Comparative study of free and bound glycolytic enzymes from sea scorpion brain

1998 ◽  
Vol 76 (4) ◽  
pp. 609-614 ◽  
Author(s):  
Volodymyr I Lushchak ◽  
Ljudmyla P Lushchak ◽  
Tetjana V Bahnjukova ◽  
Alexei V Spichenkov ◽  
Kenneth B Storey
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Substrate Inhibition ◽  
Physiological Role ◽  
Glycolytic Enzymes ◽  
Free Form ◽  
Hill Coefficients ◽  
Low Ionic Strength ◽  
Scorpaena Porcus

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45°C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.Key words: hexokinase, lactate dehydrogenase, pyruvate kinase, enzyme binding, Scorpaena porcus.

scholarly journals Targeting Pyruvate Kinase M2 and Lactate Dehydrogenase A Is an Effective Combination Strategy for the Treatment of Pancreatic Cancer

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1372 ◽  
Author(s):  
Goran Hamid Mohammad ◽  
Vessela Vassileva ◽  
Pilar Acedo ◽  
Steven W. M. Olde Damink ◽  
Massimo Malago ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Tumor Growth ◽  
Pyruvate Kinase ◽  
Glycolytic Enzymes ◽  
Pyruvate Kinase M2 ◽  
Tumor Tissues

Reprogrammed glucose metabolism is one of the hallmarks of cancer, and increased expression of key glycolytic enzymes, such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), has been associated with poor prognosis in various malignancies. Targeting these enzymes could attenuate aerobic glycolysis and inhibit tumor proliferation. We investigated whether the PKM2 activator, TEPP-46, and the LDHA inhibitor, FX-11, can be combined to inhibit in vitro and in vivo tumor growth in preclinical models of pancreatic cancer. We assessed PKM2 and LDHA expression, enzyme activity, and cell proliferation rate after treatment with TEPP-46, FX-11, or a combination of both. Efficacy was validated in vivo by evaluating tumor growth, PK and LDHA activity in plasma and tumors, and PKM2, LDHA, and Ki-67 expression in tumor tissues following treatment. Dual therapy synergistically inhibited pancreatic cancer cell proliferation and significantly delayed tumor growth in vivo without apparent toxicity. Treatment with TEPP-46 and FX-11 resulted in increased PK and reduced LDHA enzyme activity in plasma and tumor tissues and decreased PKM2 and LDHA expression in tumors, which was reflected by a decrease in tumor volume and proliferation. The targeting of glycolytic enzymes such as PKM2 and LDHA represents a promising therapeutic approach for the treatment of pancreatic cancer.

scholarly journals The brush border of rabbit kidney, a cellular compartment free of glycolytic enzymes

1978 ◽  
Vol 174 (2) ◽  
pp. 509-515 ◽  
Author(s):  
Dietrich Busse ◽  
Hans Ulrich Wahle ◽  
Harald Bartel ◽  
Barbara Pohl
Keyword(s):  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Brush Border ◽  
Gradient Centrifugation ◽  
Fluorescein Isothiocyanate ◽  
Glycolytic Enzymes ◽  
Rabbit Kidney ◽  
Cellular Compartment ◽  
Fluorescein Isothiocyanate Dextran

Activities of four enzymes of the glycolytic pathway, hexokinase, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, were determined in a vesicular brush-border preparation from rabbit kidneys. The specific activities of the enzymes were decreased several-hundredfold in the brush-border preparation compared with a kidney homogenate, but the enzymes were not totally absent. Density-gradient centrifugation of the brush-border preparation yielded brush border of even higher purity and also a characteristic pattern of distribution for each of the contaminating intracellular membranes. The presence of hexokinase in the brush-border preparation could be traced to contaminating mitochondria, and that of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase to contaminating vesicles derived from the endoplasmic reticulum. The brush-border vesicles contained some ATP. An intravesicular concentration of 0.1mm was estimated, indicating that the vesicles had retained at least a part of their original content. Experiments in which fluorescein isothiocyanate-dextran (mol.wt. 20000) was present during cell lysis revealed that much, but not all, of the brush-border contents had been exchanged with the medium. The complete absence of glycolytic enzymes from brush-border vesicles, which had retained part of their original content, indicates that the brush border does not contain glycolytic enzymes in vivo and can be thought of as a compartment of its own, somehow separated from the cytoplasm.

