Phosphoribosyl Pyrophosphate Degradation in Human Tissues

1974 ◽  
Vol 52 (12) ◽  
pp. 1162-1166 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Maximum Activity ◽  
Human Tissues ◽  
Phosphoribosyl Pyrophosphate ◽  
Phosphorylated Compounds ◽  
Tissue Homogenates ◽  
Sugar Derivatives ◽  
Hydrolysis Of ◽  
Alkaline Range

Enzymatic hydrolysis of phosphoribosyl pyrophosphate (PP-ribose-P) in dialyzed human tissue homogenates had a specific activity ranging from 0.15 to 1.37 μmol mg−1 h−1. Our observations on human placenta have characterized this reaction: (1) PP-ribose-P was degraded in the absence of Mg with stoichiometric release of Pi at 37 °C; (2) the reaction occurred from pH 5 to 10.5 with the maximum activity in the alkaline range; (3) PP-ribose-P degrading activity was localized mainly in the microsomal fraction; (4) alkaline phosphatase rather than 5′-nucleotidase was responsible for the degradation of PP-ribose-P; (5) the Km for PP-ribose-P was 0.3 mM; (6) PP-ribose-P hydrolysis was altered by many phosphorylated compounds, both nucleoside and sugar derivatives, Pi, and PPi in concentrations ranging from 0.1 to 10.0 mM.

scholarly journals Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. ex4484_79

2021 ◽  
Vol 12 ◽  
Author(s):  
Anke Chen ◽  
Dan Wang ◽  
Rui Ji ◽  
Jixi Li ◽  
Shaohua Gu ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Maximum Activity ◽  
Homologous Proteins ◽  
Glycosidic Bonds ◽  
Catalytic Sites ◽  
Potential Applications ◽  
Beta Glucosidase ◽  
Hydrolysis Of

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

scholarly journals Studies on bone enzymes. Distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates

1965 ◽  
Vol 97 (2) ◽  
pp. 389-392 ◽  
Author(s):  
G Vaes ◽  
P Jacques
Keyword(s):  
Cytochrome Oxidase ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Distribution Patterns ◽  
Mitochondrial Fraction ◽  
Acid Hydrolases ◽  
Mitochondrial Fractions ◽  
Tissue Homogenates ◽  
Isopycnic Centrifugation

1. When bone homogenates were fractionated according to the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed except cytochrome oxidase were found to occur partly in soluble and partly in particulate fractions. Among the particle-bound enzymes, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome oxidase, in the microsomal fraction for alkaline phenylphosphatase and in the light-mitochondrial fraction for eight acid hydrolases and for catalase. 2. Combined heavy-mitochondrial and light-mitochondrial fractions were subfractionated by isopycnic centrifugation in density gradients of sucrose or glycogen. In the various systems tried, cytochrome oxidase showed a relatively narrow distribution range with a sharp peak; the acid hydrolases and catalase showed flat and irregular distribution patterns, differing slightly in shape from one enzyme to the other. However, it was not possible to achieve a marked separation between the various enzymes under study. 3. It is concluded from these results that the acid hydrolases belong to special cytoplasmic particles, probably lysosomes, and that these particles are physically and enzymically heterogeneous. Catalase appears to be non-mitochondrial and could also belong to the lysosomes; but the possibility of an association with another type of particle must be kept in mind in view of what is known of liver catalase. Alkaline phenylphosphatase is largely attached to microsomal elements.

scholarly journals Identification of an Invertase With High Specific Activity for Raffinose Hydrolysis and Its Application in Soymilk Treatment

2021 ◽  
Vol 12 ◽  
Author(s):  
Juanjuan Liu ◽  
Jing Cheng ◽  
Min Huang ◽  
Chen Shen ◽  
Ke Xu ◽  
...  
Keyword(s):  
Nutritional Value ◽  
Specific Activity ◽  
High Specific Activity ◽  
Maximum Activity ◽  
Beneficial Bacteria ◽  
Human Gut ◽  
Mild Conditions ◽  
Alternative Approach ◽  
Hydrolysis Of

