Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Purification procedure and physiochemical characterization of the pure enzymes

Cytosolic epoxide hydrolase was purified from the liver of untreated and clofibrate-treated male C57Bl/6 mice. The purification procedure involves chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxyapatite, takes two days to perform and results in a 120-fold purification and approximately 35% yield of the enzyme from untreated mice. The purified enzyme is a dimer with a molecular mass of 120 kDa, a Stokes' radius of 4.2 nm, a frictional ratio of 1.0 and an isoelectric point of 5.5. The subunits behave identically upon isoelectric focusing in 8 M urea and only one band with a molecular mass of 60 kDa is seen after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The form purified from clofibrate-treated mice had very similar properties and was apparently identical to the control form as judged by amino acid analysis and peptide mapping as well. These analyses also demonstrated that the cytosolic enzyme is clearly different from microsomal epoxide hydrolase isolated from rat liver. Furthermore, Ouchterlony immunodiffusion using antibodies raised in rabbits towards the control form of cytosolic epoxide hydrolase revealed identity between the two forms of cytosolic epoxide hydrolase, but no reaction with the microsomal epoxide hydrolase was observed. These findings indicate large structural differences between the cytosolic and microsomal forms of epoxide hydrolase in the liver.     

Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s).

Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.     

Purification and characterization of glutathione transferase of human thyroid

An anionic (pI 4.6) isoenzyme of glutathione transferase was purified to homogeneity from human thyroid by affinity chromatography followed by isoelectric focusing. The content of enzyme was calculated to constitute about 0.2% of soluble proteins. The enzyme is formed by two identical subunits of 23,000 daltons approximately. The thyroid transferase did not catalyze the reduction of peroxides. Physical, catalytic and immunological analyses demonstrated extensive similarities between the thyroid transferase and the transferase from placenta, erythrocytes and breast. On the other hand, the thyroid transferase appears catalytically different from transferase 7-7, even if both cross-react with the antibodies raised against human placenta transferase.     

Purification and characterization of a trypsin-like protease in the submandibular gland of rats

A trypsin-like protease (named RSP-V) was purified to homogeneity from rat submandibular glands by isoelectric focusing and high-performance liquid chromatography. The purified enzyme had an isoelectric point of 5.3 and an apparent molecular weight of 25,000, and consisted of two subunits with molecular weights of 19,500 and 6,000. RSP-V hydrolyzed BAEE, BAPA, and TAME, but not ATEE or BTPA. It had an optimum pH at around 10.0. RSP-V was strongly inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, but not by ovomucoid trypsin inhibitor, p-CMB, or iodoacetic acid. This enzyme partly resembled, but was not identical with, tonin. It was also different from kallikrein, salivain, and glandulain in rat submandibular gland. Although the physiological role of RSP-V has not yet been clarified, this enzyme inactivated dopa decarboxylase alone among catecholamine-synthesizing enzymes.     

Metabolism of trans-Aconitic Acid in Maize : I. PURIFICATION OF TWO MOLECULAR FORMS OF CITRATE DEHYDRASE

Trans-aconitate synthesis via citrate dehydrase was determined in crude extracts of maize (Zea mays L.) coleoptiles. Two molecular forms of this enzyme were purified by substrate-specific elution from DEAE-cellulose, ammonium sulfate precipitation, and gel filtration. Each molecular form migrates as a single band in isoelectric focusing. Gel filtration and sodium dodecyl sulfate electrophoresis provided evidence that one enzyme form is composed of four 80,000-dalton subunits while the other is composed of two 60,000-dalton subunits. There was no evidence of proteolytic conversion of the large to the small molecular weight form when the former was incubated with either the 15,000_g supernatant or with proteases. The data indicate that the two molecular forms of citrate dehydrase are isozymes.     

Glutathione S-transferase from beef heart: structural and kinetic properties

A simple and rapid method for the purification of glutathione S-transferase is described. The physical and kinetic properties of purified enzyme are reported. The protein is constituted of two identical subunits with a total molecular weight of 46,000 daltons. The isoelectric focusing of crude cytosol or purified preparation gives a single peak of activity with a pI of 7.1. The kinetic analysis shows a relatively strict substrate specificity. Only 1-chloro-2,4-dinitrobenzene is conjugated to reduced glutathione at an appreciable rate. The peroxidase activity of the enzyme with respect to cumene hydroperoxide as substrate is negligible. Hemin and bilirubin are competitive inhibitors of catalytic activity.     

Acidic glutathione transferase from human heart. Characterization and N-terminal sequence determination

The anionic glutathione transferase of human heart has been purified to homogeneity by using DEAE-cellulose, affinity chromatography, and FPLC. The enzyme has an isoelectric point at pH 4.75 and has an electrophoretic mobility on SDS-PAGE identical to placental transferase pi, indicating that the heart enzyme is formed by two similar subunits of 23,000 Mr. Upon isoelectric focusing on ampholine PAG plates the enzyme recovered from FPLC gave two bands of activity at pH 4.75 and 4.9 which were reduced to essentially a single band at pH 4.75 after incubation with dithiothreitol. In the immunodiffusion experiment, the heart enzyme gave a positive precipitin line with the antibodies against transferase pi but not with antibodies prepared against the "basic" transferase of human skin or against the "near-neutral" transferase of human uterus. The substrate specificities, the sensitivities to characteristic inhibitors, the amino acid composition, together with the immunological studies, strongly indicate that the anionic enzyme of human heart is closely related to the transferase pi of human placenta. The N-terminal amino acid sequence of the first 48 residues was determined and compared with the N-terminal region of other reported human glutathione transferase sequences. The heart enzyme differs from the placental enzyme in a single residue (Trp instead of Arg in the 28th position) further supporting their similarity.     

