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PubMed1982

Phosphoenolpyruvate:sugar phosphotransferase system-mediated regulation of carbohydrate metabolism in Salmonella typhimurium.

The crr mutation was shown to affect the phosphoenolpyruvate:sugar phosphotransferase system-mediated transient repression of the lac operon, intracellular cAMP levels, and sensitivity to inducer exclusion. Our results indicate that the presumed crr gene product, factor IIIGlc, plays a direct role in the regulation of inducer exclusion. We propose a mechanism in which inducer exclusion depends on both the level and state of phosphorylation of factor IIIGlc and the level of an inducer exclusion-sensitive transport system. The results of studies on the sensitivity to inducer exclusion of glycerol and maltose in cultures induced for short periods of time on these substrates (resulting in varying degrees of activity of the respective transport systems) support this model of inducer exclusion. Previously, the crp*-771 mutation has been shown to result in an altered cAMP receptor protein, which has a changed affinity for cAMP, and to affect the sensitivity for inducer exclusion of glycerol. Changes in other functions of the altered cAMP receptor protein were indicated by our results; these changes were in the roles of this protein in (i) the cAMP-dependent initiation of transcription of the lac operon and (ii) the regulation of intracellular cAMP levels and the export of cAMP. We propose that the crp*-771 mutation has an indirect effect in relieving inducer exclusion in repressed or hypersensitive strains, in which the crp*-771 mutation allows the synthesis of inducer exclusion-sensitive transport systems to higher levels than the levels found in strains containing wild-type cAMP receptor protein.

Carbohydrate Metabolism↗
PubMed2006

Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits.

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic alpha subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK alpha subunit contained an autoinhibitory domain (alpha1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the alpha subunit (alpha1-(313-335)), which includes a predicted alpha-helix, is responsible for alpha subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for alpha1 autoinhibition. An autoinhibitory structural model of human AMPK alpha1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 A and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK alpha1-(1-394) with deletion of residues 313-335 (Deltaalpha394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Deltaalpha394, V298G, or L328Q mutants, which indicated that these AMPK alpha1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitory function.

AMP-Activated Protein Kinases↗
PubMed2006

Biochemical characterization of a recombinant von Willebrand factor (VWF) with combined type 2B and type 1 defects in the VWF gene in two patients with a type 2A phenotype of von Willebrand disease.

BACKGROUND: In a patient previously diagnosed with type 2A von Willebrand disease (VWD) [absence of high and intermediate molecular weight von Willebrand factor (VWF) multimers and markedly reduced ristocetin-induced platelet aggregation (RIPA)], an infusion test of desmopressin was followed by mild thrombocytopenia. This led to further laboratory investigations of his affected brother and of family members, who showed different phenotypic patterns compatible with type 1, 2A, 2B and an uncertain classification of VWD. The two brothers were compound heterozygotes (C275R/P1337L), whereas the others members of the family were heterozygous for C275R (a novel mutation in the D1 domain) or P1337L (a type 2B mutation in the A1 domain). OBJECTIVE AND METHODS: To evaluate the role of the combined effect of the two mutations in the two brothers, C275R and P1337L recombinant (r) VWFs were transiently expressed in COS-7 cells. RESULTS: Recombinant VWF levels secreted in cell media were similar for wild-type (WT), P1337L and hybrid P1337L/WT rVWFs, reduced for hybrids C275R/P1337L and C275R/WT rVWFs, and strongly reduced for C275R rVWF. All rVWFs had a full set of multimers except C275R rVWF, which had only dimers. P1337L rVWF and C275R/P1337L rVWF showed the highest degree of binding to glycoprotein (GP) Ibalpha and the lowest to collagen, followed by P1337L/WT rVWF (with an intermediate level of binding to both ligands), and by WT rVWF with the lowest level of binding to GPIbalpha and the highest to collagen. CONCLUSION: These results suggest that the two compound heterozygous patients have a circulating VWF mainly mutated in the A1 domain (P1337L). This peculiar type 2B VWF variant showed a remarkably high affinity for the GPIbalpha platelet receptor, leading to the loss of high and intermediate molecular weight multimers and hence to decreased RIPA, as in type 2A VWD.

Collagen↗
PubMed1996

Role of estrogen receptor/Sp1 complexes in estrogen-induced heat shock protein 27 gene expression.

Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.

Antibodies↗
PubMed1999

PU.1 and USF are required for macrophage-specific mannose receptor promoter activity.

