scholarly journals Wheat-germ aspartate transcarbamoylase. The effect of ligands on the inactivation of the enzyme by trypsin and denaturing agents

1973 ◽  
Vol 131 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Robert J. Yon
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Low Concentrations ◽  
Alkaline Conditions ◽  
High Concentrations ◽  
Very High ◽  
Resistant State

In the absence of added ligands aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ is inactivated fairly rapidly by trypsin, by heat (60°C), by highly alkaline conditions (pH11.3) and by sodium dodecyl sulphate. Addition of UMP alone, at low concentrations, decreases the rate of inactivation by each of these agents significantly. Carbamoyl phosphate alone does not alter the rate of inactivation by trypsin and by the detergent, but it antagonizes the effect of UMP in protecting the enzyme against these agents. These results have been interpreted to mean that two conformational states are reversibly accessible to the enzyme, namely an easily inactivated state favoured in the presence of carbamoyl phosphate and a more resistant state favoured in the presence of UMP. In the absence of ligands the enzyme is in the easily inactivated conformation. At very high concentrations l-aspartate also protects the enzyme but to a smaller extent than UMP. Some implications of these results are discussed.

The Development of a Radioimmunoassay for the Measurement of Urinary Tammhorsfall Glycoprotein in the Presence of Sodium Dodecyl Sulphate

1973 ◽  
Vol 44 (2) ◽  
pp. 163-179 ◽  
Author(s):  
Anne M. S. Grant ◽  
A. Neuberger
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Solid Phase ◽  
Excretion Rate ◽  
Sodium Dodecyl ◽  
Antibody Concentration ◽  
Low Concentrations ◽  
Freeze Dried ◽  
Ionic Detergent ◽  
Normal Humans

1. A specific and quantitative radioimmunoassay was developed for the measurement of low concentrations of human and rabbit Tamm—Horsfall glycoprotein in the presence of other proteins. Antibody-coated tubes were used as a solid phase in the assay and the optimum antibody concentration and duration of antibody coating were established. 2. Pure Tamm—Horsfall glycoprotein was labelled with 125I and, because of its apparent susceptibility to radiation damage, was labelled at weekly intervals. 3. Sodium dodecyl sulphate, an ionic detergent, was included in the assay at a final concentration of 0.0005% to disaggregate the glycoprotein. An overnight preincubation step in the presence of the detergent was necessary before the disaggregated glycoprotein solutions were allowed to react with the antibody. Pretreatment of the tracer with detergent was not necessary. 4. Two glycoprotein standards were prepared fresh for each assay from freeze-dried material. The average linear range of the assay was between approx. 150 ng/ml and 2.5 μg/ml. Albumin was only shown to interfere with the assay at concentrations greater than 100 μg/ml. 5. Urines were dialysed against water for 3 days before assay to remove inhibitory material. Urines were never frozen as this was found to affect the assay. 6. A recovery experiment showed that the pure freeze-dried standard behaved in an immunologically identical way to the urinary glycoprotein. 7. Human Tamm-Horsfall glycoprotein cross-reacted with guinea-pig anti-(rabbit Tamm—Horsfall) antiserum and rabbit Tamm—Horsfall glycoprotein cross-reacted with guinea-pig anti-(human Tamm—Horsfall) antiserum, but not with rabbit anti-(human Tamm—Horsfall) antiserum. This showed a partial immunological identity between Tamm-Horsfall glycoprotein from humans and rabbits which was only evident when the antiserum was raised in a third species. 8. The excretion rate of Tamm—Horsfall glycoprotein in normal humans was found to be 48.1 ± 9.6 (SD) mg/24 h for males and 50.5 ± 14.8 (SD) mg/24 h for females. The mean excretion rate of the glycoprotein in New Zealand White rabbits was 34.8 ± 7.9 mg/24 h.

scholarly journals Effects of lipids on nucleotide inhibition of wheat-germ aspartate transcarbamoylase: evidence of an additional level of control?

1996 ◽  
Vol 313 (2) ◽  
pp. 669-673 ◽  
Author(s):  
Ashan KHAN ◽  
Babur Z. CHOWDHRY ◽  
Robert J. YON
Keyword(s):  
Fatty Acids ◽  
Wheat Germ ◽  
Hill Coefficient ◽  
Aspartate Transcarbamoylase ◽  
Kinetic Effect ◽  
Carbamoyl Phosphate ◽  
Allosteric Mechanism ◽  
Additional Level ◽  
Nucleotide Inhibition ◽  
Metabolic Significance

Wheat-germ aspartate transcarbamoylase, a monofunctional trimer, is strongly inhibited by uridine 5ʹ-monophosphate (UMP), which shows kinetic interactions with the substrate, carbamoyl phosphate, suggesting a classical allosteric mechanism of regulation. Inhibition of the purified enzyme by UMP was amplified in the presence of a variety of ionic lipids at concentrations low enough to preclude denaturation. In the absence of UMP, most of these compounds had no kinetic effect or were slightly activating. Two phospholipids did not show the effect. In a homologous series of fatty acids (C6-C16), the potentiating effect was only seen with homologues greater than C8, reaching a maximum at C12. The effect of dodecanoate (C12) on kinetic cooperativity (UMP as variable ligand) was studied. At each of several fixed concentrations of carbamoyl phosphate, dodecanoate had a pronounced effect on the half-saturating concentration of UMP, which was reduced by about half in every case, indicating substantially tighter binding of UMP. However, dodecanoate had relatively little effect on the kinetic Hill coefficient for the cooperativity of UMP. The possible metabolic significance of these effects is discussed.