The hydrolyzation of raffinose into melibiose by using invertases under mild conditions improves the nutritional value of soybean products. However, this strategy has received little attention because a suitable invertase remains lacking. In this study, a novel invertase named InvDz13 was screened and purified from Microbacterium trichothecenolyticum and characterized. InvDz13 was one of the invertases with the highest specific activity toward raffinose. Specifically, it had a specific activity of 229 U/mg toward raffinose at pH 6.5 and 35°C. InvDz13 retained more than 80% of its maximum activity at pH 5.5–7.5 and 25–40°C and was resistant to or stimulated by most cations that presented in soymilk. In soymilk treated with InvDz13 under mild conditions, melibiose concentration increased from 3.1 ± 0.2 to 6.1 ± 0.1 mM due to raffinose hydrolyzation by InvDz13. Furthermore, the prebiotic property of InvDz13-treated soymilk was investigated via in vitro fermentation by human gut microbiota. Results showed that InvDz13 treatment increased the proportion of the beneficial bacteria Bifidobacterium and Lactobacillus by 1.6- and 3.7-fold, respectively. By contrast, the populations of Escherichia and Collinsella decreased by 1.8- and 11.7-fold, respectively. Thus, our results proved that the enzymatic hydrolysis of raffinose in soymilk with InvDz13 was practicable and might be an alternative approach to improving the nutritional value of soymilk.

scholarly journals Purification and properties of myo-inositol-1-phosphatase from bovine brain

1988 ◽  
Vol 253 (2) ◽  
pp. 387-394 ◽  
Author(s):  
P V Attwood ◽  
J B Ducep ◽  
M C Chanal
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Brain ◽  
Native Enzyme ◽  
Hydroxyl Groups ◽  
Inositol 1 ◽  
Substrate Structure ◽  
Purification And Properties ◽  
Phosphorylated Compounds ◽  
Hydrolysis Of

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.

Ontogeny of gastric H(+)-K(+)-ATPase in suckling rabbits

1990 ◽  
Vol 259 (6) ◽  
pp. G913-G921 ◽  
Author(s):  
J. M. Crothers ◽  
W. W. Reenstra ◽  
J. G. Forte
Keyword(s):  
Microsomal Fraction ◽  
Apical Membrane ◽  
Specific Activity ◽  
Oxyntic Cell ◽  
Age Related ◽  
Nitrophenyl Phosphate ◽  
Weaning Age ◽  
Age Range ◽  
Hydrolysis Of

Gastric mucosal homogenates were prepared from resting and stimulated stomachs of rabbits, age 3-57 days postnatal, and fractionated by differential centrifugation. Total H(+)-K(+)-adenosinetriphosphatase (ATPase) (assayed as K(+)-dependent ouabain-insensitive hydrolysis of p-nitrophenyl phosphate) was low in the first 3 wk but rapidly accumulated between days 20 and 43. Specific activity rose eightfold from day 3 to a typically adult level of 2 mumol.mg-1.h-1 by day 43. The microsomal fraction (P3) was subfractionated on sucrose gradients (20, 27, and 33% steps or 10-40% continuous gradient). H(+)-K(+)-ATPase from P3 of resting stomachs was distributed bimodally on the continuous gradients, with activity mainly in the denser peak (or on the 33% sucrose step) before day 20, but accumulating mainly in the lighter peak (or in the lighter step-gradient fractions) after day 20. Throughout the age range tested, in vivo stimulation with histamine just before removal of the stomach caused a loss of most H(+)-K(+)-ATPase from P3 and an increase in H(+)-K(+)-ATPase in a lower-speed fraction P1. Thus, even in the 1st postnatal wk, when H(+)-K(+)-ATPase is low, most of the enzyme occurs in cells with histamine H2 receptors and all the intracellular mechanisms for fusion of oxyntic cell tubulovesicles (enriched in P3) with the apical membrane (enriched in P1). These studies delineate a 3-wk period of sharply accelerated synthesis of H(+)-K(+)-ATPase before weaning. Age-related changes in distribution of H(+)-K(+)-ATPase among microsomal density subfractions suggest maturational changes either in the intracellular partitioning of the enzyme or in properties of the membranes containing the enzyme.

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

INCORPORATION OF IODIDE INTO ORGANIC COMPOUNDS IN THE GUINEA PIG THYROID

1963 ◽  
Vol 43 (1) ◽  
pp. 110-118 ◽  
Author(s):  
R. Ekholm ◽  
T. Zelander ◽  
P.-S. Agrell
Keyword(s):  
Guinea Pig ◽  
Protein Binding ◽  
Organic Compounds ◽  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Thyroid Tissue ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hydrolysis Of

ABSTRACT Guinea pigs, kept on a iodine-sufficient diet, were injected with Na131I and the thyroids excised from 45 seconds to 5 days later. The thyroid tissue was homogenized and separated into a combined nuclear-mitochondrial-microsomal fraction and a supernatant fraction by centrifugation at 140 000 g for one hour. Protein bound 131iodine (PB131I) and free 131iodide were determined in the fractions and the PB131I was analysed for monoiodotyrosine (MIT), diiodotyrosine (DIT) and thyroxine after hydrolysis of PB131I. As early as only 20 minutes after the Na131I-injection almost 100% of the particulate fraction 131I was protein bound. In the supernatant fraction the protein binding was somewhat less rapid and PB131I values above 90% of total supernatant 131I were not found until 3 hours after the injection. In all experiments the total amount of PB131I was higher in the supernatant than in the corresponding particulate fraction. The ratio between supernatant PB131I and pellet PB131I was lower in experiments up to 3 minutes and from 2 to 5 days than in experiments of 6 minutes to 20 hours. Hydrolysis of PB131I yielded, even in the shortest experiments, both MIT and DIT. The DIT/MIT ratio was lower in the experiments up to 2 hours than in those of 3 hours and over.

