Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on neuropeptides

A calcium-activated neutral protease was purified from Japanese monkey brain by ammonium sulfate fractionation and sequential column chromatographies monitored by assay of caseinolytic activity. The purified enzyme gave a single protein band on non-denaturing polyacrylamide gel electrophoresis, and consisted of two subunits with molecular weights of 74,000 and 20,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required millimolar order calcium ions for activation, and was optimally active at pH 7.5-8.0. Upon incubation with various neuropeptides as substrates, the enzyme preferentially cleaved the peptide bonds with Arg, Lys, or Tyr at the P1 position and an amino acid residue with a bulky aliphatic side chain, such as Leu, Val, or Ile, at the P2 position. The hydrolytic activity toward neuropeptides as well as casein was strongly inhibited by various thiol protease inhibitors. These results suggested that the brain calcium-activated neutral protease may participate in the degradation of neuropeptides in vivo.     

Purification of a neutral thiol protease from Paragonimus westermani metacercariae by single-step chromatography and preparation of antibodies

A neutral thiol protease from extracts of larvae of the mammalian digenean parasite Paragonimus westermani metacercariae was purified by single-step chromatography on Ultrogel AcA-54, measuring its activity on t-butyloxycarbonyl-valyl-leucyl-lysyl-4-methylcoumaryl-7-amide as a substrate. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and size exclusion-high-performance liquid chromatography analysis of the enzyme indicated that the fraction obtained by gel filtration was homogeneous. Antibodies against the purified protease were raised in rabbits by immunizing with micro quantities of the enzyme protein. The antibodies revealed a single precipitin line against the enzyme on double immunodiffusion analysis.     

Purification and properties of a thiol protease from rat liver nuclei

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.     

Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP) from rabbit skeletal muscle

Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.     

Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae

1. A neutral thiol protease was isolated from the extract of larvae of the mammalian trematode parasite, Paragonimus westermani metacercariae, by arginine-Sepharose, Ultrogel AcA-54 and DEAE-toyopearl column chromatography, measuring its activity by the hydrolysis of Boc-Val-Leu-Lys-MCA as a substrate. 2. The molecular weight of the purified enzyme was estimated to be 22,000 as a single polypeptide by SDS-polyacrylamide gel electrophoresis and was estimated to be 20,000 by size exclusion high-performance liquid chromatography. 3. The activity was suppressed by antipain, E-64, leupeptin, chymostatin, N-tosyl-L-lysine chloromethyl ketone, but was not affected by metallo protease inhibitors or serine protease inhibitors. 4. Studies on the substrate specificity showed that the enzyme hydrolyzed Boc-Val-Leu-Lys-MCA, Z-Phe-Arg-MCA, fluorescein isothiocyanate-labeled collagen, azocoll and casein. 5. The enzyme was found to hydrolyze peptide bonds of oxidized insulin B chain preferentially at the carboxy side of hydrophobic and basic amino acids.     

The purification and properties of cancer procoagulant from murine tumors.

The protease, cancer procoagulant, was isolated from three murine metastatic tumors and was purified to apparent homogeneity (SDS-PAGE) from Lewis lung cells by the sequence of (NH4)2SO4 precipitation, DE-53 anion-exchange chromatography, and Sephacryl 200 chromatography. The murine tumor enzyme has a molecular weight of 68,000 and Ca2+ is required for procoagulant and proteolytic activity; thus, the murine enzyme is very similar to that isolated from rabbit tumors. Two peptidyl chromogenic substrates of cancer procoagulant were discovered, facilitating kinetic and inhibition studies with the enzyme. The peptide substrate structures and the results of inhibition studies suggest that cancer procoagulant is thrombin-like in specificity but is a thiol protease.     

