Giovanni B. Principato

Purification and characterization of two forms of glyoxalase II from the liver and brain of Wistar rats.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms (alpha, pI = 8.0; beta, pI = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the alpha and beta forms from either source in the relative molecular mass (about 24,000) or in Km values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50 degrees C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of alpha and beta forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S-D-mandeloylglutathione, a slowly reacting substrate, suggest the presence of histidine at the active site. The alpha and beta forms show different half-life values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distinct structures (sequences) for alpha and beta forms.

Characterization of the soluble alkaline phosphatase from hepatopancreas of Squilla mantis L.

Abstract 1. 1. A soluble alkaline phosphatase (AP) present in the hepatopancreas of Squilla mantis was extracted. 2. 2. The enzyme was purified by acetone fractionation and then by DEAE-cellulose and Sephadex G-200 chromatography; a single AP form was obtained, which was characterized by studying molecular and catalytic properties. 3. 3. Kinetic studies were carried out using phosphoesters as inhibitors; all these substances led to competitive inhibition. The enzyme shows a higher affinity for ADP and ATP; glucose phosphoesters are weak inhibitors. 4. 4. Possible roles of the studied AP in vivo are discussed.

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.

Propionylcholinesterase from Murex brandaris: Comparison with other invertebrate cholinesterases

Abstract 1. A soluble propionylcholinesterase from Murex brandaris is purified by affinity chromatography on a procainamide-containing gel. 2. Purified enzyme is a protein of 260 kDa with subunits of 66 kDa. 3. On the basis of both kcat/Km and kcat, propionylthiocholine is the best substrate. Acetyl- and butyryl-thiocholine are hydrolyzed at a similar rate. 4. Tetramethylammonium, tetraethylammonium, procainamide, trimethyl(aminophenyl)-ammonium are linear competitive inhibitors. Mixed-type inhibition is shown by tetrapropylammonium and tetrabutylammonium. 5. The kinetic properties of the enzyme from Murex brandaris are compared with those of other invertebrate cholinesterases.

Propionylcholinesterase in Hirudo medicinalis: Purification, partial characterization and comparative study with a mammalian acetylcholinesterase

Abstract 1. The authors carried out the purification of a cholinesterase from Hirudo medicinalis by homogenization, ultracentrifugation, (NH4)2SO4 or DEAE-cellulose fractionation and Sephadex G-200 chromatography. 2. Certain molecular properties (mol. wt, electrophoretic pattern) were studied. 3. A comparative study of the kinetic parameters of cholinesterase from H. medicinalis and acetylcholinesterase from rabbit brain was performed, using substrates with a different composition; the enzymes show signs of marked differences in the active site conformation and/or in the catalytic mechanism. 4. The enzyme from H. medicinalis is a propionylcholinesterase, particularly active on propionylthiocholine, and, to a lower extent, on butyrylthiocholine and acetylthiocholine.

Characterization of the soluble cholinesterase from Squilla mantis

Abstract 1. 1. The partial purification of the soluble cholinesterase from Squilla mantis is described. 2. 2. Affi-Gel Blue chromatography has represented the most efficient step in the purification of the enzyme. 3. 3. A single form of cholinesterase is present in the soluble fraction (70,000 g supernatant) of Squilla mantis homogenate. The isoelectric point is 5.3. 4. 4. Although propionylthiocholine was hydrolyzed at a higher rate than other substrates, Vmax/Km values indicated substrates containing acetic acid (acetylthiocholine and acetyl-β-methylthiocholine) showed a better affinity for the enzyme. 5. 5. Tetramethyl-, tetraethyl-, tetrapropyl- and tetrabutyl-ammonium ions are all linear competitive inhibitors of the hydrolysis of acetyl- and propionylthiocholine. 6. 6. Some kinetic, structural and phylogenetic features of the enzyme are discussed.

Cholinesterases from Maia verrucosa and Palinurus vulgaris: A comparative study

Abstract 1. Soluble cholinesterases were purified from the crustaceans Maia verrucosa and Palinurus vulgaris , belonging to the same order (Decapoda) and suborder (Reptantia). 2. Purification was carried out from 100,000 g supernatants by affinity chromatography on a procainamide-containing gel. 3. Each purified enzyme is a single protein giving differently sized subunits on PAGE-SDS electrophoresis. 4. According to k cat / K m values, the enzymes are propionylcholinesterase ( M. verrucosa ) and butyryl-cholinesterase ( P. vulgaris ), even if acetylthiocholine and propionylthiocholine respectively display the highest rates of hydrolysis. 5. According to kinetic constants, the enzyme from M. verrucosa mainly discriminates among substrates (choline or p -nitrophenol esters) on the basis of steric hindrance and hydrophobic interactions. Catalytic efficiency of the enzyme from P. vulgaris is mostly affected by electrostatic forces. Charge-bearing substrates are quite better for both the cholinesterases. 6. Kinetic properties of the studied enzymes are compared with those of other Invertebrata and their possible phylogenetic and adaptive features are discussed.

Multiple forms of acetylcholinesterase in Allolobophora caliginosa: Purification and partial characterization

Abstract 1. The authors studied certain characteristics of the acetylcholinesterase present in Allolobophora caliginosa ; a good purification of the enzyme was achieved by homogenization, ultracentrifugation and then by Sephadex G-200 and DEAE-cellulose chromatographies. 2. Three enzymatic forms, probably monomeric, dimeric and tetrameric were isolated. The monomeric one is quantitatively prevailing and shows a higher specific activity; it seems to be composed of of two subunits with the same molecular weight. 3. The various active fractions are inhibited by eserine and hydrolyse acetylthiocholine more rapidly than butyrylthiocholine; besides, this latter does not cause substrate inhibition. 4. The enzyme is classifiable as acetylcholine hydrolase (E.C. 3.1.1.7).

Propionylcholinesterase from Allolobophora caliginosa

Abstract 1. Propionylcholinesterase from 100,000 g supernatant of Allolobophora caliginosa is purified by affinity chromatography on a procainamide containing gel. 2. Purified enzyme is single protein band on PAGE. SDS-electrophoresis resolved subunits of 28 and 62 kD. 3. On the basis of k cal /K m propionylthiocholine and acetylthiocholine are the best substrates. Propionylthiocholine shows the highest k cat . 4. Tetramethylammonium, tetraethylammonium, procainamide, trimethyl-( p -aminophenyl)ammonium, and d -tubocurarine are competitive inhibitors. Mixed-type inhibition is shown by tetrapropylammonium and tetrabutylammonium. 5. Inhibition by atropine is depending on both substrate and inhibitor concentrations.