Purification, crystallization and properties of triosephosphate isomerase from human skeletal muscle

1. Triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketoisomerase, EC 5.3.1.1) from human skeletal muscle was purified to homogeneity and crystallized. The crystalline enzyme preparation was resolved on polyacrylamide-gel electrophoresis into three isoenzymes. 2. The molecular weight of the enzyme estimated by gel filtration method was found to be 57,400 +/- 3000. Molecular weight determination under dissociation conditions indicated a dimeric subunit structure of the enzyme. 3. The apparent Km for D-glyceraldehyde-3-phosphate as substrate is 0.34 mM, and for dihydroxyacetone phosphate, 0.61 mM. Vmax of the reaction is, respectively, 7200 and 660 units/mg protein at 25 degrees C and pH 7.5. 4. Molecular and kinetic properties of triosephosphate isomerase from human skeletal muscle are very similar to those of rabbit muscle enzyme.     

Interactions of the components of the prothrombinase complex

1. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) of house-fly head tissue was solubilized as a 7.4-S form by autolysis for 80-100 h at 25 degrees C and pH 8.0. 2. The autolysed enzyme was purified by affinity chromatography, firstly on Con-A-Sepharose and subsequently on m-trimethylammoniumaniline-Affi-Gel 202. This sequence permitted overall purification yields of approx. 50% of the solubilized enzyme. 3. The 7.4-S purified enzyme was essentially homogeneous on polyacrylamide gel electrophoresis, and its specific activity coincided with the highest previously reported for fly-head acetylcholinesterase. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and beta-mercaptoethanol revealed two major polypeptide components of molecular weight 82 000 and 59 000. Each of these polypeptides contained diisopropylphosphofluoridate-binding sites, as shown with [3H] diisopropylphosphofluoridate. 5. The results suggest a strong structural similarity between fly-head acetylcholinesterase and the purified electric eel enzyme.     

Purification and characterization of the glucoamylase produced by a strain of Aspergillus flavus

A strain of Aspergillus flavus isolated from an agricultural soil in Egypt produced a gluycoamylase which when purified had a molecular weight of 51,300 +/- 800 Daltons. The optimum pH for activity was 4 and the optimum temperature was 60 degrees C. The enzyme was stable at 70 degrees C for 15 min but denatured at 90 degrees C over 30 min. The Km value with soluble starch was 2.85 mg ml-1, and 10 mM HgCl2 inhibited the enzyme. It was possible to store the enzyme for at least 1 year at -20 degrees C without significant loss in activity.     

Studies on glucose isomerase from a Streptomyces species

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.     

On the extracellular calcium concentration and the relaxing effect of oxytocin

3 beta-Hydroxysteroid dehydrogenase / delta 5-4 isomerase activity was demonstrated in the human amniotic epithelium from the first trimester of pregnancy. The evidence was based on the in vitro formation of [4-14C] testosterone and [4-14C] androstenedione from [4-14C] 5-androstenediol and [4-14CA1 DEHYDROEPIANDROSTERONE, RESPECTIVELY. The activity of the enzyme studied in age dependent, reaching a maximum in the 8th-9th week of pregnancy and decreasing to negligible values at the end of the second trimester of gestation.     

Comparison of technetium-99m-sestamibi scintimammography with contrast-enhanced MRI for diagnosis of breast lesions

A carboxylesterase [2,3,4,6-tetra-O-acetyl-1-[(N-acetyl-N-phenylamino)oxy]-1-deoxy-beta-D-g lucopyranoside (GPA) O-deacetylase] from a culture product of Aspergillus oryzae (Taka diastase) was purified 8500-fold with a yield of 3%. The molecular mass of the purified enzyme was shown to be 35 +/- 1 kDa by SDS/PAGE. The enzyme shows a selective O-deacetylation activity of GPA to give the fully O-deacetylated glucoside. Among the substrates tested, the enzyme did not hydrolyze benzoyl and phenylacetyl esters and acetamides. In the hydrolysis of p-nitrophenyl esters, the acyl preference is acetyl > propionyl > butyryl, judging from the Vmax/Km values. A good correlation between log(Vmax/Km) and the Taft's Es constant of the alkyl group of the acyl moiety was obtained. The optimum pH was around 7.3 at 37 degrees C, and the enzyme was inhibited by mercuric chloride, p-chloromercuribenzoate and diisopropyl fluorophosphate. This enzyme should be useful for the selective removal of acetyl groups that serve to protect hydroxyl groups during carbohydrate synthesis.     

