Showing papers on "Pyruvate kinase published in 1979"
TL;DR: The addition of naturally occurring polyamines and inorganic ions to an in vitro protein-synthesizing system improved the extent and fidelity of translation and regeneration of the nucleoside triphosphates with phosphoenolpyruvates and pyruvate kinase reduced further the missense error frequency to the in vivo level.
Abstract: The addition of naturally occurring polyamines and inorganic ions to an in vitro protein-synthesizing system improved the extent and fidelity of translation. In such an optimized system, regeneration of the nucleoside triphosphates with phosphoenolpyruvate and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) reduced further the missense error frequency to the in vivo level as well as enhanced the extent of translation. The effect of nucleoside triphosphate regeneration was shown to be due primarily to the increase in the ratio of adenosine and guanosine triphosphates to their hydrolysis products and only marginally due to the increase in the absolute concentrations of the nucleoside triphosphates.
292 citations
TL;DR: No correlation of low or high levels of fructose-1,6-bisphosphate with the degree of catabolite repression and inactivation could be found, and this observation does not support the concept that hexose metabolites are directly involved in these regulatory mechanisms in yeast.
Abstract: Saccharomyces cerevisiae mutants unable to grow and ferment glucose have been isolated. Of 45 clones isolated, 25 had single enzyme defects of one of the following activities: phosphoglucose isomerase (pgi), phosphofructokinase (pfk), triosephosphate isomerase (tpi), phosphoglycerate kinase (pgk), phosphoglyceromutase (pgm), and pyruvate kinase (pyk). Phosphofructokinase activities in crude extracts of the pfk mutant were only 2% of the wild-type level. However, normal growth on glucose medium and normal fermentation of glucose suggested either that the mutant enzyme was considerably more active in vivo or, alternatively, that 2% residual activity was sufficient for normal glycolysis. All other mutants were moderately to strongly inhibited by glucose. Unusually high concentrations of glycolytic metabolites were observed before the reaction catalyzed by the enzyme which was absent in a given mutant strain when incubated on glucose. This confirmed at the cellular level the location of the defect as determined by enzyme assays. With adh (lacks all three alcohol dehydrogenase isozymes) and pgk mutants, accumulation of the typical levels of hexosephosphates was prevented when respiration was blocked with antimycin A. A typical feature of all glycolytic mutants described here was the rapid depletion of the intracellular adenosine 5′-triphosphate pool after transfer to glucose medium. No correlation of low or high levels of fructose-1,6-bisphosphate with the degree of catabolite repression and inactivation could be found. This observation does not support the concept that hexose metabolites are directly involved in these regulatory mechanisms in yeast.
175 citations
TL;DR: In the dark chloroplasts could metabolize all the products of starch breakdown and catalyse much of the respiration of pea shoots via the oxidative pentose phosphate pathway and/or glycolysis as far as 3-phosphoglycerate, and it is suggested that pea shoot chloroplast lack phosphoglyceromutase but contain some phosphopyruvate hydratase and pyruvating kinase.
147 citations
TL;DR: Standard methods for the analysis of PK variants would make it possible to compare results obtained in different laboratories, and to identify variants on the basis of biochemical parameters as well as clinical manifestations, and comprise the major part of this report.
Abstract: Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40, PK) deficiency associated with hereditary nonspherocytic haemolytic anaemia was first reported in 1961 (Valentine et al, 1961). I t has become increasingly apparent that most, if not all, cases of PK deficiency are caused by the production of mutant enzymes with abnormal characteristics. A variety of techniques and methods have thus far been used in different laboratories for characterizing PK variants. The use of standard methods for the analysis of PK variants would make it possible to compare results obtained in different laboratories, and to identify variants on the basis of biochemical parameters as well as clinical manifestations. Such methods have been developed through a collaborative effort among members of a working group of the ICSH Expert Panel on Red Cell Enzymes, and comprise the major part of this report. As far as possible, a common buffer system, substrate and co-factor solution are proposed in order to simplify the methodology as well as the interpretation of data. The number of essential parameters for abnormal PK are minimized because of the limitation of both time required for the studies and volume of blood obtainable from a patient. This does not imply that other procedures such as urea stability (Koster et a l , 1971; Miwa et al , 1975), alanine inhibition (Staal et al, 1976), immunological methods (Lincoln et al , 1975; Miwa et al , 1975; Kahn et a l , 1976), isoelectric focusing (lbsen & Trippet, 1971; Kahn et al , 1976; Oda et a l , 1976), etc., are unimportant. Rather, it is suggested that these other studies be done optionally for further understanding of the abnormal characteristics of the variant enzymes.
