scholarly journals The purification and properties of pig spleen phosphofructokinase

1975 ◽  
Vol 151 (2) ◽  
pp. 327-336 ◽  
Author(s):  
P E Hickman ◽  
M J Weidemann
Keyword(s):  
Metal Ion ◽  
Protein Concentration ◽  
Deae Cellulose ◽  
Significant Inhibition ◽  
Free Form ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Phosphoenol Pyruvate ◽  
Sigmoidal Kinetics ◽  
Cellulose Column

Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.

scholarly journals Purification and properties of a proteolytic enzyme from French beans

1965 ◽  
Vol 97 (1) ◽  
pp. 228-235 ◽  
Author(s):  
JRE Wells
Keyword(s):  
Proteolytic Enzyme ◽  
Deae Cellulose ◽  
Basic Amino Acid ◽  
Animal Origin ◽  
Prolonged Storage ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
French Beans ◽  
Purification And Properties ◽  
Endopeptidase Activity

1. A proteolytic enzyme with some features of a carboxypeptidase has been purified some 1180-fold from the sap of French beans (Phaseolus vulgaris var. Prince). A bright blue protein, plastocyanin, was separated from the enzyme by DEAE-cellulose chromatography. 2. Unlike carboxypeptidase A or B of animal origin, there is no evidence that the enzyme is a metalloprotein. There was no stimulation of activity by a number of metal ions, reducing agents or 2-mercapto-ethanol. Neither EDTA nor 1,10-o-phenanthroline inhibited the enzyme. 3. The proteolytic enzyme from beans, readily soluble at neutral or slightly acidic pH values, has a pH optimum of pH5.6 for the hydrolysis of leucine from benzyloxy-carbonylglycyl-l-leucine. Solutions of the enzyme in 0.1m-sodium acetate, pH5.5, lose about 2% of their activity/week at 4 degrees . Virtually no loss of activity results after prolonged storage at -15 degrees . 4. Incubation of the bean enzyme with peptides indicates that the enzyme will release acidic, neutral and basic amino acid residues as well as proline, although adjacent acidic residues in a peptide appear to inhibit the enzyme. The possibility of endopeptidase activity in the purified preparation requires further examination.

Purification and properties of an extracellular proteolytic enzyme fromBacillus cereus

1973 ◽  
Vol 40 (3) ◽  
pp. 427-440 ◽  
Author(s):  
M. A. Islam ◽  
J. M. V. Blanshard
Keyword(s):  
Metal Ion ◽  
Absorption Maximum ◽  
Proteolytic Enzyme ◽  
Heavy Metal Ions ◽  
Freeze Drying ◽  
Deae Cellulose ◽  
Milk Clotting ◽  
Purification And Properties ◽  
Extracellular Proteolytic Enzyme ◽  
Metal Ion Activation

SummaryA milk-clotting proteolytic enzyme was isolated and purified from the culture filtrate ofBacillus cereusstrain x29 by fractionation with acetone or ammonium sulphate and subsequent column chromatography employing DEAE cellulose and DEAE Sephadex. The purified enzyme was found to be homogeneous by acrylamide gel electrophoresis from pH 3·5 to 8·6, with, a molecular weight of about 50000. The single absorption maximum of the native enzyme was at 277 nm and the value ofat 280 nm was 7·79. Purification resulted in a 9-fold enhancement of activity with 24 % yield. The optimum activity of the enzyme was at pH 8·0 at 40 °C with casein as the substrate. The enzyme was found to be most stable at pH 6·0 and was stable to freezing and freeze-drying. Heavy metal ions were found to inactivate the enzyme, but no metal ion activation was found. Enzyme activity was inhibited irreversibly by EDTA and reversibly by 1,10-phenanthroline. The enzyme has been identified as a Zn-containing neutral protease.

