Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated, soluble AC-dependent cAMP amplification pathway common to GPCR signalling.

BACKGROUND AND PURPOSE: H2 O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion. This study investigated signalling downstream of the receptors activated by H2 O2 . EXPERIMENTAL APPROACH: Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4 receptor agonist or ß2 -adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I ) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy. KEY RESULTS: Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated anion currents. A similar Epac-mediated pathway was seen following ß2 -adrenoceptor or forskolin stimulation. CONCLUSIONS AND IMPLICATIONS: H2 O2 initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca(2+) crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.

Steroid sensitivity of norepinephrine uptake by human bronchial arterial and rabbit aortic smooth muscle cells.

We have shown that an inhaled glucocorticosteroid (GS) causes alpha(1)-adrenergic antagonist-blockable, rapid, and transient bronchial vasoconstriction in healthy and asthmatic subjects. Steroids inhibit norepinephrine (NE) uptake by non-neuronal cells, thereby increasing NE concentration at alpha-adrenergic receptor sites. This could explain the GS-induced bronchial vasoconstriction. We therefore studied expression of the steroid-sensitive extraneuronal monoamine transporter (EMT) and steroid sensitivity of NE uptake in human bronchial artery and rabbit aorta (as a substitute for the limited supply of human bronchial artery). NE uptake was measured using a semiquantitative, sucrose-potassium phosphate-glyoxylic acid fluorescence method that we newly adapted for use in single cells. Both human bronchial arteries and rabbit aorta expressed messenger RNA for EMT, and steroids blocked NE uptake into freshly dissociated human bronchial arterial and rabbit aortic smooth-muscle cells (SMCs). In the latter, inhibition of NE uptake by steroids was not altered, either by a protein synthesis inhibitor (cycloheximide) or by a transcription inhibitor (actinomycin D), and corticosterone made membrane-impermeant by conjugation to bovine serum albumin inhibited NE uptake equipotently. These data show that NE uptake into bronchial arterial and rabbit aortic SMCs is sensitive to steroids, possibly mediated by EMT, and suggest a mechanism for GS-induced bronchial vasoconstriction.

Hyaluronan serves a novel role in airway mucosal host defense.

Enzymes secreted onto epithelial surfaces play a vital role in innate mucosal defense, but are believed to be steadily removed from the surface by mechanical actions. Thus, the amount and availability of enzymes on the surface are thought to be maintained by secretion. In contrast to this paradigm, we show here that enzymes are retained at the apical surface of the airway epithelium by binding to surface-associated hyaluronan, providing an apical enzyme pool 'ready for use' and protected from ciliary clearance. We have studied lactoperoxidase, which prevents bacterial colonization of the airway, and kallikrein, which mediates allergic bronchoconstriction that limits the inhalation of noxious substances. Binding to hyaluronan inhibits kallikrein, which is needed only in certain situations, whereas lactoperoxidase, useful at all times, does not change its activity. Hyaluronan itself interacts withthe receptor for hyaluronic acid-mediated motility (RHAMM or CD168) that is expressed at the apex of ciliated airway epithelial cells. Functionally, hyaluronan binding to RHAMM stimulates ciliary beating. Thus, hyaluronan plays a previously unrecognized pivotal role in mucosal host defense by stimulating ciliary clearance of foreign material while simultaneously retaining enzymes important for homeostasis at the apical surface so that they cannot be removed by ciliary action.

The lactoperoxidase system functions in bacterial clearance of airways.