In the current study we report the isolation of 854 base pairs of the rat mannose receptor promoter. Analysis of the sequence revealed one Sp1 site, three PU.1 sites, and a potential TATA box (TTTAAA) 33 base pairs 5' of the transcriptional start site. The tissue specificity of the promoter was determined using transient transfections. The promoter was most active in the mature macrophage cell line NR8383 although the promoter also showed activity in the monocytic cell line RAW. No activity was observed in pre-monocytic cell lines or epithelial cell lines. Mutation of the TTTAAA sequence to TTGGAA resulted in a 50% decrease in activity in transient transfection assays suggesting that the promoter contains a functional TATA box. Using electrophoretic mobility shift assays and mutagenesis we established that the transcription factors Sp1, PU.1, and USF bound to the mannose receptor promoter, but only PU.1 and USF contributed to activation. Transient transfections using a dominant negative construct of USF resulted in a 50% decrease in mannose receptor promoter activity, further establishing the role of USF in activating the rat mannose receptor promoter. Comparison of the rat, mouse, and human sequence demonstrated that some binding sites are not conserved. Gel shifts were performed to investigate differences in protein binding between species. USF bound to the rat and human promoter but not to the mouse promoter, suggesting that different mechanisms are involved in regulation of mannose receptor expression in these species. From these results we conclude that, similar to other myeloid promoters, transcription of the rat mannose receptor is regulated by binding of PU.1 and a ubiquitous factor at an adjacent site. However, unlike other myeloid promoters, we have identified USF as the ubiquitous factor, and demonstrated that the promoter contains a functional TATA box.

Animals↗
PubMed2005

Molecular characterization of two novel mutations causing factor X deficiency in a Chinese pedigree.

Factor X (FX) deficiency is a rare bleeding disorder inherited as an autosomal recessive trait. In this study, we investigated the molecular basis of FX deficiency in a Chinese pedigree. The proposita showed a markedly prolonged activated partial thromboplastin time and a mild prolongation of prothrombin time. The levels of FX antigen and FX activity were 58.6% and 2.5%, respectively. Molecular analysis revealed that the proposita was compound heterozygous for two novel mutations: IVS1 + 1G > A and G1185A (Arg347His). The aberrant transcripts from the IVS1 + 1G > A mutant allele were not detected by analyzing the splicing pattern of ectopic transcripts in leukocytes of the patient with nested polymerase chain reaction after reverse transcription. We thus hypothesize that the mRNA molecules originating from the IVS1 + 1G > A mutation were rapidly destroyed in vivo. Site-directed mutagenesis of FX cDNA was used to introduce FXG1185A mutation, and wild-type as well as mutant FX proteins were expressed by transient transfection in HEK 293 cells. Normal FX antigen levels both in the conditioned media of cells expressing the mutant and in cell lysates were detected by an enzyme-linked immunoadsorbent assay. Evaluation of wild-type and mutant coagulant activity demonstrated that the FX molecules carrying the Arg347His mutation have dramatically decreased activity.

Adolescent↗
PubMed2006

An unusual X-linked retinoschisis phenotype and biochemical characterization of the W112C RS1 mutation.

A 52-year-old subject harboring an RS1 gene W112C mutation presented with a prominent and asymmetric tapetal-like retinal sheen. Transient ERG responses were smaller and slower in the eye with the more extensive sheen, an association that, to our knowledge, had not been previously reported. An ON-pathway dysfunction explained the abnormalities of the transient but not those of the flicker ERGs. Although in vitro studies showed that the W112C mutant retinoschisin is present only in the cellular fraction and is not secreted, disease expression was remarkably mild, consistent with the notion of the existence of genetic and/or epigenetic disease modifiers.

Cells, Cultured↗
PubMed2003

Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation.

The crystal structure of recombinant hepatitis B virus (HBV) capsids formed by 240 core proteins has recently been published. We wanted to map sites on the surface of the icosahedral 35-nm particle that are important for nucleocapsid envelopment by HBV surface proteins during virion morphogenesis. For this purpose, we individually mutated 52 amino acids (aa) within the N-terminal 140 aa of the 185-aa long core protein displaying their side chains to the external surface of the capsid to alanine residues. The phenotype of the mutations with respect to virion formation was tested by transcomplementation of a core gene-negative HBV genome in transiently cotransfected cells, immunoprecipitation of nucleocapsids from cells and secreted virions from culture media, and detection of the particles by radioactive endogenous polymerase reactions. Thirteen point mutations impeded nucleocapsid detection by endogenous polymerase reactions. Twenty-seven mutations were compatible with virion formation. Among these were all capsid-forming mutations in the upper half of the spike protruding from the particle shell and two additional triple mutations at tip of the spike. Eleven mutations (S17, F18, L60, L95, K96, F122, I126, R127, N136, A137, and I139) allowed nucleocapsid formation but blocked particle envelopment and virion formation to undetectable levels. These mutations map to a ring-like groove around the base of the spike and to a small area at the capsid surface close to the pores in the capsid shell. These residues are candidate sites for the interaction with envelope proteins during virion morphogenesis.

