scholarly journals THE HISTOCHEMICAL DEMONSTRATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ACTIVITY

1961 ◽  
Vol 9 (3) ◽  
pp. 573-581 ◽  
Author(s):  
S. Ralph Himmelhoch ◽  
Morris J. Karnovsky
Keyword(s):  
Dehydrogenase Activity ◽  
Intracellular Localization ◽  
Histochemical Demonstration ◽  
Cellular Level ◽  
Tissue Component ◽  
Ph Optimum ◽  
Coenzyme Specificity ◽  
Acid Acid ◽  
Activating Agent ◽  
Hydrolysis Of

A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.

scholarly journals Microbial Isobutyronitrile Utilization under Haloalkaline Conditions

2007 ◽  
Vol 73 (17) ◽  
pp. 5574-5579 ◽  
Author(s):  
Dimitry Y. Sorokin ◽  
Sander van Pelt ◽  
Tatjana P. Tourova ◽  
Gerard Muyzer
Keyword(s):  
Carboxylic Acids ◽  
Nitrile Hydratase ◽  
Bacterial Species ◽  
Intracellular Localization ◽  
Soda Lake ◽  
Bacterial Strains ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Aliphatic Nitriles ◽  
Hydrolysis Of

ABSTRACT The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.

Polarographic study of hydrolysis of [8-lysine]vasopressin and its derivatives with blood serum of pregnant women

1980 ◽  
Vol 45 (4) ◽  
pp. 1099-1108 ◽  
Author(s):  
Mikuláš Chavko ◽  
Michal Bartík ◽  
Evžen Kasafírek
Keyword(s):  
Blood Serum ◽  
Pregnant Women ◽  
Degree Of Hydrolysis ◽  
Polarographic Study ◽  
Ph Optimum ◽  
Lysine Vasopressin ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Vasopressin Analogues

A polarographic study of the hydrolysis of [8-lysine]vasopressin and some hormonogens of the vasopressin series with the blood serum of women in the last week of pregnancy was studied. The dependence of hydrolysis on pH (pH optimum: 7.4-7.50, substrate concentration (Km 1.2 . 10-5M), pH stability and thermal stability were determined. The rate of hydrolysis of individual vasopressin analogues decreases in the order: [8-lysine]vasopressin > Nα-glycyl-prolyl[8-lysine]-vasopressin > Nα-leucyl-[8-lysine]vasopressin > Nα-alanyl-[8-lysine]vasopressin > Nα-phenyl alanyl-[8-lysine]vasopressin > Nα-diglycyl-[8-lysine]vasopressin > Nα-prolyl-[8-lysine]vasopressin > Nα-triglycyl-[8-lysine]vasopressin > Nα-sarcosyl-glycyl-[8-lysine]vasopressin. The degree of hydrolysis gradually increases to a multiple with the length of the pregnancy in consequence of the presence of oxytocine. However, vasopressin is also hydrolysed to a small extent with the enzymes from the blood sera of non-pregnant women. Under similar analytical conditions oxytocin was not hydrolysed with the sera of non-pregnant women and therefore oxytocin is a more suitable substrate than vasopressin for polarographic determination of serum oxytocinase.

scholarly journals Clathrin- and dynamin-dependent endocytosis limits canonical NF-κB signaling triggered by lymphotoxin β receptor

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Małgorzata Maksymowicz ◽  
Marta Miączyńska ◽  
Magdalena Banach-Orłowska
Keyword(s):  
Cellular Response ◽  
Target Genes ◽  
Intracellular Localization ◽  
Tumor Necrosis Factor Receptor ◽  
Transcriptional Response ◽  
Cellular Level ◽  
Effector Proteins ◽  
Receptor Internalization ◽  
Pcr Analysis ◽  
Β Receptor

Abstract Background Lymphotoxin β receptor (LTβR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTβR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTβR internalization to its signaling potential. Methods Intracellular localization of LTβR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTβR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTβR stimulation was assessed by qRT-PCR analysis. Results We demonstrated that LTβR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTβR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTβR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTβR-triggered activation of the non-canonical branch of the NF-κB pathway. Conclusions Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTβR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Graphical abstract

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori

2002 ◽  
Vol 362 (1) ◽  
pp. 131-135 ◽  
Author(s):  
Michael ARAND ◽  
Alexander M. GOLUBEV ◽  
J. R. Brandao NETO ◽  
Igor POLIKARPOV ◽  
R. WATTIEZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Solid Phase ◽  
Aspergillus Awamori ◽  
Glycoside Hydrolase Family ◽  
Orthorhombic Space Group ◽  
Ph Optimum ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

