Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.     

Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.     

Purification and characterization of a new arginine carboxypeptidase in human serum

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.     

Enhanced Co2+ activation and inhibitor binding of carboxypeptidase M at low pH. Similarity to carboxypeptidase H (enkephalin convertase).

Carboxypeptidases H and M differ in their distribution and other properties, but both are activated by Co2+ and inhibited by guanidinoethylmercaptosuccinic acid. The higher degree of activation or inhibition of carboxypeptidase H by these agents at acid pH has been employed to identify this enzyme in tissues. We found that the activation or inhibition of both purified and plasma-membrane-bound human carboxy-peptidase M depends on the pH of the medium. CoCl2 activated over 6-fold at pH 5.5, but less than 2-fold at pH 7.5. Guanidinoethylmercaptosuccinic acid inhibited the membrane-bound carboxypeptidase M more effectively than the purified enzyme, and the IC50 was about 25-30 times lower at pH 5.5. As purified human plasma carboxypeptidase N and pancreatic carboxypeptidase B were also activated more at pH 5.5, we conclude that the increased activation by CoCl2 is due to the enhanced dissociation of Zn2+ below the pKa of the ligands that co-ordinate the cofactor in the protein. Thus increased activation or inhibition at acid pH would not differentiate basic carboxypeptidases.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus

Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10(-4) M, respectively.     

Intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki and their role in protoxin activation

The intracellular proteases in sporulated Bacillus thuringiensis subsp. kurstaki were studied to identify the endogenous proteases involved in the activation of protoxin. The proteases obtained with 30% ammonium sulfate saturation were analysed by both gelatin zymography and azocasein hydrolysis. Three proteases with molecular mass 92 kDa, 78 kDa and 69 kDa were identified on gelatin gel and their gelatinolytic activity was inhibited by ethylenediamine tetraacetic acid. Significantly, 1,10-phenanthroline caused an inhibition of the azocasein hydrolytic activity by 98% and ethylenediamine tetraacetic acid by 28%. The three proteases were heat-stable at 65 °C, while the 69-kDa protease was active up to 75 °C. Intracellular protease-deficient mutants (ethyl methanesulfonate mutagenesis) could not generate the active toxin suggesting the existence of a specific enzyme affecting the conversion of protoxin to toxin.     

Phosphodiesterase from the venom of Crotalus ruber ruber

Phosphodiesterase was isolated from the venom of Crotalus ruber ruber from the U.S.A. using the gel filtration on a Sephadex G-75 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and isoelectric focusing electrophoresis. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB), but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approx. 98,000 and the isoelectric point was found to be pH 10.5 by isoelectric focusing with carrier ampholyte. This enzyme contained 1.04 mol zinc per mol. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 8.3 X 10(-3) and 1.2 X 10(-2) M, respectively.     

Maturation of isoprenylated proteins in Saccharomyces cerevisiae. Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro.

Eukaryotic polypeptides containing COOH-terminal-CXXX sequences can be posttranslationally modified by isoprenylation of the cysteine residue via a thioether linkage, proteolytic removal of the three terminal amino acids, and alpha-carboxyl methylation of the cysteine residue. Through the development of an indirect coupled assay, we have identified three in vitro activities in the yeast Saccharomyces cerevisiae that can catalyze the proteolytic cleavage of the three COOH-terminal amino acids of the synthetic peptide substrate N-acetyl-KSKTK[S-farnesyl-Cys]VIM. One of these is the vacuolar protease carboxypeptidase Y. Using a mutant strain deficient in this enzyme, we find evidence for an additional soluble activity as well as for a membrane-associated activity. These latter activities are candidates for roles in the physiological processing of isoprenylated protein precursors. They are both insensitive to inhibitors of serine and aspartyl proteinases but are sensitive to sulfhydryl reagents and 0.5 mM ZnCl2. The soluble activity appears to be a metalloenzyme, inhibitable by 2 mM o-phenanthroline but not by 1 mM N-ethylmaleimide, whereas the membrane-associated enzyme is inhibitable by 1 mM N-ethylmaleimide but not 2 mM o-phenanthroline. We show that the membrane-bound protease is not an activity of the membrane-bound methyltransferase, because protease activity is observed in membrane preparations that lack the STE14-encoded methyltransferase. The soluble activity appears to be a novel carboxypeptidase of approximately 110 kDa that catalyzes a processive removal of amino acids from the COOH terminus from both the farnesylated and non-farnesylated substrate, but not from three other unrelated peptides. Finally, we find no evidence for non-vacuolar membrane or soluble activities that catalyze the ester hydrolysis of N-acetyl-S-farnesyl-L-cysteine methyl ester.     

Studies on the characterization of the Rho(D) antigen

The Rh_o(D) antigen of red cell membranes was solubilized using ethylenediamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, a pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rh_o(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rh_o(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rh_o(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rh_o(D) molecule in its natural environment. The solubilized Rh_o(D) molecules are apparently not dependent on lipid for their Rh_o(D) activity.     