Airway mucus is a complex mixture of secretory products that provides a multifaceted defense against pulmonary infection. Mucus contains antimicrobial peptides (e.g., defensins) and enzymes (e.g., lysozyme) although the contribution of these to airway sterility has not been tested in vivo. We have previously shown that an enzymatically active, heme-containing peroxidase comprises 1% of the soluble protein in sheep airway secretions, and it has been hypothesized that this airway peroxidase may function as a biocidal system. In this study, we show that sheep airway peroxidase is identical to milk lactoperoxidase (LPO) and that sheep airway secretions contain thiocyanate (SCN(-)) at concentrations necessary and sufficient for a functional peroxidase system that can protect against infection. We also show that airway LPO, like milk LPO, produces the biocidal compound hypothiocyanite (OSCN(-)) in vitro. Finally, we show that in vivo inhibition of airway LPO in sheep leads to a significant decrease in bacterial clearance from the airways. The data suggest that the LPO system is a major contributor to airway defenses. This discovery may have significant implications for chronic airway colonization seen in respiratory diseases such as cystic fibrosis.

Bronchial tissue kallikrein activity is regulated by hyaluronic acid binding.

Tissue kallikrein (TK) is secreted by serous cells of tracheobronchial submucosal glands and plays a role in allergic airway responses. To better understand the regulation of TK, we used primary cultures of submucosal gland cells that release TK upon stimulation. Media from cultures stimulated with chymase (10(-7) M) showed increased TK activity (0.50 +/- 0.22 mU/ml mean +/- standard error) in comparison with the control group (0.08 +/- 0.02 mU/ml). The increased TK activity was significantly correlated with increases in the levels of the serous cell marker, secretory leukoprotease inhibitor. Anion exchange chromatography of the conditioned culture media showed that TK activity eluted as a broad peak between 1.6 and 1.8 M NaCl, unlike the reported elution (0.3 to 0.6 M NaCl) of kallikreins from other tissues, suggesting that secreted bronchial TK was bound to a negatively charged molecule. Hyaluronidase digestion increased TK activity in both pre- and post-chymase-stimulated culture media, whereas no such change was seen after samples were digested with heparinase or chondroitinase ABC. Further, after hyaluronidase digestion of media, TK eluted from an anion exchange column between 0.3 and 0.6 M NaCl. Enzymatic detection of TK after nondenaturing gel electrophoresis showed that hyaluronidase digestion also reduced the electrophoretic heterogeneity of TK to a single band, whereas adding back hyaluronic acid (HA) to hyaluronidase-digested samples restored the original heterogeneity. Finally, TK activity bound to HA-Sepharose and could be eluted with HA. These studies show that primary cultures of ovine submucosal gland cells secrete TK in a regulated fashion, and that secreted TK binds to HA. This binding reduces TK enzymatic activity; therefore, factors that affect HA turnover could modify the TK activity in the airway lumen. These events could be important in the regulation of kinin-mediated airway inflammation.

Cathepsin D antigenic epitopes identified by the humoral responses of ovarian cancer patients.

Previous studies implicated cathepsin D as one commonly recognized target of tumor-reactive immunoglobulins from ovarian cancer patients. These immunoglobulins are shown to be immunoreactive with both the 52-kDa procathepsin D and the 32-kDa mature cathepsin D derived from the UL-1 ovarian cancer cell line. Whether the carbohydrate domains or the core protein were associated with its immunogenicity was analyzed with cathepsin D isolated from tunicamycin-treated UL-1 cells. No significant difference was detected in the immunoreactivity of patient serum with the glycosylated and deglycosylated forms of the cathepsin D, suggesting that patient humoral responses are directed primarily against the core protein. To define the antigenic epitopes of cathepsin D, tryptic fragments were prepared from UL-1-derived procathepsin D. The epitopes of the core protein recognized by sera from more than one patient were identified using a peptide-specific enzyme-linked immunosorbent assay and microsequencing of positive immunoreactive peptides. This protocol identified four epitopes: two peptides within the propeptide, a third at the carboxy terminus and the fourth at the glycosylation site of the mature enzyme. This approach to the identification of specific antigenic epitopes may be useful in defining effective targets for directed active immunotherapy against cancer.

Isolation and characterization of a peroxidase from the airway.

