Influence of carcinogens and group-specific compounds on DNAse II

1969 ◽  
Vol 47 (10) ◽  
pp. 987-989 ◽  
Author(s):  
Marvin S. Melzer
Keyword(s):  
Enzyme Activity ◽  
Enzyme Level ◽  
Iodoacetic Acid ◽  
Dnase Ii ◽  
Histidine Residues ◽  
Carcinogenic Process

A number of cancer-causing and group-specific compounds were tested for their effects on the activity of DNAse II. The following were almost completely inhibitory: iodoacetic acid, N-bromosuccinimide (at an N-bromosuccinimide/enzyme level ≥ 24), and H2O2 (at an H2O2/enzyme level > 10 000). Either noninhibitory or less than 30% inhibitory were iodoacetamide and diisopropylfluorophosphate. Noninhibitory were such carcinogens as beta-butyrolactone, diepoxybutane, 3-hydroxyxanthine, and ascaridole. Also noninhibitory was malonaldehyde.From these results (and others in the literature), it was concluded that (1) the carcinogens tested (at least in their unmetabolized forms) might not act directly on DNAse II in the critical reaction of the carcinogenic process, and (2) tryptophan, methionine, and/or histidine residues play important roles in the enzyme activity.

QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS

1963 ◽  
Vol 42 (4) ◽  
pp. 615-624 ◽  
Author(s):  
Claes Hellerström ◽  
Bo Hellman
Keyword(s):  
Enzyme Activity ◽  
B Cells ◽  
Enzyme Level ◽  
Enzyme Reaction ◽  
Histochemical Staining ◽  
Pancreatic Tissue ◽  
Free Access ◽  
Isolated Pancreatic Islets ◽  
Insulin Synthesis ◽  
Dipeptidase Activity

ABSTRACT Microtitrimetric assays of dipeptidase activity were performed in isolated pancreatic islet tissue from mice. Considerable enzyme activity was found in both the endocrine and exocrine pancreas of normal mice, the enzyme level of the exocrine parenchyma being significantly higher. In obesehyperglycaemic mice with free access to food, isolated islets of Langerhans had a much higher enzyme activity than in normal animals. The increased islet dipeptidase activity in the obese-hyperglycaemic animals may, at least in part, be accounted for by their higher proportion of B cells. The intense insulin synthesis and renewal of B cells in these animals have been considered as alternative explanations. Histochemical staining for leucine aminopeptidase revealed a moderate enzyme reaction in both the endocrine and exocrine pancreatic tissue of normal and obese-hyperglycaemic mice.

Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes

2000 ◽  
Vol 27 (1) ◽  
pp. 29-33 ◽  
Author(s):  
G. Ataie ◽  
A.A. Moosavi-Movahedi ◽  
A.A. Saboury ◽  
G.H. Hakimelahi ◽  
J.Ru. Hwu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenosine Deaminase ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

scholarly journals Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1977 ◽  
Vol 165 (2) ◽  
pp. 385-393 ◽  
Author(s):  
Choong Yee Soon ◽  
Maxwell G. Shepherd ◽  
Patrick A. Sullivan
Keyword(s):  
Enzyme Activity ◽  
Chemical Reduction ◽  
Enzyme Inactivation ◽  
Subunit Structure ◽  
Histidine Residue ◽  
Lactate Oxidase ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Competitive Inhibitors ◽  
Active Site Histidine

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [14C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the14C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

scholarly journals Bovine inositol monophosphatase. Modification, identification and mutagenesis of reactive cysteine residues

1992 ◽  
Vol 285 (2) ◽  
pp. 461-468 ◽  
Author(s):  
M R Knowles ◽  
N Gee ◽  
G McAllister ◽  
C I Ragan ◽  
P J Greasley ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Iodoacetic Acid ◽  
Native Enzyme ◽  
Steric Effects ◽  
Cysteine Residues ◽  
Native Protein ◽  
Protein Fluorescence ◽  
Inositol Monophosphatase ◽  
Nitrobenzoic Acid ◽  
Full Activity

1. Bovine inositol monophosphatase reacts with thiol reagents such as 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA). 2. Modification by NEM results in nearly total loss of enzyme activity, whereas modification by IAA causes a slight increase in activity. 3. The loss of activity caused by NEM can be prevented by the inclusion of Ins1P, or better Ins1P and LiCl in the reaction mixture. 4. Two equivalents of p-nitrothiobenzoate (NTB2-) are released from the native enzyme on reaction with DTNB, and six equivalents of NTB2- are released from the SDS-denatured enzyme, suggesting that none of the six cysteine residues per molecule of enzyme is involved in intra- or inter-molecular disulphide bridges. 5. Both NEM and IAA react with two cysteine residues (residues 141 and 184 in the sequence) in a mutually exclusive manner. 6. NEM also reacts stoichiometrically with residue 218. 7. The NEM-induced loss of enzyme activity is accompanied by a 15% decrease in protein fluorescence. 8. A mutant of the enzyme which has an Ala-218 replacement for Cys-218 has full activity and is not sensitive to NEM, showing that the modification of this cysteine by NEM causes inhibition of the native protein by steric effects and that Cys-218 is not essential for activity.

