Isolation and properties of carboxypeptidase from leaves of wounded tomato plants

A carboxypeptidase was purified to homogeneity from upper, unwounded leaves of tomato plants in which carboxypeptidase activity had been induced to increase over three-fold by severely wounding the lower leaves. The carboxypeptidase was purified by ammonium sulfate precipitation, affinity chromatography, and finally by gel permeation chromatography. Electrophoresis at pH 4.3 and isoelectric focusing showed only a single band. The isoelectric point was 5.2 and the MW 105 000. Tomato carboxypeptidase possessed both peptidase and esterase activities and it sequentially hydrolysed amino acids from the carboxyl-terminal end of insulin chain B. It was optimally active at pH 6–7 on peptidase substrates, and at pH 8 on esterase substrates. The enzyme was inhibited by diisopropylfluorophosphate and incorporated 1 mol of DFP-[³H]. per mol of enzyme. Both peptidase and esterase activities were strongly inhibited by HgCl₂ but not by p-hydroxymercuribenzoate or iodoacetamide. Carboxypeptidase inhibitor from potatoes did not inhibit the enzyme.     

Substrate specificity of a human-specific esterase

A human species-specific esterase has been identified in tissues, cell cultures, and urine. It is the most slowly migrating (i.e., cathodal) of the esterase isoenzymes in agarose electrophoresis; it is not a choline estrase, a pseudocholine esterase, an acetyl phenylalanine-3-naphthyl esterase or N-benzoyl-arginine-3-naphthyl esterase. Hydrolysis of N-methyl indoxyl acetate caused by this esterase is not inhibited by eserine, eserine sulfate, or EDTA. Phenylmethylsulfonyl fluoride, however, did inhibit hydrolysis. Furthermore, this cathodal esterase does not show any chymotrypsin, trypsin, or leucine aminopeptidase enzyme activity.     

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.     

Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.     

Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.     

Purification and characterization of cholesterol esterase from porcine pancreas.

A protein catalyzing the hydrolysis of cholesterol esters and p-nitrophenyl acetate has been purified 200-fold from porcine pancreas. The enzyme is homogenous as judged by polyacrylamide gel electrophoresis and exhibits a molecular weight of 80 000 as determined by sodium dodecyl sulfate electrophoresis and gel filtration. Activity toward p-nitrophenylacetate exhibits a broad pH optimum and is influenced by a group with a pKa of 5.5--6.0. The enzyme is completely inhibited by diisopropylfluorophosphate at concentrations as low as 10(-5) M, suggesting that it is a serine esterase. Partial inhibition was observed with p-chloromercuribenzoate.     

Choliae esterases and choline acetyltransferase in the seeds ofAllium altaicum (Pall.) Reyse

In the seeds ofAllium altaicun (Pall.)Reyse a set of enzymes was found, metabolizing choline esters, composed of active choline esterases and choline acetyltransferase. Choline esterase cleaving acetylcholine occurs in five isoenzymes. The enzyme preparation hydrolyses strongly acetylthiocholine and sinapine, but weakly butyrylthiocholine (20%) in comparison with acetylthiocholine. The hydrolysis of the substrates mentioned is inhibited by physostigmine and neostigmine, but it is not inhibited by the specific inhibitor of acetylcholine esterase (BW 284 C51). In addition to hydrolytic activity a strong catalytic activity of choline acetyltransferase was also observed during the synthesis of sinapine from sinapic acid and choline. The detection of the mentioned enzymes in some representatives of theAllium genus indicates that choline esterases are more widely distributed in monocotyledons than previously assumed.     

Purification and Some Properties of Carboxylesterase from Arthrobacter globiformis; Stereoselective Hydrolysis of Ethyl Chrysanthemate

An esterase with excellent stereoselectivity for (+)-trans-ethyl chrysanthemate was purified to homogeneity from Arthrobacter globiformis SC-6-98-28. The purified enzyme hydrolyzed a mixture of ethyl chrysanthemate isomers stereoselectively to produce (+)-trans-acid with 100% stereoisomeric purity. The apparent molecular weight of the purified enzyme was 43,000 on SDS–polyacrylamide gel electrophoresis, and 94,000 on gel filtration chromatography. The optimum conditions for the ester hydrolysis were pH 10.0 at 45°C. The purified esterase hydrolyzed short-chain fatty acid esters, but did not have detectable activity on long-chain water-insoluble fatty acid esters. The enzyme activity was inbibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride.     