Sheep airway mucus can potently scavenge hydrogen peroxide, an important mediator of airway inflammation. Here, the scavenging activity was identified as a peroxidase produced by goblet cells of the airway epithelium and secreted into the airway lumen. Ovine airway peroxidase activity was purified approximately 100-fold from airway lavage fluid in two steps, using cation exchange and lectin affinity chromatography, yielding an apparently homogeneous 82-kD glycoprotein. Ovine airway peroxidase represented about 1% of the total protein in airway mucus and thus was an abundant enzyme in airway secretions. The absorbance spectrum of the purified peroxidase showed a major peak at 412 nm indicative of a hemoprotein. The ratio of A412/A280 of the purified enzyme was 0.86. The absorption spectrum of ovine airway peroxidase, its ability to oxidize halides, its sensitivity to inhibitors and its apparent molecular mass on sodium dodecyl sulfate gels showed that airway peroxidase was similar to lactoperoxidase but distinguished from myeloperoxidase, eosinophil peroxidase as well as from glutathione peroxidases. Based on these observations, ovine airway peroxidase is a newly isolated and abundant enzyme of airway mucus which may function to control reactive oxygen species in the airway and to prevent infection by catalyzing the formation of biocidal compounds.

Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): its mechanism and inhibition by ganglioside.

Saposins A, B, C, and D, which are required for the enzymatic hydrolysis of sphingolipids by specific lysosomal hydrolases, are produced by proteolytic processing of their common precursor protein, prosaposin. Our previous observation suggested that lysosomal cathepsin D may be involved in the proteolysis of prosaposin. Herein we report the involvement of cathepsin D in the proteolytic processing of prosaposin. An antibody against human placental cathepsin D blocked the proteolytic activity toward prosaposin in a human testicular lysosomal protease mixture (glycoprotein fraction). On immunoblot analysis using a monoclonal antibody against human saposin C, cathepsin D showed a similar proteolytic pattern as that of a human testicular glycoprotein fraction and hydrolyzed prosaposin into products of 48 and 29 kDa. The Km and Vmax values were 0.9 microM and 167 nmol/h/mg, respectively. N-Terminal sequence analysis indicated that the 48-kDa band was a mixture of two trisaposins, including domains for saposins A, B, and C and saposins B, C, and D, respectively. A similar study also showed that the 29-kDa band contained two disaposins, including domains for saposins A and B and saposins C and D, respectively. By longer treatment with cathepsin D, disaposins were further processed into mature saposin A and small fragments (14.5-17.5 kDa) containing individual saposins and portions of interdomain sequences. These small fragments were no longer processed by cathepsin D, but trimmed to fragments having similar molecular sizes (10.5-11.5 kDa) to those of mature saposins by a rat lysosome preparation. These findings indicated that cathepsin D is involved in the maturation of saposins but that, in addition to cathepsin D, other proteases appear to be involved in the maturation of saposin B, C, and D in lysosomes. Gangliosides, which specifically form complexes with prosaposin and saposins, inhibit proteolysis of prosaposin by cathepsin D. This finding indicates that prosaposin may be protected from lysosomal proteolysis by forming a complex with gangliosides in vivo.

Characterization of rat uterine matrilysin and its cDNA. Relationship to human pump-1 and activation of procollagenases.

A small uterine metalloproteinase of the rat has been shown by amino acid and cDNA sequencing to be orthologous to human pump-1. Both proteinases are now designated as matrilysin or matrix metalloproteinase 7. The properties of purified uterine metalloproteinase and recombinant pump-1 were compared. Their specificities on substrates (gelatins, fibronectin, transferrin, elastin, Azocoll, and (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3,[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2) are similar and distinct from those of the stromelysins and gelatinases. The two matrilysins have similar sensitivity to hydroxamate and pseudopeptide inhibitors. Rat matrilysin selectively cleaves the alpha 2(I) chain of rat gelatin, producing major cuts at Gly713-decreases-Ile714, Gly775-decreases-Leu776, and Gly809-decreases-Ile810. Rat matrilysin produces maximum activation of latent human interstitial collagenase 1 (pro-matrix metalloproteinase 1) when added in the presence of 4-aminophenylmercuric acetate (APMA) by cleaving the Gln80-decreases-Phe81 bond. Rat and human matrilysin do not directly activate latent rat collagenase 3 (matrix metalloproteinase 13) and do not enhance its activation when added together with APMA. Autoactivation of collagenase 3 in the presence of APMA results in cleavage at Val81-decreases-Tyr82 corresponding to the Gln80-decreases-Phe81 cleavage in collagenase 1. Thus collagenase 3 is capable of maximal autoactivation, whereas collagenase 1 is dependent upon another matrix metalloproteinase in order to be activated to its full potential.

