, while thermolysin revealed significant but less marked overall similarity. The ellipsoid-shaped structure of tACE, which has a preponderance of alpha-helices, is characterised by a core channel that has a constriction approximately 10 A from its opening where the zinc-binding active site is located. Comparison of the native protein with the inhibitor-bound form (lisinopril-tACE) does not reveal any striking differences in the conformation of the inhibitor binding site, disfavouring an open and closed configuration. However, the inhibitor complex does provide insights into the network of hydrogen-bonding and ionic interactions in the active site as well as the mechanism of ACE substrate hydrolysis. The three-dimensional structure of ACE now paves the way for the rational design of a new generation of domain-selective ACE inhibitors.

Cleavage of disulfide-bridged stalk domains during shedding of angiotensin-converting enzyme occurs at multiple juxtamembrane sites.

Shedding of the ectodomain of angiotensin-converting enzyme (ACE) and numerous other membrane-anchored proteins results from a specific cleavage in the juxtamembrane (JM) stalk, catalyzed by "sheddases" that are commonly activated by phorbol esters and inhibited by peptide hydroxamates such as TAPI. Sheddases require a stalk of minimum length and steric accessibility. However, we recently found that substitution of the ACE stalk with an epidermal growth factor (EGF)-like domain from the low-density lipoprotein receptor (LDL-R) did not abolish shedding; cleavage of the ACE-JMEGF chimera occurred at a Gly-Phe bond in the third disulfide loop of the EGF domain. We have now constructed two additional stalk chimeras, in which the native stalk in ACE was replaced with the EGF domain from factor IX (ACE-JMfIX) and with a cysteine knot motif (ACE-JMmin23). Like the ACE-JMEGF chimera, the ACE-JMfIX and -JMmin23 chimeras were also shed, but mass spectral analysis revealed that the cleavage sites were adjacent to, rather than within, the disulfide-bonded domains. Homology modeling of the LDL-R EGF domain revealed that the third disulfide loop is larger and more flexible than the equivalent loop in the factor IX EGF domain. Similarly, the NMR structure of the Min-23 motif is highly compact. Hence, cleavage within a disulfide-bonded domain appears to require an unhindered loop. Interestingly, unlike wild-type ACE and the ACE-JMEGF and -JMmin23 chimeras, shedding of the ACE-JMfIX chimera was not stimulated by phorbol or inhibited by TAPI, but instead was inhibited by 3,4-dichloroisocoumarin, indicating the activity of an alternative sheddase. In summary, the ACE shedding machinery is highly versatile, but an accessible JM sequence, in the form of a flexible stalk or an exposed loop within or adjacent to a folded domain, appears to be required. Moreover, alternative sheddases are recruited, depending on the nature of the JM sequence.

Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding.

Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.

A study of crystal matrix extract and urinary prothrombin fragment 1 from a stone-prone and stone-free population.

South African blacks are immune to urinary calculi whereas whites have an incidence rate similar to that reported in Western societies. Urinary prothrombin fragment 1 (UPTF1) and the crystal matrix extract (CME) from which it is derived have been shown to be potent inhibitors of crystal growth and aggregation in undiluted human urine. The objective of the present study was to isolate CME and UPTF1 from the urines of black and white subjects in order to assess whether either might contribute to the black population's relative stone immunity. CME was isolated from freshly precipitated calcium oxalate (CaOx) crystals and a crystallization study was conducted in synthetic urine. Coulter Counter, 14C-oxalate deposition, and scanning electron microscopy data demonstrated that the extracts from both race groups strongly inhibited CaOx nucleation. The extract derived from the black subjects inhibited nucleation to a greater extent than that from the whites. A phase conversion from COM to COD in the presence of the extracts, in support of the inhibitory effect of CME, was also observed. Purified UPTF1 isolated from both groups' CME was subjected to rigorous biochemical characterization involving matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, protein sequencing by Edman degradation, and amino acid analyses. No differences in molecular weight or amino acid sequence and composition were identified. It is suggested that the more potent inhibitory activity of the extract derived from the black subjects might be related to this group's relative stone immunity.

A point mutation in the juxtamembrane stalk of human angiotensin I-converting enzyme invokes the action of a distinct secretase.

Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn(631) to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE(NQ)) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE(NQ) was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 degrees C revealed that ACE(NQ) was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn(635)-Ser(636) bond, three residues N-terminal to the normal secretase cleavage site at Arg(638)-Ser(639). These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites.

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].

Phorbol ester-induced juxtamembrane cleavage of angiotensin-converting enzyme is not inhibited by a stalk containing intrachain disulfides.