Purification and Properties of the Soluble 17β-Hydroxysteroid Dehydrogenase of Rabbit Uterus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 726-737 ◽  
Author(s):  
Kunhard Pollow ◽  
Walter Eiger ◽  
Herrmann Heßlinger ◽  
Barbara Pollow
Keyword(s):  
Enzyme Activity ◽  
Dehydrogenase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Maximal Velocity ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Mobility Data ◽  
Purification And Properties

Abstract 17 β-Hydroxysteroid dehydrogenase activity towards estradiol-17 β has been demonstrated in the 105,000 X g supernatant of rabbit uterus. Hydroxylapatite chromatography of the enzyme activity isolated by ammonium sulfate precipitation, gel filtration and DEAE-cellulose chromato­graphy yielded a single 17 β-hydroxysteroid dehydrogenase activity. Further purification of the enzyme preparation by isoelectric focusing resulted in multiple peaks of activity. The molecular weight or the enzyme, calculated from mobility data on Sephadex gel, is approximately 64,000. Some properties of partially purified 17 β-hydroxysteroid dehydrogenase activity have been studied. Estradiol-17 β reacts at a faster rate than testosterone. The Km for estradiol is 4.16X 10-5 mol/1 for the NAD-linked enzyme activity and 4.37 X 10-5 mol/1 when NADP as cofactor was used. The ratio of the maximal velocity for NADP to that for NAD was 1.42. The pH-optimum for estradiol appears between 9.5 and 10.5 and for estrone between 5.5 and 6.5. The enzyme appears to be of the sulfhydryl type.

scholarly journals Purification and properties of arylsulphatase A from rabbit testis

1976 ◽  
Vol 159 (1) ◽  
pp. 133-142 ◽  
Author(s):  
C H Yang ◽  
P N Srivastava
Keyword(s):  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Substrate Affinity ◽  
Ph Optimum ◽  
Major Band ◽  
Time Activity ◽  
Purification And Properties ◽  
Arylsulphatase A ◽  
Beta Galactosidase ◽  
Cellulose Column

Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species.

Purification and properties of phosphoenolpyruvate carboxylase from green leaves of maize

1979 ◽  
Vol 44 (6) ◽  
pp. 1835-1840 ◽  
Author(s):  
Jaroslav Mareš ◽  
Jana Barthová ◽  
Sylva Leblová
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Chloride Ions ◽  
Magnesium Ions ◽  
Ph Optimum ◽  
Green Leaves ◽  
Purification And Properties ◽  
Fructose 1,6 Bisphosphate ◽  
No Activation

Phosphoenolpyruvate carboxylate was isolated from green leaves of maize (Zea mays L.) by a procedure including fractionation with ammonium sulphate, chromatography on DEAE-cellulose and preparative electrophoresis on polyacrylamide gel. The specific activity of the electrophoretically homogeneous enzyme was 23 U/mg. Its molecular weight was about 405 000, pH optimum was within the range 7.9 to 8.3, Km for phosphoenolpyruvate was 1.05 . 10-3 and the apparent Km for the magnesium ions was 8.0 . 10-4M. The enzyme was inhibited by malate, aspartate, citrate, pyruvate, ATP and ADP and chloride ions. It was strongly activated by glycine and glucose 6-phosphate and to a lesser degree by glucose 1-phosphate and fructose 1,6-bisphosphate; no activation by orthophosphate and 3-phosphoglycerate was observed.

Protein-Catalyzed Isotopic Exchange Reaction between Cysteine and Sulfide in Spinach Leaves

1977 ◽  
Vol 32 (3-4) ◽  
pp. 219-225 ◽  
Author(s):  
Ahlert Schmidt
Keyword(s):  
Exchange Reaction ◽  
Metal Ion ◽  
Isotopic Exchange ◽  
Rhodospirillum Rubrum ◽  
Deae Cellulose ◽  
Ph Optimum ◽  
Cysteine Synthase ◽  
Simple Method ◽  
Isotopic Exchange Reaction ◽  
Spinach Leaves