Hydrogen peroxide-scavenging properties of sheep airway mucus.

Reactive oxygen species released from luminal phagocytes in the airway can potentially injure the airway epithelium. Naturally occurring oxygen radical scavengers must therefore exist to protect the epithelium. This study was designed to determine whether the high-molecular-weight fraction of normal sheep tracheal mucus has hydrogen peroxide (H2O2)-scavenging activity. Lyophilized mucus from 10 sheep was reconstituted in phosphate-buffered saline (PBS) or Krebs-Henseleit buffer. H2O2 was added to these mucus samples to a final concentration of 15 microM, and the level of H2O2 remaining was measured over a 10 min period. From a zero-time level of 17 +/- 1.8 microM (mean +/- SD), the H2O2 concentration fell within 10 min to 8 +/- 1.7 microM in 0.05%; to 3.9 +/- 2.2 microM in 0.1%; to 2.6 +/- 2.4 microM in 0.2%; and to 1.2 +/- 1.5 microM in 0.4% mucus reconstituted in PBS. The results obtained in Krebs-Henseleit buffer were similar. The disappearance of H2O2 was not due to the transformation into hydroxyl radicals. Heat and acid denaturation and cleavage of carbohydrate-free peptides from glycoproteins by pronase E treatment abolished the scavenging potential. Fractionation of 0.4% mucus samples according to molecular weight by gel filtration revealed that only one fraction with proteins of M(r) > 110 kD contained the active scavenger. Polyacrylamide gel electrophoresis and lectin blotting with Ulex europaeus I (UEAI) showed that both the whole mucus and the actively scavenging gel filtration fraction contained a glycoprotein that comigrated with a 205 kD molecular weight marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural requirements of procathepsin D activation and maturation.

Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N-terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo.

Intermolecular association of lysosomal protein precursors during biosynthesis.

To study the mechanism involved in mannose-6-phosphate (Man-6-P) independent lysosomal proenzyme membrane association, we used a reversible cross-linker to probe radiolabeled human HepG2 cells permeabilized with saponin in the presence of Man-6-P. After immunoprecipitation of the extracted and cross-linked cells with anti-cathepsin D antibody, followed by complete reduction of the immunoprecipitates and SDS-polyacrylamide gel electrophoresis analysis, we found that procathepsin D was specifically and transiently associated, independent of Man-6-P, with two co-synthesized glycoproteins having molecular masses of 68 and 72 kDa. Pulse-chase and cell fractionation experiments showed that the Man-6-P independent association of procathepsin D with the 68-kDa protein started in the rough endoplasmic reticulum, continued in the Golgi, but had no association with either membrane. The Man-6-P independent association of procathepsin D with the 72-kDa protein and the membrane was found in compartments all the way from the Golgi to the dense lysosome, where processing of procathepsin D is believed to occur and where procathepsin D dissociated from the 72-kDa protein and the membrane. Endo H digestion of the 72-kDa protein showed that this protein was partially resistant to Endo H, suggesting that membrane association of the procathepsin D-72-kDa protein complex probably began in a late Golgi compartment. Endo F digestion of the proteins showed both have the same molecular mass around 58 kDa. Using antiserum against human saposin C, we identified the two glycoproteins as forms of prosaposin with different glycosylation. The transient, Man-6-P independent, membrane association of the procathepsin D-prosaposin complex and the presence of this complex in heavy lysosomes indicated that the proteins were transported to the lysosome as a complex. The association of two lysosomal proteins in the endoplasmic reticulum early after synthesis suggested that preassembly of some lysosomal components occurs before the earliest previously identified steps in the sorting pathway.

