Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Purification and properties of alkaline phosphatase from boar seminal plasma

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity. The pH optimum was 10 and the Km was approximately 0.2 mM with p-nitrophenyl phosphate as substrate. The enzyme was a zinc metalloenzyme as indicated by the loss of activity when treated with o-phenanthroline and the restoration of activity by zinc and magnesium ions. It also lost activity when treated with thiols. Molecular weight estimates from SDS polyacrylamide gel electrophoresis and gel filtration suggest that the enzyme is a tetramer of identical subunits, each of which has a molecular weight of 68,000.     

PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.     

A radioimmunoassay for human glucose phosphate isomerase: Measurement of immunoreactive enzyme from genetic variants

Glucose phosphate isomerase has been isolated to homogeneity from human placenta and monospecific antibody generated against the protein. The enzyme was radiolabeled with ¹²⁵I and purified by specific substrate-induced elution from cellulose phosphate. A radio-immunoassay was developed and the ratio of catalytic activity to immunoreactivity of the enzyme (molecular specific activity) was determined in hemolysates from five individuals heterozygous for genetic variant forms of the enzyme. In four cases involving point mutations no change in the molecular specific activity was noted. However, in a fifth case involving a deletion mutation, a decreased molecular specific activity was observed. Erythrocytes were fractionated on a density gradient and the molecular specific activity of the enzyme was measured in cells of different ages. A lower molecular specific activity was found in older cells, suggesting the accumulation of catalytically inactive, immunoreactive material during the aging of the erythrocyte.     

Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method.

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].     

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

Oxidation of catechol in plants. 3. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The K_m for catechol was determined as 4 × 10^?4m. The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.     

Molecular weight of starch synthetase from Oryza sativa leaves

Soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase with primed activity was extracted from rice leaves and purified by (NH₄)₂SO₄ fractionation, gradient elution on DEAE-cellulose and finally by Sephadex G200 gel filtration or amylopectin-cellulose chromatography. The purified enzyme was essentially homogeneous electrophoretically, but exhibited two peaks corresponding to MW of 22 000 and 67 000 on Sephadex G200 chromatography and five distinct bands on sodium dodecyl sulfate gel electrophoresis with MW of 11·5, 20, 35, 50 and 68 × 10³.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Isolation of N5N10 -Methylene Tetrahydrofolate Reductase From Bovine Brain

N⁵,N¹⁰ -methylene tetrahydrofolate reductase has been purified 100-fold from bovine brain. The initial fractionation with protamine sulfate and ammonium sulfate was followed by chromatography on DEAE-polyacrylamide gel (Bio Gel DM-30) and Sephadex G-200 as well as the selective adsorption and elution of the enzyme on calcium phosphate gel. The purified enzyme required FADH₂ and catalyzed the reduction of the methylene group of N⁵,N¹⁰ -methylene tetrahydrofolate to the methyl group of N⁵ -methyl tetrahydrofolate. The pH optimum of the bovine brain reductase occurred at a pK of 6.5. The enzyme proved quite unstable. Both air oxidation and prolonged periods of storage at -20° inactivated the enzyme.     

Purification and characterization of phytase from cotyledons of germinating soybean seeds

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.     

Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the presence of calcium, lysozyme treatment also resulted in the release of a 180-kDa enzyme molecule. The total proteinase activity released by lysozyme treatment (in the presence or absence of calcium) was not only greater than that released by phosphate buffer but was also greater than that initially detectable on the surface of whole cells, suggesting an unmasking of enzyme on the cell surface. The presence of calcium during release treatment resulted in increased stability of the crude enzyme preparations. For the proteinase preparation released by using lysozyme with 50 mM CaCl₂, the half-life of proteinase activity at 37°C was 39 h, compared with 0.22 h for the calcium-free phosphate buffer-released preparation. In all cases, maximum stability was observed at pH 5.5. Comparison of β-casein hydrolysis by the three forms of the enzyme showed that the products of short-term (5- to 30-min) digestions were very similar, although subtle differences were detected with the 180-kDa form.     

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.     

Ornithine Carbamoyltransferase from Spinacea oleracea: Purification and Characterization

Ornithine carbamoyltransferase (OCT) from spinach (Spinacea oleracea L.) was purified to homogeneity and studied for some kinetic and structural properties. The enzyme showed a specific activity of 436 U mg^–1, its molecular mass was approximately 118 kDa as estimated by Sephacryl S-200 gel filtration chromatography, the purified protein ran as a single band of 38 kDa in sodium dodecyl sulfate-polyacryamide gel electrophoresis. The enzyme catalyses an ordered bi-bi-sequential reaction in which carbamoyl phosphate binds first, followed by L-ornithine; L-citrulline leaves first, followed by phosphate. The Michaelis constant was 0.19 mM for L-ornithine and 13.1 µM for carbamoyl phosphate; the dissociation constant for the enzyme and carbamoyl phosphate complex was of 19 µM. The K_m of the reaction decreases from pH 6.0 to pH 10.4. The enzyme is heat-labile, but it was protected from thermal inactivation by substrates; more by ornithine alone than by two substrates acting together.     

Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

Purification of creatine kinase from beef heart mitochondria]

53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification.