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PubMed1992

Correlation between plasma alpha-cysteine proteinase inhibitor level and pyruvate kinase activity in vitamin E-deficient rats.

Weaning rats were divided into two groups, one group being fed a vitamin E-deficient diet, and the other an alpha-tocopherol-containing (50 mg/kg) control diet. Rats were killed at 1, 2, 3 and 4 months of feeding. The following results were obtained. 1. Both plasma and liver alpha-tocopherol levels decreased greatly by feeding vitamin E-deficient diet for 1 month, and thereafter decreased gradually by continued feeding. 2. Somehow different results were obtained concerning liver peroxidation value by the method of analysis. In the case of chemiluminescence, the value increased by vitamin E deficiency during the first 2 months, but thereafter, the value was almost unchanged. On the contrary, in the case of TBA-RS, the value increased gradually throughout the entire 4 months of feeding period. 3. Both plasma alpha-CPI level and pyruvate kinase activity increased by vitamin E deficiency, showing similar pattern of change with feeding period. Especially, marked increase of these values was observed in vitamin E-deficient rats fed for longer than 2 months, and differences from control groups were highly significant (in both cases, at 2 months, p < 0.005, and at 3 and 4 months, p < 0.001). And, in vitamin E-deficient group, including all the rats fed on test diet for 1 to 4 months, correlation between both values was very high, and was highly significant (r = 0.9060, p < 0.001).

Animals↗
PubMed2006

Hexose-specificity of hexokinase and ADP-dependence of pyruvate kinase play important roles in the control of monosaccharide utilization in freshly diluted boar spermatozoa.

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.

Adenosine Diphosphate↗
PubMed2006

ChREBP binding to fatty acid synthase and L-type pyruvate kinase genes is stimulated by glucose in pancreatic beta-cells.

Pancreatic beta-cell dysfunction is central to the pathogenesis of type 2 diabetes and may involve secretory failure through glucolipotoxity. The relative importance of the transcription factors carbohydrate-responsive element binding protein (ChREBP), sterol-responsive element binding protein-1c (SREBP-1c), and upstream stimulatory factor (USF) in the induction of lipogenic genes by glucose remains unclear. By confocal imaging, we show that ChREBP translocates to the nucleus in MIN6 beta cells in response to glucose. Both ChREBP and SREBP-1c were required for the induction of the fatty acid synthase (FAS) promoter by glucose, and chromatin immunoprecipitation (ChIP) assay revealed that glucose induced the binding of both ChREBP and SREBP-1c to the FAS promoter without affecting USF2 binding. By contrast, ChIP assay revealed that high glucose prompted direct binding of ChREBP, but not SREBP-1c or USF2, to the liver-type pyruvate kinase (L-PK) promoter. This event was indispensable for the induction of the L-PK gene by glucose, as demonstrated by RNA silencing, single-cell promoter analysis, and quantitative real-time PCR. We conclude that ChREBP is a critical regulator of lipogenic genes in the beta cell and may play a role in the development of glucolipotoxicity and beta cell failure through alteration of gene expression in type 2 diabetes.

Active Transport, Cell Nucleus↗
PubMed1985

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.

Amino Acid Sequence↗
PubMed1998

Zinc deficiency changes preferred macronutrient intake in subpopulations of Sprague-Dawley outbred rats and reduces hepatic pyruvate kinase gene expression.

Macronutrient selection patterns of male rats were analyzed using a 3-choice macronutrient selection system providing either adequate (+Zn) or deficient (-Zn) levels of zinc (30 or 1 mg Zn/kg). In study 1, rats were provided +Zn and -Zn diets for 28 d. All rats preferred carbohydrate (>50% carbohydrate intake) at the onset, consuming an average of 71% carbohydrate (cho), 17% protein (pro), and 12% fat. By the end of the study, 25% of the -Zn rats switched preference from cho to fat, whereas no +Zn rats changed. In study 2, -Zn rats preferring fat increased their total intake to normal levels, but only 50% reverted to carbohydrate preference after 35 d of zinc repletion. Hypothalamic concentrations of galanin were measured in groups of +Zn and -Zn cho- and fat-preferring rats. Galanin, which may be regulated with fat intake, was not different in -Zn rats preferring fat vs. -Zn rats preferring cho. Galanin concentrations were higher in +Zn than in -Zn rats (P < 0.05) and higher in +Zn rats preferring fat than in +Zn rats preferring cho (P < 0.05). Hepatic pyruvate kinase (PK) mRNA concentrations were related to cho preference, regardless of zinc status. When PK mRNA levels were measured in rats consuming a single AIN- 93-based diet, PK mRNA levels were significantly reduced by zinc deficiency (P < 0.05). Because PK is highly regulated by insulin, the effect of insulin may be reduced by zinc deficiency, making it more difficult for -Zn rats to catabolize dietary cho. This may explain why some -Zn rats switched from preferring cho to fat after developing zinc deficiency.

