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Biomedical subjects

N Taniguchi

Publications and source records attributed to N Taniguchi.

At least 55 records · Page 3Linked to original sources

[Responses to a questionnaire on laboratory examinations performed by graduates of Jichi Medical School in rural practice].

Graduates from Jichi Medical School are obligated to work at rural clinics or hospitals, where most of them are the only medical doctor in the house. Our need to continuously improve medical education, including the learning that takes place in the clinical setting, requires us to understand how these graduates actually use laboratory examinations and what examinations they find most important in their practice. We designed a questionnaire to address these questions. Three hundred sixty-eight graduate physicians working at clinics or hospitals in both rural or urban areas were asked to complete the questionnaire, providing information on the size of their institution, the laboratory equipment and examinations that they have direct access to, and what examinations they find most important. Two hundred seventy-eight (75.5%) of the 368 recipients responded. More than seventy percent of the respondents reported that their institutions had electrocardiographs, abdominal and cardiac ultrasonographs, urinalysis test paper, and portable blood glucose meters; and more than half of them reported having used these instruments without assistance in emergency situations and outside of ordinary office hours. Moreover, a majority of the respondents said that they considered it important that a physician is able to use these instruments without the help of other staff members. These responses clearly show the importance and usefulness of covering examination techniques and the principles of laboratory medicine in medical education.

Attitude of Health Personnel↗

Fusion hybrids with macrophage and melanoma cells up-regulate N-acetylglucosaminyltransferase V, beta1-6 branching, and metastasis.

It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed enhanced metastatic potential in vivo, increased motility in vitro, increased ability to produce melanin, and responsiveness to melanocyte stimulating hormone compared with the parental Cloudman S91 melanoma cells. These hybrids also showed altered N-glycosylation consistent with a slower migration pattern of lysosome-associated membrane protein (LAMP-1) on electrophoretic gels. Because LAMP-1 is the major carrier of polylactosamine sugar structures, and synthesis of this complex sugar moiety indicates the extent of beta1,6 branch formation by beta1,6-N-acetyl-glucosaminyltransferase V (GnT-V), we analyzed the expression of GnT-V and beta1,6 branching in highly metastatic macrophage-fusion hybrids and compared with poorly metastatic ones. GnT-V was up-regulated in regard to both mRNA levels and enzymatic activity specifically in metastatic hybrids as well as parental macrophages compared with weakly metastatic hybrids and parental melanoma cells. Macrophages and metastatic hybrids also showed increased binding of the lectin L-phytohemagglutinin, which specifically binds to the beta1,6-branched sugar moiety. In addition, in metastatic hybrids there was increased cell surface expression of LAMP-1 and beta1 integrin, two prominent substrates for GnT-V also known to be associated with metastasis. Finally, exposure of metastatic hybrids in vitro to L-phytohemagglutinin or LAMP-1 completely eliminated melanocyte stimulating hormone/ isobutylmethyl xanthine-induced motility, suggesting a role for GnT-V in the motility of these cells. In summary, macrophage fusion with melanoma cells often increased metastatic potential, which was associated with enhanced expression of GnT-V and beta1,6-branching in glycoproteins. It is suggested that the known correlation with elevated GnT-V in both human and animal metastasis could, at least in some cases, reflect previous fusion of tumor cells with tumor-infiltrating macrophages, which, similar to malignant cells, show elevated expression of GnT-V and beta1,6-branched polylactosamines.

Animals↗

Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors.

N-Acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. In this study we investigated the effect of GnT-III on epidermal growth factor (EGF) signaling in HeLaS3 cells. Although the binding of EGF to the epidermal growth factor receptor (EGFR) was decreased in GnT-III transfectants to a level of about 60% of control cells, the EGF-induced activation of extracellular signal-regulated kinase (ERK) in GnT-III transfectants was enhanced to approximately 1.4-fold that of the control cells. A binding analysis revealed that only low affinity binding of EGF was decreased in the GnT-III transfectants, whereas high affinity binding, which is considered to be responsible for the downstream signaling, was not altered. EGF-induced autophosphorylation and dimerization of the EGFR in the GnT-III transfectants were the same levels as found in the controls. The internalization rate of EGFR was, however, enhanced in the GnT-III transfectants as judged by the uptake of (125)I-EGF and Oregon Green-labeled EGF. When the EGFR internalization was delayed by dansylcadaverine, the up-regulation of ERK phosphorylation in GnT-III transfectants was completely suppressed to the same level as control cells. These results suggest that GnT-III overexpression in HeLaS3 cells resulted in an enhancement of EGF-induced ERK phosphorylation at least in part by the up-regulation of the endocytosis of EGFR.