scholarly journals Mechanism of neutrophil chemiluminescence induced by wheat germ agglutinin: partial characterization of the antigens recognized by wheat germ agglutinin

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1094-1102 ◽  
Author(s):  
Y Ozaki ◽  
J Iwata ◽  
T Ohashi
Keyword(s):  
Membrane Proteins ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Binding Property ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Cell Extracts ◽  
High Concentrations

Abstract Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N- acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.

scholarly journals Detection of endotoxins with the Limulus test in burned and unburned mice infected with different species of gramnegative bacteria

1975 ◽  
Vol 75 (1) ◽  
pp. 99-112 ◽  
Author(s):  
R. J. Jones ◽  
E. A. Roe ◽  
R. E. Dyster
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Low Concentrations ◽  
High Concentrations ◽  
Very High ◽  
Limulus Test

SUMMARYThe Limulus test detected endotoxins in the plasma of burned and unburned mice infected with different species of gram-negative bacteria. Individual strains of different species of gram-negative bacteria produced different amounts of endotoxin in the plasma of infected mice. Plasma from mice given lethal infections showed very high concentrations of endotoxin. Low concentrations of endotoxin in the plasma were tolerated by mice but high concentrations were invariably fatal. A polyvalent pseudomonas vaccine reduced endotoxin in the plasma of mice given lethal infections of Pseudomonas aeruginosa.

scholarly journals The use of sodium perchlorate in deproteinization during the preparation of nucleic acids

1973 ◽  
Vol 135 (3) ◽  
pp. 559-561 ◽  
Author(s):  
John Wilcockson
Keyword(s):  
Nucleic Acids ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sodium Perchlorate ◽  
High Concentration ◽  
High Concentrations

Sodium perchlorate in high concentrations will remove from solution the detergent sodium dodecyl sulphate and protein complexed with it. This and the failure of proteins to be precipitated by ethanol from solutions containing a high concentration of sodium perchlorate can be utilized as efficient, rapid and simple deproteinization procedures during the preparation of nucleic acids.

Copper, nickel, and thallium uptake by the lichen Cladina rangiferina

1981 ◽  
Vol 59 (1) ◽  
pp. 91-100 ◽  
Author(s):  
M. A. S. Burton ◽  
P. LeSueur ◽  
K. J. Puckett
Keyword(s):  
Metal Uptake ◽  
Deionized Water ◽  
Metabolic Inhibitors ◽  
Temperature Dependent ◽  
Low Concentrations ◽  
Nickel Uptake ◽  
High Concentrations ◽  
Uptake Studies ◽  
Cladina Rangiferina ◽  
Very High

Metal uptake studies with Cladina rangiferina showed that the affinity for nickel was much lower than for copper or thallium. Nickel uptake was not decreased by the absence of light or oxygen or by pretreatment with metabolic inhibitors. Nickel uptake was not temperature dependent but was very dependent upon pH.Cation-exchange studies demonstrated that there was a stoichiometric exchange of Ni2+ for Sr2+, and Cu2+ for Sr2+. The exchange of Tl+ for Sr2+ was not stoichiometric, excess Tl+ was accumulated in relation to the Sr2+ released. The ratio of Sr2+:Tl+ exchange increased with increasing Tl+ availability from 1:9 (12.5 μmol Tl+ available/g of lichen) to 1:2 (500 μmol Tl+ available). Acid-treated lichen gave the expected exchange ratio of 1:2. Washing of the thalli with deionized water resulted in the continued loss of Tl+ from acid-treated and live C. rangiferina. Copper and nickel were not released in this manner.Increasing concentrations of copper and thallium produced a corresponding loss of potassium from the thallus. The potassium loss was initiated at low concentrations of copper and thallium whereas very high concentrations of manganese and nickel were required to bring about the same response.

scholarly journals Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility

1984 ◽  
Vol 221 (2) ◽  
pp. 289-296 ◽  
Author(s):  
S C J Cole ◽  
R J Yon
Keyword(s):  
Conformational Changes ◽  
Wheat Germ ◽  
Binding Pocket ◽  
Order Rate Constant ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Peptide Bonds ◽  
First Order ◽  
Mediated Effects ◽  
Arginine Residues

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.

scholarly journals Wheat-germ aspartate transcarbamoylase. Kinetic behaviour suggesting an allosteric mechanism of regulation

1972 ◽  
Vol 128 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Robert J. Yon
Keyword(s):  
Wheat Germ ◽  
Initial Rate ◽  
Aspartate Transcarbamoylase ◽  
Kinetic Properties ◽  
Quasi Equilibrium ◽  
Carbamoyl Phosphate ◽  
Partial Explanation ◽  
Enzyme Binding ◽  
Hill Coefficients ◽  
The Hill

1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25°C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both Vmax. and Km in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting Vmax.. Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.

scholarly journals Etude, dans le raisin, de l'activité β-glucosidase

OENO One ◽  
1988 ◽  
Vol 22 (2) ◽  
pp. 125
Author(s):  
C. Biron ◽  
Robert Cordonnier ◽  
Ollivier Glory ◽  
Ziya Günata ◽  
Jean-Claude Sapis
Keyword(s):  
Plant Material ◽  
Grape Berry ◽  
Cabernet Sauvignon ◽  
Mature Fruit ◽  
Glucosidase Activity ◽  
Low Concentrations ◽  
High Concentrations ◽  
Grape Leaf ◽  
Method Of Extraction ◽  
Very High

<p style="text-align: justify;">Dans ce travail, on étudie l'activité β-glucosidase du raisin. Une méthode d'extraction de l'enzyme à partir du matériel végétal a été mise au point et optimisée. L'activité enzymatique est mesurée spectrophotométriquement avec le paranitrophénylglucopyranoside comme substrat. Dans la baie de raisin, la β-glucosidase est concentrée dans les parties solides, pellicule et pulpe. Dans le jus, elle est en faible quantité. Dans la feuille de vigne, l'activité est élevée dans le limbe, faible dans le pétiole. Au cours de la maturation du raisin l'activité β-glucosidase augmente jusqu'à la maturité. Au-delà, jusqu'au stade de surmaturation, elle reste constante. Dans les raisins mars, la β-glucosidase est présente dans toutes les variétés étudiées, aussi bien dans les variétés aromatiques (Muscat d'Alexandrie, Muscat de Frontignan, Muscat de Hambourg) que dans celles non aromatiques (Carignan, Grenache, Cinsaut, Baroque, Cabernet-Sauvignon, Syrah, Merlot). Pour un millésime donné, l'activité β-glucosidase varie selon la variété. Les activités les plus élevées ont été rencontrées dans les Muscats, le Carignan, la Syrah et le Merlot. Elles sont environ 2,8 fois plus élevées que les activités les plus faibles. Pour une variété donnée, les niveaux d'activité varient fréquemment selon le millésime.</p><p style="text-align: justify;">+++</p><p style="text-align: justify;">β-glucosidase activity in grape was studied. A method of extraction of the enzyme from plant material was optimized and glucosidase activity measured spectrophotometrically using paranitrophenylglucopyranoside as substrate. High concentrations of β-glucosidase were found in grape berry solids (skin and pulp) and low concentrations in the juice. This distribution is similar to that of free terpenols in the same parts of the berry. The β-glucosidase content were very high in grape leaf blades and low in stems. Activity of the enzyme increased during maturation of the fruit. It was found in mature fruit of both aromatic (Muscat of Alexandria, Muscat of Frontignan, Muscat of Hamburg) and non-aromatic (Carignane, Grenache, Cinsaut, Baroque, Cabernet-Sauvignon, Sirah, Merlot) varieties. β-glucosidase activity varied according to variety for a given vintage. The highest activities (approximately 2.8 times higher than the lowest observed) were found in the differents Muscats and also in non-aromatic varieties such as Sirah, Merlot and Carignane. β-glucosidase activity in a given variety was frequently found to vary considerably from one vintage to another.</p>

Mitotic blocking agents for suspension cultures of maize 'Black Mexican Sweet' cell lines

Genome ◽  
1989 ◽  
Vol 32 (3) ◽  
pp. 475-478 ◽  
Author(s):  
J. Stadler ◽  
R. Phillips ◽  
M. Leonard
Keyword(s):  
Mitotic Index ◽  
Culture Medium ◽  
Colchicine Treatment ◽  
Mitotic Arrest ◽  
Suspension Cultures ◽  
Metaphase Arrest ◽  
Low Concentrations ◽  
High Concentrations ◽  
Very High ◽  
Black Mexican Sweet

Good metaphase arrest (25% mitotic index) in maize (Zea mays L.) 'Black Mexican Sweet' suspension cultures can be obtained with colchicine treatment alone, but only if very high concentrations (0.5%; 12.5 mM) are used. This colchicine concentration is 5- to 10-fold greater than that required for optimum mitotic arrest in cell cultures of other plant species. In contrast, we report that the herbicide amiprophos methyl (APM) is a much more efficient metaphase inhibitor for 'Black Mexican Sweet' suspension cultures than colchicine. Low concentrations (50 μm) of APM applied for 21–28 h produce a similar 25% mitotic index, and the growth-inhibiting effects of this treatment are undetectable 24 h after the removal of APM from the culture medium, APM, therefore, may be a useful agent for mitotic arrest in experiments which require survival of the treated cells. Another antitubulin herbicide, trifuralin, also was tested for ability to promote metaphase arrest in 'Black Mexican Sweet' cultures, but it proved to be ineffective.Key words: mitotic arrest, colchicine, phosphoric amide herbicide.

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