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PubMed1976

Pyruvate kinase isozymes in various tissues of rat, and increase of spleen-type pyruvate kinase in liver by injecting chromatins from spleen and tumor.

Pyruvate kinase [EC 2.7.1.40] in various tissues of rats was separable into seven kinds of pI-isozymes by isoelectric separation with Ampholine carrier ampholytes; pI 5.4-isozyme, pI 5.6-isozyme, pI 6.2-isozyme (2 kinds), pI 6.6-isozyme, pI 7.4-isozyme, and pI 7.8-isozyme. Some of these pI-isozymes contained bound fructose 1,6-diphosphate (FDP). The bound FDP was completely dissociated when the pI-isozymes were salted out with ammonium sulfate. In the FDP-free form, pyruvate kinase was classified into three types, liver-type (type L) of pI 6.2, muscle-type (type M) of pI 7.4, and spleen-type (type M2) of pI 7.8. The liver-type isoenzyme had two kinds of FDP-binding sites; the pI 5.6-isozyme and pI 5.4-isozyme were obtained when one and two kinds of sites were bound with FDP, respectively. The association and dissociation of FDP at both sites were reversible in the presence and absence of 0.15 M KC1 (high ionic strength). The muscle-type isoenzyme had no FDP-binding site. The spleen-type isoenzyme had two kinds of FDP-binding sites, like the liver-type isoenzyme. When the ionic strength of solutions containing the enzyme and FDP was sufficiently low, one and two kinds of the sites could bind with FDP, converting the enzyme into pI 6.6-isozyme and pI 6.2-isozyme, respectively. FDP bound with one kind of site (the 2nd site) was easily dissociable, but FDP bound with the other kind of site (the 1st site) was not. Provided that the 1st site carried bound FDP, the 2nd site was associable at high ionic strength. The liver-type isoenzyme free of FDP and the spleen-type isoenzyme bound with FDP at both sites had similar pI values of 6.2 and were not separable by isoelectric separation. Some properties of these pI-isozymes were compared. When Rhodamine sarcoma was transplanted in rats, the content of spleen-type isoenzyme in the livers increased. When rats were injected with chromatin prepared from either Rhodamine sarcoma or spleen, the content of spleen-type isoenzyme in the livers again increased. This was not observed on the injection of chromatin prepared from liver, indicating that the factor capable of controlling the gene expression was present in chromatins of sarcoma and spleen but barely or not at all in chromatin of liver.

Animals↗
PubMed1989

Comparative study of human M2-type pyruvate kinases isolated from human leukocytes and erythrocytes of a patient with red cell pyruvate kinase hyperactivity.

M2-type pyruvate kinases (M2-PK) have been isolated from human leukocytes and from the erythrocytes of a patient with erythrocyte PK hyperactivity. The kinetic characteristics of the patient erythrocyte M2-PK were similar to those of leukocyte M2-PK except for the Hill coefficient of phosphoenol pyruvate kinetics that showed little difference in the values. The patient erythrocyte M2-PK displayed complete immunological identity with leukocyte M2-PK in immunodiffusion, immunoblotting and immunoneutralization. The sensitivity to proteolysis by trypsin and the electrophoretic migration in different conditions were similar for the M2-PK of both origins. These results suggest an identity between this M2-PK abnormally present in erythrocytes and the M2-PK from leukocytes.

Chromatography, Ion Exchange↗
PubMed1989

Subunit associations in the mammalian pyruvate dehydrogenase complex. Structure and role of protein X and the pyruvate dehydrogenase component binding domain of the dihydrolipoyl transacetylase component.