Endocytosis↗

alpha1,6fucosyltransferase is highly and specifically expressed in human ovarian serous adenocarcinomas.

An elevated level of alpha1,6fucosylation in N-glycans represents one of the cancer-related alterations of oligosaccharides and is associated with the metastatic potential of hepatoma cells. However, expression of alpha1,6fucosyltransferase (alpha1,6FucT), which is involved in this aberrant glycosylation, has not been intensively explored in other malignant tumors. We report on a study of the expression of alpha1,6FucT in various types of epithelial ovarian carcinoma tissue, as well as normal ovary, benign and borderline ovarian tumors. The activity assay showed that alpha1,6FucT is highly and specifically elevated in serous adenocarcinomas but not in normal and other ovarian tumor tissues. This elevation was due to enhancement of mRNA expression, as evidenced by Northern blot analysis. Furthermore, we have shown immunohistochemically that alpha1,6FucT expression is localized predominantly in cancer cells. Lectin blot analysis using Lens culinaris agglutinin, which preferentially recognizes alpha1,6fucose residue, suggested that several glycoproteins were likely targets for modification by alpha1, 6fucosylation in serous adenocarcinoma tissues. These findings suggest that the elevated expression of alpha1,6FucT and the resulting modification of N-glycans are distinctive features of this type of ovarian cancer and may be related to the progression of this malignancy.

Adult↗

Molecular cloning and expression of cDNA encoding chicken UDP-N-acetyl-D-glucosamine (GlcNAc): GlcNAcbeta 1-6(GlcNAcbeta 1-2)- manalpha 1-R[GlcNAc to man]beta 1,4N-acetylglucosaminyltransferase VI.

A cDNA that encodes UDP-N-acetyl-d-glucosamine (GlcNAc):GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R[GlcNA c to Man]beta1, 4N-acetylglucosaminyltransferase VI (GnT VI), which is responsible for the formation of pentaantennary asparagine-linked oligosaccharides (N-glycans), has been cloned from a hen oviduct cDNA library based on the partial amino acid sequences of the purified enzyme. The isolated cDNA clone contained an open reading frame encoding 464 amino acids, including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. The primary structure was found to be significantly similar to human GnT IV-homologue, the gene for which was cloned from the deleted region in pancreatic cancer, and to human and bovine GnT IVs. Chicken GnT VI-transfected COS-1 cells showed a high GnT VI activity (26.8 pmol/h/mg protein), whereas nontransfected, mock-transfected, or human GnT IV-homologue-transfected COS-1 cells had no activity. Northern blot analysis using poly(A)(+) RNA from hen oviduct indicated that the size of GnT VI mRNA is 2.1 kilobases. Reverse transcription-polymerase chain reaction analysis showed that GnT VI mRNA was relatively highly expressed in oviduct, spleen, lung, and colon.

Amino Acid Sequence↗

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells.

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.

Animals↗

Purification and characterization of UDP-GlcNAc: GlcNAcbeta 1-6(GlcNAcbeta 1-2)Manalpha 1-R [GlcNAc to Man]-beta 1, 4-N-acetylglucosaminyltransferase VI from hen oviduct.

