Diacetyl reductase of Lactobacillus casei

1970 ◽  
Vol 16 (10) ◽  
pp. 947-951 ◽  
Author(s):  
A. L. Branen ◽  
T. W. Keenan
Keyword(s):  
Lactobacillus Casei ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Cell Extracts ◽  
Alumina Gel ◽  
Nadh Oxidase Activity ◽  
Nadh Oxidoreductase

Diacetyl reductase (diacetyl:reduced nicotinamide adenine dinucleotide (NADH) oxidoreductase, EC. 1.1.1.5) has been isolated from Lactobacillus casei. Cell sonication, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography, and alumina gel adsorption were used to obtain the partially purified enzyme. Both NADH oxidase and diacetyl reductase activity were associated with the same fraction at all stages in purification. Growth in media containing added pyruvate resulted in a 10-fold increase in the NADH oxidase activity and a 3-fold increase in the diacetyl reductase activity of crude cell extracts on a protein basis. Purified preparations showed maximal reductase and oxidase activities at pH 4.5 and 5.0, respectively. Lineweaver–Burke plots yielded intersecting lines when NADH and diacetyl concentrations were varied, suggesting a flavin-linked reaction. The absorption spectrum of the purified preparation was characteristic of that of a flavoprotein. The product of the reduction of diacetyl was identified as acetoin. Acetoin and methylene blue were inactive as acceptors.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

scholarly journals Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

Archaea ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Sherwin Shabdar ◽  
Bukuru Anaclet ◽  
Ana Garcia Castineiras ◽  
Neyissa Desir ◽  
Nicholas Choe ◽  
...  
Keyword(s):  
Nadh Oxidase ◽  
Reductase Activity ◽  
Sulfur Metabolism ◽  
Archaeoglobus Fulgidus ◽  
Bacterial Enzymes ◽  
Detectable Activity ◽  
Metabolic Role ◽  
Nadh Oxidase Activity

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H2S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S0 and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting k cat / K m on the order of 104 M-1 s-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2 ′ -phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S0 or serve in another metabolic role that has yet to be identified.

scholarly journals Identification of the NADH-oxidase gene in Trichomonas vaginalis

2019 ◽  
Vol 119 (2) ◽  
pp. 683-686 ◽  
Author(s):  
Aline Lamien-Meda ◽  
David Leitsch
Keyword(s):  
Oxidase Activity ◽  
Trichomonas Vaginalis ◽  
Nadh Oxidase ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Oxidase Gene ◽  
Reading Frame ◽  
Sexually Transmitted ◽  
Cell Extracts ◽  
Nadh Oxidase Activity

AbstractThe microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.

scholarly journals A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

1977 ◽  
Author(s):  
F.S. Markland ◽  
J. Chou ◽  
Y. Shih ◽  
H. Pirkle
Keyword(s):  
Sodium Chloride ◽  
Large Scale ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Tris Buffer ◽  
High Yield ◽  
Crude Venom ◽  
Diamondback Rattlesnake

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.

I. Isolierung, Reinigung und Charakterisierung der Polynucleotid-Phosphorylase aus der Blaualge Anacystis nidulans

1967 ◽  
Vol 22 (2) ◽  
pp. 204-215 ◽  
Author(s):  
Ingrid Capesius ◽  
Gerhard Richter
Keyword(s):  
Rna Polymerase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Anacystis Nidulans ◽  
Lag Phase ◽  
Blue Green Alga ◽  
Polynucleotide Phosphorylase ◽  
Isolation And Purification

A method for the isolation and purification of polynucleotide phosphorylase from the blue-green alga Anacystis nidulans is described. It consists in an initial fractional centrifugation which yields up to 70% of the enzyme associated with the ribosomal fraction while the RNA-polymerase together with the bulk DNA is mainly confined to the supernatant. The ribosomal fraction is then subjected to column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) to obtain a 120 — 150 fold increase in specific activity over the crude extract. The enzyme is essentially free from nucleic acids, phosphatases, kinases and RNA-polymerase. With ADP, UDP or CDP as substrate the corresponding homopolymers poly-A, poly-U and poly-C, respectively, are synthesized with good yields; no lag phase occurs. Equimolar mixtures of these nucleoside diphosphates give rise to heteropolymers. The optimal formation of poly-G and of AGUC polymer, both of which have rather low yields under standard conditions is achieved by incubating at higher temperatures (45° and 60°) and by replacing Mg2⊕ by Mg2⊕. In the presence of inorganic phosphate the highly purified enzyme also catalyzes the cleavage, to nucleoside diphosphates, of synthetic polynucleotides and of native high molecular RNA from various organisms. The possible association of polynucleotide phosphorylase with the ribosomes and its function is discussed.