Vitamin A and carotenoids. The enzymic conversion of β-carotene into retinal in hog intestinal mucosa

The conversion of beta-carotene into retinal was studied in vitro with enzyme preparations from homogenates of hog intestinal mucosa. The hog mucosal enzyme was purified about 27-fold by precipitation with ammonium sulphate, chromatography on DEAE-Sephadex and gel filtration on Sephadex G-200. The reaction displayed a narrow optimum pH range (approx. 7.8-8.2). The enzyme was stimulated strongly by the addition of thiols, and was inhibited by thiol inhibitors and by the chelating agents alphaalpha'-bipyridyl and o-phenanthroline. The reaction required the addition of an appropriate detergent (or bile salt); maximal activity was obtained by addition of an appropriate combination of detergents and lipid (specifically Tween 40, sodium glycocholate and sphingomyelin). The reaction displayed Michaelis kinetics with K(m)1.3x10(-6)m and V(max.)1.1nmole of retinal formed/hr. (for 0.7mg. of enzyme protein). The properties of the hog enzyme are similar to those previously reported for a less purified rat enzyme preparation.     

Multiple forms of atrial natriuretic factor receptor in human placenta

Multiple forms of atrial natriuretic factor receptor have been identified in human placental membranes. Atrial natriuretic factor binds specifically to placental membranes and the binding activity could be solubilized using non ionic detergent, Triton X-100. Binding to the detergent solubilized preparation was inhibited 80% by the addition of 0.5 M sodium chloride. Affinity cross-linking analysis indicated that this binding was associated with a single protein band of molecular weight 170-kDa. On the other hand, if sodium chloride was added together with a chelator, o-phenanthroline, ANF binding to this preparation was stimulated 300%. Binding under these conditions was not to the 170-kDa protein but was associated with a broad band in the region of 100/110-kDa and a minor band at 200-kDa. These observations clearly indicated that in human placental membranes, atrial natriuretic factor binds to distinctly different molecular species depending on the presence or absence of certain ions and chelators. The two types of binding could be conveniently assayed in the presence of each other by elimination or inclusion of sodium chloride and o-phenanthroline in the assay system.     

Human placental steryl-sulfatase. Enzyme purification, production of antisera, and immunoblotting reactions with normal and sulfatase-deficient placentas

The steryl-sulfatase of normal human placental microsomes was solubilized and enriched about 350-fold. Chromatography on Sepharose 6B of the purified enzyme preparation revealed a single protein peak which eluted according to an apparent molecular mass of 270 +/- 30 kDa; when electrophorized on sodium dodecyl sulfate polyacrylamide gel the sulfatase migrated according to a molecular mass of 64 +/- 4 kDa. Estrogensulfatase activity was co-purified with the steryl-sulfatase activity; obviously, both activities belong to the same enzyme species. The purified sulfatase was injected into three rabbits. Antisera produced by the rabbits yielded a single sharp immunoprecipitation line in Ouchterlony double diffusion experiments when tested with the isolated sulfatase or with a solubilized microsomal fraction of normal placentas. The activity of sulfatase preparations incubated with antiserum was precipitated by addition of polyethylene glycol followed by centrifugation; none of the antibodies reacting with the sulfatase therefore appeared to interfere with its enzymatic activity. Using these antisera, steryl-sulfatase protein could be detected by immunoblotting analysis in solubilized microsomal fractions of normal placentas but not in solubilized microsomal fractions of three steryl-sulfatase activity-deficient placentas. This finding argues in favour of human placental steryl-sulfatase deficiency being due to extremely diminished or absent enzyme protein in the placenta.     

Kinin metabolism in human nasal secretions during experimentally induced allergic rhinitis

We have previously shown that both bradykinin and lysylbradykinin are generated in nasal secretions upon nasal challenge of allergic individuals with appropriate allergen and have suggested that these potent pro-inflammatory peptides may contribute to the pathogenesis of the allergic response. In this study we used a variety of synthetic substrates together with both thin layer and high performance liquid chromatography systems to examine the metabolism of these peptides in nasal secretions obtained by lavage. We now demonstrate that in addition to low levels of angiotensin-converting enzyme, nasal lavages contain an aminopeptidase activity that converts lysylbradykinin to bradykinin, and a carboxypeptidase that removes the C-terminal arginine from bradykinin and lysylbradykinin. The levels of all these activities are significantly increased after allergen challenge of allergic, but not nonallergic, individuals. The aminopeptidase and carboxypeptidase activities present in post-challenge lavages from allergic individuals convert lysylbradykinin to intermediate products (bradykinin and des (Arg10) lysylbradykinin) and eventually to des (Arg9) bradykinin. The nasal carboxypeptidase was activated 475% by 0.1 mM CoCl2 and was inhibited by the carboxypeptidase N inhibitor, MERGETPA (D-L-mercaptomethyl-3-guanidino-ethylthiopropanoic acid) (IC50 = 10 microM). The aminopeptidase activity was not affected by MERGETPA but was potently inhibited by amastatin and bestatin (IC50 = 0.05 microM and 3.0 microM, respectively). The activity of the aminopeptidase against its synthetic substrate was also inhibited by lysylbradykinin (IC50 = 50 microM). Both the carboxypeptidase and aminopeptidase activities had neutral pH optima and were inhibited by o-phenanthroline, but were unaffected by inhibitors of neutral endopeptidases (phosphoramidon) or angiotensin-converting enzyme (Captopril). The Km of bradykinin for the nasal carboxypeptidase was 139 +/- 14 microM (n = 3). We conclude that during the allergic response, nasal secretions contain aminopeptidase and carboxypeptidase activities that convert lysylbradykinin and bradykinin (B2 agonists) to des (Arg9) bradykinin (a B1 agonist). Because the nature of the kinin receptors in the nasal mucosa are currently unknown, it remains to be determined whether this metabolism results in the termination of biologic activity or the production of a biologically active moiety.     