Capsid↗
PubMed2004

Functional characterization of SIX3 homeodomain mutations in holoprosencephaly: interaction with the nuclear receptor NR4A3/NOR1.

Holoprosencephaly (HPE) is a relatively common brain malformation resulting in an incomplete separation of the two cerebral hemispheres. A number of mutations in different genes have been linked to this malformation, including three missense mutations in the homeodomain of the transcription factor SIX3. In this study, we investigated the functional consequences of these SIX3 mutations with respect to the ability of the protein to interact with and stimulate the transcriptional activity of the nuclear receptor NOR1 (NR4A3). Using glutathione S-transferase fusion protein pull-down assays and transient cotransfections of Neuro-2a cells with expression and reporter vectors, we found that one mutation, c.676C>G (p.L226V), does not alter the properties of SIX3 toward NOR1. Another mutation, c.749T>C (p.V250A), results in the production of a highly unstable protein in Neuro-2a cells. The third mutation, c.770G>C (p.R257P), results in a mutant SIX3 protein that no longer interacts with NOR1 in vivo. These observations suggest that different SIX3 mutations in HPE2 may affect different signaling pathways, and that one of these pathways may involve the nuclear receptor NOR1.

Cell Line↗
PubMed2005

The human secretin gene in children with autistic spectrum disorder: screening for polymorphisms and mutations.

We screened 29 children with autism for mutation in the human secretin gene using single-strand conformation polymorphism. No mutation was detected in exon 2, 3, or 4. Polymerase chain reaction and DNA sequence of 5' variable number of tandem repeats showed two polymorphisms with deletion or duplication of a repeat unit that failed to show any gene expression with transient transfection assay. We did not find evidence of a relationship between human secretin gene mutation and autism.

Adolescent↗
PubMed1997

Startle responses in hereditary hyperekplexia.

BACKGROUND: Patients with hereditary hyperekplexia have excessive startle responses that are accompanied by transient stiffness and also continuous stiffness in infancy. A point of mutation has been identified for the major form of hereditary hyperekplexia in the gene encoding the alpha 1 subunit of the glycine receptor. OBJECTIVE: To measure startle reflexes and autonomic responses in the major form of hereditary hyperekplexia in the original Dutch pedigree. DESIGN: Startle reflexes and autonomic responses were examined by administering 3 series of 20 auditory stimuli at intervals of 10 seconds (90 and 113 dB) and 60 seconds (113 dB). SETTING: The Department of Neurology and Clinical Neurophysiology at the Leiden University Hospital, Leiden, the Netherlands. SUBJECTS: Nine patients with the major form of hyperekplexia and 20 healthy controls. Of the 9 patients, 5 took medication. The patients are part of the Dutch hyperekplexia pedigree. MAIN OUTCOME MEASURES: Startle responses were quantified with latency periods and areas of electromyographic bursts of the orbicular muscle of the eye, sternocleidomastoid and biceps muscles, and the thenar muscles. Autonomic reactions were measured with psychogalvanic responses and beat to beat changes of blood pressure and heart rate. RESULTS: The electromyographic bursts of the 4 muscles occurred in similar order in both patients and controls. The onset of the latency periods in the patient group was significantly (P < .001) prolonged in patients who took medication. Without medication, patients had shorter latency periods of the sternocleidomastoid muscle (P = .003) than controls. The electromyographic burst occurred significantly more often in patients than in controls (P < .001). The areas of the bursts were significantly larger in patients than in controls (P < .001); the degree of habituation was significantly stronger in patients than in controls (P < .001). The amplitude of the psychogalvanic response was increased in hyperekplexia, and the degree of habituation was significantly weaker in patients than in controls. Blood pressure and heart rate did not clearly react in either group. CONCLUSIONS: Motor startle responses are stronger and show more habituation in patients with hereditary hyperekplexia than in controls. The excessive responses include the psychogalvanic response. Increased responses do not necessarily indicate decreased habituation in hyperekplexia.