Abstract A protein has been isolated from spinach leaves which catalyzes the following isotopic exchange reaction: Cys-SH+H235S ↔ Cys-35SH + H2S . This enzyme has an pH-optimum above 9.0; its molecular weight has been estimated on a Sephadex-G-100 column to be around 64 000 daltons. During purification this exchange reaction activity follows cysteine synthase activity on DEAE-cellulose and Sephadex-G-100 column chromato­ graphy; however this enzyme fraction has not been purified to homogeneity to prove that both activities are catalyzed by the same protein. The apparent Km for a) H2S has been determined to be 0.86 mM using cysteine as substrate and 0.6 mM using O-acetylserine as substrate; b) for cysteine was found to be 3.3 mM; and c) for O-acetylserine to be 3.3 mM. For catalysis no metal ion is required and the reaction proceeds without addition of pyridoxalphosphat. This exchange reaction might prove to be a simple method to prepare labelled cysteine from non-labelled cysteine and labelled H2S. The exchange reaction was found too in Chlorella pyrenoidosa and in Rhodospirillum rubrum.

scholarly journals Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2

1972 ◽  
Vol 130 (1) ◽  
pp. 121-132 ◽  
Author(s):  
J. P. Kitcher ◽  
P. W. Trudgill ◽  
J. S. Rees
Keyword(s):  
Pseudomonas Putida ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Visible Region ◽  
Deae Cellulose ◽  
Close Correlation ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Wide Range ◽  
Potential Inhibitors

1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH4)2SO4 fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Δ2-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27×106 and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5–9.5, a Km for 2-furoyl-CoA of 20.2μm and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.

scholarly journals Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis

1978 ◽  
Vol 172 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A Akrigg
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Duplex Dna ◽  
Ph Optimum ◽  
Endonucleolytic Cleavage ◽  
Single Strand Breaks ◽  
Purification And Properties

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.

scholarly journals Purification and properties of hyaluronidase from human liver. Differences from and similarities to the testicular enzyme

1982 ◽  
Vol 205 (1) ◽  
pp. 69-74 ◽  
Author(s):  
E W Gold
Keyword(s):  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Human Umbilical Cord ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Significant Difference ◽  
High Concentrations ◽  
Different Response

Human liver hyaluronidase was purified to homogeneity by (NH4)2SO4 fractionation, chromatography on hydroxyapatite and DEAE-cellulose, and preparative disc polyacrylamide-gel electrophoresis. The enzyme had a pH optimum of 3.8-4.0, a molecular weight (determined by gel filtration) of 76000, and a Km of 0.05 mg/ml for purified human umbilical-cord hyaluronic acid. It generally resembled hyaluronidases studied in other tissues which are believed to be lysosomal, but shared a number of characteristics with a partially purified bovine testicular hyaluronidase. Neither enzyme exhibited inhibition by high concentrations of substrate, but both were competitively inhibited by dermatan sulphate and keratan sulphate. Both enzymes exhibited increased activity in the presence of albumin, probably owing to an increased susceptibility of substrate to enzyme action. The liver enzyme was inhibited by NaCl, but the testicular enzyme exhibited an increase in activity in the presence of the salt which was similar to the effect observed with albumin. The different response toward Cl- ion appeared to be the most significant difference between the two enzymes.

scholarly journals β-Fructofuranosidases from roots of dandelion (Taraxacum officinale Weber)

1972 ◽  
Vol 126 (3) ◽  
pp. 569-573 ◽  
Author(s):  
P. P. Rutherford ◽  
A. C. Deacon
Keyword(s):  
General Formula ◽  
Soluble Protein ◽  
Hydrolytic Enzymes ◽  
Deae Cellulose ◽  
Physiological Role ◽  
Taraxacum Officinale ◽  
The Other ◽  
Ph Optimum ◽  
Cellulose Column

1. Three β-fructofuranosidases were separated by chromatography on a DEAE-cellulose column from the soluble protein extracted from dandelion (Taraxacum officinale Weber) roots. 2. One enzyme, which acted on sucrose, was characterized as an invertase, with a Km of 2.00×10-2M and pH optimum of 7.5. 3. The other two enzymes are hydrolases (A and B), which act on the inulin series of oligosaccharides [general formula glucose-fructose-(fructose)n]. They both have a pH optimum of 4.0 and Km of 1.54×10-2M but differ in their chromatographic behaviour on DEAE-cellulose. Neither of the hydrolases is inhibited by sucrose. 4. The physiological role of these three hydrolytic enzymes is discussed.

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