A new beta1,4-N-acetylglucosaminyltransferase (GnT) responsible for the formation of branched N-linked complex-type sugar chains has been purified 64,000-fold in 16% yield from a homogenate of hen oviduct by column chromatography procedures using Q-Sepharose FF, Ni(2+)-chelating Sepharose FF, and UDP-hexanolamine-agarose. This enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to tetraantennary oligosaccharide and produces pentaantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-6 arm. It requires a divalent cation such as Mn(2+) and has an apparent molecular weight of 72,000 under nonreducing conditions. The enzyme does not act on biantennary oligosaccharide (GnT I and II product), and beta1,6-N-acetylglucosaminylation of the Manalpha1-6 arm (GnT V product) is essential for its activity. This clearly distinguishes it from GnT IV, which is known to generate a beta1-4-linked GlcNAc residue only on the Manalpha1-3 arm. Based on these findings, we conclude that this enzyme is UDP-GlcNAc:GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R [GlcNAc to Man]-beta1,4-N-acetylglucosaminyltransferase VI. This is the only known enzyme that has not been previously purified among GnTs responsible for antenna formation on the cores of N-linked complex-type sugar chains.

Animals↗

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

Alanine↗

The postmitotic growth suppressor necdin interacts with a calcium-binding protein (NEFA) in neuronal cytoplasm.

Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca(2+)-binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca(2+) binding activity as determined by (45)Ca(2+) overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca(2+) levels. Thus, necdin and NEFA might be involved in Ca(2+) homeostasis in neuronal cytoplasm.

Animals↗

A pivotal role of Zn-binding residues in the function of the copper chaperone for SOD1.

A Cu chaperone for SOD1 (CCS) is required for the incorporation of copper ion into the protein. To investigate the roles of the conserved metal-binding residues in CCS, we introduced amino acid substitutions into human CCS and examined the function of the mutant CCS by transforming a mutant yeast strain, SY2950, which lacks the lys7 gene, a yeast orthologue of the mammalian CCS. Mutant CCS in which amino acid residues His147 and Asp167 were substituted by Ala exhibited a decreased ability to complement the growth of SY2950 under Lys-deficient conditions. This is because the mutations made the human CCS function in a less efficient manner, especially under metal-restricted conditions, leaving Cu,Zn-SOD in an apo-form. Since the His and Asp residues are both responsible for binding Zn which would serve to maintain the folded structure, the structural integrity supported by the coordinated Zn ion would be essential for CCS function.

Amino Acid Sequence↗

Actions of brain-derived neurotrophic factor on evoked and spontaneous EPSCs dissociate with maturation of neurones cultured from rat visual cortex.

To address the question of whether brain-derived neurotrophic factor (BDNF) directly enhances excitatory synaptic transmission, we recorded excitatory postsynaptic currents (EPSCs) from solitary neurones cultured on glial microislands for 7-38 days, and observed changes in EPSCs after the application of BDNF. In this preparation the possible action of BDNF on GABAergic inhibition was not involved, and evoked and spontaneous (miniature) EPSCs were derived from the same group of synapses (autapses). The application of BDNF at a concentration of 200 ng ml-1 rapidly enhanced the frequency of miniature EPSCs (mEPSCs) in almost all the neurones tested. On the other hand, the amplitude of mEPSCs did not change at all, suggesting that the site of BDNF action is presynaptic. In contrast to the enhanced frequency of mEPSCs, evoked EPSCs were not potentiated in 61 % of the cells tested. Most of these BDNF-insensitive EPSCs had a peak amplitude larger than 1 nA, while most of the other BDNF-sensitive EPSCs had a smaller amplitude. The former EPSCs had smaller coefficients of variation (CVs) of amplitude, while the latter had larger CVs, suggesting the possibility that the presynaptic release probability for the former groups of EPSCs might have beeen saturated so that the BDNF action was occluded. To test this possibility we applied a low Ca2+ solution to 17 cells and reduced the amplitude of their evoked EPSCs to less than or near to 1 nA. It was found, however, that BDNF did not enhance these EPSCs. Rather, evoked EPSCs of almost all the cells cultured for less than 15 days were enhanced by BDNF, while those of most of the cells cultured for longer than 16 days were not enhanced. These results suggest that BDNF enhances transmitter release from presynaptic sites through its action on the release machinery, which can be differentiated into a BDNF-insensitive form for evoked release and a BDNF-sensitive form for spontaneous release with maturation of synapses.