Angiotensin I converting enzyme of rat intestinal and vascular surface membrane

High levels of angiotensin I converting enzyme are present in rat intestinal mucosa and in intestinal arteries. Homogenates of both tissues were subfractionated and fractions enriched in vascular plasma membrane or intestinal brush border were prepared. The preparations were identified and their purities established by marker enzyme enrichment and/or electron microscopy. Converting enzyme activity was highly enriched on both the vascular plasma membrane and the intestinal brush border. Subsequently the properties of these membrane-bound enzymes were compared. Both surface membrane-bound enzymes were highly sensitive to inhibition by captopril (SQ 14225) and teprotide (SQ 20881). Similar to converting enzyme isolated from other sources, they were also inhibited by bradykinin, angiotensin I, EDTA and o-phenanthroline. Finally, both membrane-bound enzymes were relatively resistant to activation by sonication, freezing and thawing or detergent. These results demonstrate significant similarities between surface membrane-bound converting enzyme from vascular and non-vascular sites. In addition, in view of the possible relationship of kinins and angiotensins to gastrointestinal function and blood flow, inhibition of gastrointestinal converting enzyme by captopril may affect some aspects of intestinal physiology.     

Mammalian signal peptidase: partial purification and general characterization of the signal peptidase from microsomal membranes of porcine pancreas

Signal peptidase has been enriched extensively from microsomal membranes of porcine pancreas. Microsomal membranes were washed with 1 M KCl and Brij 35, and then solubilized with 1% Nonidet P-40. The solubilized signal peptidase was purified by DEAE-cellulose chromatography and Sepharose CL-6B filtration. Cleavage of pre-human placental lactogen with the partially purified enzyme gave the mature form, whose NH2-terminus was identified as valine. The signal peptidase is heat-labile and approximately 90% of the enzymatic activity was lost at 60 degrees C within 1 min. The pH optimum of the activity was 7 to 8. Chymostatin and o-phenanthroline at concentrations of 2.5 mM inhibited the signal peptidase activity by 62% and 30%, respectively.     

An elastinolytic enzyme detected in the culture medium of human arterial smooth muscle cells

The culture medium of human arterial smooth muscle cells exhibits an elastinolytic activity with 68 and 64 kDa on elastin substrate gels. The enzymatic activities are inhibited by ethylenediamine tetraacetic acid, a metalloproteinase inhibitor, but not by other inhibitors of serine, cysteine and aspartic proteinases. The proteinase in the culture medium is activatable by 4-aminophenylmercuric acetate and degrades insoluble elastin. Compared to other matrix metalloproteinases (MMP), the activity shows the similar elastinolytic pattern to that by MMP-2 purified from human rheumatoid synovium, while MMP-3 and MMP-9 have different lytic patterns and MMP-1 possesses no elastinolytic activity. An immunoblot analysis demonstrated that the 68-kDa enzyme is MMP-2. An immunofluorescence study illustrates that MMP-2 is localized within the cytoplasm of the smooth muscle cells. These findings suggest that the elastinolytic enzyme secreted by human arterial smooth muscle cells is MMP-2.     

Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes

The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-ATPase and K+-pNPPase activities.     

Distribution and subcellular localization of acylpeptide hydrolase and acylase I along the hog gastro-intestinal tract

The distribution of acylase I and acylpeptide hydrolase along the hog small intestine was investigated. No significant changes in their respective specific activity was found when the intestine was cut off and divided into eight segments (taken every 200 cm) so as to specifically study the duodenum, jejunum and ileum. Upon performing subcellular fractionation of hog enterocytes, it was observed that acylpeptide hydrolase is a soluble enzyme, while acylase I is essentially a soluble protein accounting for only 5% of the activity associated with the whole membrane fraction. The membrane-bound acylase I was neither solubilized by phosphatidylinositol-specific phospholipase C from Bacillus cereus nor by detergents which are commonly used to solubilize alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein. When a phase separation was carried out in Triton X-114, all the anchored-membrane proteins of the intestinal membranes were located in the detergent-rich phase, while acylase I was present in the detergent-poor phase. Finally, the immunolabeling of intestinal cells with specific antibodies definitively established the cytoplasmic localization of acylase I. Acylpeptide hydrolase and acylase I therefore both are located in the enterocyte cytoplasm.     

Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85 degrees C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40 degrees C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.