Animals↗
PubMed1995

[Diagnosis of pyruvate kinase deficiency in the presence of an elevated reticulocyte count].

HISTORY AND CLINICAL FINDINGS: Three members of one family (mother, now aged 44 years; daughter, now aged 23 years, and son, now aged 19 years) have had chronic haemolytic anaemia since their infancy. All three have had several blood transfusions a year because of haemoglobin (Hb) concentrations as low as 5 g/dl due to intermittent increases in haemolysis. Despite an unclear diagnosis all three had splenectomies: their spleens had been enlarged to about 5-7 cm below the rib margin. After splenectomy their haemoglobin concentration rose to 9-12 g/dl and the previously raised reticulocyte count of 150-250/1000 rose further to 400-700/1000. None of the patients had symptoms other than a slight decrease in exercise tolerance. They were admitted to hospital for further evaluation. Physical examination was unremarkable except for slight jaundice and well healed splenectomy scar. TESTS: All three patients had normochromic haemolytic anaemia (Hb 10-12 g/dl), bilirubin 2-3 mg/dl, lactate dehydrogenase activity 400-600 U/l). The reticulocyte count was raised to 420-690/1000. In all of them pyruvate kinase (PK) activity was normal or slightly increased if related to Hb concentration. Reticulocytes from healthy persons were isolated by density-gradient centrifugation so that samples could be prepared with different numbers of reticulocytes: PK activity was found to be dependent on the sample's reticulocyte count. In the three patients PK deficiency could, therefore, only be diagnosed by taking into account the raised reticulocyte count. CONCLUSION: As reticulocytes have a higher PK activity than erythrocytes the reticulocyte count must be taken into account when assessing the significance of a given value of this enzyme's activity.

Adult↗
PubMed2001

Congenital insensitivity to pain with anhidrosis (CIPA): novel mutations of the TRKA (NTRK1) gene, a putative uniparental disomy, and a linkage of the mutant TRKA and PKLR genes in a family with CIPA and pyruvate kinase deficiency.

Congenital insensitivity to pain with anhidrosis is an autosomal recessive hereditary disorder characterized by recurrent episodic fever, anhidrosis (inability to sweat), absence of reaction to noxious stimuli, self-mutilating behavior, and mental retardation. The human TRKA gene (NTRK1), located on chromosome 1q21-q22 encodes the receptor tyrosine kinase for nerve growth factor. We reported that TRKA is the gene responsible for CIPA and we developed a comprehensive strategy to screen for TRKA mutations and polymorphisms, as based on the gene's structure and organization. Here we report eight novel mutations detected as either a homozygous or heterozygous state in nine CIPA families from five countries. Mendelian inheritance of the mutations was confirmed in seven families for which samples from either parent were available. However, non-mendelian inheritance seems likely for the family when only samples from the mother and siblings, (but not from the father) were available. A paternal uniparental disomy for chromosome 1 is likely to be the cause of reduction to homozygosity of the TRKA gene mutation in this family. Interestingly, a Hispanic patient from the USA has two autosomal genetic disorders, CIPA and pyruvate kinase deficiency, whose genetic loci are both mapped to a closely linked chromosomal region. A splice mutation and a missense mutation were detected in the TRKA and PKLR genes from the homozygous proband, respectively. Thus, concomitant occurrence of two disorders is ascribed to a combination of two separate mutant genes, not a contiguous gene syndrome. This finding suggests a mechanism responsible for two autosomal genetic disorders in one patient. All these data further support findings that TRKA defects can cause CIPA in various ethnic groups. This will aid in diagnosis and genetic counseling of this painless but severe genetic disorder.

Adolescent↗
PubMed1997

Erythrocyte pyruvate kinase- and glucose phosphate isomerase deficiency: perturbation of glycolysis by structural defects and functional alterations of defective enzymes and its relation to the clinical severity of chronic hemolytic anemia.