Animals↗

Involvement of cytokines in the mechanism of whole-body hyperthermia-induced cardioprotection.

BACKGROUND: Hyperthermia increases cardiac tolerance to ischemia/reperfusion injury and activates manganese superoxide dismutase (Mn-SOD), an intrinsic radical scavenger, in myocardium in a biphasic manner. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) induced a biphasic cardioprotection that corresponded to the activation of Mn-SOD. However, a direct association between Mn-SOD activation in myocardium and the acquisition of tolerance to ischemia/reperfusion injury induced by hyperthermia and the involvement of the cytokines in the signal transduction pathway for the hyperthermia-induced cardioprotection have not yet been elucidated. METHODS AND RESULTS: Hyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. At 0.5 and 72 hours after hyperthermia, ischemia was induced by occlusion of the left coronary artery for 20 minutes, followed by reperfusion for 48 hours. Inhibition of the increases in Mn-SOD content and activity 72 hours after hyperthermia by the administration of antisense oligodeoxynucleotides to Mn-SOD abolished the expected decrease in myocardial infarct size. The simultaneous administration of neutralizing antibodies to TNF-alpha and IL-1beta before hyperthermia abolished the biphasic cardioprotection and increase in Mn-SOD activity. CONCLUSIONS: The increase in Mn-SOD activity mediated through the production of TNF-alpha and IL-1beta by whole-body hyperthermia is important in the acquisition of early- and late-phase cardioprotection against ischemia/reperfusion injury in rats.

Animals↗

The Asn-420-linked sugar chain in human epidermal growth factor receptor suppresses ligand-independent spontaneous oligomerization. Possible role of a specific sugar chain in controllable receptor activation.

To elucidate a role(s) of Asn-linked sugar chain(s) in the function of epidermal growth factor receptor (EGFR), a series of the EGFR mutants were prepared in which potential glycosylation sites in the domain III were eliminated by site-directed mutagenesis. Although the wild-type and mutants of Asn-328, Asn-337, and Asn-389 underwent autophosphorylation in response to epidermal growth factor (EGF), the Asn-420 --> Gln mutant was found to be constitutively tyrosine-phosphorylated. This abnormal ligand-independent phosphorylation of the mutant appears to be due to a ligand-independent spontaneous oligomer formation, as shown by a cross-linking experiment using the purified soluble extracellular domain (sEGFR). As revealed by the dissociation of the Asn-420 --> Gln sEGFR oligomer by simple dilution, it seems likely that the equilibrium is shifted toward oligomer formation to an unusual degree. Furthermore, it was also found that the mutation caused a loss of the ability to bind EGF. These findings suggest that the sugar chain linked to Asn-420 plays a crucial role in EGF binding and prevents spontaneous oligomerization of the EGFR, which may otherwise lead to uncontrollable receptor activation, and support the view of a specific role of an Asn-linked sugar chain in the function of a glycoprotein.

Asparagine↗

Asymmetric Synthesis of Axially Chiral Anilides by Enantiotopic Lithiation of Tricarbonyl(N-methyl-N-acyl-2,6-dimethylanilide)chromium Complex.

Axially chiral N-methylanilides were synthesized by enantioselective lithiation of prochiral tricarbonyl(N-methyl-N-pivaloyl-2,6-dimethylaniline)chromium (1) with the lithium amide of the 4-methylpiperazinylethylamine derivative 13 followed by electrophilic quenching up to 97% ee in good yields. The resulting axially chiral chromium-complexd anilides 2 were oxidized under air to give the axially chiral anilides 14 in enantiomerically active form without axial bond rotation at room temperature.

Journal Article↗

Redox capacity of cells affects inactivation of glutathione reductase by nitrosative stress.

