scholarly journals Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

1980 ◽  
Vol 66 (6) ◽  
pp. 1258-1264 ◽  
Author(s):  
A J Hance ◽  
E D Robin ◽  
L M Simon ◽  
S Alexander ◽  
L A Herzenberg ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Monoclonal Antibodies ◽  
Pyruvate Kinase ◽  
Chronic Hypoxia ◽  
Glycolytic Enzyme ◽  
Enzyme Content ◽  
Rate Limiting

Heterogeneity of Glycolytic Enzyme Activity and Isozyme Composition of Pyruvate Kinase in Breast Cancer

Tumor Biology ◽  
1988 ◽  
Vol 9 (4) ◽  
pp. 178-189 ◽  
Author(s):  
A. Hennipman ◽  
B.A van Oirschot ◽  
J. Smits ◽  
G. Rijksen ◽  
G.E.J. Staal
Keyword(s):  
Breast Cancer ◽  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Glycolytic Enzyme

scholarly journals Comparison of glycolytic enzyme and isoenzyme activity in breast cancers and dysplasia

2012 ◽  
Vol 65 (5-6) ◽  
pp. 200-205 ◽  
Author(s):  
Katica Bajin-Katic
Keyword(s):  
Breast Cancer ◽  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Enzyme Concentration ◽  
Glycolytic Enzyme ◽  
Disc Electrophoresis ◽  
Breast Cancers ◽  
Lactate Dehydrogenase Activity ◽  
Total Enzyme Activity

The study was aimed at assessing the total enzyme activity and the profile of breast cancer and dysplasia on the human material. In addition, the validity of data was evaluated from the aspect of improving diagnostics. Lactate dehydrogenase activity, as well as the profile of its isoenzymes, pyruvate kinase and hexokinase, were measured. The study included 60 samples of breast cancer, out of which 20 were benign breast tumours and 40 were 1st and 2nd degree dysplasia of the breast. The samples were collected from the patients operated at the Institute for Oncology of Faculty of Medicine in Sremska Kamenica. Lactate dehydrogenase isoenzymes were separated by the vertical polyacrylamide gel disc electrophoresis according to the slightly modified Brewer and Ashworth?s method. The activity of all the tested enzymes was measured under the conditions of linear kinetics in the function of time and enzyme concentration. Lactate dehydrogenase-5 was found in 88% of the analyzed breast cancer samples, whereas it was not detected in breast dysplasia. Pyruvate kinase (4.-isoenzyme) was about 50 times higher and the activity of hexokinase was 3 times higher in breast cancer than in breast dysplasia. Lactate dehydrogenase-5 and pyruvate kinase (4.-isoenzyme) are particularly important and reliable markers of malignity. The results obtained for quantitative and qualitative changes in the enzyme activity can be used to improve diagnostics and early diagnostics of malignant breast neoplasm.

Mechanisms of lung polyamine accumulation in chronic hypoxic pulmonary hypertension

1990 ◽  
Vol 259 (6) ◽  
pp. L351-L358 ◽  
Author(s):  
R. T. Shiao ◽  
H. B. Kostenbauder ◽  
J. W. Olson ◽  
M. N. Gillespie
Keyword(s):  
Ventricular Hypertrophy ◽  
Chronic Hypoxia ◽  
Pulmonary Arterial Pressure ◽  
Hypoxic Exposure ◽  
Maximal Velocity ◽  
Pulmonary Vascular Remodeling ◽  
Hypoxic Environment ◽  
Elevated Activity ◽  
Pulmonary Arterial ◽  
Rate Limiting