104 citations
TL;DR: It is shown that glycerol kinase and acetate kinase proceed by pathways that result in inversion of the configuration at phosphorus, which provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
Abstract: We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
95 citations
TL;DR: In this paper, the regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone.
85 citations
TL;DR: An indication of the metabolic capacity of the plastids was determined by monitoring the ribulose-1,5-bisphosphate carboxylase activity in the endosperm.
Abstract: During castor oil seed development, changes occur in the activities of enzymes involved in fatty acid biosynthesis, glycolysis, and the pentose phosphate pathways. The activities of acetyl-CoA carboxylase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase per seed increase during the phase of rapid oil synthesis in the endosperm. As the seed matures and the rate of fatty acid synthesis decreases, there is a corresponding diminution in the activities of these enzymes. An indication of the metabolic capacity of the plastids was determined by monitoring the ribulose-1,5-bisphosphate carboxylase activity in the endosperm.
82 citations
TL;DR: It is demonstrated that synthetic peptide analogs of the local phosphorylation site sequences of natural substrates may differ widely in their capacity to act as substrates for this protein kinase.
77 citations
71 citations
TL;DR: The observation that catecholamines do not increase gluconeogenesis to the same extent as glucagon was not due to a differential effect on mitochondrial CO2 fixation, but to a smaller inhibition of pyruvate kinase activity than did glucagon.
71 citations
TL;DR: Studies in the perfused liver indicate that only alanine, serine, proline, threonine, glutamine, asparagine, glutamate, aspartate, and arginine yield significant amounts of carbohydrate.
Abstract: Gluconeogenesis is the process by which glucose and glycogen are synthesized in the animal body from noncarbohydrate precursors. The liver and the kidney are the two organs which carry out gluconeogenesis and gluconeogenic substrates include lactate, pyruvate, glycerol, and the glucogenic amino acids. Although all the natural amino acids except leucine and lysine are potentially glucogenic by virtue of the fact that they yield pyruvate, oxalacetate, aketoglutarate, succinyl-CoA, or fumarate during their catabolism, studies in the perfused liver indicate that only alanine, serine, proline, threonine, glutamine, asparagine, glutamate, aspartate, and arginine yield significant amounts of carbohydrate (Ross, Hems, and Krebs, 1967).
TL;DR: It is concluded that Mg2+-induced inhibition of the ATPase activity of F1 is due to the formation of an inactive F1, and unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem.
TL;DR: Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography, finding the plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides.
Abstract: Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localized in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity.
TL;DR: A continuous fluorimetric method using auxiliary-coupling enzymes such as pyruvate kinase and lactate dehydrogenase for measuring ADP production to assay ATPase activity is described, which is simpler, more rapid, and more sensitive than the previously used spectrophotometric method.
TL;DR: It is reported here that not only are ATP levels under genetic control, but they are also highly correlated with lactate dehydrogenase (LDH) phenotype, the first such correlation reported.
Abstract: ERYTHROCYTE organic phosphate concentration is known to be an heritable trait in both humans and rats1–3. In humans this trait has been correlated with genetic variability of both pyruvate kinase and hexokinase2. However, similar phenomena have not been reported in lower vertebrates. During our studies on the allosteric regulation of fish haemoglobins by ATP (ref. 4) we observed significant variability in red cell ATP concentrations among individuals of the killifish Fundulus heteroclitus, and wondered whether this variability in organic phosphate could be the result of a genetic component. Consequently, we analysed F. heteroclitus for red cell ATP, concomitantly screening for enzyme variants. We report here that not only are ATP levels under genetic control, but they are also highly correlated with lactate dehydrogenase (LDH) phenotype. To our knowledge, this is the first such correlation reported. Moreover, as ATP is the major allosteric modifier of F. heteroclitus haemoglobin–oxygen affinity5,6, this study provides a basis for an association between LDH and the fish's external environment through the physiological role of haemoglobin at the organism–environment interface.
TL;DR: Fetal hyperinsulinemia with euglycemia increases the activity of enzymes that participate in lipogenesis, decreases some of those controlling gluconeogenesis, and has no effect on the enzymes of glycolysis.