Acoustic Stimulation↗
PubMed1999

The same xenobiotic response element is required for constitutive and inducible expression of the mammalian aldehyde dehydrogenase-3 gene.

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively express ALDH3 at very high levels. Other tissues, such as normal liver, do not express ALDH3. In liver, ALDH3 is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near -3.0 kb in the ALDH3 5' flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boesch et al., J. Biol. Chem. 271, 5150-5157, 1996) to study the molecular basis of constitutive ALDH3 expression. Transient transfection assays of corneal epithelium using a battery of ALDH3 5' flanking region-CAT reporter gene constructs indicate that high constitutive ALDH3 expression involves the same cis-acting elements as xenobiotic-induced ALDH3 expression in liver. These elements include a strong basal promoter region and the XRE located near -3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The -3.0-kb ALDH3 XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression of ALDH3 in cornea involves binding of ARNT, HNF1, and HNF4 to the ALDH3-XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection of ALDH3-CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4-DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutive ALDH3 expression.

Aldehyde Dehydrogenase↗
PubMed1994

Role of the A/B region of the human mineralocorticoid receptor in aldosterone response selectivity.

In transfection experiments performed with wild-type MR, the maximal transcriptional activation of a mineralocorticoid response induced by glucocorticoid was generally similar to that of aldosterone, the natural mineralocorticoid hormone. However, compared to aldosterone, glucocorticoid activity decreased when the A/B region of MR was absent. We describe in this study the synthesis and biological activities of seven mutated MRs differently truncated in the N-terminal region. Using transient expression conditions in MCF-7 cells, the N-terminal domain of MR has been shown to contain a region (residues 254-390) whose deletion led to an apparent "aldosterone selectivity". These results suggest that this region could help to maintain the most transcriptionally active conformation of MR even in the presence of ligands which are not by themselves able to fully induce such a conformation.

Aldosterone↗
PubMed1998

Erx, a novel retina-specific homeodomain transcription factor, can interact with Ret 1/PCEI sites.

Our previous studies on the transcriptional regulation of rod opsin gene expression had defined a strikingly conserved element, Ret 1/PCEI, present in the upstream regulatory regions of opsin and other photoreceptor-specific genes. This element interacts with a 40 kDa, developmentally regulated, retina-specific protein. In this study we report the cloning of the novel retina-specific homeodomain protein Erx. Erx contains a homeodomain that is 79% homologous to that of Drosophila empty spiracles. This 40 kDa protein can interact with the Ret 1 element in electrophoretic mobility shift assays. Mutation of key residues in Ret 1 eliminates all Erx binding. Transient transfection of Y79 retinobalstoma cells with Erx leads to significant transcriptional activation of a reporter gene via Ret 1 elements. We conclude that Erx is the Ret 1 binding activity. This is the first example of a Q50 homeodomain protein expressed in retinal photoreceptors.

Amino Acid Sequence↗
PubMed1999

Involvement of NF-kappaB p50/p65 heterodimer in activation of the human pro-interleukin-1beta gene at two subregions of the upstream enhancer element.

A region between-3134 and -2729 bp upstream from the transcription site of the human pro-interleukin 1beta (proIL-1beta) gene was identified as an LPS-responsive enhancer element. In this study, the influence of the sequences located between -3134 and -2987 on the transcriptional activity of the proIL-1beta gene in LPS-stimulated Raw 264.7 cells was examined in detail. The results obtained by transient transfection of fos -CAT constructs that contained serial 5'-deletion mutations showed that the region between -3134 and -3059 appears to be required for the induction of transcription by LPS. Gel shift assay studies with synthetic oligonucleotides corresponding to partial sequences of the latter region and nuclear extracts from stimulated cells revealed specific protein binding sites between -3110 and -3090 and between -3079 and -3059. These specific bindings were time and LPS dose dependent. The results of supershift analysis using specific antibodies against transcription factors suggested that both binding complexes contained the NF-kappaB components p50 and p65, and did not contain other NF-kappaB proteins (p52, c-Rel, Rel B), AP-1 proteins (c-Fos, C-Jun), CREB or C/EBPbeta (NF-IL6). Mutation of either of the putative NF-kappaB-binding sites in the enhancer element decreased the LPS-stimulated transcriptional activity. These data indicated that two NF-kappaB-binding sites, which are located between -3134 and -3059, are critical for the activation of proIL-1beta gene transcription.

Animals↗
PubMed2001

Nitric oxide suppresses IL-8 transcription by inhibiting c-Jun N-terminal kinase-induced AP-1 activation.