Primary structure of the canine CD4 antigen.

After PCR amplification two overlapping cDNA clones encoding the dog homologue of the human CD4 glycoprotein were identified. This internal fragment of the mature protein including the complete extracellular domains, consists of 1297 bp with a deduced amino acid sequence of 432 residues. The dog CD4 molecule differs from the corresponding protein of other species including human in the second domain. We found nine extra residues in the beginning of that domain, a cysteine at position 138, usually involved in a disulfide bridge, is substituted by tryptophan, and three new glycosylation sites are present.

Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling.

The family of aspartic proteinases includes several human enzymes that may play roles in both physiological and pathophysiological processes. The human lysosomal aspartic proteinase cathepsin D is thought to function in the normal degradation of intracellular and endocytosed proteins but has also emerged as a prognostic indicator of breast tumor invasiveness. Presented here are results from a continuing effort to elucidate the factors that contribute to specificity of ligand binding at individual subsites within the cathepsin D active site. The synthetic peptide Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu has proven to be an excellent chromogenic substrate for cathepsin D yielding a value of kcat/Km = 0.92 x 10(-6) s-1 M-1 for enzyme isolated from human placenta. In contrast, the peptide Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu and all derivatives with Ala-Lys in the P3-P2 positions are either not cleaved at all or cleaved with extremely poor efficiency. To explore the binding requirements of the S3 and S2 subsites of cathepsin D, a series of synthetic peptides was prepared with systematic replacements at the P2 position fixing either Ile or Ala in P3. Kinetic parameters were determined using both human placenta cathepsin D and recombinant human fibroblast cathepsin D expressed in Escherichia coli. A rule-based structural model of human cathepsin D, constructed on the basis of known three-dimensional structures of other aspartic proteinases, was utilized in an effort to rationalize the observed substrate selectivity.

The role of the cathepsin D propeptide in sorting to the lysosome.

The propeptides of lysosomal enzymes have been implicated in membrane association and mannose 6-phosphate-independent sorting to the lysosome (Rijnboutt, S., Aerts, H., Geuze, H. J., Tager, J. M., and Strous, G. J. (1991) J. Biol. Chem. 266, 4862-4868; McIntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445). In this report, the function of the propeptide of procathepsin D in sorting to the lysosome was directly assessed using a cathepsin D deletion mutant lacking the propeptide, and using a chimeric cDNA encoding the cathepsin D propeptide fused to the secretory protein alpha-lactalbumin. Proteins encoded by these cDNAs were expressed in mouse Ltk- cells and in human hepatoma Hep G2 cells, and then immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The deletion mutant was glycosylated but was rapidly degraded in a chloroquine-independent fashion and did not assume an active conformation. Thus the propeptide appeared to be necessary for correct folding. The chimeric protein was glycosylated and secreted. The coincidence of complex oligosaccharide modification and secretion of the chimeric protein suggested that it was slowly released from the endoplasmic reticulum and rapidly passed through the cell to the extracellular compartment. This was confirmed by immunofluorescent localization of the proteins. The data indicated that the propeptide appeared to be necessary for folding of cathepsin D but, unlike the yeast vacuolar propeptides, was not sufficient to direct a secretory protein to the lysosome in fibroblasts or in epithelial cells.

Pharmacologic characterization of the novel ligand [4,5-3H-Leu9]neurokinin-A binding to NK-2 receptors on hamster urinary bladder membranes.