The HIF-1 response to simulated ischemia in mouse skeletal muscle cells neither enhances glycolysis nor prevents myotube cell death

2007 ◽  
Vol 293 (4) ◽  
pp. R1693-R1701 ◽  
Author(s):  
Nathalie Dehne ◽  
Uta Kerkweg ◽  
Teresa Otto ◽  
Joachim Fandrey
Keyword(s):  
Skeletal Muscle ◽  
Cell Death ◽  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Vegf Mrna ◽  
Isolated Cells ◽  
Simulated Ischemia

Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1α protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1α accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization.

Bimodal substrate inhibition of lactate dehydrogenase. Factors affecting the enzyme in vivo

Biochemistry ◽  
1978 ◽  
Vol 17 (9) ◽  
pp. 1646-1653 ◽  
Author(s):  
J. W. Burgner ◽  
G. R. Ainslie ◽  
W. W. Cleland ◽  
W. J. Ray
Keyword(s):  
Lactate Dehydrogenase ◽  
Substrate Inhibition ◽  
Factors Affecting

scholarly journals The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue

1969 ◽  
Vol 115 (3) ◽  
pp. 405-417 ◽  
Author(s):  
E. D. Saggerson ◽  
A. L. Greenbaum
Keyword(s):  
Adipose Tissue ◽  
Lactate Dehydrogenase ◽  
Pyruvate Kinase ◽  
High Fat Diet ◽  
Alloxan Diabetes ◽  
Phosphoglycerate Kinase ◽  
Glycolytic Enzymes ◽  
Glucose Phosphate Isomerase ◽  
High Fat ◽  
Glucose Phosphate

1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ‘key’ glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.

scholarly journals Relationships between concentration of hepatic intermediary metabolites and induction of the key glycolytic enzymes in vivo

1973 ◽  
Vol 136 (3) ◽  
pp. 455-465 ◽  
Author(s):  
J. M. Gunn ◽  
C. B. Taylor
Keyword(s):  
Pyruvate Kinase ◽  
Time Course ◽  
Adenine Nucleotides ◽  
Metabolite Profile ◽  
Glycolytic Enzymes ◽  
British Library ◽  
Dihydroxyacetone Phosphate ◽  
Intermediary Metabolites ◽  
Metabolite Concentrations

1. The time-course for the induction of hepatic glucokinase, hexokinase, phosphofructokinase, liver-type and muscle-type pyruvate kinases in reponse to various diets and insulin has been investigated over the first 48h of change in both diabetic and non-diabetic rats. 2. The results are consistent with there being separate regulatory mechanisms for the induction of each of the three key enzymes, that is for glucokinase, phosphofructokinase and liver-type pyruvate kinase. 3. To investigate the possibility that induction of these enzymes is mediated through specific metabolites a full metabolite profile has been determined under conditions identical with those in the induction experiments and the results examined for correlations between metabolite concentrations and enzyme activities. 4. Several such relationships were detected and those between glucokinase activity and the phosphorylation state of the adenine nucleotides and between liver-type pyruvate kinase activity and the concentrations of dihydroxyacetone phosphate and pyruvate are discussed in relation to the concept of inducing metabolites. 5. It is suggested that the induction of glycolytic enzymes by insulin may be secondary to the changes in the concentration of specific hepatic metabolites brought about by the acute effects of the hormone. 6. The details of the metabolite concentrations in the various experimental states have been deposited as Supplementary Publication SUP 50021 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

1997 ◽  
Vol 78 (02) ◽  
pp. 864-870 ◽  
Author(s):  
Hideki Nagase ◽  
Kei-ichi Enjyoji ◽  
Yu-ichi Kamikubo ◽  
Keiko T Kitazato ◽  
Kenji Kitazato ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Free Form ◽  
Initial Reaction ◽  
Extrinsic Pathway ◽  
Factor Xa Inhibition ◽  
Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

120-LB: Small Molecule Activator of Pyruvate Kinase Regulates In Vivo Glucose Homeostasis

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 120-LB
Author(s):  
ABUDUKADIER ABULIZI ◽  
REBECCA L. CARDONE ◽  
STEPHAN SIEBEL ◽  
CHARLES KUNG ◽  
RICHARD KIBBEY
Keyword(s):  
Pyruvate Kinase ◽  
Small Molecule ◽  
Glucose Homeostasis

scholarly journals Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1978 ◽  
Vol 174 (3) ◽  
pp. 979-987 ◽  
Author(s):  
Victor A. Zammit ◽  
Eric A. Newsholme
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Marine Invertebrates ◽  
Adductor Muscle ◽  
Sea Anemone ◽  
Anaerobic Conditions ◽  
Fructose Bisphosphate ◽  
Low Concentrations ◽  
Optimum Ph ◽  
Hill Coefficients

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13μm produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the Km values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn2+ in addition to Mg2+ for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.

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