Angiotensin II-producing proteases from granulomatous tissue reaction in mice infected with Schistosoma mansoni

1. Angiotensin I hydrolases, Mr 140,000 and Mr 70,000 were separated by gel filtration from Tris-HCl buffer extract of hepatic granulomas developed in mice with schistosomiasis. Two enzymes had different substrate specificity. 2. Mr 140,000 hydrolase activity was inhibited by captopril as reported for angiotensin converting enzyme (ACE), while that of Mr 70,000 hydrolase activity was inhibited by potato carboxypeptidase inhibitor. 3. An intermediary, des-Leu10-angiotensin I and then angiotensin II were formed from angiotensin I by Mr 70,000 hydrolase. 4. The findings suggest that Mr 70,000 enzyme is tissue carboxypeptidase A, and it generates angiotensin II in granulomatous inflammation as does ACE.     

Angiotensin-converting enzyme: immunologic, structural, and developmental aspects

Immunization of dog and rat high pure rabbit pulmonary angiotensin-converting enzyme elicited, in some individuals, antibodies that inhibited their own converting enzyme. Active immunization with an immunologically related enzyme is thus a plausible approach for developing biologically based inhibitors of enzymes that are either in or accessible to the circulation. Rabbit testicular peptidyldipeptide hydrolase was purified to homogeneity and found to be a considerably smaller (Mr approximately 100,000) glycoprotein than pulmonary converting enzyme (Mr approximately 140,000). The two enzymes differed at their amino- and carboxy-termini. However, they exhibited identical catalytic properties, and antibodies prepared against either inhibited both similarly. In competition radioimmunoassays, antibodies against the pulmonary enzyme preferred it to the testicular species, whereas those against the latter did not distinguish between the two molecules. The testicular isozyme thus resembles an internal part of the pulmonary polypeptide, which includes its active site. In a reticulocyte lysate, mRNA from the lungs of immature and mature rabbits comparably primed the synthesis of a polypeptide (Mr approximately 129,000) that reacted with anticonverting enzyme antibodies. In contrast, an immunoreactive species was programed only by mRNA from the testis of mature animals, and this protein was much smaller (Mr approximately 85,000). Maturation dependence and a shorter polypeptide chain, the regulatory and structural properties that distinguish the testicular isozyme, are thus each pretranslationally determined.     

Angiotensin converting enzyme: implications from molecular biology for its physiological functions.

1. The two isozymes of human angiotensin converting enzyme (ACE; EC 3.4.15.1) have recently been cloned and sequenced. 2. The larger, endothelial isozyme has two highly similar internal domains each bearing a putative catalytic site. In contrast the smaller, testicular isozyme has a single catalytic site corresponding to the C-terminal domain of endothelial ACE and represents the ancestral, non-duplicated form of the gene. 3. Both isozymes are anchored in the plasma membrane by a single hydrophobic transmembrane polypeptide located near the C-terminus, and both are extensively N-glycosylated. 4. The testicular isozyme may also be O-glycosylated. 5. The soluble form of ACE in plasma, seminal fluid and other body fluids appears to be derived from the membrane-bound endothelial isozyme by a post-translational modification. 6. ACE has a complex substrate specificity with peptidyl tripeptidase or endopeptidase action on certain peptides, as well as the classical peptidyl dipeptidase activity. 7. Numerous potent inhibitors of the enzyme have been developed and used successfully in the treatment of hypertension, but some of the observed side effects may be due to inhibition of other zinc metalloenzymes. 8. Both endothelial and testicular ACE are highly conserved between species, indicative of the essential role(s) of the enzyme in blood pressure regulation and other physiological processes.     

Purification and properties of beta-galactosidase from chicken seminal plasma

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.     

Cysteine proteinase inhibitors in human and rat male sex glands

The testis and prostate of men had two types of cysteine proteinase inhibitors of different isoelectric point (pI = 4.5 and 6.3) and molecular masses (90,000 and 12,000). The seminal vesicle contained not only these two inhibitors but also a basic inhibitor with a pI = 9.5 and Mr 11,000. In rats, the inhibitors in the testis and prostate consisted of two types with pI values of 4.3 and 4.8 and Mr 90,000 for both. In addition to these two inhibitors, the seminal vesicle contained large amounts of two basic inhibitors with pI values of 7.5 and 8.3 and Mr of 11,000 for both. We suggest that the basic cysteine proteinase inhibitor found in semen might be excreted from or be modified in the seminal vesicle.     

Functional heterogeneity of monoclonal antibodies obtained using different screening assays

Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed.     

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.     

Gelatinolytic proteinase activities in human seminal plasma

Proteinase activities in human seminal plasma were detected using gelatin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. Three prominent bands of activity of Mr 60,000, 66,000 and 90,000 were observed as well as 9 other bands of less intensity (34,000-158,000). These proteinases were dependent upon calcium for optimal activity, did not hydrolyse casein, and were predominantly in the soluble portion of seminal plasma. Examination of seminal plasma of men with different sperm concentrations, split ejaculates, and prostatic secretions indicated that the prostate gland was a source of most of these activities. Proteinase activities of Mr 34,000, 37,000, 82,000 and 120,000 were expressed more frequently in seminal plasma from normozoospermic men than from seminal plasma of oligo- or azoospermic men, indicating that they may also arise from spermatozoa in the semen sample. The proteinases of Mr 60,000 and 66,000 were found in all seminal plasmas whereas there was variation in the expression of the other molecular forms of enzyme, even in the normozoospermic samples. There are multiple forms of gelatinolytic proteinase activities in human seminal plasma which appear to arise from multiple sources in the reproductive tract including the Cowper's/urethral glands, the prostate gland, seminal vesicle and/or spermatozoa. Their function(s) in semen remains to be established.     

Substance K and substance P as possible endogenous substrates of angiotensin converting enzyme in the brain

In the brain angiotensin converting enzyme is highly localized to a striatonigral pathway, which contains no endogenous angiotensin. Substance P, also localized to a striatonigral pathway, is degraded by ACE via two different pathways. The lung and striatal isozymes of angiotensin converting enzyme exhibit differential cleavage of substance P, with lung preferring an initial tripeptide cleavage, and striatum an initial dipeptide cleavage. Substance K is degraded by the striatal isozyme but is not cleaved by the lung isozyme. Substance P 5-11 is not cleaved by either form of angiotensin converting enzyme.     

Angiotensin-converting enzyme from human tissues. Physicochemical, catalytic, and immunological properties

Angiotensin-converting enzyme was purified from human lung, kidney, testis, blood plasma, and seminal plasma using a facile two-step protocol which included affinity chromatography on Sepharose-bound lisinopril followed by either gel filtration or hydroxylapatite chromatography. Molecular mass for converting enzyme from all sources except testis was 140 kDa. That from testis consisted of both a 90- and a 140-kDa form in a 4:1 ratio. Detergent-extracted membrane-bound converting enzyme aggregated on gel filtration chromatography, while trypsin-extracted and soluble converting enzyme did not. Comparison of detergent-extracted and trypsin-extracted membrane-bound converting enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane binding sequence contributed minimally to the size and charge of the enzyme. Catalytic and kinetic properties assessed by interaction with substrates, inhibitors, and anti-converting enzyme immunoglobulin were similar for all forms and sources of converting enzyme. Enzyme-linked immunosorbent assay revealed only partial homology between the 90- and 140-kDa forms of the enzyme.     

Guanine nucleotides allow the trypsin solubilization of an active Mr = 68,000 guanylate cyclase

It has been previously shown that trypsin treatment of rat liver plasma membranes causes the solubilization of a guanylate cyclase of Mr = 140,000 (Lacombe, M. L., Haguenauer-Tsapis, R., Stengel, D., Ben Salah, A., and Hanoune, J. (1980) FEBS Lett. 116, 79-84). In this study, we observed that addition of Mn-GTP during this step greatly protected the enzyme from proteolytic degradation. This effect was specific for guanine nucleotides, being weaker for other nucleotides triphosphate and GDP, and absent for cyclic GMP and GMP. Metal-GTP complex was required with a strict specificity for Mn2+. In addition to the Mr = 140,000 enzyme, trypsin solubilization in the presence of Mn-GTP led to the formation of a small and active form of guanylate cyclase. Based on its behavior on Ultrogel AcA 34 and sucrose gradients, its apparent Mr was calculated to be 68,000. Both forms could be well separated by high performance liquid chromatography and were shown to be sequentially solubilized (the larger appearing before the smaller species). Mr = 140,000 species, but not the cytosolic enzyme, was able to generate the Mr = 68,000 enzyme upon tryptic treatment in the presence of Mn-GTP. The Mr = 140,000 and 68,000 enzymes exhibited Michaelis-Menten kinetics (Hill coefficient = 1) with Km for Mn-GTP of 130 and 70 microM, respectively. The proteolytically solubilized enzymes were strickingly heat labile and highly protected by Mn-GTP. These results support the hypothesis that the rat liver membrane-bound guanylate cyclase has a dimeric structure similar to that of the cytosolic enzyme. They also suggest a possible role for GTP in limiting the degradation rate of membrane guanylate cyclase in vivo and, thus, in regulating the active enzyme concentration.     

Generation of a 90 000 molecular weight fragment from human plasma angiotensin-I-converting enzyme by enzymatic or alkaline hydrolysis

A catalytically active Mr 90 000 fragment was generated from native Mr 140 000 human plasma angiotensin-I-converting enzyme after treatment with reagents that induced a perturbation of the native tertiary conformation. Treatment of converting enzyme with 6 M urea produced an aggregation of molecules that was susceptible to proteolysis by either trypsin, chymotrypsin or Staphylococcus aureus V8 proteinase to generate the Mr 90 000 converting enzyme. Also, 1 M ammonium hydroxide, pH 11.3, or 0.01 M sodium hydroxide, pH 11.3, cleaved converting enzyme to the Mr 90 000 fragment. Degradation was not an autocatalytic phenomenon, since it was not prevented by inhibition of converting enzyme with EDTA. The enzymatically mediated, but not the alkaline mediated, cleavage was inhibited by specific converting enzyme inhibitors captopril and Merck L-154,826. This suggests that captopril and Merck L-154,826 can prevent converting-enzyme degradation by preserving a conformation that does not have sites exposed to proteolytic enzymes. This conformation may mimic the native conformation which is quite resistant to serine proteinases.     

Molecular heterogeneity of angiotensin converting enzyme in human cerebrospinal fluid.

Three different molecular forms of angiotensin converting enzyme (ACE) (approximately Mr 150,000, 80,000 and 40,000, respectively), have been recovered from human cerebrospinal fluid. All three enzymes were inhibited by captopril and enalapril and their activity was potentiated by chloride ions. They were capable of degrading Leu-enkephalin-Arg6 and substance -P, but gave no conversion of neurokinin A. In all these aspects, the CSF enzymes were identical with the human pulmonary enzyme. The Mr 40,000 form of ACE is the smallest active form of the enzyme hitherto reported and is likely to represent a fragment of the C-terminal part of native ACE, where its active center is located.     

Molecular and catalytic properties of angiotensin converting enzyme-I from bovine seminal plasma

Angiotensin converting enzyme [EC 3.4.15.1] was shown to exist in two distinct forms in bovine seminal plasma. The higher molecular weight form of the enzyme (angiotensin convering enzyme I) was purified to homogeneity by Sephadex G-200 gel filtration, and DEAE-Sepharose, blue Sepharose, and concanavalin A-Sepharose column chromatography. Final recovery of the enzyme was 9.0. The molecular weight of the enzyme was estimated to be 8 x 10(5) by the gel filtration method. A value of 4.6 x 10(5) was obtained for the reduced and denatured enzyme by dodecylsulfate polyacrylamide gel electrophoresis. The Stokes' radius, diffusion coefficient, and intrinsic viscosity of the purified enzyme were determined to be 95 A, 2.3 x 10(-7) cm2/s, and 6.76 ml/g. The enzyme had a specific activity of 105.12 mumol/min/mg protein for hippurylhistidylleucine. The Km value for hippurylhistidylleucine was found to be 20 mM. Studies with EDTA suggest that metal ions which are tightly bound are required for its activity. The enzyme was inhibited by some heavy metal ions but did not required sulfhydryl groups for its activity. Trypsin treatment of the urea-denatured enzyme produced a catalytically active fragment with an Mr of 30,000. Chemical hydrolysis of the native enzyme did not produce any active fragment.     

Initial events involved in the synthesis of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum

In Dictyostelium discoideum the lysosomal enzyme alpha-mannosidase is initially synthesized in vivo as a 140,000 Mr protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 Mr. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the alpha-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K alpha-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.     

Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin

Treatment of skins of newborn mice with the neutral protease Dispase in order to separate dermis and epidermis causes pronounced changes in the levels of transglutaminase activity in the epidermis. Two soluble transglutaminases, one anionic enzyme and one cationic enzyme, of Mr approximately 90,000 and approximately 50,000, respectively, are extracted from epidermis; and the activities of both enzymes increase as a function of the time of Dispase treatment of skin. When the anionic Mr approximately 90,000 enzyme is incubated with Dispase after its chromatographic isolation from epidermal extracts, it is converted to a lower molecular weight enzyme. Hair follicles isolated from dermis prepared by a 12-h Dispase treatment of the skin of newborn mice contain two soluble cationic transglutaminases, one of which is indistinguishable from that of epidermis and the other which is not seen in epidermis. Both of these hair follicle enzymes are of Mr approximately 50,000 and appear to exist in monomeric form. They have been partially purified. Based upon these findings, we suggest that transglutaminase processing and control occur during normal differentiation of keratinocytes in epidermis and of hair follicle epidermal cells in dermis and that production of the proper forms of the enzyme may be essential to the formation of mature cornified envelopes and hair shafts, respectively.     

Comparative studies of two cathepsin B isozymes from porcine spleen. Isolation, polypeptide chain arrangements, and enzyme specificity

Our previous studies on carbohydrate structures of purified porcine spleen cathepsin B indicated that there are two cathepsin B isozymes, each containing a different carbohydrate (Takahashi, T., Schmidt, P.G., and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). We have now isolated these two enzymes and carried out a comparative study on their structures and enzymic properties. The major isozyme (CB-I) is a two-chain enzyme (Mr = 28,000) with a light chain (Mr = 5,000) and a heavy chain (Mr = 23,000), whereas the minor enzyme (CB-II) is a single chain enzyme (Mr = 27,000). The NH2-terminal amino acid residues of CB-I were leucine and valine for the light and heavy chain, respectively. However, the NH2-terminal residue of CB-II was not available for automated Edman degradation. In addition, peptide mapping experiments indicated a difference in the primary structure of these two proteins. Despite such structural differences, they are similar in many enzymic properties. CB-I was more catalytically efficient than CB-II toward synthetic substrates, except for the substrate benzoyl-L-arginine beta-naphthylamide for which the relative catalytic efficiency is reversed. Both isozymes degraded glucagon by a dipeptidyl carboxypeptidase activity. Under the same conditions, CB-I was 4-5 times more efficient than CB-II. The results indicate that the cathepsin B isozymes are two separate gene products, but they are similar in enzymic properties.     

Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles

Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.     

Ascaris suum: electrophoretic characterization of reproductive tract and perienteric fluid polypeptides, and effects of seminal and uterine fluids on spermiogenesis.

Fluids isolated from the testis, seminal vesicle, uterus, and pseudocoelomic cavity of Ascaris suum were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured for protein concentration, pH, and osmolarity. The testis and seminal fluids display much homology and share major polypeptide components having molecular weights of 15,000 and 35,000. A cytoplasmic extract of spermatids from the seminal vesicle exhibited a banding pattern nearly identical to that of testis fluid. The seminal fluid has unique major components of 57,000 and 150,000, and seminal fluid from individual worms showed differences in major band concentration and distribution of minor components. The uterine fluid has major polypeptides of 14,000, 16,000, 66,000, 74,000, 120,000, and 140,000, and exhibits more similarity to the perienteric fluid then either the seminal or testis fluids. Electrophoretic comparisons of four uterine regions revealed nearly identical banding patterns although somewhat higher concentrations of four major components occurred in certain segments. The male and female perienteric fluids have major bands at 40,000, 120,000, and 140,000, and the female fluid has more intense minor components of 90,000 and 115,000. Perienteric fluid from individual worms differed only in minor band distribution. The reproductive fluids have numerous minor components mostly from 20,000 to 70,000, while the perienteric fluid minor bands are mainly located in the 80,000 to 120,000 range. The pH of the seminal fluid (6.5) differs from that of the uterine fluid (7.7), and both seminal and uterine fluids are of lower osmolarity than the perienteric fluid. In vitro studies demonstrate that uterine fluid does not induce spermatid transformation into bipolar, ameboid spermatozoa, while the seminal fluid induces only lipid granule coalescence in either seminal vesicle or terminal testis spermatids.