Purification of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase

An NAD-linked 15-hydroxyprostaglandin dehydrogenase has been purified 13, 100-fold from human placental tissue. The specific activity of the purified enzyme ranges from 6900 to 8300 mU/mg protein depending on the method used to determine the protein concentration. On discontinous electrophoresis in sodium dodecyl sulfate more than 95% of the protein migrates as a single band; its estimated molecular weight is 25.5–26.0 kDa. This is half the value obtained when the molecular weight is estimated under non-denaturing conditions and suggests that the enzyme is composed of two identical or nearly identical subunits.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Purification and general properties of AMP deaminase from sheep brain

AMP deaminase from sheep brain was purified to homogeneity on SDS-PAGE and its general properties were investigated. The native enzyme has a molecular weight of approximately 350,000 as estimated by gel filtration and it is composed of four identical subunits with a molecular weight of 85,000 each. The purified enzyme had a specific activity of 500 units/mg protein and shows a sigmoid-shaped AMP saturation curve in the presence of 100 mM KCl. This deaminase is strongly activated by ATP and inhibited by GTP. It slightly catalyzes the hydrolysis of adenosine monosulfate (AMS), dAMP, and adenosine phosphoramidate (APA). These catalytic properties resemble those of AMP deaminase from human liver.     

Purification of betaine-aldehyde dehydrogenase from spinach leaves and preparation of its antibody

Betaine-aldehyde dehydrogenase was purified from spinach leaves and characterized. The molecular weight of the enzyme was estimated to be 120 kDa by a gel filtration chromatography. The enzyme was judged to consist of two identical pieces of the monomeric subunit with molecular weight of 60 kDa. A specific polyclonal antibody was raised against the enzyme subunit.     

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12. 1.Purification and subunit structure.

1. 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity. The purified enzyme has a specific activity of 67 units/mg which is about 1000 times that found in cell-free extracts of wild-tupe E. coli K12. 2. The minimum molecular weight of the enzyme was estimated by dodecylsuphate-gel electrophoresis to be 33000. Re-estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000. 3.Amino acid anaktsus abd tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical. 4.The purified enzyme does not contain Co2+.     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.     

Purification of Cytosine Deaminase from Pseudomonas aureofaciens

Cytosine deaminase from Pseudomonas aureofaciens was purified about 480-fold by means of ammonium sulfate fractionation, ethyl alcohol fractionation, chromatography on columns of DEAE-Sephadex A–50 and hydroxylapatite and by gel filtration on a Sephadex G–200 column. The enzyme was homogeneous by the criteria of sedimentation and electrophoretic analysis. The molecular weight of the purified enzyme was estimated to be 630,000 by gel filtration and consisted of twelve to sixteen identical subunits having a molecular weight of about 45,000. The enzyme catalyzed the deamination of cytosine and 5-methylcytosine.     

Immunoaffinity purification and identification of the molecular chaperone calnexin.

We have developed a method for the immunoaffinity purification of calnexin, an endoplasmic reticulum molecular chaperone, and analyzed the molecular weight of purified calnexin using matrix-assisted laser adsorption ionization time of flight mass spectrometry (MALDI TOF-MS). Calnexin was thereby found to have a molecular weight of 66.1 x 10(3), which is nearly identical to the molecular weight estimated from the protein sequence.     

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.     

Purification and Properties of Extracellular Acid Phosphatase from Zizania latifolia-pavasite,stilago esculenta

An extracellular acid phosphatase from Ustilago esculenta was purified to homogeneity on the basis of polyacrylamide gel electrophoresis. It was a glycoprotein with an isoelectric point of 4.7. The molecular weight of the enzyme was estimated to be about 343,000 by gel filtration on Sephadex G-200, whereas on SDS-polyacrylamide gel electrophoresis, the enzyme gave a single protein band with a molecular weight of 116,000. This result suggests that the enzyme consists of three identical subunits. The enzyme showed an optimum activity at pH 4.5, retained 90% of its activity for 10 min at 55°C and had a Km value of 0.25 mm for p-nitrophenylphosphate. No definite substrate specificity of the enzyme was observed.     

Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition

Antiserum against calf thymus dUTPase was raised in rats by injection of the partially purified enzyme. The antiserum did not react with dUTPase purified from rat spleen, while antibody against rat spleen dUTPase partially reacted with calf thymus enzyme. Native molecular weight of the calf thymus dUTPase was estimated at 46,000 daltons by gel filtration, and the denatured form of the enzyme was about 22,000, as judged by immunoblot analyses using the antibodies. These results indicate that the calf thymus dUTPase is composed of two identical subunits.     

Purification and Characterization of Ornithine Transcarbamylase from Pea (Pisum sativum L.)

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using δ-N-(phosphonacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37°C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.     

Purification and properties of phosphoserine aminotransferase from bovine liver

L-Phosphoserine aminotransferase was purified from bovine liver to apparent homogeneity as judged by nondenaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical ultracentrifugation, and immunochemical analysis. The purification procedure described involves the specific elution of the enzyme from Cibacron blue-agarose by micromolar concentrations of its substrate, phosphohydroxypyruvate. The purified enzyme had a specific activity of approximately 13 mumol of phosphohydroxypyruvate formed min-1 mg-1 of protein at 38 degrees C. Determinations of the native molecular weight and the subunit molecular weight indicated that the phosphoserine aminotransferase from bovine liver was a dimer composed of two subunits with identical molecular weights of 43,000.     

Purification and properties of alpha-aminoadipate aminotransferase from rat kidney.

Previous studies with rat kidney preparations indicated that alpha-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are associated with a single protein. However, recent studies from our laboratory demonstrated that AadAT and KAT activities belong to two different proteins. AadAT from rat kidney supernatant fraction was purified by affinity chromatography to electrophoretic homogeneity. This rapid and efficient procedure improved the yield and the degree of purification over previously published methods and separated AadAT from KAT. The molecular weight of the enzyme was estimated to be 89,000 by Sephadex G-200 gel filtration chromatography. SDS-PAGE indicated that the enzyme is composed of two apparently identical subunits. Absorption spectra and the kinetic properties of AadAT are reported.