Effect of Near-Ultraviolet Light on the Respiratory Chain of Escherichia coli

1971 ◽  
Vol 49 (5) ◽  
pp. 492-495 ◽  
Author(s):  
P. D. Bragg
Keyword(s):  
Ultraviolet Light ◽  
Cytochrome B ◽  
Respiratory Chain ◽  
Nadh Oxidase ◽  
Reductase Activity ◽  
State Level ◽  
Progressive Increase ◽  
E Coli ◽  
Near Ultraviolet ◽  
Nadh Oxidase Activity

Irradiation of a particulate fraction from E. coli with near-ultraviolet light destroyed NADH oxidase activity. This treatment did not affect markedly the levels of cytochromes b1 and o, and ubiquinone in this preparation. Cytochrome a2 was destroyed by irradiation. A progressive increase in the aerobic steady state level of cytochrome b1 reduction during irradiation confirmed that irradiation affected the cytochrome oxidase region of the respiratory chain. There was a second site of inactivation between substrate and cytochrome b1. This was indicated by lowered NADH:cytochrome b1 reductase activity. Partial reactivation of this activity was obtained by addition of ubiqumone-2 but not ubiquinone-8.

2-Phosphoglycerate phosphatase and serine biosynthesis in Veillonella alcalescens

1981 ◽  
Vol 27 (8) ◽  
pp. 808-814 ◽  
Author(s):  
J. J. Pestka ◽  
E. A. Delwiche
Keyword(s):  
Biosynthetic Pathway ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Ammonium Sulfate Precipitation ◽  
Ph Optimum ◽  
Phosphoserine Aminotransferase ◽  
Cell Extracts ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Biosynthesis

The constituent enzymes for the phosphorylated and nonphosphorylated serine biosynthetic pathways in Veillonella alcalescens were identified and included phosphoserine phosphatase, 3-phosphoglycerate dehydrogenase, glycerate dehydrogenase, phosphoserine aminotransferase, and serine–pyruvate aminotransferase. Cell extracts of the organism were also found to cause the specific dephosphorylation of 2-phosphoglycerate. The phosphatase was purified 39-fold by manganese chloride precipitation, ammonium sulfate precipitation, and DEAE-cellulose chromatography. Sephadex G-200 gel filtration data established an apparent molecular weight of 50 000 for the enzyme. The 2-phosphoglycerate phosphatase had a pH optimum of 5.5 and was distinct from phosphoglyceromutase. Assays conducted with the purified enzyme on a number of other phosphorylated intermediates indicated that the phosphatase was most specific for 2-phosphoglycerate. Glucerate, hydroxypyruvate, and serine inhibited the enzyme, whereas succinate stimulated activity. Veillonella 2-phosphoglycerate phosphatase is the first such enzyme to be described in a prokaryote and is probably involved in glycerate generation for the nonphosphorylated serine biosynthetic pathway.

scholarly journals Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

2018 ◽  
Vol 22 (2) ◽  
pp. 47
Author(s):  
Akhmad Solikhin ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Wendry Setiyadi Putranto
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Iodoacetic Acid ◽  
Aspartic Protease ◽  
Partial Purification ◽  
Antioxidant Agents

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

scholarly journals Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts.

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012 ◽  
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

scholarly journals Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei

1976 ◽  
Vol 157 (3) ◽  
pp. 559-571 ◽  
Author(s):  
J G Dann ◽  
G Ostler ◽  
R A Bjur ◽  
R W King ◽  
P Scudder ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Resistant Strain ◽  
Lactobacillus Casei ◽  
Large Scale ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Binding Constants ◽  
Affinity Column ◽  
Protein Fluorescence ◽  
Turnover Number

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.

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