scholarly journals Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2

2001 ◽  
Vol 358 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Ignacio G. BRAVO ◽  
Sofía BARRALLO ◽  
Miguel A. FERRERO ◽  
Leandro B. RODRÍGUEZ-APARICIO ◽  
Honorina MARTÍNEZ-BLANCO ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Specific Activity ◽  
Polysialic Acid ◽  
Kinetic Mechanism ◽  
Pasteurella Haemolytica ◽  
Kinetic Properties ◽  
Acid Activation ◽  
Acetylneuraminic Acid ◽  
Apparent Homogeneity ◽  
Encapsulated Bacteria

Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77mM for CTP and 1.82mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.

scholarly journals Isolation, characterization and the role of rabbit testicular arysulphatase A in fertilization

1979 ◽  
Vol 181 (2) ◽  
pp. 331-337 ◽  
Author(s):  
A A Farooqui ◽  
P N Srivastava
Keyword(s):  
Specific Activity ◽  
Deae Cellulose ◽  
Cumulus Cells ◽  
Kinetic Properties ◽  
Neutral Sugar ◽  
Acetylneuraminic Acid ◽  
Crude Homogenate ◽  
Step Procedure ◽  
Arylsulphatase A

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.

scholarly journals The fate of orally administered sialic acid: First insights from patients with N-acetylneuraminic acid synthase deficiency and control subjects

2021 ◽  
Vol 28 ◽  
pp. 100777
Author(s):  
Christel Tran ◽  
Licia Turolla ◽  
Diana Ballhausen ◽  
Sandrine Cornaz Buros ◽  
Tony Teav ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Acetylneuraminic Acid ◽  
Control Subjects ◽  
And Control

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

scholarly journals N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 815
Author(s):  
Cindy M. Spruit ◽  
Nikoloz Nemanichvili ◽  
Masatoshi Okamatsu ◽  
Hiromu Takematsu ◽  
Geert-Jan Boons ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Animal Models ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Upper Respiratory Tract ◽  
Human Influenza ◽  
Influenza A Viruses ◽  
Sialic Acid Content ◽  
Acetylneuraminic Acid

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)-/- knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

Interaction of Mycoplasma gallisepticum with sialyl glycoproteins

1980 ◽  
Vol 30 (2) ◽  
pp. 353-361
Author(s):  
L R Glasgow ◽  
R L Hill
Keyword(s):  
Sialic Acid ◽  
Mycoplasma Gallisepticum ◽  
Binding Assays ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Direct Binding ◽  
Membrane Bound ◽  
Strict Specificity ◽  
Human Glycophorin ◽  
Acid Structure

The binding of several glycoproteins to freshly grown and harvested cells of Mycoplasma gallisepticum was examined. Only human glycophorin, the major sialoglycoprotein of the erythrocyte membrane, bound tightly as judged by direct binding assays with 125I-labeled glycoproteins. Neuraminidase-treated glycophorin did not bind, suggesting that binding is mediated through sialic acid groups. Although other sialoglycoproteins did not appear to bind M. gallisepticum by direct binding assays, some inhibited the binding of glycophorin. The best inhibitors had a mucin-like structure, with high molecular weights and high sialic acid contents. N-acetylneuraminic acid appeared to be the favored sialic acid structure for binding, but there was no strict specificity for its anomeric linkage. Neuraminidase activity could not be detected on the surface of M. gallisepticum, suggesting that this enzyme is not involved in the mechanism of adherence of sialoglycoproteins. Binding of sialoglycoproteins was time dependent, however, and markedly diminished with increasing ionic strength, but was largely unaffected between pH 4 and 9.

Porcine Sugar Nucleotide: Glycoprotein Glycosyltransferases. I. Blood Serum and Liver Sialyltransferase

1971 ◽  
Vol 49 (7) ◽  
pp. 829-837 ◽  
Author(s):  
Roger L. Hudgin ◽  
Harry Schachter
Keyword(s):  
Sialic Acid ◽  
High Speed ◽  
Liver Homogenate ◽  
Acetone Powder ◽  
Acid Glycoprotein ◽  
Acetylneuraminic Acid ◽  
Pork Liver ◽  
Terminal Galactose ◽  
And Function ◽  
Powder Extract

The properties of CMP-N acetylneuraminic acid: glycoprotein sialyltransferase have been studied in pork serum, a crude pork liver homogenate, and a soluble acetone powder extract prepared from pork liver. Whereas the crude liver homogenate enzyme is activated by the detergent Triton X-100, this detergent has no effect on the activities of either serum or acetone powder extract; since high-speed centrifugation does not sediment the enzyme activities of the latter two preparations, it is concluded that they are soluble. Comparison of the membrane-bound and soluble liver enzymes indicates that the membrane modifies kinetic behavior only to a limited extent. In both liver and serum, a single sialyltransferase is responsible for incorporation of sialic acid into α1-acid glycoprotein, fetuin, and N-acetyllactosamine, and sialic acid incorporation occurs whenever a terminal galactose linked (β, 1 → 4) to a penultimate N-acetylglucosamine is presented to the enzyme. Although the serum enzyme resembles the liver enzyme, both the source and function of serum sialyltransferase are unknown.

Purification to homogeneity and characterization of nonproteolyzed potato (Solanum tuberosum) tuber hexokinase 1

Botany ◽  
2011 ◽  
Vol 89 (5) ◽  
pp. 289-299 ◽  
Author(s):  
Marie-Claude Moisan ◽  
Jean Rivoal
Keyword(s):  
Solanum Tuberosum ◽  
Specific Activity ◽  
Solanum Tuberosum L ◽  
Extraction Procedure ◽  
Hydrophobic Interaction Chromatography ◽  
Solanum Chacoense ◽  
Kinetic Properties ◽  
Chromatographic Separations ◽  
The Stability ◽  
Fast Flow

We have developed an extraction procedure that improves the stability of potato ( Solanum tuberosum L.) tuber hexokinase (HK) after extraction. Using this protocol, we showed that at least four HK isoforms are present in this tissue, and they can be separated by hydrophobic-interaction chromatography on a butyl-Sepharose™ 4 Fast Flow column. One of the main HK isoforms was purified to homogeneity using further chromatographic separations on red dye, DEAE Fractogel, hydroxyapatite, cibacron blue, and MonoQ matrices. HK-specific activity of this fraction (10.2 U·mg protein–1) corresponds to an enrichment of more than 5500-fold, with a yield of 0.9%. This is the highest reported HK-specific activity from a plant source. The purified enzyme consisted of a monomer with a subunit apparent Mr of 51 kDa when analyzed by SDS–PAGE. This polypeptide was recognized by affinity-purified anti- Solanum chacoense Bitt. recombinant HK IgGs. The protein was digested with trypsin and its digestion products were subjected to MS – MS sequencing after HPLC separation. The sequences of these tryptic peptides matched the predicted coding sequence of the S. tuberosum HK1 gene with a coverage of 57%. Examination of the kinetic properties of the purified protein HK1 indicates that it may be regulated by the internal O2 concentration of the tuber because of its sensitivity to acidic pHs and inhibition by ADP.

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