We have further distinguished the structures and roles of the two lipoyl-bearing components of the pyruvate dehydrogenase complex, the dihydrolipoyl transacetylase (E2) component and the component designated as protein X. The amino acid sequences of the NH2-terminal regions of the lipoyl-bearing domain of the E2 component and protein X are different but related. The dihydrolipoyl dehydrogenase (E3) component but not the pyruvate dehydrogenase (E1) component protected protein X against proteolytic degradation by trypsin and protease Arg C. Protein X-specific polyclonal antibodies inhibit reconstitution of the overall reaction catalyzed by the complex (E2-X subcomplex recombined with the E1 and E3 components). The rate of development of this inhibition was reduced by pretreatment of E2-X subcomplex with the E3 component. These data strongly suggest the E3 component associates with protein X. The E1 component (an alpha 2 beta 2 tetramer), but not the E3 component, reduced trypsin cleavage of E2 subunits at 4 degrees C and altered the patterns of cleavage at 22 degrees C. At 22 degrees C a large (Mr congruent to 49,000) outer domain (E2LB) of the E2 component was produced. E2LB had the same NH2-terminal amino acid sequence as the smaller (Mr congruent to 38,000) lipoyl-bearing domain (E2L). E2LB, in contrast to E2L, interacted with both the E1 component and the beta subunit of the E1 component. Thus the E1 component is bound through an E1-binding domain that is located in E2 subunits between the inner domain and the outer, lipoyl-bearing domain.

Acetyltransferases↗
PubMed1988

Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences.

The structural gene of pyruvate formate-lyase (pfl) and that of pyruvate-formate-lyase-activating enzyme were shown to be adjacent on the chromosomal map of Escherichia coli. DNA sequencing was performed along a stretch of 3592 nucleotides to obtain the amino acid sequences of both proteins. The derived primary structures (759 and 245 residues) were confirmed by partial structure analyses on the purified proteins. The open reading frames are separated by a 194-nucleotide stretch, and their flanking regions include signal elements that are compatible with separate control of protein synthesis from the two genes.

Acetyltransferases↗
PubMed1986

Identity of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase.

Identification of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase has been done. When a mitochondria extract was subjected to isoelectric focusing, the two enzyme activities were identically focused. This procedure and DEAE-Sepharose chromatography revealed multiple forms of the enzyme, in which the main form was purified. In the various purification steps the two enzyme activities appeared in the same fraction. The enzyme of the final preparation step gave a single band in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. During the purification, a similar increase of the specific activity and yield were obtained in the two activities. Phenylalanine was found to be a competitive inhibitor of asparagine transaminase. These results suggest the identity of the two enzymes.

Animals↗
PubMed1979

Interaction of the chiral pyruvate analog, 2-keto-3-bromobutyrate, with pyruvate lyases. 2-Keto-3-deoxygluconate-6-phosphate aldolase of Pseudomonas putida.

The enzyme 2-keto-3-deoxygluconate-6-P aldolase of Pseudomonas putida is inactivated by one of the chiral forms of 2-keto-(3RS)-3-bromobutyric acid (bromoketobutyrate). The inactivation shows saturation kinetics and competition with pyruvate. The minimal inactivation half-time is 4 min and that concentration of bromoketobutyrate half-saturating the enzyme is 2 mM. (3RS)-[3-3H]bromoketobutyrate is catalytically detritiated during enzyme inactivation. A kinetic analysis of rates gave data consistent with both catalysis and inactivation occurring at a single protein site, the catalytic site. The enzyme only detritiates one of the two optical isomers of bromoketobutyrate, and that form which is detritiated also alkylates the catalytic site. The inactive isomer of reagent degrades, with inversion, to L-lactate so that the chiral form specific for the enzyme is 2-keto-(3S)-3-bromobutyrate. Thus, as is the case with bromopyruvate, the enzyme catalyzes protonation of the re face at C-3 of the enzyme-reagent eneamine. As a result, bromoketobutyrate could serve as a chiral probe for stereochemical constraints of selected pyruvate-specific lyase active sites.

Aldehyde-Lyases↗
PubMed1988

Inactivation of pyruvate dehydrogenase complex in heart muscle mitochondria of gold-thioglucose-induced obese mice is not due to a stable increase in activity of pyruvate dehydrogenase kinase.

