scholarly journals Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences

1998 ◽  
Vol 331 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Robert G. SPIRO ◽  
Vishnu D. BHOYROO
Keyword(s):  
Periodate Oxidation ◽  
Maximal Activity ◽  
Sialic Acid Residue ◽  
Lymphoid Tissues ◽  
Ph Optimum ◽  
Bivalent Cation ◽  
Selectin Ligands ◽  
Strict Requirement ◽  
Lectin Chromatography ◽  
Triton X

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.

The Uptake of Adenine by Isolated Human Platelet Membranes

1974 ◽  
Vol 32 (02/03) ◽  
pp. 457-464
Author(s):  
Paul C. French ◽  
Jan J. Sixma ◽  
Holm Holmsen
Keyword(s):  
Human Platelet ◽  
Facilitated Diffusion ◽  
Adenine Phosphoribosyltransferase ◽  
Ph Optimum ◽  
Intact Cells ◽  
Platelet Membranes ◽  
Group Translocation ◽  
Triton X

SummaryAdenine uptake into isolated platelet membranes had about the same Km (151 ± 21 • 9 nM) as uptake into intact cells (159 ± 21 nM) and was also competitively inhibited by papaverine and hypoxanthine. No uptake occurred at 0° and accumulated adenine was converted to AMP. AMP was not firmly bound to protein as judged by chromatography of triton X-100 solubilized membranes on Sephadex G25. The pH optimum for adenine uptake was at pH 5-5. Exogenous 5-phosphoribosyl-l-pyrophos- phate strongly stimulated uptake. These data may be explained by uptake of adenine by facilitated diffusion followed by conversion to AMP by adenine phosphoribosyltransferase but group translocation cannot be entirely excluded.

scholarly journals Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

1977 ◽  
Vol 75 (1) ◽  
pp. 135-147 ◽  
Author(s):  
A L Blitz ◽  
R E Fine ◽  
P A Toselli
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Coated Vesicles ◽  
Potassium Oxalate ◽  
Triton X ◽  
Trapping Agent ◽  
The Brain

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

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.

scholarly journals The effects of diphenyleneiodonium and of 2,4-dichlorodiphenyleneiodonium on mitochondrial reactions. Mechanism of the inhibition of oxygen uptake as a consequence of the catalysis of the chloride/hydroxyl-ion exchange

1976 ◽  
Vol 158 (2) ◽  
pp. 317-326 ◽  
Author(s):  
S J Gatley ◽  
H S A Sherratt
Keyword(s):  
Oxygen Uptake ◽  
Absolute Rate ◽  
Proton Extrusion ◽  
Ph Optimum ◽  
Succinate Oxidation ◽  
Hydroxyl Ion ◽  
The Matrix ◽  
Rate Of Decay ◽  
Triton X ◽  
Reactions Mechanism

1. Increasing the substrate concentration only decreased the inhibition of mitochondrial oxidations by diphenyleneiodonium or by 2,4-dichlorophenyleneiodonium by a small amount. 2. Diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium lowered the amounts of succinate, citrate and glutamate accumulated in the matrix of mitochondria in the presence of Cl-, but not in its absences. 2,4-Dichlorodiphenyleneiodonium decreased the accumulation of substrates by mitochondria oxidizing glycerol 3-phosphate. 3. Diphenyleneiodonium caused an alkalinization of the medium with an anaerobic suspension of mitochondria, which was only partly reversed by Triton X-100. 4. The rate of proton extrusion by mitochondria oxidizing succinate was not altered by diphenyleneiodonium or by 2,4-dichlorodiphenyleneiodium, although the rate of decay of proton pulses was increased. 5. 2,4-Dichlorodiphenyleneiodonium shifted the pH optimum for succinate oxidation by intact mitochondria from pH 7.2 to 8.0, whereas there was no effect on that of freeze-thawed mitochondria, which was pH 8.0. 6. The concentration of 2,4-dichlorophenyleneiodonium required to inhibit respiration by 50% is less the higher the absolute rate of oxygen uptake. 7. EDTA, but not EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid] increased the inhibition of respiration by diphenyleneiodonium, 2,4-dichlorodiphenyleneiodonium and by tri-n-propyltin. 8. It is concluded that diphenyleneiodonium and 2,4-dichlorodiphenyleneiodonium limit respiration in Cl--containing medium by causing an acidification of the matrix, and that there are pH-sensitive sites in the respiratory chain between NADH and succinate, and between succinate and cytochrome c.

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.

scholarly journals Identification and characterization of L-selectin ligands in porcine lymphoid tissues

Immunology ◽  
2002 ◽  
Vol 105 (4) ◽  
pp. 441-449 ◽  
Author(s):  
Adil I. Khan ◽  
Dorian O. Haskard ◽  
Rajneesh Malhotra ◽  
R. Clive Landis
Keyword(s):  
Lymphoid Tissues ◽  
Selectin Ligands ◽  
Identification And Characterization

scholarly journals Development, characterization, and subcellular location of DNAse activity in HL-60 cells and monocytes

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 976-983 ◽  
Author(s):  
PJ Roberts
Keyword(s):  
Subcellular Location ◽  
Maximal Activity ◽  
Bimodal Distribution ◽  
Dnase Activity ◽  
Phagocytic Cells ◽  
Ph Optimum ◽  
Free System ◽  
Bacterial Dna ◽  
A Cell ◽  
Acid Dnase