Bleomycin hydrolase is a unique thiol aminopeptidase

Bleomycin hydrolase, which hydrolyzes the carboxamide bond in the pyrimidoblamic acid moiety of the bleomycin molecule, also cleaved several p-nitroanilide substrates with a neutral or basic amino acid residue and dipeptide substrates such as L-leucyl-glycine. The activity of bleomycin hydrolase was inhibited by two thiol protease inhibitors, E-64 and leupeptin, as well as by N-ethylmaleimide. These results suggest that bleomycin hydrolase is a thiol aminopeptidase. Magnesium ion, sodium chloride, ethylenediaminetetraacetic acid and 1,2-dihydroxybenzene-3,5-disulfonic acid specifically activated the enzymatic hydrolysis of L-arginine-p-nitroanilide, but did not that of L-leucine-p-nitroanilide. Lineweaver-Burk plots showed that Km values of the enzymatic activity for L-arginine-p-nitroanilide were altered by these reagents, although Vmax values were almost unaltered.     

Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.     

Purification and characterization of a neutral serine protease from non-sulfur purple photosynthetic bacterium

A neutral serine protease was purified as a homogeneous protein from the culture broth of photosynthetic bacterium T-20 by sequential chromatographies on columns of DEAE-cellulose, Toyopearl HW 55F, hydroxyapatite, and CM-cellulose. The molecular weight was estimated to be approximately 44,000 by SDS-PAGE, while the value of approximately 80,000 was obtained when the Hedrick-Smith method was used; this suggested that the enzyme consists of two identical subunits. The isoelectric point was determined to be 6.3 by isoelectric focusing. The enzyme had a pH optimum at 7.8. Maximal enzyme activity was detected at 50°C, and the activity was stable up to 50°C for 5 min at pH 7.0–7.2. The substrate specificity of the protease was investigated with a series of synthetic peptidyl-p-nitroanilide. The best substrate examined was Suc-Ala-Ala-Pro-Phe-pNA. The protease activity was inhibited by various inhibitors of serine protease such as chymostatin, PMSF, and DFP. EDTA, which is an inhibitor of metal protease, also inhibited the protease activity, whereas inhibitors of thiol and aspartic proteases had no significant effect.     

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.     

Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and in-aminohexanoate (in-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells.     

Comparative studies of mouse liver cathepsin B and an analogous tumor thiol proteinase

Cathepsin B (EC 3.4.22.1) and an analogous thiol proteinase were isolated from mouse liver and from a transplantable tumor induced by methylcholanthrene, respectively, by a sequence of steps involving salt fractionation and ion exchange and gel permeation chromatography. Both enzymes are capable of hydrolyzing N-benzyloxycarbonyl-L-Ala-L-Arg-L-Arg-4-methoxy-2-naphthylamide but are weakly active towards N-benzoyl-DL-arginine-2-naphthylamide. The specific activity of the liver enzyme towards these substrates is approximately 14 times greater than that of the tumor enzyme. Both enzymes show a single band with slight difference in mobility when subjected to gel electrophoresis at pH 4.5, but both exhibit a multiple banding pattern when examined by isoelectric focusing. The tumor enzyme has a somewhat higher molecular weight than the liver enzyme (33,000 versus 30,000) and possesses a slightly higher helical content (48% versus 40%) based on CD spectra. Both enzymes display maximum activity in the pH range of 5.5 to 7.0 and are irreversibly denatured above pH 7 and below pH 4. Both enzymes cross-react with antiserum towards the tumor enzyme. The liver enzyme displays a higher catalytic efficiency towards a series of oligopeptide substrates than the tumor enzyme, but is only one-third as active towards N-benzoyl-L-arginine-2-naphthylamide. Both proteinases exhibit similar patterns of inhibition by iodoacetate, chloroquine, leupeptin, antipain, and several peptide chloromethylketones. Despite what appear to be subtle differences in physical properties, amino acid composition data and peptide mapping revealed significant differences between these two enzymes reflective of extensive regions of non-identity. These results suggest that the tumor thiol protease and liver cathepsin B are products of separate genes and that the tumor enzyme is not likely an immediate precursor of the liver enzyme produced by post-translational modification.     