Specialized proteases, referred to as sheddases, secretases, or membrane-protein-solubilizing proteases (MPSPs), solubilize the extracellular domains of diverse membrane proteins by catalyzing a specific cleavage in the juxtamembrane stalk regions of such proteins. A representative MPSP (tumor necrosis factor-alpha convertase) was cloned recently and shown to be a disintegrin metalloprotease that is inhibited by peptide hydroxamates including the compound TAPI. Substrate determinants that specify cleavage by MPSPs remain incompletely characterized, but may include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We constructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain (ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open, comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a typical MPSP activity. We found that ACE-JMEGF was solubilized inefficiently and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester and inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cleavage occurred at a Gly-Phe bond between the fifth and sixth cysteines within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell-associated via disulfide tethering. The mechanism for the release of ACE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is prevented by disulfide tethering to the TM domain.

Limited proteolysis of human kidney angiotensin-converting enzyme and generation of catalytically active N- and C-terminal domains.

The somatic form of angiotensin converting enzyme is a class I ectoenzyme that is bound to the surface of endothelial calls. It consists of two homologous, catalytic domains of approximately 600 residues each; a juxtamembrane "stalk" region; a transmembrane, hydrophobic sequence; and a 30 residue, C-terminal cytosolic domain. We have used limited proteolysis to probe the structural and functional properties of the enzyme. Endoproteinase Asp-N cleaves both the Thr615-Asp616 and the Leu1219-Asp1220 peptide bonds to generate the two catalytic domains which were isolated by a combination of immunoaffinity and lisinopril Sepharose affinity chromatography. The enzymatic characteristics of the N and C fragments were examined with angiotensin I, hippuryl-His-Leu, and luteinizing hormone-releasing hormone and indicate that both fragments contain catalytically active sites that retain their individual functional integrity.

Identification of N-linked glycosylation sites in human testis angiotensin-converting enzyme and expression of an active deglycosylated form.

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.

Assignment of free and disulfide-bonded cysteine residues in testis angiotensin-converting enzyme: functional implications.

Human testicular angiotensin-converting enzyme (tACE) is an extracellular protein that contains seven cysteine residues. The cysteines occur in a sequential distribution that is precisely mimicked in the tACE from rabbit and mouse, and in both domains of all known species of somatic ACE. One of the cysteines in human tACE, Cys496, is present in the reduced form as shown by labeling it with 5-[[2-(iodoacetyl)amino]ethylamino]naphthalene-1-sulfonic acid, isolating the fluorescent peptide from enzymatic digests by HPLC, and analyzing its sequence by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thiol reagents have no significant effect on the activity of tACE, indicating that this Cys is not involved in catalysis. The other six cysteines exist as three disulfides. Mass spectral analysis of cyanogen bromide peptides has established that the cystine connectivities follow a nearest-neighbor, aabbcc, pattern i.e., Cys152-Cys158, Cys352-Cys370, and Cys538-Cys550, in which the disulfides form three small loops of five, 17, and 11 residues, respectively. Although these disulfide loops constitute less than 5% of the total sequence of the protein, they contribute to the overall structural stabilization of tACE.

Identification of two new mutations in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) or Günther's disease is an inborn error of heme biosynthesis transmitted as an autosomal recessive trait and characterized by a profound deficiency of uroporphyrinogen III synthase (UROIIIS) activity. Six missense mutations in the UROIIIS gene, a deletion and an insertion have already been described in CEP. This work brings further evidence for the heterogeneity in the genetic defect found in CEP. Two new mutations are described, a point mutation (V99A) and a frame-shift mutation (633insA) in the same patient who had a mild to moderate form of Günther's disease. The mutation (V99A) had a detectable residual activity when expressed in Escherichia coli while the insertion (633insA), which introduced a premature stop, had no activity. In the patients studied in our laboratory, the mutation C73R, associated with a severe phenotype, remains the most frequently seen.

Subcellular distribution of asialoglycoprotein receptor in liver regeneration.

Asialoglycoprotein (ASGP) receptor specific activity was determined directly using a radiolabelled ligand (asialoorosomucoid) binding assay in various fractions of rat liver cells before and 24 hours after two-thirds partial hepatectomy. The specific activity of ASGP receptor in the plasma membrane fraction was significantly reduced 24 hours after partial hepatectomy. In an attempt to identify a subgroup of receptor involved in intercellular recognition the calcium-dependent ASGP receptor was re-assayed after pre-incubation in ethylenediaminetetra-acetic acid. This revealed significant amounts of additional receptor in microsomal preparations but not in plasma membrane fractions. Cryptic receptor levels were not affected by partial hepatectomy. Plasma membrane-bound receptor was significantly decreased in regenerating liver.