The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Substrate-dependent dissociation of malate thiokinase.

Malate thiokinase has been purified to apparent homogeneity by employing conventional purification techniques along with affinity chromatography. The enzyme is composed of two nonidentical subunits (alpha subunit Mr=34,000, beta subunit Mr=42,500) to yield an alpha 4 beta 4 structure for the native enzyme. Phosphorylation of the enzyme by ATP occurs exclusively on the alpha subunit. The phosphorylated enzyme is acid labile and base stable consistent with phosphorylation of a histidine residue. Dephosphorylation of the enzyme is promoted by ADP, succinate, malate, and coenzyme A plus inorganic phosphate. Phosphorylation of the enzyme leads to a reversible change in the sedimentation properties of the enzyme; the native enzyme exhibits an S20,w of approximately 10, whereas the phosphoenzyme exhibits an S20,w of approximately 7. Formation of the 7 S form of the enzyme is also observed when coenzyme A and succinyl-CoA interact with the enzyme. The ratio of alpha to beta subunits in both the 10 S and 7 S forms of the enzyme is approximately 1.0, suggesting that the 7 S form of the enzyme has an alpha 2 beta 2 structure.     

Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes

The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such ‘isoelectrically immobilized’ reactor, the enzyme β-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5β-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3β-hydroxy-7,12-dioxo-5β-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD⁺) collected, separately, into the two neighbouring chambers at the anodic side. Advantages: in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.     

Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

Activities in Crude Porcine Pancreatic Lipase: Enantioselectivity in Hydrolysis of the Diacetate of 2-Phenylpropane-1,3-Diol

The hydrolysis of a prochiral diacetate by porcine pancreatic lipase is catalysed by the purified enzyme, not by an enzyme present in the crude enzyme but absent from the purified enzyme, as previously reported.     

Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).     

Pro-opiomelanocortin and pro-vasopressin converting enzyme in pituitary secretory vesicles

Peptide hormones are synthesized from larger precursors by cleavages at paired basic residues. We have isolated a pro-hormone converting enzyme from bovine neural and intermediate lobe secretory vesicles that cleaves pro-vasopressin and pro-opiomelanocortin at Lys-Arg residues to yield vasopressin, and adrenocorticotropin/endorphin-related peptides, respectively. The enzyme from both lobes is an aspartyl protease of approximately 70,000 Da, is a glycoprotein and has an optimum pH range of 4.0-5.0. Present within the same secretory vesicles is an aminopeptidase B-like enzyme which is a metalloprotease that is inhibited by Co2+ and Zn2+. This enzyme may play a role in trimming off the N-terminal extended basic residues from peptides liberated by the pro-hormone converting enzyme.     

Analysis of slow-binding enzyme inhibitors at elevated enzyme concentrations

The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.     

Purification of nitric oxide synthase from rat macrophages.

Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and gel filtration chromatography on a Superose 12 HR 10/30 column. The apparent molecular weight is 300,000 by gel filtration. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-biopterin, and dithiothreitol. Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme. The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme).     

蚯蚓纤溶酶的分离纯化及部分序列的测定

以新鲜蚯蚓为原料,经过保温抽提、乙醇沉淀、DEAE-SepharoseFastFlow离子交换层析、Lysine-Sepharose4B亲和层析以及SDS-PAGE制备电泳等纯化步聚,得到一种纯度达95％以上的蚯蚓纤溶酶．该酶具有强烈的溶解纤维蛋白的作用及蛋白酶活性,平板法测得其比活性为90OUK单位／毫克蛋白,TAME法测得其比活性为2500O单位／毫克蛋白．酶学性质研究表明其最适反应温度为65℃,最适反应PH值为8．5．该酶的分子量为33kD,等电点为pH3．5．还对该酶进行了氨基酸组成分析,并测定了其N端部分序列．     

Isolation and properties of porcine thyroid fucokinase.

A 23 000-fold purification of porcine fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) has been achieved using a combination of ion-exchange, hydrophobic ligand, affinity, hydroxyapatite and molecular sieve chromatography. The enzyme was determined to have a subunit molecular weight of 78 180 +/- 4260 by sodium dodecyl sulfate chromatography and a tetrameric molecular weight of 309 200 +/- 4100 in the active state as determined by molecular sieve chromatography. The enzyme exhibits a single pH optimum at a pH value of 6.5 and gives evidence of a high order of specificity for L-fucose and ATP. The enzyme requires a divalent metal ion and this need is best satisfied by Mg2+. The activity of the enzyme is modified by a number of nucleotides. ADP is an enzyme inhibitor competitive with ATP. GDP-beta-L-fucose is also an inhibitor and appears to compete with L-fucose. GDP-alpha-D-mannose stimulates the enzyme. A possible role for the actions of these nucleotide sugars is discussed.     

Oligomerization and regulation of higher plant phosphoenolpyruvate carboxylase

The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6-phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K_m, and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed.     

Gas-liquid Chromatographic Determination of Carbohydrates in Tobacco

An enzyme which catalyzes the degradation of polyvinyl alcohol) (PVA) oxidized by secondary alcohol oxidase, in which hydroxyl groups of PVA are partially converted to carbonyl groups, has been purified from a fraction adsorbed on DEAE-Sephadex at pH 7.0 from PVA-degrading enzyme activities produced by a bacterial symbiotic mixed culture in a minimal medium containing PVA as a sole source of carbon and energy. The purified enzyme was electrophoretically homogeneous in the absence and presence of SDS.

The enzyme is a single polypeptide with a molecular weight of about 36,000 and has an isoelectric point of 5.1. The N- and C-terminal amino acid residues are both alanine. The enzyme is most active at pH 6.5 and at 40°C and is stable between pH 6.0 and 9.0 and at temperatures below 45°C. The enzyme is inhibited by Hg²⁺ and is restored by the addition of reduced glutathione, although p-chloromercuribenzoate has no effect.