We synthesized a novel ligand [4,5-3H-Leu9]-Neurokinin A (3H-NKA, S.A 117-144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 +/- 4%) and protein-dependent manner. Binding was rapid (k1 = 0.037 nM-1*min-1) and saturable (Bmax = 1210 +/- 177 fmol/mg protein), to a single population of high-affinity sites (KD = 2.41 +/- 0.15 nM, nH = 0.99 +/- 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [beta-Ala8]-NKA(4-10) > [Nle10]-NKA(4-10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar9-Met (O2)11]-SP, (+/-) CP96,345 and Senktide respectively, did not inhibit binding at 10 microM, whereas, the selective NK-2 antagonists: (+/-) SR-48,968 >> L-659,877 > R396 >> MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (< 30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1-10 microM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.

Isolation and characterization of a stable activation intermediate of the lysosomal aspartyl protease cathepsin D.

Procathepsin D is the intracellular aspartyl protease precursor of cathepsin D, a major lysosomal enzyme. Procathepsin D is rapidly processed inside the cell, and, thus, examination of its proteolyic activation and structure has been difficult. To study this proenzyme, a nonglycosylated form of the human fibroblast procathepsin D was expressed in Escherichia coli, refold in vitro, and purified by affinity chromatography on pepstatinyl agarose. Sequence analysis of the refolded, autoactivated enzyme allowed determination of the autoproteolytic cleavage site. The sequence surrounding this cleavage site between residues LeuP26 and IleP27 (in the "pro" region) resembled the first cleavage site found during activation of other aspartyl proteases. Thus, the autoactivated procathepsin D is analogous to the pepsin activation intermediate, which has been termed pseudopepsin. The enzymatic activity, thermal and pH stability, and fluorescence spectra of pseudocathepsin D were compared to mature, predominantly two-chain, cathepsin D isolated from human placenta. The results indicated that pseudocathepsin D and mature enzyme have a similar Km toward a peptide substrate and cleave a protein substrate at identical sites. Temperature stability of the recombinant enzyme was similar to that of the tissue-derived enzyme. However, the recombinant enzyme had increased stability at low pH when compared to the glycosylated tissue-derived two-chain cathepsin D. Fluorescence spectra of the recombinant and tissue-derived enzymes were identical. Thus, the absence of asparagine-linked oligosaccharides and the presence of the remaining segment of propeptide did not significantly alter the structural and enzymatic properties of the enzyme.

Proteolytic activation of human procathepsin D.

Procathepsin D is a short-lived inactive precursor of the lysosomal aspartyl protease, cathepsin D. Pulse-chase analysis using radiolabeled amino acids demonstrated the existence of several biosynthetic intermediates during formation of mature cathepsin D (summarized in Figure 1). Procathepsin D is capable of autocatalytic cleavage to pseudocathepsin D. This was demonstrated using small quantities of procathepsin D isolated from cell culture media as well as using a non-glycosylated form of procathepsin D synthesized in a bacterial expression system. Complete conversion to the single-chain cathepsin D appears to require a second enzyme which is inhibited by leupeptin. This conclusion was drawn from the inability to produce single-chain enzyme from either procathepsin D or pseudocathepsin D in vitro as well as observations from addition of protease inhibitors to cell cultures. It appears that the conversion of procathepsin D to active single-chain enzyme falls between the paradigms of pepsinogen autoactivation and prorenin conversion by a separate enzyme.

Importance of fasting in the lymphocyte calcium test for malignant hyperthermia.

Anaesthetic-induced increases in cytoplasmic free Ca2+ have been reported to be greater in lymphocytes from malignant hyperthermia (MH) susceptible patients than in those from controls, suggesting that this may be the basis for a less invasive test for MH susceptibility. In the present study the cytoplasmic Ca2+ concentrations of lymphocytes were monitored with indo-1 in 14 control subjects (nine fasted and five nonfasted) and five fasted MH susceptible and three fasted nonsusceptible patients, diagnosed by the halothane and caffeine contracture tests. No relationship was observed between MH susceptibility and Ca2+ concentrations in lymphocytes in the absence or presence of halothane. There was, however, a relationship in control subjects between fasting and the response of lymphocytes to halothane, with the halothane-induced Ca2+ increase being considerably larger in nonfasted subjects.