Purification and characterization of a carboxylesterase associated with organophosphate resistance in the greenbug,Schizaphis graminum (Homoptera: Aphididae)

The Type II esterase associated with organophosphate resistance in the greenbug, Schizaphis graminum, was purified by column chromatography and preparative electrophoresis resulting in over 100-fold purification and approximately 11% recovery. The native enzyme appears to exist as a heterodimer with the subunits equal to 52 and 56 kDa. The mass of the native enzyme was estimated at 102 kDa by gel filtration chromatography and the isoelectric focusing point was 4.8. The enzyme was inhibited by both paraoxon and mercuric chloride, suggesting that it is a serine hydrolase, although it was not inhibited by carbamate insecticides or eserine. The enzyme was active toward both β- and α-naphthol esters, although the length of the side chain (C-2 or C-4) also affected activity. The enzyme displayed no activity toward acetylthiocholine. N-Terminal amino acid sequence analysis of the enzyme subunits indicates that residues Val-4 and Gly-10 of the larger fragment were highly conserved among 11 other carboxylesterase sequences. Sequence data from the smaller fragment did not reveal any similarity with other esterase sequences. Arch. Insect Biochem. Physiol. 36:229–240, 1997. © 1997 Wiley-Liss, Inc.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Substrate specificity of carboxypeptidase from Watermelon.

The substrate specificity of carboxypeptidase (F-II) purified from watermelon for various synthetic peptides and esters was examined kinetically. The enzyme showed a broad substrate specificity against various carbobenzoxy- and benzyl-dipeptides. Peptides containing glycine or proline were hydrolyzed slowly by the enzyme. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal resulted in an increase in the rate of hydrolysis. Inhibition studies with diisopropyl flurophosphate and diastereomers of carbobenzoxy-Phe-Ala demonstrated that the peptidase and esterase activities of the enzyme are both catalyzed by the same site of the enzyme molecule, but the binding sites for peptides and esters seem not to be the same. The enzyme also had amidase activity, which was optimal at pH 7.0.     

Macrophage esterase: identification, purification and properties of a chymotrypsin-like esterase from lung that hydrolyses and transfers nonpolar amino acid esters.

A chymotrypsin-like esterase was purified from beef lung. This lysosomal enzyme, not previously characterized, seemed to be composed of two or more forms with molecular weights of about 52 000. It hydrolysed N-benzoyl-DL-phenylalanine beta-naphthol ester at acid and neutral pH; it polymerized L-phenylalanine methyl ester(Phe-OMe) at neutral pH; and it transferred the Phe-residue from Phe-OMe to hydroxylamine at neutral pH. Phenylmethanesulfonyl fluoride, an inhibitor of hydrolytic enzymes with serine in their catalytic site, inhibited this enzyme, but pepstatin, the cathepsin D (EC 3.4.4.23) inhibitor, did not. Sulfhydryl reagents were not required for activity. Macrophages, especially pulmonary alveolar macrophages, were a rich source of this esterase, so it is likely that the enzyme purified from lung came from its macrophages. The esterase hydrolysed and transferred monoamino acid esters, especially those of the aromatic type. Cathepsin C, the dipeptidyl peptide hydrolase (EC 3.4.14.1), acted only on dipeptide esters and amides. Pancreatic chymotrypsin acted on both monoamino acid and dipeptide esters. The chymotrypsin-like esterase did not hydrolyse hemoglobin, casein, or plasma albumin. Thus its proteolytic activity, if present, must be limited to specific substrates, as yet unknown.     

Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Characterization of tumor cell membrane serine proteases by polyacrylamide gel electrophoresis

Studies in our laboratory have indicated that tumor cell membrane-bound proteases are responsible for the ability of tumor cells to lyse normal cells in vitro. In order to evaluate the tumor cell membrane enzymes, a purified tumor cell membrane preparation was prepared and the nonionic detergent Triton X-100 was used to extract active enzymes from the cell membranes. The solubilized membrane enzymes were then studied by Triton X-100 polyacrylamide gel electrophoresis under non-denaturing conditions. Using this technique the tumor cell membranes were shown to contain esterproteases that reacted with the substrates alpha-naphthyl acetate and naphthol-AS-aminocaproate. These esterproteases were inhibited by diisopropyl fluorphosphate and tosyl lysine chloromethyl ketone but not by tosylamide phenylethyl chloromethyl ketone, soybean trypsin inhibitor p-chloromercuribenzene sulfonic acid; N-ethylmaleimide choline iodide, alpha-1-anti-trypsin. NaF, epsilon-aminocaproic acid, ethylenediamine tetraacetic acid, or eserine. SBTI affinity chromatography of the tumor cell membrane extract revealed that some of the serine esterproteases bound to the SBTI column. The proteolytic activity of the tumor cell membrane extract and a fraction eluted from the SBTI affinity column was demonstrated using casein. We conclude that the tumor cell membranes contain previously undescribed serine proteases that are identifiable by their esterase activity and inhibitor profiles in polyacrylamide gels.     

鸭血清胆碱酯酶的纯化及性质研究

首次采用新技术双水相萃取方法作为鸭血清胆碱酯酶(EC.3.1.1.8 CHE) 纯化的第一步,后经 DEAE-Sephadex A50,sephadex G200 柱层析,获得电泳纯鸭血清胆碱酯酶,提纯倍数1018倍,酶活力回收43.4%,比活274.9U/mg。鸭血清胆碱酯酶性质研究表明:此酶是糖蛋白和酸性蛋白水解酶,等电点 4.2 左右,最适 pH7.5 左右;对底物碘化硫代丁酰胆碱的 K_m=9.8×10^-5mol/L;SDS-PAGE 电泳和聚丙烯酰胺梯度电泳表明,鸭血清胆碱酯酶以相同亚基组成的不同聚合体形式存在,亚基分子量 78000,具有完整的酶活性.不同聚合体带电状态相同.     

Purification and Properties of Hydroxycinnamic Acid Ester Hydrolase from Aspergillus japonicus

Hydroxycinnamic acid ester hydrolase from the wheat bran culture medium of Aspergillus japonicus was purified 255-fold by ammonium sulfate fractionation, DEAE-Sephadex treatment and column chromatographies on DEAE-Sephadex, CM-Sephadex and various other Sephadexes. The purified enzyme was free from tannase and found to be homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was estimated to be 150,000 by gel filtration and 142,000 by SDS-gel electrophoresis. The isoelectric point of the enzyme was pH 4.80. As to its amino acid composition, aspartic acid and glycine were abundant. The optimum pH and temperature for the enzyme reaction were, respectively, 6.5 and 55°C when chlorogenic acid was used as a substrate. The enzyme was stable between pH 3.0 to 7.5 and inactivated completely by heat treatment at 70°C for 10 min.

All metal ions examined did not activate the enzyme, while Hg⁺⁺ reduced its activity. The enzyme was markedly inhibited by diisopropylfluorophosphate and an oxidizing reagent, iodine, although it was not affected so much by metal chelating or reducing reagents. The purified enzyme hydrolyzed not only esters of hydroxycinnamic acids such as chlorogenic acid, caffeoyl tartaric acid and p-coumaroyl tartaric acid, but also ethyl and benzyl esters of cinnamic acid. However, the enzyme did not act on ethyl esters of crotonic acid and acrylic acid or esters of hydroxybenzoic acids.     