The enzyme was active on oxidized PVA, but not on PVA and on various low molecular weight carbonyl compounds examined. The enzyme reaction on oxidized PVA resulted in a rapid decrease in viscosity, a fall of pH, and production of carboxylic acids. The enzyme, therefore, is considered to be an oxidized PVA hydrolase.

The enzyme shows a common antigenicity in immunodiffusion and neutralization reactions with antisera to an oxidized PVA hydrolase previously purified from another fraction adsorbed on SP-Sephadex at pH 7.0 from the PVA-degrading enzyme activities [Agric. Biol. Chem., 45, 63 (1981)]. The relations between these two oxidized PVA hydrolases are discussed.     

Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects

The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.     

Glucose analysis utilizing immobilized enzymes

The four commercial instruments that measure glucose by incorporation of immobilized reagents are described and compared. The design of the immobilized enzyme or enzymes is shown to be related to the type of instrument. Three of the instruments are of the partitioned enzyme-sensor type requiring an immobilized enzyme capable of rapid, constant flow rate when inserted in a flowing stream. Moderately high enzyme loading is required if the instrument is designed to operate in the equilibrium mode while lower enzyme loading can be tolerated in kinetic mode. Only one instrument is an enzyme electrode in which the immobilized glucose oxidase is in the immediate vicinity of the electrochemical detector. In that case the immobilized enzyme must have very high enzyme activity per unit volume, but need not have high physical durability. The design of the instrument and immobilized enzyme(s) is also affected by whether the instrument is to be used in an industrial or a clinical laboratory.     

Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site

Two types of conformational changes are mediated in Escherichia coli ornithine transcarbamoylase by the metal ion zinc. Upon binding of zinc in rapid equilibrium, the enzyme undergoes an allosteric transition. In the absence of substrates, the zinc-bound enzyme further undergoes a slow isomerization with a concomitant activity loss. Three metal ions are tightly complexed in the isomerized enzyme as determined by gel chromatography and atomic absorption spectroscopy. Since the enzyme is a trimer composed of identical subunits, one zinc ion is bound per enzyme monomer. With the application of site-directed mutagenesis, the cysteinyl residue at position 273 of the enzyme has been identified as a metal ligand. When this residue is replaced by an alanine, zinc is no longer a tight-binding inhibitor and does not promote isomerization. The alteration in the action of zinc on the mutant enzyme is attributed to a reduced metal affinity. The mutant enzyme, when saturated by the metal, displays an intrinsic allostery unchanged from that of the wild-type; an identical Hill coefficient of 1.5 is found for zinc binding to the Ala273 and wild-type enzymes. Cys273 is also a binding site of L-ornithine. At pH 8.5, the Ala273 enzyme binds the substrate analog L-norvaline ten times more weakly and exhibits a kcat/Kmorn that is 27 times less than that of the wild-type enzyme. This finding supports our earlier interpretation that the zinc-induced ornithine co-operativity of ornithine transcarbamoylase is caused by direct competition between L-ornithine and the metal for the same site. As controls, each of the remaining three cysteinyl residues of the bacterial ornithine transcarbamoylase has also been replaced with alanine. These sulfhydryl groups are found not to be related to zinc complexation, ornithine binding or enzyme allostery.     

Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.

Two aminotransferases from Escherichia coli were purified to homogeneity by the criterion of gel electrophoresis. The first (enzyme A) is active on L-aspartic acid, L-tyrosine, L-phenylalanine, and L-tryptophan; the second (enzyme B) is active on the aromatic amiono acids. Enzyme A is identical in substrate specificity with transaminase A and is mainly an aspartate aminotransferase; enzyme B has never been described before and is an aromatic amino acid aminotransferase. The two enzymes are different in the Vmax and Km values with their common substrates and pyridoxal phosphate, in heat stability (enzyme A being heat-stable and enzyme B being heat-labile at 55 degrees) and in pH optima with the amino acid substrates. They are similar in their amino acid composition, each enzyme appears to consist of two subunits, and enzyme B may be converted to enzyme A by controlled proteolysis with subtilsin. The conversion was detected by the generation of new aspartate aminotransferase activity from enzyme B and was further verified by identification by acrylamide gel electrophoresis of the newly formed enzyme A. The two enzymes appear to be products of two genes different in a small, probably terminal, nucleotide sequence.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.     

A high molecular weight protease in the cytosol of rat liver. II. Properties of the purified enzyme

The properties of a soluble endoprotease from rat liver were studied. The enzyme was purified in a latent form. It sedimented as a single component with a sedimentation coefficient (S(0)20,w) of 19.8 S. Measurement by quasi-elastic light scattering gave a diffusion coefficient (D(0)20,w) of 2.5 X 10(-7) cm2 X s-1 and an effective hydrodynamic radius of 85 A. The enzyme had an unusually high molecular weight, estimated as 743,000 by sedimentation equilibrium and 722,000 by sedimentation velocity and diffusion measurements and as 760,000 by a recently developed low-angle laser light scattering method. Judging from electron microscopic observation and the calculated frictional and axial ratios, the enzyme molecule is disc-shaped. Analysis of the far-ultraviolet circular dichroic spectrum showed that the enzyme contains 50% alpha-helical, 25% beta-sheet, and 15% unordered structures with 10% beta-turns. The isoelectric point of the enzyme is 5.0. These properties indicate that the purified enzyme is a homogeneous molecule. In addition, the enzyme is a simple protein since it contains no measurable amounts of nucleic acid carbohydrate or lipid.