Purification and characterization of the Rieske iron-sulfur protein from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

The previously detected Rieske iron-sulfur protein from the membranes of the thermoacidophile Sulfolobus acidocaldarius [Anemüller, S., et al. (1993) FEBS Lett. 318, 61-64] was purified to electrophoretic homogeneity and the N-terminal amino acids determined. The apparent molecular weight was estimated to be 32 kDa. The reduced protein displays a rhombic EPR spectrum with gxyz = 1.768, 1.895, 2.035. The average g-value of 1.902 is typical for nitrogen ligand-containing clusters. EPR spin quantification and the iron content indicate the presence of one [2Fe-2S] cluster. The purified protein displays ubiquinol cytochrome c reductase activity. The pH optimum of this reaction is temperature dependent and was determined to be pH 7 at 56 degrees C. The results presented in this study clearly prove that the Sulfolobus Rieske protein belongs to the family of the true Rieske iron-sulfur proteins.     

Purification and cloning of a thermostable manganese catalase from a thermophilic bacterium

We have purified a heat-stable catalase from a thermophilic bacterium, Thermus species strain YS 8-13. The enzyme was purified 160-fold from crude cellular extracts and possessed a specific activity of 8000 units/mg at 65 degrees C. The purified enzyme displayed the highest activity at pH 7 to 10 and temperatures around 85 degrees C. The catalase was determined to be a manganese catalase, based on results from atomic absorption spectra and inhibition experiments using sodium azide. The enzyme was composed of six identical subunits of molecular weight 36,000. Amino acid sequences determined from the purified protein were used to design oligonucleotide primers, which were in turn used to clone the coding gene. The nucleotide sequence of a 1.4-kb fragment of Thermus sp. YS 8-13 genomic DNA containing a 909-bp open reading frame was determined. The gene encoded a 302-residue polypeptide of deduced molecular weight 33,303. The deduced amino acid sequence displayed a region-specific homology with the sequences of the manganese catalase from a mesophilic organism, Lactobacillus plantarum.     

The isolation and partial characterization of transferrin binding components of the rabbit reticulocyte plasma membrane

An affinity chromatograpy method utilising transferrin liganded agarose has been developed for the partial purification of transferrin binding components from Triton X-100 solubilised rabbit reticulocyte plasma membranes. A protein of molecular weight 30-35 000, shown to be located at the reticulocyte extra-cellular surface by lactoperoxidase 125I labelling, was isolated by the affinity method. The protein appeared to form a dimer of molecular weight 65-70 000 in Triton X-100 solution and was shown to associate with both 125I-labelled and unlabelled rabbit transferrin to form a high molecular weight complex in the same solution. N-[14C]Ethylmaleimide appeared to disrupt this association with transferrin and inhibit the formation of the dimer in Triton X-100 by binding to the protein. The protein appeared as a broad band of molecular weight 40 000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis.     

Purification and properties of human N-acetylgalactosamine-6-sulfate sulfatase

1. Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.     

Purification and some properties of human erythrocyte hexokinase

1. Human erythrocyte hexokinase (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified 50 000--100 000-fold with a final specific activity of about 25--50 units/mg protein using gel-filtration, ion-exchange chromatography and affinity chromagraphy. 2. After isoelectrofocusing ofthe preparation one major protein band could be detected besides a minor band. THe isoelectric point of the major protein band was found to be 4.7. 3. After purification the enzyme could be stabilized in a medium containing inorganic phosphate, glucose, glycerol and mercaptoethanol. 4. The molecular weight was determined by gel-filtration and was found to be 132 000+/-8000. 5. The enzyme shows a broad pH optimum ranging from 7.0 to 8.4. 6. The kinetic behavior of the purified enzyme at 37 degrees C was somewhat different from the normal Michaelis-Menten kinetics due to its instability. The affinity constants were 0.048--0.080 mM for glucose and 0.57--1.0 mM for Mg-ATP. 7. The enzyme was specific for Mg- ATP as the nucleotide substrate. Mg-UTP, Mg-ITP,Mg-GTP and Mg-CTP were not converted to corresponding diphosphates. Several hexoses could be phosphorylated by the enzyme. Mannose could be phosphorylated at the same rate as glucose, although the affinity for the enzyme was lower (5m=0.60mM). Much lower rates and lower affinities were found with 2-deoxy-D-glucose (5m=1.0mM), D(+)-glucosamine (5m=4.5 mM) and fructose (5m=10 mM). N-acetyl-D-glucosamine , galactose andsorbose were not phosphorylated at all.     

