scholarly journals Purification and properties of arylsulphatase B of human liver

1976 ◽  
Vol 153 (2) ◽  
pp. 415-421 ◽  
Author(s):  
S I O Agogbua ◽  
C H Wynn
Keyword(s):  
Active Site ◽  
Human Liver ◽  
Specific Activity ◽  
Cold Treatment ◽  
Apparent Molecular Weight ◽  
Acid Group ◽  
Kinetic Properties ◽  
Amino Acid Group ◽  
Purification And Properties ◽  
Purification Scheme

1. A purification scheme for an arylsulphatase B from human liver is described. Specificity of purification was achieved by the use of the affinity chromatography on an agrose-4-hydroxy-2-nitrophenyl sulphate derivative. The scheme provides a rapid and convenient method for preparation of a highly purified enzyme. 2. The purified enzyme was examined by isoelectric focusing electrophoresis on polyacrylamide gel and by ultracentrifugation and was found to be catalytically homogenous, with an apparent molecular weight of 50000 and a specific activity of 93.3 units/mg of protein. 3. The kinetic properties of the purified preparation and the effect of various amino acid group-specific reagents on the catalysis of the enzyme are described. The involvement of histidine residues in the active site of the enzyme is suggested. 4. The purified enzyme lost activity rapidly on freezing. The implication of this observation is discussed in terms of a possible dissociation-reaggregation phenomenon induced by cold treatment.

scholarly journals Properties of oxidized and reduced spinach (Spinacia oleracea) chloroplast fructose-1,6-bisphosphatase activated by various agents

1991 ◽  
Vol 278 (3) ◽  
pp. 787-791 ◽  
Author(s):  
T Chardot ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Acid Group ◽  
Lag Phase ◽  
Kinetic Properties ◽  
Amino Acid Group ◽  
Progress Curve ◽  
Fructose 1,6 Bisphosphatase ◽  
Specificity Constant ◽  
The Stability ◽  
Fructose 1,6 Bisphosphate

Fructose-1,6-bisphosphatase (FBPase) can be reduced and activated by either dithiothreitol or reduced thioredoxin. This activation is pH-dependent. An amino acid group with a pK value of 5.55 is involved in this process. Both enzyme forms can also be stimulated by agents such as fructose 1,6-bisphosphate, Mg2+, Ca2+ and Ca2+/fructose 1,6-bisphosphate. FBPase reduced by dithiothreitol is more strongly activated than the enzyme reduced by thioredoxin. The specificity constant (kcat./Km) is enhanced over 2.5-25-fold and 1.5-2-fold (depending on the agent used) for FBPase reduced by dithiothreitol and thioredoxin respectively. In both cases, no new kinetic properties appeared. The pH-activity profile of the stimulated enzyme is slightly shifted towards the acidic side with respect to the reduced enzyme. A lag phase is observed in the progress curve of both enzymic forms, treated or untreated. Each agent used to stimulate must induce a new conformation of the enzyme, more active than the initial one, characterized by a specificity constant and a relaxation time. This lag phase tends to disappear when the assay temperature is increased. Temperature has the same effect on the activity of oxidized, reduced and stimulated FBPase, but different effects on the stability of the different forms.

scholarly journals Purification and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids

1990 ◽  
Vol 267 (1) ◽  
pp. 179-183 ◽  
Author(s):  
G G Preston ◽  
J D Wall ◽  
D W Emerich
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Pyridoxal Phosphate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Permeation Chromatography ◽  
Acetyl Coa ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Purification Scheme ◽  
A Subunit

Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively.

Purification and properties of chlorocatechol 1,2-dioxygenase from Alcaligenes denitrificans BRI 6011

1993 ◽  
Vol 39 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Carlos B. Miguez ◽  
Charles W. Greer ◽  
Jordan M. Ingram
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Logarithmic Growth Phase ◽  
Chlorobenzoic Acid ◽  
Alcaligenes Denitrificans ◽  
Purification And Properties ◽  
Chlorinated Catechols ◽  
Logarithmic Growth

The specific activity of chlorocatechol 1,2-dioxygenase from Alcaligenes denitrificans BRI 6011 was found to be maximal in the early logarithmic growth phase. The enzyme was purified from cultures at mid-log phase of growth using ammonium sulfate fractionation, and phenyl-Sepharose and DEAE-Sepharose chromatography. The protein gave a single band by SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 33 000, and the temperature and pH optima were 30 °C and 7.5, respectively. Catechol, 3-chlorocatechol (3-CC), 4-CC, 3,4-dichlorocatechol (3,4-DCC), 3,5-DCC, 3,6-DCC, 3-methylcatechol (3-MC), and 4-MC served as substrates for the enzyme. The Vmax values for the dichlorocatechols were similar, while those for the monochlorinated and methylated catechols were higher. The Km values for all the chlorinated catechols were typically below 1 μM, while those for catechol and the methylated catechols were above 10 μM.Key words: chlorocatechol 1,2-dioxygenase, Alcaligenes denitrificans, purification, characterization, chlorobenzoic acid degradation.

scholarly journals The purification and properties of human liver ketohexokinase. A role for ketohexokinase and fructose-bisphosphate aldolase in the metabolic production of oxalate from xylitol

1985 ◽  
Vol 230 (1) ◽  
pp. 53-60 ◽  
Author(s):  
R Bais ◽  
H M James ◽  
A M Rofe ◽  
R A Conyers
Keyword(s):  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Properties ◽  
Fructose Bisphosphate ◽  
Reaction Sequence ◽  
Purification And Properties ◽  
Phosphorylated Compounds ◽  
Fructose Bisphosphate Aldolase ◽  
A Minor ◽  
Inhibition Studies

Ketohexokinase (EC 2.7.1.3) was purified to homogeneity from human liver, and fructose-bisphosphate aldolase (EC 4.1.2.13) was partially purified from the same source. Ketohexokinase was shown, by column chromatography and polyacrylamide-gel electrophoresis, to be a dimer of Mr 75000. Inhibition studies with p-chloromercuribenzoate and N-ethylmaleimide indicate that ketohexokinase contains thiol groups, which are required for full activity. With D-xylulose as substrate, ketohexokinase and aldolase can catalyse a reaction sequence which forms glycolaldehyde, a known precursor of oxalate. The distribution of both enzymes in human tissues indicates that this reaction sequence occurs mainly in the liver, to a lesser extent in the kidney, and very little in heart, brain and muscle. The kinetic properties of ketohexokinase show that this enzyme can phosphorylate D-xylulose as readily as D-fructose, except that higher concentrations of D-xylulose are required. The kinetic properties of aldolase show that the enzyme has a higher affinity for D-xylulose 1-phosphate than for D-fructose 1-phosphate. These findings support a role for ketohexokinase and aldolase in the formation of glycolaldehyde. The effect of various metabolites on the activity of the two enzymes was tested to determine the conditions that favour the formation of glycolaldehyde from xylitol. The results indicate that few of these metabolites affect the activity of ketohexokinase, but that aldolase can be inhibited by several phosphorylated compounds. This work suggests that, although the formation of oxalate from xylitol is normally a minor pathway, under certain conditions of increased xylitol metabolism oxalate production can become significant and may result in oxalosis.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

scholarly journals Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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