Pig lens glutathione S-transferase belongs to class Pi enzyme

Class Pi glutathione S-transferase was purified to homogeneity from pig lens cytosol. This enzyme was composed of two identical 22 kDa subunits and had isoelectric point of 8.5 from the results of SDS gel electrophoresis, gel filtration, amino acid sequence analysis and isoelectric focusing. Amino acid sequence of N-terminal 15 residues was almost identical to class Pi enzymes from human, rat and mouse. Antibody against the pig enzyme crossreacted to human glutathione S-transferase-pi and anti-rat glutathione S-transferase-P antibody crossreacted to pig enzyme.     

Purification and properties of 3-ketothiolase fromAlcaligenes latus

Summery 3-Ketothiolase was purified fromAlcaligenes latus by ion exchange chromatography and isoelectric focusing. The purified enzyme was a tetramer with identical subunits (Mr, 42,000). The kinetic data for cleavage and condensation reactions with 3-ketothiolase were obtained at various substrate concentrations, and compared with those from other PHB-accumulating bacteria.     

Proteolytic activity in the maize pollen wall

A new protease from maize ( Zea mays L.) pollen is described. It was purified using gel filtration, ion exchange and high performance liquid chromatography. SDS-PAGE and HPLC showed that the enzyme has a dimeric structure of M, ca 60,000. Inhibitor investigations indicated an aspartic acid residue in its active site. The optimum pH for maize pollen aspartic proteinase activity was 5.6, and the optimum temperature was 45°C. The enzyme is easily eluted from the pollen grains and, as confirmed by enzymoblotting after isoelectric focusing, it is located in the pollen wall. Similar to metallo-proteinases, its activity is inhibited by Zn₂₊. The pL value for purified aspartic proteinase, as estimated after IEF, was 5.0. Two-dimensional electrophoresis analysis of proteins eluted from maize pistils suggests that the enzyme digests the proteins and may be involved in pollen-tube germination. The properties of serine and aspartic proteinases from maize pollen are compared.     

Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli

Abstract We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the p I points of B subunits of the LThs were identical to each other, the p I points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli .     

Purification and some properties of a novel ethylene-forming enzyme produced by Penicillium digitatum

We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the pI points of B subunits of the LThs were identical to each other, the pI points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli.     

Studies on the subunits of Escherichia coli coenzyme A transferase. Reconstitution of an active enzyme.

The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.     

Purification and characterization of neuraminidase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.     

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.     

Wild-type and mutant forms of homoisocitric dehydrogenase in the yeast Saccharomycopsis lipolytica

Homoisocitric dehydrogenase (EC 1.1.1.155) has been purified 525-fold from the yeast Saccharomycopsis lipolytica with a yield of 25%. The preparation was judged to be homogeneous by electrophoresis under denaturing and non-denaturing conditions and by isoelectric focusing; it consisted of a single protein with molecular weight of 48000. In the presence of homoisocitric acid, a higher molecular weight was observed, suggesting a dimeric structure for the native enzyme. Complementing mutants devoid of homoisocitric dehydrogenase activity mapped at two closely linked loci (lys9 and lys10). Lys10 mutants displayed NAD-reducing activity, whereas lys9 mutants retained some carboxylating activity. Our results are best explained by the assumption that the active enzyme is a dimer of identical subunits involved in successive dehydrogenation and decarboxylation steps.     

Studies on the biosynthesis of cyclitols, XXXVI. Purification of myo-inositol-1-phosphate synthase of the duckweed, Lemna gibba, to homogeneity by affinity chromatography on NAD-Sepharose molecular and catalytic properties of the enzyme.

Using the technique of affinity chromatography on NAD-Sepharose the myo-inositol-1-phosphate synthase of Lemna gibba was purified to homogeneity. The molecular and catalytic properties of this enzyme differ very much from those of myo-inositol-1-phosphate synthase from animal sources. Thus the specific activity of the duckweed enzyme is more than two orders of magnitude higher than that of the enzyme from rat testes. It is inhibitied by EDTA and can be reactivated by Mn2. Its molecular weight (135000 +/- 5000), its subunit composition (3 subunits with identical electrophoretic behaviour) and its isoelectric point (pH 7.7) are also very different from the corresponding parameters for the animal enzyme.     

Derivatization of the catalytic subunits of the chloroplast ATPase by 2-azido-ATP and dicyclohexylcarbodiimide. Evidence for catalytically induced interchange of the subunits

Modifications of the catalytic beta subunits of the chloroplast ATPase (CF1-ATPase) are reported which support the proposal that all three subunits participate sequentially during catalysis. The beta subunits of the CF1-ATPase are sufficiently homogeneous to allow detection of their derivatization with dicyclohexylcarbodiimide (DCCD) or the substrate analog 2-azido-ATP by two-dimensional isoelectric focusing. Whether the DCCD reacts with the same beta subunit that tightly binds ATP has not been known. Our results show that when CF1-ATPase is covalently labeled with 2-azido-ATP followed by reaction with DCCD, different beta subunits are labeled. The DCCD labeling does not stop catalytic cooperativity of the CF1-ATPase and allows slow enzyme turnover. When the DCCD-modified enzyme catalyzes 2-azido-ATP cleavage and the enzyme with tightly bound nucleotide is photolyzed, both DCCD-modified and unmodified subunits are randomly labeled by the azido nucleotide. This result is as expected if during the catalytic cycle one beta subunit with unique properties is replaced by another subunit that gains these properties. The participation of all three subunits in the catalytic cycle is suggested by the apparent retention of catalytic cooperativity by the two remaining subunits after one subunit has already catalyzed 2-azido-ATP cleavage and been labeled.