Partial purification and properties of the fatty acid elongation systems in the outer and inner membranes of beef liver mitochondria

Purified outer membrane of beef liver mitochondria was found to elongate medium chain fatty acyl-CoA primer by the incorporation of [1-14C]acetyl-CoA. This enzymic activity, extracted by Triton X-100, was purified 8-fold by ammonium sulfate fractionation followed by chromatography on a Sephadex column. Purified inner membrane, when processed through an identical purification procedure, yielded a second enzyme system which incorporated [1-14C]acetyl-CoA into long chain fatty acids in the presence of medium chain fatty acyl-CoA primer. This enzyme preparation was about four times as active as the preparation from the outer membrane, and used NADH as the reductant for the synthesis. The molecular weights of the inner and the outer membrane enzyme systems, estimated by gel filtration as well as sucrose density gradient centrifugation, were approx. 57 000 and 126 000, respectively. The partially purified outer membrane enzyme system required NADH and a medium chain acyl-CoA primer for the incorporation of [1-14C]acetyl-CoA into long chain fatty acids. KNC stimulated the reaction. NADPH could substitute for NADH only to a limited extent. Malonyl-CoA was ineffective as a substrate in this reaction. The optimum pH of the reaction was 7.2-7.6 in 0.1 M potassium phosphate buffer. Dithiothreitol, beta-mercaptoethanol, N-ethylmaleimide and high concentrations of ATP and acyl-CoA primer inhibited the reaction. The specificity for the acyl-CoA primer in the reaction was very broad. All the primers tested, C8 to C16, incorporated acetyl-CoA significantly. However, maximum incorporation was observed with dodecanoyl-CoA. Decanoyl-CoA was the best primer for the enzyme system isolated from the inner membrane. About 42% of the radioactivity in the fatty acids synthesized by the outer membrane enzyme system, from myristoyl-CoA and [1-C14]acetyl-CoA, was in palmitic acid. Of the remaining activity, 41% was in stearic acid and about 38% in longer-chain acids. Hence, the elongation of the primer fatty acid by one C2 unit appeared to be the predominant process in this synthesis. In the elongation of myristoyl-C0A by the inner membrane enzyme system, palmitic acid which constituted nearly 78% of the fatty acids synthesized, was the primary product.     

Prevalence of Toxoplasma gondii in Canadian market-age pigs

Triacylglycerol lipase (L3) was purified from Aspergillus oryzae RIB128 by ammonium sulfate fractionation, acetone precipitation, anion-exchange chromatography, and gel filtration. The purified enzyme was formed from a glycoprotein and a monomeric protein with molecular masses of 25 and 29 kDa, by SDS-PAGE and gel filtration, respectively. The optimum pH at 40 degrees C was 5.5 and the optimum temperature at pH 5.5 was 40 degrees C. The enzyme was stable between a pH range of 4.0-7.5 at 30 degrees C for 24 h, and at up to 30 degrees C at pH 5.5 for 1 h. Heavy metal ions, detergents, DFP, and DEP strongly inhibited the enzyme activity. The lipase hydrolyzed not only triacylglycerols but also monoacylglycerols and diacylglycerols. The enzyme had higher specificity toward triacylglycerols of middle-chain saturated fatty acids than short-chain or long-chain fatty acids. The enzyme had 1,3-positional specificity. The N-terminal amino acid sequence of the enzyme was not significantly similar to that of other lipases with published sequences.     

The interaction of an anionic photoreactive probe with the anion transport system of the human red blood cell

N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]sulfonate is employed as a photoreactive probe for the anion transport system in the human erythrocyte. In the dark and at 37 degrees C the probe penetrates the membrane via a pathway sensitive to specific inhibitors of anion permeability. It reversibly inhibits sulfate and chloride fluxes but the inhibition is reduced by higher concentrations of sulfate. Upon photolysis to produce a reactive nitrene (at 0 degrees C to minimize penetration), the probe inhibition of anion permeability. Under appropriate conditions the degree of inhibition after photoactivation (irreversible) is almost the same as that in the dark (reversible). The binding sites for the radioactive probe are largely found in proteins of 95 000 apparent molecular weight (band 3). After pronase treatment of the labelled cells, most of the probe is found in a 65 000 molecular weight segment derived from the 95 000 molecular weight protein. In this respect the photoreactive probe resembles another potent irreversible inhibitor of anion transport, 4, 4'-diisothiocyano-2, 2' stilbene disulfonate. In fact, most of the binding sites for each probe are common to both. Thus, in the dark, the azido derivative protects the anion system from inhibition by DIDS and substantially reduces the binding of DIDS to band 3 protein. Conversely, pretreatment with DIDS substantially reduces the binding of the photoreactive probe to the same protein. The fact that an apparent substrate for the anion permeation system competes for binding sites with a specific non-penetrating inhibitor of anion permeability suggests that the inhibitory and transport sites may be closely related and implicates the 95 000 molecular weight protein as the element of the anion transport system which contains the substrate binding site.     

