scholarly journals The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme

1993 ◽  
Vol 293 (2) ◽  
pp. 369-375 ◽  
Author(s):  
F L González Flecha ◽  
P R Castello ◽  
A J Caride ◽  
J J Gagliardino ◽  
J P Rossi
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Deleterious Effect ◽  
Kinetic Properties ◽  
Control Activity ◽  
End Products ◽  
Effect Of Glucose ◽  
Inside Out

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.

The soluble adenosine triphosphatase of Thiobacillus ferrooxidans

1975 ◽  
Vol 21 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Catherine Adapoe ◽  
Marvin Silver
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Gel Electrophoresis ◽  
Inorganic Phosphate ◽  
Adenosine Triphosphatase ◽  
Thiobacillus Ferrooxidans ◽  
Polyacrylamide Gel Electrophoresis ◽  
Histochemical Analysis ◽  
Ph Optimum ◽  
Major Band

Adenosine triphosphatase (ATPase) from Thiobacilhis ferrooxidans was purified 55-fold. Polyacrylamide gel electrophoresis of the most purified fraction showed only one major band; histochemical analysis showed that the ATPase activity was associated with this band. The pH optimum is 9–10. The enzyme hydrolyzed ATP stoichiometrically to ADP and inorganic phosphate, the Km for this substrate being 7.75 × 10−3 M. GTP and ITP are alternate substrates, the Km values for these being 6.71 × 10−3 M and 3.12 × 10−3 M, respectively. ADP is slightly hydrolyzed. Magnesium, manganese, and calcium can serve as cofactors; Km values for these are 2.0 × 10−3 M, 9.4 × 10−4 M, and 8.0 × 10−4 M, respectively. The enzyme activity was not activated by either sodium or potassium, but a combination of the two ions were inhibitory. Azide and p-hydroxymercuribenzoate strongly inhibited the enzyme activity, whereas cyanide, dinitrophenol, and N, N′-dicyclohexylcarbodiimide (DCCD) were without effect. The enzyme was cold labile at 0 °C, but was more stable at 18–24 °C.

Effects of fatty acid intermediates on Na+-K+-ATPase activity of cardiac sarcolemma

1982 ◽  
Vol 242 (3) ◽  
pp. H456-H461 ◽  
Author(s):  
K. Owens ◽  
F. F. Kennett ◽  
W. B. Weglicki
Keyword(s):  
Enzyme Activity ◽  
Fatty Acid ◽  
Atpase Activity ◽  
Fatty Acyl ◽  
Cardiac Sarcolemma ◽  
Control Activity ◽  
Initial Concentration ◽  
Palmitoyl Carnitine ◽  
Membrane Bound ◽  
Sarcolemmal Vesicles

The effect of amphiphilic lipid intermediates on the Na+-stimulatable activity of the Na+-K+-ATPase of sarcolemma from adult canine cardiac myocytes was studied. Sarcolemma (mean Na+-stimulatable ATPase activity of 73 mumol.mg sarcolemmal protein-1.h-1) was preincubated (37 degrees C for 10 min at pH 7.2) or rapidly mixed at 0 degrees C with amphiphilic lipid intermediates prior to dilution and assay of enzyme activity. Their effects were dependent on temperature, initial concentration, and the ratio of bound amphiphile to sarcolemmal protein. In particular, pretreatment of freshly prepared sarcolemma at 0 degrees C with arachidonyl CoA (up to 0.25 mM) caused 110% stimulation above control activity; palmitoyl CoA or palmitoyl carnitine under the same conditions caused no significant effect. Despite strong binding to the sarcolemmal vesicles, palmitoyl carnitine (up to 0.4 mM or 5 mumol/mg protein) and palmitoyl CoA (0.1 mM or 1.0 mumol of membrane-bound palmitoyl CoA/mg protein) were ineffective even with preincubation. Palmitoyl CoA was inhibitory above this level. Preincubation (22 degrees C for 10 min) with lysophosphatidylcholine only produced inhibition (40% at 0.75 mM). Thus fatty acyl thioesters of CoA and lysophosphatidyl choline but not palmitoyl carnitine perturb sarcolemmal Na+-K+-ATPase activity.

Determination of Na-K-ATPase activity in single segments of the mammalian nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F105-F113 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz ◽  
F. Morel
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Rapid Freezing ◽  
Phosphate Release ◽  
Large Numbers ◽  
Hydrolysis Of ◽  
Single Tubules

A micromethod for the determination of Na-K-ATPase in discrete segments of nephrons from rabbit, rat, and mouse kidneys is described. To facilitate tubule microdissection, the kidneys were perfused with collagenase after it had been verified that collagenase had no effect on ATPase activity. Individual tubule segments were dissected under stereomicroscopic observation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 microliter assay medium. Enzyme activity was determined by direct measurement of labeled inorganic phosphate release by the hydrolysis of [gamma-32P]ATP and was expressed as a function of tubule length. This method is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine its activity in each region of the nephron. Correlation of Na-K-ATPase activity in single tubules with functional measurements obtained in the corresponding segment of the nephron with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.

Optimal Conditions for Measurement of Na+, K+-ATPase Activity of Human Leucocytes

1985 ◽  
Vol 68 (2) ◽  
pp. 143-149 ◽  
Author(s):  
D. N. Baron ◽  
F. A. Khan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Coefficient Of Variation ◽  
Healthy Subjects ◽  
Inorganic Phosphate ◽  
Inhibitory Effect ◽  
Cell Disruption ◽  
Optimal Conditions ◽  
Maximal Inhibition ◽  
Hypotonic Lysis

1. The Na+, K+-ATPase activity of human leucocytes was assayed by measuring the release of inorganic phosphate (Pi) from ATP. 2. The maximum enzyme activity was achieved under the following conditions: concentration (mmol/l), Tris/HCl 50, Na 100, K 15, ATP 5, Mg 7, EDTA 1; pH 7.2 and temperature 37°C, were optimal. Ouabain showed maximal inhibition at a concentration of 10-100 μmol/l. Ethanol, the solvent for ouabain, had a dose-related inhibitory effect. 3. Heparin or citrate used as an anticoagulant gave similar results. Leucocyte samples could be stored at −20°C for up to 6 days without loss of activity. Hypotonic lysis had advantages over sonication as the technique for cell disruption. 4. The leucocyte Na+, K+-ATPase enzyme activity in healthy subjects was 186 μmol of Pi h−1 g−1 of protein (median) with a range 136-243 μmol of Pi h−1 g−1 of protein. The within-batch coefficient of variation was 6.4% and the between-batch precision was 9.6%.

scholarly journals Lipoxygenase in Wheat: Genetic Control and Impact on Stability of Lutein and Lutein Esters

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1149
Author(s):  
Daryl J. Mares ◽  
Judy Cheong ◽  
Shashi N. Goonetilleke ◽  
Diane E. Mather
Keyword(s):  
Enzyme Activity ◽  
Product Quality ◽  
Genetic Control ◽  
Nutritional Value ◽  
Lipoxygenase Activity ◽  
Lipoxygenase Inhibitor ◽  
Wheat Varieties ◽  
Post Harvest ◽  
End Products

Preservation of lutein concentrations in wheat-based end-products during processing is important both for product quality and nutritional value. A key constituent involved in lutein degradation is endogenous lipoxygenase. Lutein and lutein ester concentrations were compared at intervals during storage of noodle sheets prepared from flour of wheat varieties representing a range in lipoxygenase activity, as well as in different mill streams and in different grain tissues. Higher lipoxygenase concentration was associated with an increased loss of free lutein and lutein mono-esters whereas lutein diesters appeared to be more resistant to degradation. Lutein degradation was reduced in the presence of a lipoxygenase inhibitor, when noodle sheets were heated to destroy enzyme activity or when pH was increased. In addition, three populations were used to investigate the genetic control of lipoxygenase. A previously reported mutation of Lpx-B1.1 was associated with a reduction in activity from high to intermediate whilst a new locus on chromosome 4D was associated with variation between intermediate and near-zero. The gene underlying the 4D locus is a putative lipoxygenase. Stability of lutein could be improved by deployment of the mutations at the 4B and 4D loci and/or by post-harvest storage of grain under conditions that promote esterification.

Phosphate Binding to Isolated Chloroplast Coupling Factor (CF1)

1988 ◽  
Vol 43 (3-4) ◽  
pp. 213-218 ◽  
Author(s):  
Bernhard Huchzermeyer
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Inorganic Phosphate ◽  
Coupling Factor ◽  
Binding Domain ◽  
Atp Binding ◽  
Phosphate Binding ◽  
Chloroplast Coupling Factor ◽  
Single Binding Site ◽  
Site Binding

A single binding site for phosphate was found on isolated chloroplast coupling factor in the absence of nucleotides. In our experiments the phosphate binding site showed a Kd of 170 μᴍ. We did not observe any differences whether the ATPase activity of CF] had been activated or not. If the enzyme was incubated with [γ-32P]ATP the amount of 32P bound per CF1 depended on the pretreatment of the enzyme: In the presence of ADP no ATP or phosphate was bound to CF,. After activation of ATPase activity one mol of ATP per mol CF, was rapidly bound and hydrolyzed while there was a slowly occurring binding of another phosphate without concomitant nucleotide binding. We conclude that there are two different types of phosphate binding observed in our experiments: 1) Inorganic phosphate can be bound by one catalytic site per mol of CF1 2) The γ-phosphate of ATP is able to bind to an ATP binding domain of the enzyme if this domain can exchange substrates with the incubation medium. This ATP binding domain appears to differ from the site binding inorganic phosphate, because at least a portion of the coupling factor contains more than one labelled phosphate during our ATPase tests.

1H n.m.r. studies of the kinetic properties expressed by erythrocyte enzymes in situ and in vitro

1983 ◽  
Vol 11 (3) ◽  
pp. 280-281 ◽  
Author(s):  
KEVIN M. BRINDLE ◽  
IAIN D. CAMPBELL ◽  
ROBERT J. SIMPSON
Keyword(s):  
Kinetic Properties ◽  
Erythrocyte Enzymes

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.

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