Glutathione reductase (GR) plays a pivotal role in maintaining glutathione (GSH) in its reduced form. We have isolated a cDNA for rat GR and constructed a baculovirus system to produce recombinant GR on a large scale. This protein was purified by simple, two-step chromatographic procedure using DE52 and 2',5'-ADP Sepharose. Tissue distributions of GR were examined by Northern and Western blotting with a rabbit antibody to purified GR. GR was expressed in the order of reactivity; kidney, colon, liver, stomach, etc. Western blot analysis showed that both the cytosolic and the mitochondrial fractions of liver homogenate gave immunoreactive bands of similar size. This indicates that the same gene products exist in these fractions. Since nitric oxide (NO) produced under inflammatory conditions causes nitrosative stress and affects the redox states of surrounding tissues, we investigated the effects of NO donors on the enzymatic activities of purified GR. S-nitrosoglutathione (GSNO), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 mM gave 39, 15, and 12% inhibitions, respectively. In RAW 264.7 cells the GR activity was reported to be inhibited by GSNO. In A549 cells, however, no such change in the activity, protein levels and mRNA of GR was noted. Since these cells have a much higher redox capacity than RAW 264.7 cells as judged by GR activity and thioredoxin reductase activity it wound minimize cellular damage, including inactivation of GR caused by nitrosative stress.

Amino Acid Sequence↗

A potent inhibitor of inducible nitric oxide synthase, ONO-1714, a cyclic amidine derivative.

(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K(i) of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID(50) value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent.

Amidines↗

Brain-derived neurotrophic factor blocks long-term depression in solitary neurones cultured from rat visual cortex.

1. To address questions of whether long-term depression (LTD) in the visual cortex is expressed in pre- or postsynaptic sites, whether brain-derived neurotrophic factor (BDNF) exerts its LTD-blocking action without involvement of GABAergic inhibition, and whether the action of BDNF is pre- or postsynaptic, we observed excitatory postsynaptic currents (EPSCs) from solitary neurones cultured on glial microislands. In this preparation GABAergic inhibition is not involved and a group of synapses (autapses) which generate evoked EPSCs is thought to be the same as those generating spontaneous EPSCs. 2. A short depolarising voltage step to the soma generated Na+ spikes which were followed by autaptic EPSCs. When this somatic activation was paired with prolonged depolarisation for 100 ms to -30 mV and repeated at 1 Hz for 5 min, LTD was induced in all of the nine cells tested. Then, the frequency of spontaneous EPSCs decreased, but the amplitude did not change, suggesting that the site of LTD expression is presynaptic. 3. Application of BDNF at 50 ng ml-1 blocked the depression of evoked EPSCs and the decrease in the frequency of spontaneous EPSCs. An inhibitor for receptor tyrosine kinases, K252a, antagonised the action of BDNF, suggesting an involvement of BDNF receptors, TrkB. 4. These results suggest that BDNF prevents low-frequency inputs from inducing LTD of excitatory synaptic transmission through presynaptic mechanisms in the developing visual cortex.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Dominant negative role of the glutamic acid residue conserved in the pyruvate kinase M(1) isozyme in the heterotropic allosteric effect involving fructose-1,6-bisphosphate.

Pyruvate kinase M(1), a nonallosteric isozyme, lacks heterotropic allosteric effect involving fructose-1,6-bisphosphate (FBP). To explore the molecular basis for this, a series of mutants were prepared and characterized, in which the possible candidate, Glu-432, was replaced in the rat M(1) isozyme and its allosteric mutant with the replacement of Ala-398 by Arg. Although these single mutants of Glu-432 remained nearly fully active, similar to the wild type, only the mutants with replacements by Lys and Ala were more efficiently activated by FBP when the enzymes were inhibited by L-phenylalanine. Kinetic analyses and ligand-induced fluorescence quenching studies using the allosteric double mutants indicated that the loss of a negative charge at residue 432 led to a dramatic decrease in the apparent activation constant and apparent K(d) for FBP. Furthermore, this enhancement was found to be associated with the modification of the FBP-binding site rather than the alteration of the subunit assembly. These findings suggest that Glu-432 hinders the heterotropic allosteric effect by preventing the binding of FBP through a repulsive electrostatic interaction and thereby contributes to its unique unregulated properties, independent of the shifted allosteric transition.

Allosteric Regulation↗