The pathogenesis of two metabolic disorders caused by enzyme defects in the red blood cell leading to hemolytic anemia, and in some cases of glucose phosphate isomerase (GPI) deficiency additionally to neurological impairment was investigated. Rheological studies were performed to determine the influence of a shortage of energy on the deformability of the erythrocytes. The functions of the enzymes were determined by studying the enzyme kinetics, the temperature dependence of the enzyme activity and the migration of the proteins in an electric field. A detailed molecular genetic analysis of the gene encoding for the given protein allowed the detection of mutations involving amino acid exchanges which cause alterations of the protein structure. For both enzyme deficiencies, a good correlation was found between the structural changes (usually caused by single point mutations in the gene), the altered function of the enzymes and the severity of the clinical picture. The exchange of amino acids close to either the active site or the regulatory domain results in a decreased turnover as well as an alteration of the regulatory properties of the enzymes; this usually leads to an increased severity of the disease. Increased concentrations of glucose-6-phosphate (G-6-P), found in all red blood cells of patients suffering from hemolytic anemia caused by pyruvate kinase (PK) and GPI deficiency, correlate well with the severity of the clinical picture, apparently reflecting the degree of the perturbation of glycolysis. This results in a lack of the energy donor adenosine triphosphate (ATP); this leads then to a destabilization of the red cell membrane which causes earlier lysis of the red blood cell, which in turn gives rise to hemolytic anemia of variable degrees. One patient with neurological symptoms has been studied so far biochemically and at the molecular genetic level. The point mutations found in this patient's GPI gene support the idea that GPI may have a neurological function in addition to its role in the carbohydrate metabolism; this is due to the presence of a monomeric sequence analogue called neuroleukin (NLK). The mutations apparently lead to the incorrect folding of this neurotrophic factor, and thus destroy the neurological activity.

Anemia, Hemolytic↗
PubMed1998

Requirements for the adaptor protein role of dihydrolipoyl acetyltransferase in the up-regulated function of the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.

The dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembled via its COOH-terminal domain with exterior E1-binding domain and two lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Human E2 structures were prepared with only one lipoyl domain (L1 or L2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown to be essential for markedly enhanced PDP function and were required for greatly up-regulated PDK function. The complete absence of the L1 domain reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. With nonlipoylated L2 present, lipoylated L1 supported a lesser enhancement in PDK function with significant stimulation upon acetylation of L1. Prevention of L1 lipoylation in K46AE2 removed this competitive L1 role and enhanced L2-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of stimulatory effectors. Thus, the E2-L2 domain has a paramount role in facilitating enhanced PDK and PDP function but inclusion of E2-L1 domain, even in a noninteracting (nonlipoylated) form, contributes to the marked elevation of these activities.

Acetyltransferases↗
PubMed1999

Photosynthesis and carbon partitioning in transgenic tobacco plants deficient in leaf cytosolic pyruvate kinase

Whole-plant diurnal C exchange analysis provided a noninvasive estimation of daily net C gain in transgenic tobacco (Nicotiana tabacum L.) plants deficient in leaf cytosolic pyruvate kinase (PKc-). PKc- plants cultivated under a low light intensity (100 &mgr;mol m-2 s-1) were previously shown to exhibit markedly reduced root growth, as well as delayed shoot and flower development when compared with plants having wild-type levels of PKc (PKc+). PKc- and PKc+ source leaves showed a similar net C gain, photosynthesis over a range of light intensities, and a capacity to export newly fixed 14CO2 during photosynthesis. However, during growth under low light the nighttime, export of previously fixed 14CO2 by fully expanded PKc- leaves was 40% lower, whereas concurrent respiratory 14CO2 evolution was 40% higher than that of PKc+ leaves. This provides a rationale for the reduced root growth of the PKc- plants grown at low irradiance. Leaf photosynthetic and export characteristics in PKc- and PKc+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data suggest that PKc in source leaves has a critical role in regulating nighttime respiration particularly when the available pool of photoassimilates for export and leaf respiratory processes are low.

Journal Article↗
PubMed1990

Utilization of an enzyme heat stability test and erythrocyte glycolytic intermediate assays to assist in the diagnosis of canine pyruvate kinase deficiency.

A macrocytic hypochromic anemia, with marked reliculocytosis and large numbers of circulating nucleated erythrocytes, was recognized in a 3 1/2-year-old male Beagle-cross dog. A presumptive diagnosis of erythrocyte pyruvate kinase (PK) deficiency was made, even though PK activity from the patient was within the range of activities measured for normal dogs, because the PK activity should have been several times normal in light of the large number of reticulocytes present. The PK activity in a hemolysate from the patient was heat-labile compared to activities in hemolysates from normal dogs, a finding consistent with results from a previous study of PK-deficient Basenji dogs. Seven phosphorylated glycolytic intermediates and 2,3-diphosphoglycerate were measured in protein-free extracts of erythrocytes from the patient. All were present in concentrations above that present in normal canine erythrocytes, further supporting the diagnosis of PK deficiency.

Journal Article↗