The β-glucosidases of porcine kidney

1977 ◽  
Vol 55 (2) ◽  
pp. 140-145 ◽  
Author(s):  
Julian N. Kanfer ◽  
Richard A. Mumford ◽  
Srinivasa S. Raghavan
Keyword(s):  
Sodium Taurocholate ◽  
Hydrolytic Activity ◽  
Acidic Ph ◽  
Porcine Kidney ◽  
Ph Optimum ◽  
Pig Kidney ◽  
Competitive Inhibitors ◽  
Triton X ◽  
Hydrolysis Of ◽  
Estradiol 17Β

Some of the properties of a partially purified particle bound and soluble β-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble β-glucosidase (1) hydrolyzed 4-methylumbelliferyl-β-D-glucoside (4-MU-β-D-glucoside) 17α-estradiol 3β-glucoside, 17α-estradiol 17β-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5–7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone β-glucoside being the most effective. In contrast, a particulate β-glucosidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-β-glucoside and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-β-D-glucoside or glucosylceramide as the glucosyl donor, and [l4C]ceramide as acceptor.

HYDROLYSIS OF GLYCEROLPHOSPHATIDES BY PLASTID PHOSPHATIDASE C

1956 ◽  
Vol 34 (5) ◽  
pp. 967-980 ◽  
Author(s):  
Morris Kates
Keyword(s):  
Ethyl Ether ◽  
Phosphatidyl Choline ◽  
Phosphatidyl Ethanolamine ◽  
Enzymatic Reaction ◽  
Phosphatidyl Serine ◽  
Ph Optimum ◽  
Soybean Phosphatide ◽  
Optimum Ph ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Studies of the influence of structural variation in the glycerolphosphatide molecule on the hydrolysis of this class of compounds by plastid phosphatidase C showed that the presence of both fatty acid ester groups is necessary for enzymatic reaction; that release of nitrogenous bases occurred, in the presence of ethyl ether, from phosphatidyl cholines, phosphatidyl ethanolamine, and phosphatidyl serine; and that a phosphatidyl choline was hydrolyzed more rapidly than the corresponding phosphatidyl ethanolamine or phosphatidyl serine. The rate of hydrolysis of phosphatidyl choline was influenced greatly by the chain length and degree of unsaturation of the fatty acids. The corresponding phosphatidic acid formed in the hydrolysis of (dipalmitoyl)- or (dipalmitoleyl)-lecithin by carrot phosphatidase C was isolated. Studies on the hydrolysis of crude soybean phosphatide by phosphatidase C showed that both choline and ethanolamine were liberated in the absence of ethyl ether, at an optimum pH of 4.8; in the presence of ether, the rate of liberation of each base was increased, and the pH optimum was between 4.8 and 6. Soybean phosphatide probably contains a substance that stimulates the enzymatic hydrolysis.

HYDROLYSIS OF GLYCEROLPHOSPHATIDES BY PLASTID PHOSPHATIDASE C

1956 ◽  
Vol 34 (1) ◽  
pp. 967-980 ◽  
Author(s):  
Morris Kates
Keyword(s):  
Ethyl Ether ◽  
Phosphatidyl Choline ◽  
Phosphatidyl Ethanolamine ◽  
Enzymatic Reaction ◽  
Phosphatidyl Serine ◽  
Ph Optimum ◽  
Soybean Phosphatide ◽  
Optimum Ph ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Studies of the influence of structural variation in the glycerolphosphatide molecule on the hydrolysis of this class of compounds by plastid phosphatidase C showed that the presence of both fatty acid ester groups is necessary for enzymatic reaction; that release of nitrogenous bases occurred, in the presence of ethyl ether, from phosphatidyl cholines, phosphatidyl ethanolamine, and phosphatidyl serine; and that a phosphatidyl choline was hydrolyzed more rapidly than the corresponding phosphatidyl ethanolamine or phosphatidyl serine. The rate of hydrolysis of phosphatidyl choline was influenced greatly by the chain length and degree of unsaturation of the fatty acids. The corresponding phosphatidic acid formed in the hydrolysis of (dipalmitoyl)- or (dipalmitoleyl)-lecithin by carrot phosphatidase C was isolated. Studies on the hydrolysis of crude soybean phosphatide by phosphatidase C showed that both choline and ethanolamine were liberated in the absence of ethyl ether, at an optimum pH of 4.8; in the presence of ether, the rate of liberation of each base was increased, and the pH optimum was between 4.8 and 6. Soybean phosphatide probably contains a substance that stimulates the enzymatic hydrolysis.

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