Enzymatic characterization of arginine esterase from dog seminal plasma

Previously purified arginine esterase from dog seminal plasma was characterized enzymatically. The enzyme was found to have a rather narrow specificity for arginine esters, much less for lysine esters and was practically devoid of activity towards tyrosine esters, casein, albumin and azocoll. It had a broad optimum pH between 8 and 9. It presented no kallikrein-like activities either in the blood pressure test in dog or in the rat uterus contraction test. It was inhibited by bovine pancreas trypsin inhibitor, aprotinin, phenylalanylprolyl arginine chloromethyl ketone, diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, sodium dodecyl sulfate and leupeptin, but not by soybean trypsin inhibitor, tosyllysine chloromethyl ketone, tosylamide-2-phenylethyl chloromethyl ketone, iodoacetamide, Triton X-100 and EDTA. Experiments involving incubation of prostatic cytosol with purified arginine esterase showed that actin was the only important prostatic protein that was extensively hydrolyzed by this enzyme. It is not known presently whether the hydrolysis of actin is related to a true physiological function of the enzyme and whether actin and arginine esterase ever come into contact with each other in vivo. These properties indicate that arginine esterase from dog seminal plasma is different from other known proteinases including classical kallikreins, although it presents many similarities with this class of enzyme.     

Esterases in Folsomia candida (Collembola: Isotomidae). Characterization of enzymes among parthenogenic strains

Esterase enzymes from four strains of Folsomia candida were investigated using polyacrylamide gel electrophoresis. Up to 12 bands of enzymatic activity were present in each strain. Esterase bands were classified as choline esterases or as one of two groups of carboxyl esterases, based on mobility, on substrate specificity and on activity remaining after inhibition by class-specific chemicals. One strain-specific choline esterase was discovered which resisted the effects of many organophosphate inhibitors. Organophosphate inhibitor concentrations had to be 10 to 100 times greater to reduce the staining activity of this resistant choline esterase to the level of comparable esterases in other strains.     

Intracellular X-prolyl dipeptidyl peptidase from Lactococcus lactis spp. lactis: purification and properties

An X-prolyl dipeptidyl peptidase (EC 3.4.14.5) has been purified from a crude intracellular extract from Lactococcus lactis spp. lactis NCDO 763 by ion-exchange chromatography and gel filtration. One protein band was detected after electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulphate. The enzyme is a 190 kDa dimer composed of identical subunits. Optimal activity occurs at pH 8.5 and 40–45°C and the enzyme is inhibited by reagents specific for serine proteases, such as diisopropylfluorophosphate. The enzyme hydrolyzes p -nitroanilide- or β-naphthylamide-substituted X-Pro dipeptides, as well as β-casomorphin.     

Purification and characterization of a kallikrein-like T-kininogenase

A T-kininogenase has been purified to homogeneity from rat submandibular gland extracts by DEAE-Sepharose chromatography and preparative gel electrophoresis. The purified protein has an apparent Mr of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and splits into heavy and light chains with Mr of 22,000 and 6,000 in the presence of dithiothreitol. It is an acidic glycoprotein with pI of 4.65-4.75. The carbohydrate moiety is located on the light chain and binds concanavalin A and wheat germ agglutinin. The active site serine residue of the heavy chain is labeled with [14C]diisopropylfluorophosphate and visualized by fluorography. NH2-terminal amino acid sequences of the light and heavy chains reveal 74-84% identity to rat tissue kallikrein, tonin, and other kallikrein-related enzymes. The enzyme cleaves T-kininogen to release T-kinin which was separated by high performance liquid chromatography on a reverse phase C18 column and identified by a kinin radioimmunoassay. Its T-kininogenase but not N-tosyl-L-arginine methyl ester esterase activity can be enhanced 10-fold in the presence of dithiothreitol. The esterolytic activity of the enzyme is inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, and antipain; whereas lima bean and ovomucoid trypsin inhibitors stimulate its activity. The enzyme is localized at the granular convoluted tubule and striated duct cells in rat submandibular glands by immunohistochemistry. The results indicate that T-kininogenase belongs to the group of structurally similar yet distinct kallikrein-like serine proteases.