The proportion of pyruvate dehydrogenase (PDH) complex in the active dephosphorylated form was decreased (compared with fed lean control mice) in heart muscle mitochondria after the induction of obesity with gold-thioglucose (by 54%) or starvation of lean mice for 48 h (by 81%). The effects of obesity to inactivate PDH complex were demonstrable 4 weeks after administration of gold-thioglucose, and occurred despite significant hyperinsulinaemia in obese animals. Phosphorylation and inactivation of PDH complex in mouse heart muscle in starvation was attributed to a stable increase (2.7-fold) in the activity of PDH kinase as measured in extracts of mitochondria mediated by increased specific activity of a protein activator of PDH kinase (KAP) [Denyer, Kerbey & Randle (1986) Biochem. J. 239, 347-354]. In obese mice no such increase in kinase activity was observed, and we conclude that phosphorylation and inactivation of PDH complex in heart muscle in obesity is not mediated by KAP, but rather is a consequence of increased lipid oxidation.

Animals↗
PubMed1991

Evidence that rat liver pyruvate dehydrogenase kinase activator protein is a pyruvate dehydrogenase kinase.

It is shown here that rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP) catalyses ATP-dependent inactivation and [32P]phosphorylation of pig heart PDHE1 and of yeast (Saccharomyces cerevisiae) PDH complex devoid of PDH kinase activity, that fluorosulphonylbenzoyladenosine inactivates rat liver KAP and the intrinsic PDH kinase of rat liver PDH complex, and that KAP, like PDH kinase, is inactivated by thiol-reactive reagents. It is concluded that KAP is a free PDH kinase.

Adenosine Triphosphate↗
PubMed1991

Long-term regulation of pyruvate dehydrogenase complex. Evidence that kinase-activator protein (KAP) is free pyruvate dehydrogenase kinase.

The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified approx. 2250-fold from high-speed supernatants of mitochondrial extracts from the liver of 48 h-starved rats. Purified KAP demonstrates kinase activity towards both the E1 component of PDC and towards a synthetic peptide corresponding to the major phosphorylation site on E1. Furthermore, the activities of KAP and PDC kinase co-fractionate through several stages of purification and have the same apparent mass. We conclude that KAP is not a distinct protein, but is kinase which has dissociated from the complex.

Animals↗
PubMed1992

Mechanisms of fructose-induced hypertriglyceridaemia in the rat. Activation of hepatic pyruvate dehydrogenase through inhibition of pyruvate dehydrogenase kinase.

1. The effects of purified diets containing 70% glucose or 70% fructose on the activation state of hepatic pyruvate dehydrogenase (PDHa), activity of mitochondrial PDH kinase, plasma triacylglycerols (TG) and hepatic lipogenesis de novo in rats were measured. 2. Plasma TG were significantly increased in the fructose-fed compared with the glucose-fed group (125 +/- 45 mg/dl versus 57 +/- 19 mg/dl; P less than 0.002) after 3-5 weeks on the diet despite less daily food intake. 3. Hepatic PDHa in fructose-fed rats was 144% of the value in glucose-fed rats (15.4 +/- 1.2% versus 10.7 +/- 0.5%; P less than 0.002), whereas cardiac muscle PDHa was not different (45.5 +/- 6.6% versus 41.0 +/- 7.8%). 4. Intrinsic hepatic PDH kinase activity was decreased to 34% of glucose-fed values by fructose feeding (-k = 3.56 +/- 0.39 versus 10.41 +/- 1.85 min-1; P less than 0.005). 5. The fractional contribution to very-low-density-lipoprotein palmitate from hepatic lipogenesis de novo, measured by a stable-isotope mass-spectrometric method, was 10.49 +/- 2.42% (n = 8) in fructose-fed rats versus 5.55 +/- 1.38% (n = 9) in glucose-fed rats (P less than 0.05), and 2.66 +/- 2.39% (n = 3) in chow-fed rats (P less than 0.05 versus fructose-fed group). The absolute contribution to circulating TG from lipogenesis de novo was also significantly higher in the fructose-fed than in the glucose-fed group (14.9 +/- 5.1 mg/dl versus 2.9 +/- 0.6 mg/dl; P less than 0.05) 6. Portal insulin concentrations were significantly higher in the fructose-fed rats (206 +/- 49 mu-units/ml versus 81 +/- 15 mu-units/ml; P less than 0.05). 7. In conclusion, dietary fructose appears to have a specific activating effect on hepatic PDH, mediated at least in part by inhibition of PDH kinase. These results are consistent with increased flux through hepatic PDH and synthesis of new fat, not just increased re-esterification of non-esterified fatty acids.