Chronic hypoxia causes polyamine-dependent hypertensive pulmonary vascular remodeling (J. E. Atkinson. J. W. Olson, R. J. Altierre, and M. N. Gillespie, J. Appl. Physiol. 62: 1562–1568, 1987), but mechanisms by which lung polyamine contents are elevated have not been established. This study measured polyamine contents, biosynthetic activities, and transport in lungs of rats exposed to hypobaric hypoxia (simulated altitude: 4,570 m) for 4–14 days. Hypoxia increased lung contents of spermidine and spermine within 40 h and of putrescine within 4 days. These changes preceded hypoxia-induced increases in pulmonary arterial pressure and development of right ventricular hypertrophy. Additional experiments determined whether increased lung polyamine contents could be ascribed to elevated activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in conversion of ornithine to putrescine. Lung ODC activity did not differ from controls at 40 h posthypoxia and was reduced below control levels from 4–14 days of exposure. Putrescine transport kinetics were assessed in isolated, salt solution-perfused lungs. Apparent Km for putrescine uptake was increased from 10.4 microM in control lungs to 16.9 microM in lungs from rats maintained for 7 days in an hypoxic environment. Maximal velocity (Vmax) of lung putrescine transport was increased from 1.67 nmol.g-1.min-1 in controls to 2.65 in hypoxic lungs. Putrescine efflux also was altered by hypoxic exposure; T1/2 for loss of diamine from a slowly effluxing pool was increased from 60.6 min in controls to 91.5 min in hypoxic lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Kinetics and mechanism of action of muscle pyruvate kinase

1978 ◽  
Vol 169 (1) ◽  
pp. 39-54 ◽  
Author(s):  
Leighton G. Dann ◽  
Hubert G. Britton
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Pyruvate Kinase ◽  
Alternative Pathway ◽  
Slow Step ◽  
Velocity Data ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Muscle Pyruvate Kinase ◽  
Rate Limiting

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg2+. 2. Methods of preparing [32P]phosphoenolpyruvate from [32P]Pi in high yield and determining [32P]-phosphoenolpyruvate and [8-14C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [32P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-14C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [32P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg2+, with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-14C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-14C]-pyruvate or [γ-32P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.

scholarly journals Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 607-613 ◽  
Author(s):  
W Nijhof ◽  
PK Wierenga ◽  
GE Staal ◽  
G Jansen
Keyword(s):  
Pyruvate Kinase ◽  
Gradient Centrifugation ◽  
Recovery Period ◽  
Erythroid Differentiation ◽  
Glycolytic Enzyme ◽  
Minor Amount ◽  
Type I ◽  
In Vitro Differentiation ◽  
Percoll Density Gradient Centrifugation

Late committed progenitor cells of erythropoiesis, CFU-E (colony- forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6- phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU- E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU- E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.

scholarly journals Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

2018 ◽  
Author(s):  
Jamie A. Macpherson ◽  
Alina Theisen ◽  
Laura Masino ◽  
Louise Fets ◽  
Paul C. Driscoll ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
High Activity ◽  
Pyruvate Kinase ◽  
Cross Talk ◽  
Glycolytic Enzyme ◽  
Computational Framework ◽  
Allosteric Inhibitor ◽  
Fructose 1,6 Bisphosphate ◽  
Low Activity

ABSTRACTAllosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

The activities of three enzymes of haem synthesis during hepatic erythropoiesis in the mouse embryo

Development ◽  
1971 ◽  
Vol 26 (2) ◽  
pp. 313-322
Author(s):  
R. I. Freshney ◽  
J. Paul
Keyword(s):  
Enzyme Activity ◽  
Maximal Activity ◽  
Cell Types ◽  
Population Changes ◽  
Adult Liver ◽  
Foetal Liver ◽  
Different Cell Types ◽  
Rate Limiting ◽  
Haemoglobin Synthesis ◽  
Liver Cell Population

Aminolaevulinate synthetase, aminolaevulinate dehydratase, and haem synthetase, three enzymes which may have a regulatory role in haem synthesis, have been determined in liver extracts from different foetal stages of the mouse. Haemoglobin synthesis increases rapidly from early on the 14th day, after fertilization, to reach a maximum late on the 15th day. Aminolaevulinate synthetase reaches a maximum on the 14th day, 24–36 h before the peak of haemoglobin synthesis, aminolaevulinate dehydratase on the 15th day, about 12 h before the peak of haemoglobin synthesis, and haem synthetase on the 17th day. Maximal activity of aminolaevulinate synthetase and aminolaevulinate dehydratase is of only a few hours' duration. Throughout embryonic development the activities of all three enzymes are higher than in the adult liver. The absence of a correlation of enzyme activity with foetal liver cell population changes implies that fluctuations in enzyme activity cannot be explained solely by changes in the proportions of different cell types. The high levels of activity relative to those of adult liver may be related to the high proportion of erythroid cells in the foetal liver. It is concluded that these enzymes are unlikely to form rate-limiting steps during the increase in haemoglobin synthesis between 14 and 15 days.

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