The role of activator protein-1 (AP-1) in tumor necrosis factor-alpha (TNF-alpha)-induced interleukin-8 (IL-8) gene expression was evaluated. We showed that TNF-alpha activates AP-1 in the transformed endothelial cell line ECV304 by transient transfections of IL-8 promoter construct pGL-3BF(2). Mutation of either the AP-1 site or the NF-IL-6 site on the IL-8 promoter suppressed the TNF-alpha-induced activation, suggesting cooperation between these transcription factors and transcription factor NF-kappaB. Overexpression of dominant negative mutants of c-Jun suppressed AP-1-driven transcription of the IL-8 promoter following stimulation by TNF-alpha, suggesting that cooperative interaction between AP-1 and NF-kappaB is essential for IL-8 transcription in the presence of TNF-alpha. We also showed that nitric oxide (NO), in the form of an exogenous NO donor, suppressed the level of activation of the AP-1 subunit, c-Jun, by down-regulation of c-Jun NH2 terminal kinase. This down-regulation could be the putative mechanism of action for NO-mediated inhibition of IL-8 secretion in activated endothelium. These observations suggest for the first time that NO has broad suppressive activities on various proinflammatory effectors in activated endothelium.

CCAAT-Enhancer-Binding Protein-beta↗
PubMed1998

Cloning and characterization of the promoter of baboon XRCC1, a gene involved in DNA strand-break repair.

The DNA repair gene XRCC1 was the first cloned human DNA repair gene involved in resistance to ionizing radiation. Previous studies have shown that rodent and baboon homologs of XRCC1 are expressed in all tested tissues with significantly higher levels in testis. Furthermore, expression of murine XRCC1 is most abundant in pachytene spermatocytes and round spermatids. To begin to study regulation of XRCC1 expression, the 5' region of baboon XRCC1 was cloned and characterized. 400 bp of 5'-flanking region showed the greatest promoter activity, while -194 to -8 bp of the 5'-flanking region displayed core promoter activity in transient transfection assays. A comparison between baboon and human 5'-flanking sequences in the core promoter region revealed a potential CAAT-box, an imperfect CREB-binding site and two putative Sp1-binding sites. Results from transient transfection assays in which each putative binding site was individually mutated, indicated that the distal Sp1-binding site has a functional role in transcription. In comparison, both putative Sp1-binding sites bound protein(s) from HeLa cell nuclear extracts in vitro. In vitro binding was lost when mutated Sp1 sites were used in gel mobility shift assays. Finally, anti-Sp1 antibodies produced mobility supershifts, thereby indicating Sp1 or an Sp1-like protein bound to the DNA fragment in vitro.

Animals↗
PubMed2002

Amino acids involved in differences in the pharmacological profiles of the rat and human noradrenaline transporters.

It has been suggested from a previous study in our laboratory that differences in the pharmacology of the species variants of the noradrenaline transporter (NET) are the result of four non-conservative amino acid exchanges from the total of 26 amino acids that are divergent between the rat NET (rNET) and human NET (hNET). The aim of this study was to examine the effects of changing the rNET at each of these four amino acid residues, which markedly alter local charge distribution, to the amino acid found in hNET.Site-directed mutagenesis was used to create mutant cDNAs from rNET cDNA. The mutant NETs (rK7D, rE62K, rK375N and rR612Q), rNET and hNET were expressed in transiently transfected COS-7 cells to determine the effects of the mutations on the differing pharmacological properties of the species variants. The ratios of V(max) for noradrenaline uptake and B(max) for nisoxetine binding (which are a measure of the turnover number of the transporter, i.e. the number of transport cycles per min) were greater for rNET and rR612Q than for hNET, rK7D, rE62K and rK375N. The K(m) of noradrenaline was lower for hNET, rK7D, rE62K and rK375N than for rNET or rR612Q. There were no differences between the K(i) values for inhibition of noradrenaline uptake by nisoxetine for rNET, hNET or the mutants, but the K(i) values of cocaine were lower for hNET, rE62K and rR612Q than rNET or rK375N.Hence, the study showed that: (1) the aspartate 7, lysine 62 and asparagine 375 amino acid residues are important in determining the lower substrate translocation by hNET than rNET; (2) the aspartate 7 and lysine 62 residues in the N-terminus of hNET determine the higher affinities of substrates for the hNET than the rNET; and (3) the lysine 62 and glutamine 612 residues in the N- and C-termini, respectively, of hNET are determinants of the higher cocaine affinity for the hNET than rNET.

1-Methyl-4-phenylpyridinium↗