Abstract: Hyperinsulinemia was produced in fetal rhesus monkeys for 21 days in the last third of gestation by subcutaneous pork insulin injected at 19 U a day. Plasma insulin concentrations in treated fetuses ( N = 4) were 3525 μU/ml compared with controls' ( N = 6) 51 μ/ml. There was no difference in paired pre- and post-treatment fetal plasma glucose concentration. Activity of the hepatic enzymes that promote glucose utilization (glucokinase and hexokinase) and glycolysis (phos-phofructokinase, pyruvate kinase, and pyruvate dehy-drogenase) was unaffected. Similarly, glycogen metabolism enzymes (active and inactive synthase and phosphorylase) were unaltered. Two gluconeogenic enzymes (PEPCK and glucose-6-phosphatase) were diminished in the treated group compared with controls. Fetal hyperinsulinemia enhanced lipogenic and NADPH-producing enzyme activities, as evidenced by a twofold increase in fatty acid synthase and in citrate cleavage enzyme activity. Malic enzyme was absent. Hyperinsulinemia with euglycemia (1) increases the activity of enzymes that participate in lipogenesis, (2) decreases some of those controlling gluconeogenesis, and (3) has no effect on the enzymes of glycolysis.
TL;DR: Results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. Lane.
TL;DR: Quantitatively, anaerobic glycolysis accounts for a maximum of 28% of basal aerobic ATP generation, suggesting that prolonged diving is also accompanied by a reduction in brain energy requirements, and the adaptation subserving short-term (natural) diving is an increase in brain Glycolytic capacity.
Abstract: The adaptations in the freshwater turtle that permit survival despite prolonged loss of mitochondrial ATP generation were investigated by comparing the bioenergetics of turtle brain slices with rat brain slices. Aerobic turtle brain shows no significant difference in basal levels of total ATP generation compared to rat brain; levels in turtle brain and rat brain were 18.4 +/- 2.8 (SD) and 19.4 +/- 2.2 mumol (100 mg of tissue)-1 hr-1, respectively. However, in turtle brain, a significantly greater fraction of ATP is derived from glycolysis both under aerobic and anaerobic conditions [aerobic turtle (24%) and rat (13%), P less than 0.02; anaerobic, turtle (28%) and rat (18%), P less than 0.05]. The increased glycolytic capacity is related to high levels of rate-limiting glycolytic enzymes, such as pyruvate kinase (EC 2.7.1.40). Turtle brain operates close to glycolytic capacity even under aerobic conditions, and no Pasteur effect can be demonstrated. Quantitatively, anaerobic glycolysis accounts for a maximum of 28% of basal aerobic ATP generation, suggesting that prolonged diving is also accompanied by a reduction in brain energy requirements. The adaptation subserving short-term (natural) diving is an increase in brain glycolytic capacity. The adaptation subserving prolonged diving (days to weeks) may be a reduction in the energy requirements of brain (and other cells).
Journal Article•
TL;DR: A direct relationship between the differences was found for the activities of some enzymes belonging to carbohydrate metabolism, namely, hexokinase, pyruvate kinase, aldolases A and B, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogensase and the differences found for glucose utilization by the different cell lines.
Abstract: Carbohydrate metabolism by four rat hepatoma cell lines in culture, namely, Reuber H35, MH1C1, RLC, and HTC, has been investigated. Glucose utilization by H35 and MH1C1 cells is lower than that by RLC and HTC cells. The four cell lines also differ with respect to the accumulation of lactic acid in the growth medium; in particular, H35 cells show uptake of lactic acid, rather than accumulation in the medium. Specific activities of a number of enzymes involved in glycolysis, gluconeogenesis, pentose phosphate pathway, and glycogen formation were determined in the four cell lines. A direct relationship between the differences was found for the activities of some enzymes belonging to carbohydrate metabolism, namely, hexokinase, pyruvate kinase, aldolases A and B, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase and the differences found for glucose utilization by the different cell lines.
TL;DR: There is ever-increasing literature pointing out that gene expression in tumour cells is different from that in normal cells from which or in which the tumour originates.
Abstract: There is ever-increasing literature pointing out that gene expression in tumour cells is different from that in normal cells from which or in which the tumour originates. In general, neoplasia is characterized by misprogramming of protein synthesis (16).
TL;DR: The drastic reduction of liver tissue after partial hepatectomy was compensated by an increase of gluconeogenesis in the liver remnant and in kidney cortex, both accompanied by an enhancement of the phosphoenolpyruvate carboxykinase level, in order to maintain the glucose homeostasis.