Animals↗
PubMed1979

Computer simulation of metabolism in pyruvate-perfused rat heart. III. Pyruvate dehydrogenase.

A physiologically and biochemically realistic model of the regulation of pyruvate dehydrogenase complex (PDH) was constructed for the perfused rat heart. It includes conversion between inactive (phospho) and active (dephospho) forms by a specific protein kinase (PDHK) and phosphoprotein phosphatase (PDHP). The activity of the tightly bound PDHK is influenced by synergistic activation/inhibition by acetyl CoA/CoASH and NADH/NAD. PDHK in this simulation was more sensitive to the fraction of ADP that was Mg2+-chelated than to the ATP-to-ADP ratio. Ca2+ stimulates binding of Mg2+-dependent PDHP to the complex; the bound enzyme was considered to be the active species. The fraction of PDH in the active form, rather than substrate and inhibitor levels, determines PDH activity under these conditions. This fraction depends on the present value and recent history of the difference between PDHK and PDHP activities. Both of these are active continuously and continuously control PDH.

Animals↗
PubMed1991

Pyruvate dehydrogenase deficiency due to a 20-bp deletion in exon II of the pyruvate dehydrogenase (PDH) E1 alpha gene.

A 20-bp deletion in the last exon of the pyruvate dehydrogenase (PDH) E1 alpha gene was found in a severely affected female patient diagnosed with PDH deficiency. PDH-complex activity in the patient's fibroblasts was 22% of that in normal controls. The mutation was characterized using PCR techniques with both patient cDNA and genomic DNA, followed by sequencing of the products. E1 beta cDNA sequence was found to be the same as that in controls. The deletion causes a frameshift and the occurrence of a premature stop codon. Western blot analysis revealed an extra band migrating just above the PDH E1 beta band. Northern blot analysis showed normal levels of both E1 alpha and E1 beta message when probed with the respective cDNAs. However, a larger intermediate-size transcript was observed for this patient in the E1 beta blot. The 20-bp deletion was not found in either parent's genomic DNA, and hence we conclude that the mutation must have occurred de novo, either in the germ-line cells or immediately following fertilization.

Amino Acid Sequence↗
PubMed1986

Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L from Rana pipiens.

The regulatory properties of type L pyruvate kinase from Rana pipiens are intermediate between those of the mammalian K and L isozymes. As with mammalian type L, the levels of the frog isozyme are affected by the animal's nutritional state. The mammalian and amphibian isozymes show similar sensitivities to fructose 1,6-bisphosphate activation and amino acid inhibition. By contrast, the frog L isozyme shares several properties of the K class: i.e. irreversible inactivation by oxidized glutathione and lack of response to a cyclic AMP stimulated phosphorylation. Furthermore, as for some mammalian K isozymes, frog type L shows a high PEP affinity and a low cooperativity of PEP binding. Insofar as the properties of this present day enzyme reflect those of its counterpart in the amphibian ancestor of higher vertebrates, our results suggest that at its first expression, the type L resembled the type K. Many important regulatory properties of the L isozyme, especially the sensitivity to phosphorylation, were acquired more recently perhaps in association with an increased importance of constant blood glucose.

Animals↗
PubMed1988

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex.

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.

Amino Acid Sequence↗
PubMed1992

Purification and partial characterization of rat liver pyruvate dehydrogenase kinase activator protein (free pyruvate dehydrogenase kinase).

Rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP), a free PDH kinase readily separable from PDH complex and its intrinsic kinase, has been purified to apparent homogeneity from liver mitochondria of fed and 48-h starved rats. On SDS-PAGE an apparently single band of M(r) 45 kDa was obtained. N-Terminal amino acid sequence analyses (8-10 cycles) confirmed the presence of a single peptide in each case. The specific activity of the purified KAP from 48-h starved rats (14,413 U/mg protein) was 4.5-fold greater than that from fed rats.

Amino Acid Sequence↗
PubMed1991

cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia.

cDNA clones for human R-type pyruvate kinase (PK) were isolated from a human reticulocyte cDNA library, constructed by PCR with a single gene-specific primer. The full-length cDNA was 2060 base pairs long, and the cDNA encoded 574 amino acids, the same number as that by rat R-type PK. Compared with human L-type PK, R-type PK was 31 amino acids longer at the amino terminus. We also cloned and characterized R-type PK cDNA clones from patients with hereditary hemolytic anemia from a PK deficiency, PK Tokyo. A single nucleotide substitution (ACG to ATG) was found at nucleotide 1151 of the coding sequence of the R-type PK, which caused an amino acid substitution, Thr384----Met. Dot blot hybridization of PCR-amplified genomic DNA from patients and their parents by allele-specific oligonucleotide probes showed that the parents, who were second cousins, were heterozygous. To confirm that the nucleotide change was responsible for the variant phenotype, we expressed the L-type PK with the single amino acid change in Escherichia coli and characterized the enzyme. The variant PK was thermolabile and moved slowly in the polyacrylamide gel buffered in 10 mM Tris.HCl, pH 8.3; these characteristics were fully compatible with data obtained from the patient's PK. From these results, we concluded that enzymatic stability of the variant was affected by the point mutation of the PK-encoding gene.

Amino Acid Sequence↗
PubMed1976

Chronic haemolytic anaemia in two patients heterozygous for erythrocyte pyruvate kinase deficiency. Electrofocusing and immunological studies of erythrocyte and liver pyruvate kinase.

Two patients with mild chronic haemolytic anaemia, a mother and her son, were found to be heterozygous for erythrocyte pyruvate kinase deficiency. In the red blood cells the enzymatic activity was reduced by about 50% and the residual PK had normal kinetic properties, stability and electrofocusing pattern. The PK antigen concentration was also decreased by half, so that the ratio of the enzymatic activity to the immunological reactivity (i.e. the molecular specific activity) was normal. In the son's liver PK enzymatic activity was slightly reduced and, above all, an abnormal active form, more anodic than normal PK, was detected by electrofocusing. The propositus's liver PK was also slightly thermo-unstable. It is suggested that the patients were heterozygous for an unstable PK variant which is found in liver, nucleated tissue actively synthesizing proteins, but which disappeared from the erythrocytes because of its unstability.

Adult↗
PubMed1991

Biochemical and molecular characterization of variant pyruvate kinase enzymes and genes from three patients with red blood cell pyruvate kinase deficiency.

Pyruvate kinase (PK) from red blood cells (RBC) of three patients with nonspherocytic hemolytic anemia due to PK deficiency was characterized according to internationally standardized methods. The variant enzymes, which were designated PK 'Memphis', PK 'Bartlett', and PK 'Pontotoc', had 11, 60, and 61%, respectively, of the normal enzyme activity. All variant PK enzymes had increased thermolability. Compared with control, Km (PEP) were 200-300% greater for PK 'Memphis', 50% less for PK 'Bartlett' and 300-400% greater for PK 'Pontotoc'. The Km (ADP) were 40 and 300% greater than normal for PK 'Bartlett' and PK 'Pontotoc', respectively. All variants required higher than normal concentrations of the allosteric modifier, fructose-1,6-diphosphate, to achieve 50% activation of maximal enzyme activity. To define the molecular basis of the gene defect, DNA samples from these patients were examined for restriction-fragment-linked polymorphisms. No differences were observed in the structure of the patients' PK genes compared with a normal control. These results are consistent with a mutation in coding sequences, rather than a large insertion, deletion or rearrangement of genetic information, as the underlying genetic defect that accounts for the altered enzyme properties in these PK-deficient patients.

Enzyme Stability↗