scholarly journals The role of histidine residues in glutamate dehydrogenase

1972 ◽  
Vol 129 (2) ◽  
pp. 419-425 ◽  
Author(s):  
N. Tudball ◽  
R. Bailey-Wood ◽  
P. Thomas
Keyword(s):  
Enzyme Activity ◽  
Glutamate Dehydrogenase ◽  
Rose Bengal ◽  
Marked Decrease ◽  
Total Loss ◽  
Appreciable Effect ◽  
Normal Response ◽  
Original Activity ◽  
Histidine Residues

1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD+in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD+had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.

scholarly journals Glucose-dependent induction of acetyl-CoA carboxylase in rat hepatocyte cultures

1984 ◽  
Vol 221 (2) ◽  
pp. 343-350 ◽  
Author(s):  
S Giffhorn ◽  
N R Katz
Keyword(s):  
Enzyme Activity ◽  
Enzyme Level ◽  
Enzyme Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Enzyme Protein ◽  
Atp Citrate Lyase ◽  
Acetyl Coa ◽  
Adult Rats ◽  
Hepatocyte Cultures

The carbohydrate-dependent long-term regulation of acetyl-CoA carboxylase was studied in primary hepatocyte cultures from adult rats. (1) The enzyme activity was increased 2-fold either by elevation of the glucose concentration to 20mM or by enhancement of the insulin concentration to 0.1 microM. Simultaneous increases in glucose and insulin resulted in a 5-fold increase in the enzyme activity. (2) As shown by immunochemical titration, the enhancement of the enzyme activity was due to an increase in the enzyme protein. (3) Incorporation of [35S]methionine and immunoprecipitation of the enzyme revealed that the increase in enzyme protein was due to an increased rate of enzyme synthesis. The rate of enzyme degradation remained essentially unchanged. (4) The glucose- and insulin-dependent induction of acetyl-CoA carboxylase was prevented by the addition of alpha-amanitin (10 microM) or cordycepin (10 microM), indicating a transcriptional regulation of the enzyme level. (5) The parallel induction of acetyl-CoA carboxylase and of ATP citrate lyase indicates a co-ordinate long-term regulation of lipogenic enzymes.

scholarly journals Identification of three crucial histidine residues (His115, His132 and His297) in porcine deoxyribonuclease II

2006 ◽  
Vol 398 (2) ◽  
pp. 177-185 ◽  
Author(s):  
Yu-Che Cheng ◽  
Chin-Chen Hsueh ◽  
Shao-Chun Lu ◽  
Ta-Hsiu Liao
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Enzyme Activity ◽  
Catalytic Mechanism ◽  
Enzyme Properties ◽  
Dnase Ii ◽  
Exogenous Dna ◽  
Mutant Proteins ◽  
General Base ◽  
Constructed Plasmids

DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.

scholarly journals Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme

1990 ◽  
Vol 270 (2) ◽  
pp. 319-323 ◽  
Author(s):  
A K Drabikowska ◽  
G Woźniak
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Site ◽  
Order Rate Constant ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Uridine Phosphorylase ◽  
High Concentration ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

Cysteine proteinase of Phascolomyces articulosus: purification and properties

1986 ◽  
Vol 64 (11) ◽  
pp. 2441-2445 ◽  
Author(s):  
R. Balasubramanian ◽  
M. S. Manocha
Keyword(s):  
Enzyme Activity ◽  
Cysteine Proteinase ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Iodoacetic Acid ◽  
Phenylmethylsulfonyl Fluoride ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Salting Out ◽  
Purification And Properties

A proteinase from the mycelial extracts of Phascolomyces articulosus has been purified by salting out with ammonium sulphate, gel filtration, hydroxyapatite adsorption, and affinity chromatography. The proteinase rapidly hydrolysed haemoglobin but failed to hydrolyse any of the synthetic peptides tested. The enzyme is a glycoprotein with an apparent molecular weight of 12 800. The carbohydrate content was estimated to be 65%. It has a temperature optimum of 20 °C, pH optimum of 3.0, and has a Km value of 6.6 mg∙mL−1 for denatured haemoglobin. Iodoacetic acid, iodoacetamide, benzamidine, as well as all the heavy metals tested inhibited the enzyme activity. The enzyme activity was not enhanced by reducing agents such as cysteine, ethylenediaminetetra acetic acid, and dithiothreitol, the latter, however, reversed inhibition by phenylmethylsulfonyl fluoride. The inhibitor studies suggest that the enzyme belongs to the group of cysteine proteinases.

scholarly journals Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human γ-glutamyl hydrolase

1999 ◽  
Vol 343 (3) ◽  
pp. 551-555 ◽  
Author(s):  
Karen J. CHAVE ◽  
John GALIVAN ◽  
Thomas J. RYAN
Keyword(s):  
Enzyme Activity ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Iodoacetic Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Mutant Proteins ◽  
Key Enzyme

γ-Glutamyl hydrolase (GH), which hydrolyses the γ-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

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