Abstract: The metabolic rates and key enzymes of gluconeogenesis and glycolysis were determined and correlated in perfused livers, isolated hepatocyte suspensions and kidney cortex slices of rats after two-thirds hepatectomy. Fed unoperated, sham operated and fasted animals served as controls.
In liver the following results were obtained. Firstly, after partial hepatectomy [14C]glucose formation from [14C]lactate was increased in perfused livers to 223% after 24 h and to 173% after 48 h and in hepatocyte suspensions to 222% after 48 h. [14C]Glucose production from [14C]fructose, however, was unchanged. These metabolic rates correlated well with an increase of the level of phosphoenolpyruvate carboxykinase to 206% after 24 h and to 184% after 48 h. The levels of fructose-1,6-bisphosphatase and glucose-6-phosphatase were unaltered. Secondly, after a 24-h fast gluconeogenesis from lactate was enhanced to 282% in perfused livers and to 300% in hepatocyte suspensions. Gluconeogenesis from fructose was slightly increased to 140%. These altered rates were accompanied by an increase of phosphoenolpyruvate carboxykinase to 213% and of glucose-6-phosphatase to 190%. Thirdly, after partial hepatectomy [14C]lactate formation from [14C]glucose was decreased in hepatocyte suspensions to less than 20% after 48 h. [14C]Lactate production from [14C]fructose was unchanged. These rates coincided with a decrease of the level of glucokinase to 40% and with unaltered levels of phosphofructokinase and pyruvate kinase. Fourthly, after a 24-h fast glycolysis from glucose was lowered to below 30%. This rate was paralleled by a decrease of the level of glucokinase to 60%.
In kidney cortex the following results were obtained. Firstly, after partial hepatectomy gluconeogenesis from lactate was enhanced in tissue slices to 217% after 24 h. Gluconeogenesis from fructose was unchanged. These rates coincided with an increase of the level of phosphoenolpyruvate carboxykinase to 193% and of glucose-6-phosphatase to 151%. Secondly, after a 24-h fast similar changes for the gluconeogenic rate to 200% and of phosphoenolpyruvate carboxykinase to 240% as well as of glucose-6-phosphatase to 136% were observed. Thirdly, neither the rates of glycolysis nor the levels of hexokinase, phosphofructokinase and pyruvate kinase were altered after partial hepatectomy or a 24-h fast.
No significant changes neither in liver nor in kidney cortex occurred after sham operation. Thus the drastic reduction of liver tissue after partial hepatectomy was compensated by an increase of gluconeogenesis in the liver remnant and in kidney cortex, both accompanied by an enhancement of the phosphoenolpyruvate carboxykinase level, in order to maintain the glucose homeostasis.
TL;DR: The integrity of isolated plastids was demonstrated by their capacity for protein synthesis as well as during regreening pyruvate kinase and phosphofructokinase in the developing proplastid decreased, neither enzyme being present in the mature chloroplast.
Abstract: Organelles were isolated from dark-grown Euglena gracilis Klebs by sucrose density gradient centrifugation. Plastids, identified by triosephosphate isomerase and NADP glyoxylate reductase were present at an equilibrium density of 1.24 grams per cubic centimeter clearly separated from mitochondria at an equilibrium density of 1.22 grams per cubic centimeter. Assay for choline phosphotransferase and glucose-6-phosphatase showed that endoplasmic reticulum membranes were present at a density of 1.12 grams per cubic centimeter. The plastid fraction contained phosphofructokinase, pyruvate kinase, triosephosphate isomerase and aldolase indicating the operation of a glycolytic pathway. During regreening pyruvate kinase and phosphofructokinase in the developing proplastid decreased, neither enzyme being present in the mature chloroplast. However, plastids were present in the photosynthetic cell as shown by a peak of glycolysis enzymes at an equilibrium density of 1.24 grams per cubic centimeter. The integrity of isolated plastids was demonstrated by their capacity for protein synthesis. Plastids isolated from dark-grown cells rapidly incorporated [ 35 S]methionine into protein with an absolute dependence on added ATP. The large subunit of ribulose diphosphate carboxylase was the major polypeptide synthesized by these isolated plastids.
TL;DR: Data suggest that certain purines and pyrimidine compounds may exert their stimulatory effect on hexokinase activity, resulting in an augmentation of cow red cell glycolysis.