In vitro 19-norandrogen synthesis by equine placenta requires the participation of aromatase

1995 ◽  
Vol 144 (3) ◽  
pp. 517-525 ◽  
Author(s):  
S Moslemi ◽  
P Silberzahn ◽  
J-L Gaillard
Keyword(s):  
Silica Gel ◽  
Aromatase Inhibitors ◽  
Column Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Term Placenta ◽  
Flow Detector

Abstract Explants of equine full-term placenta have been shown to synthesize 19-norandrogens from labelled androgens. Steroid metabolites were purified by silica-gel column chromatography then analysed and quantified by C18-reverse-phase HPLC coupled to a radioactive flow detector. 19-Norandrostenedione was subsequently recrystallized to constant specific activity, providing unequivocal evidence of its synthesis by the equine placenta. 19-Norandrostenedione synthesis appeared to be localized in the microsomal fraction. Regardless of the substrate used, formation of 19-norandrogens was far weaker than that of oestrogens; moreover, the yield of 17-oxosteroids produced was much greater than that of 17β-hydroxysteroids, suggesting the presence of a dehydrogenase with predominant oxidative activity. Sulphoconjugated steroids formed were less than 0·5% of total steroids. Although 19-nortestosterone could not be generated by equine purified aromatase incubated with labelled testosterone, the synthesis of 19-norandrogens and oestrogens by equine placental explants was blocked by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole. Our results provide evidence for a placental origin of at least a part of the 19-norandrogens previously identified in the blood of the pregnant mare. Furthermore, it is suggested that 19-norandrogen biosynthesis would involve the enzymatic metabolism of 19-oxygenated androgens formed by equine aromatase. Journal of Endocrinology (1995) 144, 517–525

scholarly journals Biochemical characterisation of α-glucosidase and β-glucosidase in the alimentary canal of larval Leptinotarsa decemlineata SAY, 1824 (Coleoptera: Chrysomelidae)

2014 ◽  
Vol 83 (4) ◽  
pp. 281-294 ◽  
Author(s):  
Majid Kazzazi ◽  
Fahimeh Dehghanikhah ◽  
Hossein Madadi ◽  
Vahid Hossseininaveh
Keyword(s):  
Incubation Time ◽  
Leptinotarsa Decemlineata ◽  
Digestive Enzymes ◽  
Specific Activity ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Pest Population ◽  
Biochemical Characterisation ◽  
Leptinotarsa Decemlineata Say ◽  
Environmentally Safe

ABSTRACT Host plant resistance is an environmentally safe method used for reducing a pest population. Basically, when developing resistant cultivars one needs to study the biochemical characteristics of the digestive enzymes in the insect’s midgut. In this study, the activities of α- and β-glucosidase were determined from Leptinotarsa decemlineata midgut using p-nitrophenyl-α-Dglucopyranoside and p-nitrophenyl-β-D-glucopyranoside as substrates respectively. The results showed that the specific activity of α- and β-glucosidase from 4th instar larvae midguts of L. decemlineata were 5.14 and 5.48 Umg-1 protein respectively. The activity of α-glucosidase was optimal at pH 4, whereas the maximum activity of β-glucosidase in the midgut of L. decemlineata occurred at pH 4-5.5. Both enzymes were stable at pH 3-8 over an incubation time of 8 hours. The respective activities of α- and β-glucosidase were at their highest at 45 °C and 50 °C, but they were not stable at 50 °C during an incubation time of 8 days. Furthermore, our data showed that MgCl2, Tris and urea have a moderate but SDS a severe inhibitory effect on enzyme activity. Biochemical characterisation revealed one and three bands of α- and β-glucosidase activities in the midgut of L. decemlineata respectively.

Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density

1988 ◽  
Vol 8 (10) ◽  
pp. 4169-4173
Author(s):  
M Hoshino ◽  
M Kawakita ◽  
S Hattori
Keyword(s):  
Gene Product ◽  
Cell Density ◽  
Cultured Cells ◽  
Specific Activity ◽  
Gtpase Activating Protein ◽  
Ras Gene ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Almost All ◽  
Hydrolysis Of

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

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