Cathepsin L Plays an Important Role in the Lysosomal Degradation of L-Lactate Dehydrogenase

A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Simultaneous isolation of protein C activator,fibrin clot promoting enzyme (fiprozyme) and phospholipase A2 from the venom of the southern copperhead snake

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A₂.     

Cathepsin L plays an important role in the lysosomal degradation of L-lactate dehydrogenase

A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Isolation,Purification and Some Properties of Invertase from Leaves of Mandarin Orange

Highly active acid invertase was found in the young leaf extract of mandarin orange Citrus reticulata Blanco). The invertase was isolated and purified from the young leaf extract of mandarin orange through the procedures of ammonium sulphate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. 6.4% of the invertase activity was recovered. Invertase was 179.2-folds purified. The purified invertase preparation was homogeneous as shown in polyacrylamide gel electrophoresis and Sephacryl S-200 molecular sieve chromatography. The molecular weight of the native invertase determined by gel filtration was 80 kD. The invertase consists of two identical subunits with apparent equal subunit weight of 40 kD as determined on SDS-PAGE. The invertase followed typical Michaelis-Menten Kinetics with apparent Km Of 1. 6 × 10-2 mol/L for sucrose. Vmax of the invertase was 100 mg reducing sugar · mg-1 protein · h-1 The optimum pH was 5.0 (stable from 4.5—5.5). The optimum temperature was 55℃.     

Purification and Characterization of Soluble (Cytosolic) and Bound (Cell Wall) Isoforms of Invertases in Barley (Hordeum vulgare) Elongating Stem Tissue

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (β-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organomercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, K_m of 12 millimolar, and a V_max of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, K_m of millimolar, and a V_max of 9 micromole per minute per milligram of protein.     

Purification and immunological properties of cathepsin B in carpCyprinus carpio

Cathepsin B was purified from the crude extract of carp (Cyprinus carpio) hepato-pancreas by the method involving ammonium sulfate fractionation and five sequential chromatographies monitored the activity with Z-Arg-Arg-MCA as a substrate, and the specific activity increased about 11,400 fold with a 2% recovery. Although the homogeneity of the purified cathepsin B was established on Native-PAGE, it migrated as two bands of 29,000 and 25,000 molecular weights by the single and heavy chains on SDS-PAGE, respectively. The monospecific antibody against the homogeneous cathepsin B was purified by the affinity chromatography on cathepsin B-Sepharose 4B, and did not immunologically react with rat cathepsin B, carp cathepsins H and L but only with carp cathepsin B by immunoelectrophoretic blot analysis. As the result of the tissue and liver distributions of cathepsin B, the remarkable immunological reactivities in the extracts of spleen, kidney and hepato-pancreas in carp and those of pacific cod, yellow fin tuna, skip jack tuna and common mackerel in pisces were detected with the anti-carp hepato-pancreas cathepsin B at molecular weight of nearby 29,000 or 25,000.     

Large Scale Purification of Isoinhibitors of Trypsin from Swine Colostrum Using Zinc Chelate Chromatography and Chromatofocusing

Low molecular weight trypsin inhibitors were purified from swine colostrum on a large scale under mild conditions. Ammonium sulfate fractionation and metal chelate chromatography on zinc chelate Sepharose and phenyl Sepharose were used for removal of the bulk of proteins. The inhibitors showed only a weak hydrophobic interaction with phenyl Sepharose even in the presence of 1 M (Nll₄)₂SO4, and advantage was taken of this property to remove the inhibitors from contaminating colostrum proteins which remained tightly adsorbed to phenyl Sepharose under these conditions. The low and high molecular weight inhibitors were then separated by gel filtration on Bio-Gel P-300. The low molecular weight material was eluted in three major inhibitor fractions on DEAE-Sepharose.

Chromatofocusing of these fractions provided greater resolution of the inhibitors, and several previously unreported inhibitor peaks were detected. The six major inhibitors purified by chromatofocusing were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. These inhibitors were composed of a single polypeptide chain with a molecular weight of 18,000 as determined by Sephacryl S-200 gel filtration and polyacrylamide qel electrophoresis in the presence of sodium dodecyl sulfate and e-mercaptoethanol. The specific activities of the pure inhibitors were approximately 30% higher than those previously reported.     