Abstract: Cow red cells, under in vitro incubation conditions, exhibit a comparatively low glycolytic rate of 0.56 +/- 0.05 micromol/(ml cells.h), with a ratio of lactate formed to glucose consumed of 1.58. It has been found that this low glycolytic rate can be stimulated 50--60% above the basal level in the presence of a variety of purine and pyrimidine compounds including adenosine, inosine, adenine, hypoxanthine, xanthine, and uracil. In contrast, calf red cells, which have a much higher glycolytic rate, display no discernible response to these agents. In attempts to elucidate the mechanism by which this stimulation takes place, both glucose transport and glycolytic enzyme activities were determined in the presence of these stimulators. Glucose influx in cow red cells, measured using the glucose analog 3-O-methyl-glucose, exhibits both a low Km of 117 microM and a Vmax of 0.38 micromol/(ml cells.min), and is unaltered in the presence of adenosine. On the other hand, hexokinase, which in normal hemolysates of cow red cells has an activity of 0.49 +/- 0.03 micromol/(g Hb.min). was found to be stimulated to 0.73 micromol/(g Hb.min) in the presence of adenine. Both pyruvate kinase and phosphofructokinase were unaffected by this compound. These data suggest that certain purines and pyrimidine compounds may exert their stimulatory effect on hexokinase activity, resulting in an augmentation of cow red cell glycolysis.
TL;DR: The rate of microbiological ring-expansion of penicillin N to deacetoxycephalosporin C using protoplast lysates of the antibiotic-negative mutant Cephalosporium acremonium M-0198 has been increased some 70-fold over that of earlier system and the stimulatory effects of FeSO4 and ascorbate were confirmed.
Abstract: The rate of microbiological ring-expansion of penicillin N to deacetoxycephalosporin C using protoplast lysates of the antibiotic-negative mutant Cephalosporium acremonium M-0198 has been increased some 70-fold over that of our earlier system. We confirmed the stimulatory effects of FeSO4 and ascorbate described by HOOKet al. (Biochem. Biophys. Res. Commun. 87: 258, 1979); the optimum concentrations found were 0.04 mM FeSO4 and 0.67 mM ascorbate. Adenosine triphosphate concentration was lowered to 0.83 mM; phosphoenolpyruvate and pyruvate kinase were eliminated. The optimum pH and temperature for the reaction were 7.2 and 25°C, respectively. α-Ketoglutarate and MnCl2 showed no marked effect on the reaction, MgSO4 and KCl were mildly stimulatory, and CUSO4 and ZnSO4 were very inhibitory. Penicillin N was optimal at a concentration of 0.07 mM. Specific ring-expansion activity reached its peak 13 hours after growth ceased and then disappeared rapidly.
TL;DR: Results suggest that anin vivo proteolytic modification that yields the 51,000-dalton subunit of Liver pyruvate kinase could be derived from rats fed a high carbohydrate, low protein diet or limited digestion of the 56,000 daltons subunit.
TL;DR: The data support the conclusion that total CO2, fructose-1,6-diphosphate concentration, the adenylate energy charge, and-only to a limited extent-the intracellular pH are regulatory signals which govern PEP metabolism in Tubifex.
Abstract: 1.
During anaerobic exposure ofTubifex, lactate and alanine increase only within the first 24 h, while concentrations of succinate, propionate and also acetate continually increase under prevailing anaerobic conditions. Enzymes involved in anaerobic energy metabolism were isolated, and the effects of various metabolites and inorganic compounds on their catalytic properties studied.
2.
The specific activities of the cytosolic enzymes LDH, PK, MDH, and PEPCK, and of mitochondrial malic enzyme and MDH are high.
3.
Under conditions of high HCO3− (HCO3−+CO2 system), PEPCK is maximally active while PK is inhibited.
4.
The catalytic properties of mitochondrial malic enzyme indicate that in vivo this enzyme operates in the direction of malate decarboxylation.
5.
The cytosolic oxidoreductases LDH and MDH were examined with regard to the maintenance of redox state. Function of MDH is only subject to substrate availability, while the enzyme-substrate affinity of LDH might be affected by pH to a limited extent.
6.
The data support the conclusion that total CO2, fructose-1,6-diphosphate concentration, the adenylate energy charge, and-only to a limited extent-the intracellular pH are regulatory signals which govern PEP metabolism inTubifex.
TL;DR: A method for measuring potassium is described which is based on the requirement of pyruvate kinase for this cation and results were in agreement with those obtained using a flame photometer.