Abstract The digestion of DNA from intact bacteria by human phagocytic cells was measured by the release of solubilized radiolabeled DNA. Two subclones from the human promyelocytic HL-60 cell line were unable to digest bacterial DNA unless they were previously induced to mature by incubation for several days with 1.25% dimethylsulfoxide (DMSO). The maximal capacity of DMSO-induced HL-60 cells to digest DNA was similar to that of monocytes purified from peripheral blood (PB) and much greater than that of neutrophils. The increasing capacity to digest DNA during maturation was associated with the development of acid DNAse activity, measured in a cell-free system, and slightly preceded development of 12-O-tetradecanoyl phorbol 13-acetate-stimulated respiratory burst activity. The acid DNAse had a pH optimum of 5.0 and did not require the presence of calcium or 2-mercaptoethanol (2-ME). A third subclone of HL-60 cells was able to digest DNA from intact bacteria without previous maturation, however, and this was associated with the presence of an alkaline DNAse which had a pH optimum between 7.0 and 8.0 and showed a dependence on calcium and 2-ME for maximal activity. The subcellular location of acid DNAse in DMSO-induced HL-60 cells was similar to that of monocytes in having a bimodal distribution on fractionated sucrose density gradients. The dense peak (mean density 1.195 g/mL) was located in the same region of the gradient as primary granule enzymes but the light peak (mean density 1.137 g/mL) did not codistribute with either plasma membrane, endoplasmic reticulum, or mitochondria, suggesting accumulation in a different organelle.

scholarly journals Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles

1987 ◽  
Vol 244 (1) ◽  
pp. 219-224 ◽  
Author(s):  
J M Jacobs ◽  
N J Jacobs
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chlorophyll Synthesis ◽  
Yeast Mitochondria ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Haem Biosynthesis ◽  
Triton X ◽  
Polypeptide Band

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.

scholarly journals The permeability to calcium of pigeon erythrocyte ‘ghosts’ studied by using the calcium-activated luminescent protein, obelin

1975 ◽  
Vol 152 (2) ◽  
pp. 255-265 ◽  
Author(s):  
Anthony K. Campbell ◽  
Robert L. Dormer
Keyword(s):  
Pyruvate Kinase ◽  
Time Course ◽  
Small Concentration ◽  
Ionophore A23187 ◽  
Erythrocyte Ghosts ◽  
Experimental Conditions ◽  
Bivalent Cation ◽  
Low Concentration ◽  
Triton X ◽  
Obelia Geniculata

1. Obelin, the Ca2+-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ‘ghosts’ in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca2+ within the ‘ghosts’ were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ‘ghosts’ during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ‘ghosts’. Less than 10% of the inulin or pyruvate kinase sealed within the ‘ghosts’ was released under any of the experimental conditions. 3. Triton X-100 (0.1–10%, v/v) made the ‘ghosts’ highly permeable to Ca2+. In the presence of 1mm-Ca2+ and Triton, 95–100% of the obelin was utilized within 10–20s. 4. A time-course of resealing ‘ghosts’ at 37°C showed that over a period of 90min, the ‘ghosts’ became gradually less permeable to Ca2+. ‘Ghosts’ which remained at 0°C retained only a small concentration of obelin and ATP, and were highly permeable to Ca2+. 5. Erythrocyte ‘ghosts’ resealed for 30min at 20°C rather than 37°C were more permeable to Ca2+, as shown by the fact that 92% of the obelin in the ‘ghosts’ was utilized during the first 60s after the addition of 1mm-Ca2+, as opposed to 44% for ‘ghosts’ resealed at 37°C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ‘ghosts’ which were highly permeable to Ca2+ after resealing for 60min at 37°C. Of the obelin in the ‘ghosts’, produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca2+ compared with 23% for ‘ghosts’ produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ‘ghosts’ to Ca2+. Maximum effects of the ionophore (16μg/ml) were obtained by preincubating the ‘ghosts’ with the ionophore A23187 (16μg/ml) in the presence of a low concentration of Mg2+ and in the absence of Ca2+.

scholarly journals The serum opacity reaction of Streptococcus pyogenes: general properties of the streptococcal factor and of the reaction in aged serum

1968 ◽  
Vol 66 (1) ◽  
pp. 37-47 ◽  
Author(s):  
M. J. Hill ◽  
Lewis W. Wannamaker
Keyword(s):  
Streptococcus Pyogenes ◽  
Divalent Cations ◽  
Horse Serum ◽  
Sodium Deoxycholate ◽  
Heat Stability ◽  
Maximal Activity ◽  
Free Enzyme ◽  
Ph Optimum ◽  
Serum Opacity Factor ◽  
High Concentrations

SUMMARYThe capacity of certain strains of Streptococcus pyogenes to produce opacity in aged horse serum has been studied. Cells from all stages of the growth cycle are able to produce opacity. Maximal activity is reached towards the end of the exponential phase of growth.Examination of cell fractions obtained by mechanical breakage and differential centrifugation suggested that the cell-bound activity is predominantly associated with the membrane fraction. Extraction with sodium deoxycholate yields a soluble fraction of high activity.There is considerable strain variation in heat stability of the serum opacity factor. Cell-bound activity is often quite resistant to heat, whereas extracted activity is less stable.Low concentrations of divalent cations have an activating effect, whereas high concentrations inhibit the serum opacity reaction. High concentrations of uni-valent cations are without effect on the cell-free enzyme but have an activating effect on the cell-bound enzyme.For both the cell-bound and the cell-free enzyme the pH optimum was 5·8.Although sensitive to trypsin and pepsin, the serum opacity factor appears to be resistant to streptococcal proteinase. Its activity is destroyed by formaldehyde and by periodate but is unaffected by a number of reducing agents.Pre-heating of the serum or the addition of iodoacetate did not affect the serum opacity reaction. The enhanced cholesterol esterification previously described with fresh serum appears to be a secondary reaction. Even when isolated by relatively gentle methods, α-lipoprotein serves as a substrate only in the presence of crystalline serum albumin.

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