Auxin-Binding Protein in Etiolated Mung Bean Seedlings: Purification and Properties of Auxin-Bindmg Protein-II

An auxin-binding protein (ABP-II) was purified from the extractof etiolated mung bean seedlings by affinity chromatographyon 2,4-D-linked Sepharose 4B and by gel filtration on Sepharose4B and Sephacryl S-200. The molecular weight was estimated tobe about 190,000 by gel filtration on Sephacryl S-200. ABP-IIgave a single band corresponding to a molecular weight of about48,000 on SDS-polyacrylamide gel electrophoresis. The dissociationconstants of ABP-II for 2,4-D determined by amrnonium sulfateprecipitation and equilibrium dialysis were 9.5?10^–6 Mand 1.1?10^–5 M, respectively. ¹⁴C-2,4-D-binding to ABP-IIwas reversible and inhibited by addition of IAA, naphthalene-1-aceticacid, 2,4,5-trichlorophenoxyacetic acid or p-chlorophenoxyisobutylicacid to the assay mixture. (Received September 5, 1984; Accepted November 5, 1984)     

SAR studies of some acetophenone phenylhydrazone based pyrazole derivatives as anticathepsin agents

Cathepsins have emerged as promising molecular targets in a number of diseases such as Alzeimer’s, inflammation and cancer. Elevated cathepsin’s levels and decreased cellular inhibitor concentrations have emphasized the search for novel inhibitors of cathepsins. The present work is focused on the design and synthesis of some acetophenone phenylhydrazone based pyrazole derivatives as novel non peptidyl inhibitors of cathepsins B, H and L. The synthesized compounds after characterization have been explored for their inhibitory potency against cathepsins B, H and L. The results show that some of the synthesized compounds exhibit anti-catheptic activity with K_i value of the order of 10⁻¹⁰ M. Differential inhibitory effects have been observed for cathepsins B, H and L. Cathepsin L is inhibited more pronounced than cathepsin B and cathepsin H in that order.     

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzedAbz-QVVAGA-EDDnp andAbz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10⁷M^–1 sec^–1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10⁵ M^–1 sec^–1 range due to lowerk _cat values, with theK _m 's being identical to those with papain. OnlyAbz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN₂ was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k ₊₀= 1,100,000 M^–1 sec^–1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN₂. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.     

Purification and Characterization of Cadmium-Binding Protein from Unicelluar Alga Chlorella sorokinian

The unicellular green alga Chlorella sorokiniana ANA9 is highly resistant to heavy metals, and its metal-binding proteins are induced in the presence of cadmium. A novel cadmium-binding protein in C. sorokiniana cultured in 100 mg/l cadmium ions for 4 days was isolated and characterized. The crude protein extract was obtained by cell disruption and partly purified by ammonium sulfate precipitation. After purification by anion-exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, the protein was further purified by gel filtration with Sephacryl S-100, followed by Sephadex G-75. The molecular weight of the purified protein was determined to be 11.5 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cadmium binding capacity of the purified protein was 119 μg/mg. The involvement of thiol coordination in metal-ion binding was confirmed by measuring the ultraviolet spectrum. This article is the first to describe the metallothionein-like cadmium-binding protein from Chlorella species, the expression of which is induced by cadmium exposure.     

Purification and properties of a heat-stable exoinulinase isoform from Aspergillus fumigatus

An inducible extracellular exoinulinase (isoform II) was purified from the extracellular extract of Aspergillus fumigatus by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel, Octyl-Sepharose (HIC), Sephacryl S-200, affinity chromatography on ConA-CL Agarose and Sephacryl S-100 columns. The enzyme was purified 75-folds with 3.2% activity yield from the starting culture broth. The purified isoform II was a monomeric 62 kDa protein with a pI value of 4.5. The enzyme showed maximum activity at pH 6.0 and was stable over a pH range of 4.0-7.0, whereas the optimum temperature for enzyme activity was 60 degrees C. The inulinase isoform II showed exo-inulinolytic activity and retained 72% and 44% residual activity after 12 h at 60 degrees C and 70 degrees C, respectively. The inulin hydrolysis activity was completely abolished with 5 mM Hg2+ and Fe2+, whereas K+ and Cu2+ enhanced the inulinase activity. As compared to sucrose, stachyose and raffinose the purified enzyme had a lower Km (1.25 mM) and higher catalytic center activity (Kcat = 3.47 x 10(4) min(-1)) for inulin. As compared to exoinulinase isoform I of A. fumigatus, purified earlier, the isoform II is more thermostable and is a potential candidate for commercial production of fructose from inulin.     

Purification and characterization of cathepsin H from hepatopancreas of carp Cyprinus carpio.

1. Cathepsin H was purified about 5400-fold from hepatopancreas of carp (Cyprinus carpio) by the method involving ammonium sulfate fractionation, and chromatography on S-Sepharose, DEAE-Sephacel, Ultrogel AcA54, Concanavalin A-Sepharose 4B and GPC on Protein-Pak 125. 2. The purified cathepsin H gave a single protein band on analytical-PAGE, but migrated as two bands of 27,000 and 23,000 mol. wt on SDS-PAGE. 3. Cathepsin H had a pH and temperature optimum of 6.5 and 45 degrees C using Arg-MCA as a substrate, respectively, and was activated by sulfhydryl compounds and inhibited by cysteine protease inhibitors and metal compounds having high reactivities at cysteine residue. 4. The carp hepatopancreas cathepsin H immunoreacted with the monospecific antibody against rat liver cathepsin H, and did not react with the antibodies against carp hepatopancreas cathepsins B and L by the method of immunoelectrophoretic blotting.     

Heat-Moisture Treatment of Cereal Starch Observed by X-ray Diffraction

Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by 8D8 polyacrylamide gel electrophoresis to be 112,000 and 115,000 for I and II respectively, while gel filtration showed the molecular weight to be 114,000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag⁺ and iodoacetic acid.     

Biochemical Characterization of a β-1,3-Glucanase from Trichoderma koningii Induced by Cell Wall of Rhizoctonia solani

Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of β-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of β-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The K_m and V_max values for β-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL⁻¹ and 0.159 U.min⁻¹, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50°C. Hg²⁺ inhibited the purified enzyme.     

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.     

Aspartate aminotransferase from Panicum maximum Jacq. var. trichoglume Eyles, a C4 plant: purification, molecular properties, and preparation of antibody

Extracts of the leaf tissue of Panicum maximum Jacq. var. trichoglume Eyles (a phosphoenolpyruvate carboxykinase type of C4 plant) were examined and at least two isoforms of aspartate aminotransferase (EC 2.6.1.1), with different electrophoretic mobilities, were detected. The predominant isoform was purified to homogeneity from mesophyll cells. The purification procedure included fractionation with ammonium sulfate followed by chromatography on diethylaminoethyl-cellulose, Sephacryl S-300, and hydroxyapatite. The purified enzyme had specific activities of 182 and 165 mumol/min/mg protein, measured in terms of the synthesis of oxaloacetate and aspartate, respectively, at pH 8.0. The enzyme, with an apparent molecular size of 100 kDa, appears to be a dimer of a single polypeptide with a molecular size of 42 kDa. Mono specific polyclonal antibodies were raised against the 42-kDa polypeptide. Only a single stained band was detected in extracts of whole leaves by immunoblot analysis with this antibody after two-dimensional polyacrylamide electrophoresis. Furthermore, no difference in mobility was observed between the enzymes extracted from mesophyll and bundle sheath cells on native polyacrylamide gels. These findings are discussed in relation to the other isoform in the leaves of this species.     

Glycosulphatase from Pseudomonas carrageenovora. Purification and some properties

A glycosulphatase present in the soluble fraction of disrupted Pseudomonas carrageenovora has been purified 500-fold by gel filtration on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose CL-6B. By dodecylsulphate/polyacrylamide gel electrophoresis the enzyme is practically homogeneous and has a molecular weight of 55 000. Conditions of optimal sodium chloride concentration and pH at 25 degrees C were 0.25--0.50 mol dm-3 and pH 7.0 respectively. The purified enzyme was inhibited by inorganic phosphate. Preparation is described of neocarrabiose 4-O-[35S]sulphate and neocarratetraose 4-O-[35S]sulphate from labelled Chondrus crispus. The purified glycosulphatase is active against both these substrates although only one of the two sulphate esters in the tetrasaccharide is hydrolysed. Analysis of the reaction products was by gel filtration, electrophoresis and 13C nuclear magnetic resonance spectroscopy. The results are consistent with the products of desulphation being respectively neocarrabiose and neocarratetraose 4-O-monosulphate with the sulphate ester proximal to the reducing end [3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-D-galactose 4-O-sulphate].     

Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.