A comparative study of soluble calcium-dependent proteolytic activity in brain

Recent studies have shown that soluble calcium activated proteases (calpains) in brain degrade proteins associated with the cytoskeleton and vary markedly in activity across regions and as a function of development. It was suggested that the observed differences in calpain activity reflect differences in the turnover rate of structural elements. The present study extends this analysis by measuring the properties and activity of calpain in representatives of the five classes of vertebrates with particular emphasis on the mammals. No evidence for proteolysis was found in soluble fractions of fish brains at neutral pH in the presence or absence of added calcium. A substantial calcium-independent proteolytic activity was found in amphibian brains—the effects of a variety of protease inhibitors indicated that it is also a neurtral thiol (cysteine) protease. Reptilian brains exhibited both calcium-independent and calcium-dependent proteolytic activity. Virtually all proteolytic activity in birds (5 species) and mammals (9 species) measured at neutral pH was calcium-dependent. The endogenous substrates for the calcium activated proteases were very similar in several species of birds and mammals as were the effects of a variety of protease inhibitors. However, the activity of the enzyme, expressed per mg of soluble protein, was highly and negatively correlated with brain size in the mammals. The allometric expression for this relationship was similar to that found for the density of neurons in cerebral cortex as a function of absolute brain size. These results indicate that soluble proteolytic enzymes in brain are differentially expressed among classes of vertebrates and suggest that the turnover of cytoskeletal elements in birds and mammals differs in important ways from that found in fish and amphibians. The results obtained for mammals raise the possibility of a relationship between brain size and the rate at which structural elements are broken down and replaced in this vertebrate class.     

Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins

The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5-8.0. The protease appeared to be a thiol protease, since it was inhibited by sulfhydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free sulfhydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of Alzheimer's ß-Amyloid Protein by Human and Rat Brain Peptidases: Involvement of Insulin-Degrading Enzyme

We examined the degradation of Alzheimer's ß-amyloid protein (1–40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the ß-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble ß-amyloid degrading enzyme at neutral pH in human brain. The highest ß-amyloid protein degrading activity in the soluble fractions occurred between pH 4–5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower ß-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases –24.11, –24.15, –24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

Enzymic properties of a Ca2+-dependent protease in rat ventral prostate: differences in distribution between lobes of the prostatic complex

Soluble extracts of rat ventral prostate contain a calcium-dependent, neutral thiol protease which is separated from an endogenous inhibitor by DEAE-cellulose chromatography. The Ca2+-dependent protease had a high calcium requirement (half maximal activation at 0.19 mM CaCl2), a pH optimum in the neutral range (pH 7-8), and it was inhibited by increased ionic strength (30% inhibition at 0.2 M NaCl). Leupeptin and antipain were strong inhibitors of the enzyme. Ca2+-activated protease activities of the coagulating gland (anterior prostate) were about 40% of those of the ventral prostate and were not detectable in the dorsolateral prostatic lobe. There was no difference in specific activities of this enzyme in chromatographed extracts of prostatic lobes from young sexually mature adults and 12 month old retired breeders. In addition, Ca2+-dependent protease activity was not detectable in chromatograms of rat ventral prostate and coagulating gland secretions. Therefore, the Ca2+-activated protease does not appear to be a secretory protein and probably acts at some intracellular site(s).     

Proteases of Senescing Oat Leaves: II. Reaction to Substrates and Inhibitors

Two proteases isolated from senescent oat (Avena sativa) leaves have been subjected to further study. One of these, an acid protease active at pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by iodoacetamide (IAc). The other, active at pH 6.6, is inhibited by both PMSF and IAc. These results, together with previously reported evidence that mercaptoethanol stimulates the activity of only the neutral protease, are taken to indicate that the acid protease is probably of the serine type, whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are inhibited by irradiation in the presence of rose bengal, a selective histidine modification reagent. The acid protease was completely unaffected by chelators, but data on the neutral protease were equivocal.

All protein substrates tested were attacked by both enzymes, though at strikingly different rates. Characterization of the digestion products, with denatured hemoglobin as substrate, indicated that the acidic enzyme is an endoprotease, while the neutral one is an exoprotease. Evidence is presented that these proteases undergo autolysis in vitro.

     

Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.     

A novel metalloprotease from Bacillus cereus for protein fibre processing

A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 °C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn³, Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴, Glu²¹, after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P₁ position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a K_m values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool.