Re-Evaluation of the Effects of Glucose-6-Phosphate and Malate on the Catalytic Properties of Phosphoenolpyruvate Carboxylase From Cynodon dactylon Under Physiological Assay Conditions

1990 ◽  
Vol 17 (4) ◽  
pp. 407 ◽  
Author(s):  
SK Stamatakis ◽  
I Skaliora ◽  
NA Gavalas ◽  
Y Manetas
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Cynodon Dactylon ◽  
Catalytic Properties ◽  
Enzyme Concentration ◽  
Light Activation ◽  
Apparent Affinity ◽  
Assay Medium ◽  
High Concentrations ◽  
Regulatory Properties ◽  
Assay Conditions

Some catalytic and regulatory properties of the day- and night-form of phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Cynodon dactylon (L.) Pers. leaves were re-evaluated in the presence of high concentrations of glycerol in the assay medium. This cosolute, known to be preferentially excluded from the hydration sphere of some proteins, apparently promotes aggregation, through an increase of the enzyme concentration in a fraction of the total volume. In the presence of this cosolute in the assay medium: (a) glucose-6-phosphate does not activate the enzyme, (b) inhibition of the Vmax by malate is increased, particularly with the night-form of phosphoenolpyruvate carboxylase, whereas the day-form is more responsive at the Km levels, (c) the light activation, detected as an increase in the apparent affinity of the substrate phosphoenolpyruvate is maintained under the new assay conditions.

Organic Cosolutes as Stabilizers of Phosphoenolpyruvate Carboxylase in Storage: an Interpretation of Their Action

1987 ◽  
Vol 14 (2) ◽  
pp. 203 ◽  
Author(s):  
E Selinioti ◽  
D Nikolopoulos ◽  
Y Manetas
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Cynodon Dactylon ◽  
Dependent Manner ◽  
Native Protein ◽  
Complete Protection ◽  
Concentration Dependent Manner ◽  
Polymeric Form ◽  
C4 Grass ◽  
High Concentrations ◽  
Protein Bovine Serum Albumin

Phosphoenolpyruvate carboxylase (EC 4.1.1.3 1) is rapidly inactivated in dilute extracts from leaves of the C4 grass Cynodon dactylon (L.) Pers. Physiological osmotica (glycerol, betaine, proline, sorbitol and sucrose) and synthetic inert polymers like polyvinylpyrrolidone and polyethylene glycol prevent inactivation in a concentration-dependent manner. In a partially purified preparation (10.5 �mol CO2 min-1 mg-1), lability to inactivation and the minimum levels of cosolvents needed for complete protection become higher as the protein is diluted. Exogenous protein (bovine serum albumin) was much less effective than native protein in stabilising activity. The results could be interpreted on the basis of the exclusion volume theory and the assumption that the catalytic activity is mainly due to a polymeric form which is maintained only at high concentrations of the enzymic protein.

Phosphoenolpyruvate Carboxykinase in C4 Plants: Its Role and Regulation

1997 ◽  
Vol 24 (4) ◽  
pp. 459 ◽  
Author(s):  
Robert P. Walker ◽  
Richard M. Acheson ◽  
László I. Técsi ◽  
Richard C. Leegood
Keyword(s):  
Molecular Mass ◽  
Malic Enzyme ◽  
Phosphoenolpyruvate Carboxykinase ◽  
C4 Plants ◽  
Panicum Maximum ◽  
C4 Plant ◽  
Cam Plants ◽  
Crude Extracts ◽  
High Concentrations ◽  
Regulatory Properties

Some of the recent findings which revise our view of the role and regulation of phosphoenolpyruvate carboxykinase (PEPCK) in C4 plants are discussed. Evidence is presented that PEPCK is present at appreciable activities in the bundle-sheath of some NADP-malic enzyme-type C4 plants, such as maize, but it was not detectable in NAD-malic enzyme-type C4 plants. PEPCK is rapidly inactivated in crude extracts of leaves of the C4 plant, Panicum maximum. This inactivation could be prevented by high concentrations of dithiothreitol or by the inclusion of ADP or ATP, suggesting the involvement of thiols at the active site. PEPCK is also subject to rapid proteolysis in crude extracts of a range of C4 plants, resulting in cleavage to a smaller (62 kDa) form. This can be reduced by extraction at high pH and by the inclusion of SDS, but it means that intact PEPCK has never been purified from a C4 plant. The molecular mass of PEPCK varies considerably in C4 plants, unlike C3 and CAM plants in which it is usually 74 kDa. PEPCK is phosphorylated during darkness (and reversed by light) in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plant, Sporobolus pyramidalis (69 kDa). The known regulatory properties of PEPCK are discussed in relation to its role in C4 photosynthesis, in particular its sensitivity to regulation by adenylates and by Mn2+.

scholarly journals Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

1977 ◽  
Vol 162 (3) ◽  
pp. 671-679 ◽  
Author(s):  
P S Agutter ◽  
J R Harris ◽  
I Stevenson
Keyword(s):  
Nuclear Envelope ◽  
Specific Activity ◽  
Assay Medium ◽  
Exogenous Rna ◽  
Mammalian Liver ◽  
High Concentrations ◽  
Enzymic Marker ◽  
Stimulation Of ◽  
Nucleoside Triphosphatase

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

scholarly journals Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities

1980 ◽  
Vol 189 (2) ◽  
pp. 277-284 ◽  
Author(s):  
T P Karpetsky ◽  
G E Davies ◽  
K K Shriver ◽  
C C Levy
Keyword(s):  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ribonuclease A ◽  
Polyacrylamide Gel ◽  
Muscle Enzymes ◽  
The Matrix ◽  
Pancreatic Ribonuclease A ◽  
High Concentrations ◽  
Positively Charged

A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the detection of polycationic ribonuclease activity rather than simply the presence of protein. Because use is made of the catalytic properties of ribonucleases, polynucleotide/polyacrylamide-gel electrophoresis is apparoximately 10(5) times more sensitive for the detection of these enzymes than conventional gel electrophoresis with the use of protein-staining dyes. Initial studies showed that the poor migration, in the gels, of highly charged polycationic ribonucleases in the presence of negatively charged synthetic polynucleotides could be overcome by high concentrations of spermine. The positively charged polyamine, by neutralizing the polyanionic polynucleotide, enabled these basic enzymes to migrate considerable distances in the gel. Electrophoresis of the RNAases under conditions of low pH, and incubation of the gel at neutral pH followed by staining for polynucleotide, resulted in coloured gels containing clear bands that define regions of enzyme activity. Alterations in spermine concentration or substrate identity caused changes in the positions of these bands, suggesting a dynamic interaction among the enzyme, polyamine and polynucleotide. Because of the advantages, in terms of selectivity and sensitivity of polynucleotide/polyacrylamide-gel electrophoresis, this technique was used to demonstrate the nuclease homogenity of three purified bovine muscle enzymes, and to compare these enzymes with each other, as well as with bovine pancreatic ribonuclease A.

Reliability of Clinical Assays for Evaluating Platelet Hypofunction.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2183-2183
Author(s):  
Donald L. Yee ◽  
Carol W. Sun ◽  
Angela L. Bergeron ◽  
Jingfei Dong ◽  
Paul F. Bray
Keyword(s):  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Laboratory Evaluation ◽  
Repeated Testing ◽  
Aggregation Assay ◽  
Aggregation Response ◽  
Agonist Concentration ◽  
High Concentrations ◽  
Few Data ◽  
Assay Conditions

Abstract Platelet hypofunction occurs commonly in various settings and may contribute to clinically important bleeding; however, its laboratory evaluation remains a rather imprecise science. Although some platelet assays are well established in clinical practice, concerns about their reliability abound. Furthermore, few data exist on which to base laboratory criteria for identifying individuals with platelet hypofunction. To address these areas of need, we studied 449 healthy adults using assays commonly used in clinical laboratories to evaluate for impaired platelet function. These assays included optical aggregometry (using high concentrations of agonists in citrated platelet-rich plasma) and the PFA-100. In addition, we assessed the reproducibility of these assays by performing them on a subgroup of 27 individuals four times each on a weekly basis in a controlled setting. For each agonist and agonist concentration studied, we observed that a platelet aggregation response >60% clearly distinguished those subjects with complete aggregation from those who did not; we then identified minimal agonist concentrations at which ≥ 80% of healthy individuals demonstrated this degree of response (see table). Regarding reproducibility, a significant number of subjects showed widely discrepant results on repeated testing (see figure for example); the percentage of subjects who did so varied with the particular agonist employed (see table). Furthermore, our data obtained using the PFA-100 were less reproducible than has previously been reported and reproducibility varied with the cartridge employed (within-subject coefficient of variation=20.3% for epinephrine versus 8.5% for ADP). Aggregometry assays using different agonists correlated only partially with each other and with the PFA-100; the highest correlation coefficient was r=0.42 (p<.001, between ADP and epinephrine). In summary, our data provide a rational basis for specifying assay conditions and criteria (<60% aggregation in response to high concentrations of agonist) by which individuals with platelet hypofunction can be identified. However, despite studying only healthy subjects in a controlled research setting, we found the reproducibility of some commonly used platelet function tests to be rather poor; aggregometry using high concentrations of ADP or ristocetin and the PFA-100 using the ADP cartridge provided the most reliable results and are preferred assays in evaluating patients with suspected platelet hypofunction. Given the incomplete correlation between different assays, the other, less reliable assays may still provide useful information for an individual patient, but caution should be exercised in interpreting the results. Such assays may bear repeating, especially when the aggregation response to agonist is marginal (near 60%). Aggregation assay characteristics Platelet agonist Required* concentration % of subjects with discrepant** results *to yield complete aggregation in ≥ 80% of subjects; **>30% difference in assay results on separate occasions ADP 4 μ M 7 Arachidonic acid 0.5 mg/mL 25 Collagen-related peptide 0.02 μg/mL 19 Collagen 50 μ g/mL 15 Epinephrine 10 μ M 29 Ristocetin 1 mg/mL 7 Reproducibility of aggregation response in 27 subjects 50 μg/mL collagen Reproducibility of aggregation response in 27 subjects 50 μg/mL collagen

scholarly journals Induction and suppression of the key enzymes of glycolysis and gluconeogenesis in isolated perfused rat liver in response to glucose, fructose and lactate

1973 ◽  
Vol 134 (1) ◽  
pp. 143-156 ◽  
Author(s):  
Janet M. Wimhurst ◽  
K. L. Manchester
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Pyruvate Carboxylase ◽  
Lactate Concentration ◽  
Sugar Concentration ◽  
Constant Infusion ◽  
Isolated Perfused Rat Liver ◽  
Key Enzymes ◽  
Serine Dehydratase ◽  
High Concentrations ◽  
Blood Sugar Concentration

1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.

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