Synthesis of zwitterionic acrylic acid buffers for isoelectric focusing in immobilized pH gradients

A range of zwitterionic acrylic acid derivatives, buffering in the neutral and basic pH ranges, have been synthesized by the Mannich reaction of malonic acid, formaldehyde and a secondary amine. These compounds include 2-(4-morpholinomethyl)propenoic acid pK2 7.59 +/- 0.03 (23 degrees C), 2-[bis(2-hydroxyethyl)aminomethyl]propenoic acid pK2 approximately 8.6 (20 degrees C), 2-[bis(2-hydroxypropyl) aminomethyl]propenoic acid PK2 approximately 8.7 (20 degrees C), 2-[N-(2-hydroxyethyl)-N-methylaminomethyl]propenoic acid pK2 9.22+/-0.08 (22 degrees C), 2-[N-ethyl-N-(2hydroxyethyl)aminomethyl]-propenoic acid pK2 approximately 9.6 (20 degrees C), and 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid, which has a sigmoidal buffering profile over the pH range 3-10. These zwitterionic acrylic acid buffers were successfully copolymerized with acrylamide to prepare immobilized pH gradients (IPGs) in the neutral to alkaline portion of the pH range. Bovine erythrocyte carbonic anhydrase isozymes were resolved on a pH 5-8 IPG prepared using 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid as the immobilized buffer, and horse heart myoglobin was focused on pH 7.1-8.1 and pH 7.5-7.7 IPGs, using 2-(4-morpholinomethyl)propenoic acid as the immobilized buffer. In both cases the pK 9.3 Immobiline compound was used as the strongly basic titrant. These new compounds, besides possessing more hydrophilic residues than the corresponding commercial basic acrylamido buffers (Immobilines), resist hydrolysis at alkaline pH values.     

Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60 degrees C. It showed maximum activity over a broad pH range (7.0-9.0). The molecular mass of the monomer was about 50,000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53,620 Da. A relative mass estimation of 100,000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer. The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5'untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.     

Purification and properties of RNA polymerase I from Ehrlich ascites cells

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.     

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Pantothenate synthetase of Escherichia coli B. I. Physicochemical properties

Using a new apparatus for preparative polyacrylamide gel electrophoresis, pantothenate synthetase (D-pantoate: beta-alanine ligase (AMP-forming), [EC 6.3.2.1] was purified about 500-fold from Escherichia coli B. It was found to be homogeneous in analytical disc gel electrophoresis and sedimentation ultracentrifugation (so20, w=4.9). From sedimentation equilibrium ultracentrifugation, a molecular weight of 70,100 was obtained, which is in good agreement with the value obtained by the Sephadex G-150 gel filtration method (69,000); the diffusion constant was calculated to be 5.88X10(-7) cm2/sec. The minimum molecular weight calculated from the amino acid composition of this enzyme protein was 19,700, a value in reasonable accord with molecular weight of the enzyme subunit, 18,000, obtained by gel electrophoresis in the presence of sodium dodecylsulfate. The partial specific volume, v, was calculated to be 0.71 cm3/g. The enzyme had an amino-terminal glycyl residue and a Leu-Ala-Ser-OH sequence at the carboxyl end. Electrophoresis of the enzyme with carrier ampholine gave an isoelectric point of pH 4.6.     

Purification and properties of a beta-mannanase from alfalfa seeds

A beta-mannanase (EC 3.2.1.25) has been purified from germinating Alfalfa seeds by successive chromatography steps; on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. The enzyme preparations were homogeneous as judged by gel electrophoresis. A 5000-fold increase in specific activity (from the crude extract) was obtained. The purified enzyme has a molecular weight of 40 000. Several of its properties were determined: pH optimum 5.2 and optimal temperature of activity 50 degrees C. The hydrolysis of galacto- and gluco-mannans (with various ratio of mannose to galactose and glucose) as well as that of mannooligosaccharides was studied in detail. A prefered point of attack at the third position from the non-reducing end was shown. Comparative results from the hydrolysis of intact galactomannans, of galactomannans previously hydrolysed by galactosidase, suggest that galactose hinders the accessibility of the mannan backbone to the enzyme.