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PubMed2000

Variant detection at the delta opioid receptor (OPRD1) locus and population genetics of a novel variant affecting protein sequence.

The three opioid receptor genes, and in particular the mu and delta loci (OPRM1 and OPRD1, respectively), are compelling candidates to influence risk for substance dependence. Previous study of a variant at the OPRD1 locus, T921C, has shown association with opioid dependence. This variant does not alter protein sequence, and could not be directly responsible for a physiologic effect. We sequenced the OPRD1 coding region in six individuals with differing T921C alleles, to identify new common variants more likely to explain the association with phenotype. We identified one novel variant in exon 1, 80T-->G, which predicts a change in amino acid sequence from phenylalanine (80T) to cysteine (80G) (F27C). We present here basic population genetics of this variant, and population genetic data for the T921C variant. We found significant differences in allele frequency between populations, and a maximum frequency of the 80G allele of 9%, in each of two European populations. This variant could contribute to the previously reported association results.

Alleles↗

PubMed1982

Immunogenic variants obtained by mutagenesis of mouse Lewis lung carcinoma. Recognition of variant-specific antigens by cytolytic T lymphocytes.

By mutagenesis of a cell line derived from Lewis lung carcinoma (3LL), it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic mice. The possibility of obtaining a cytolytic T cell (CTL) response directed specifically against these tum- variants was examined. With the four variants that were analysed, a significant cytolytic activity was obtained with peritoneal cells from immune mice collected shortly after an intraperitoneal boost and also with spleen cells after a secondary stimulation in vitro. The CTL populations preferentially lysed the immunizing tum- variant, while also showing a cross-reactive lysis against the other variants and the original 3LL cells. Highly active CTL clones could be isolated from limiting dilution microcultures of these CTL populations. The clonal analysis clearly showed the existence of two distinct CTL populations, one directed exclusively against the immunizing variant and another that lysed all 3LL targets equally. This CTL specificity analysis therefore demonstrates directly the presence of new antigens on the 3LL tum- cell variants.

Animals↗

PubMed2025

Polygenic variants in DNA repair genes are associated with neurodevelopmental disorders, regression and increased burdens of somatic variants and short tandem repeat expansions.

PURPOSE: Developmental regression, characterized by the loss of acquired milestones, occurs in some individuals with neurodevelopmental disorders (NDDs); yet, its molecular basis remains unclear. Studies suggest that DNA damage repair (DDR) genes, such as FAN1, may protect against neurological dysfunction by modulating the somatic stability of short tandem repeats (STRs). This study explores the contribution of DDR gene variants in NDD cases presenting with regression. METHODS: We analyzed 1087 NDD patients, focusing on those carrying variants in DDR genes and presenting regression. We assessed the sensitivity to DNA damage using mitomycin C on lymphoblastoid cells. Somatic variants and STR expansions were evaluated through high-depth short-read genome sequencing. To further investigate the pathogenetic role of STR expansions, we performed long-read genome sequencing on the most severely affected proband. RESULTS: Probands with regression carried multiple DDR gene variants, several within the Fanconi anemia pathway. Their lymphoblastoid cells showed increased sensitivity to mitomycin C-induced cytotoxicity compared with parental and control samples. Probands with severe phenotypes and regression exhibited an accumulation of somatic variants and STR instability, enriched in neurodevelopmental genes. CONCLUSION: Our findings suggest that polygenic DDR gene variants may contribute to developmental regression in NDDs by promoting the accumulation of somatic variants and STR expansions.

Humans↗

PubMed2002

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.

Alternative Splicing↗

PubMed1998

Persistence of human herpesvirus 6 according to site and variant: possible greater neurotropism of variant A.

Little is known of the persistence and pathogenicity of human herpesvirus 6 (HHV-6) after primary infection, including the role of strain variant. Over 2 to 5 years, 2,716 children and 149 families were studied. Peripheral blood mononuclear cell (PBMC), saliva, and cerebrospinal fluid (CSF) specimens were examined for HHV-6 DNA and variant. Ninety-nine percent of isolates causing primary infection were HHV-6 variant B (HHV-6B), which predominated in 95%-98% of the variants persisting in PBMC and saliva specimens from children and adults. Of 668 CSF samples, 13% contained HHV-6 DNA; of 77 children examined after primary infection, 61% had HHV-6 DNA detected only in their CSF and 39% had HHV-6 DNA in both CSF and PBMCs. HHV-6 variant A (HHV-6A) was detected significantly (P = .0001) more frequently in CSF than in PBMCs or saliva. In children for whom HHV-6 was identified in both CSF and PBMCs, PBMCs contained only HHV-6B, while CSF contained HHV-6A or HHV-6B, not both. Thus, in patients with dual infection, only HHV-6A persisted in CSF, which suggests that HHV-6A has greater neurotropism. Findings for adults indicate that dual infection occurs; variant persistence is similar to that for children. The frequency of HHV-6A infection increased little with age, thereby indicating that HHV-6A infection remains uncommon into adulthood. This study suggests that HHV-6 variants have different immunobiologic courses and neurotropism.

Adult↗

PubMed1977

Emergence of variant forms of Staphylococcus aureus after exposure to gentamicin and infectivity of the variants in experimental animals.

Exposure of a large inoculum of Staphylococcus aureus in vitro to concentrations of gentamicin that exceeded the minimal bactericidal concentration regularly resulted in the emergence of aminoglycoside-resistant bacterial variants. Variants lacked typical properties that are associated with S. aureus: they produced small, nonhemolytic colonies that were mostly coagulase-, deoxyribonuclease-, and mannitol-negative. In some instances phage type also differed from that of the parent forms. Animal models of subcutaneous and intravenous infection were studied with use of parent and variant forms of S. aureus. Subcutaneous injection of variants into rats readily produced abscesses, and intravenous inoculation into mice caused pyelonephritis, although in each experimental model in which equivalent bacterial inocula were used, parents produced more extensive disease. These data show that variants of S. aureus cause infection in experimental animals, although these variants appear to be somewhat less virulent than the parents from which they are derived. Preliminary studies in our laboratory have also shown that gentamicin-resistant variants of gram-negative bacilli can be induced by a single in vitro exposure to gentamicin. The virulence of these organisms and their role in antibiotic-susceptibility patterns of hospital flora warrant further investigation.

Animals↗

PubMed2004

The G336S variant in the human neurofilament-M gene does not affect its assembly or distribution: importance of the functional analysis of neurofilament variants.

The human neurofilament medium (hNFM) subunit is one of the 3 neurofilament (NF) polypeptides, which are the most abundant intermediate filament (IF) proteins in post-mitotic neurons. The formation of neurofilamentous aggregates is a pathological hallmark of many neurodegenerative diseases, including the Lewy bodies found in Parkinson disease (PD). A Gly336Ser (G336S) variant in the rod domain of hNFM has recently been described in a patient with early-onset autosomal-dominant PD. In this study, we have generated a mammalian expression vector encoding the variant hNFM cDNA and characterized its effects on the formation of heteropolymeric IFs in heterologous cell lines. We have also investigated the distribution of the (G336S) hNFM variant protein in neuronal CAD cells, as well as the effects of the variant on the distribution of other cellular organelles and proteins. Our results demonstrate that the G336S variant does not affect the formation of IF networks nor the distribution of the variant hNFM protein. Our data suggest that if the G336S variant is involved in the development of PD, it does not appear to be due to defects in the assembly and distribution of NFs.

Amino Acid Substitution↗

PubMed1977

Genetic variants of human erythrocyte glucose 6-phosphate dehydrogenase: new variants in West Africa characterized by column chromatography.

Five electrophoretically slow-moving genetic variants of glucose 6-phosphate dehydrogenase are described: four are from Nigeria and one is from Togo. All variants have normal or moderately reduced activity, and they are not associated with adverse clinical or haematological manifestations. Three variants have been fully characterized and are different from all previously described ones. Two variants have been partially characterized and at least one of them is also probably new. The overall population incidence of sporadic variants of G6PD in the Nigerian population is 0-3%. In the course of this study a previously described ion-exchange chromatographic technique for the characterization of G6PD variants has been extensively evaluated. Data are given on ten different variants to demonstrate the high resolving power of this technique.

Chromatography, Ion Exchange↗

PubMed2004

Functional characterization of four naturally occurring variants of human pregnane X receptor (PXR): one variant causes dramatic loss of both DNA binding activity and the transactivation of the CYP3A4 promoter/enhancer region.

Metabolism of administered drugs is determined by expression and activity of many drug-metabolizing enzymes, such as the cytochrome P450 (P450s) family members. Pregnane X receptor (PXR) is a master transcriptional regulator of many drug/xenobiotic-metabolizing enzymes, including P450s and drug transporters. In this study, we describe the functional analysis of four naturally occurring human PXR (hPXR) variants (R98C, R148Q, R381W, and I403V) that we have recently identified. By a reporter gene assay using the CYP3A4 promoter/enhancer reporter in COS-7 or HepG2 cells, it was found that the R98C variant failed to transactivate the CYP3A4 reporter. The R381W and I403V variants also showed varying degrees of reduction in transactivation, depending on the dose of PXR activators, rifampicin, clotrimazole, and paclitaxel. The transcriptional activities of the R148Q variant were not significantly different from that of the wild-type hPXR. The electrophoretic mobility shift assay revealed that only the R98C variant lacked DNA binding. Furthermore, the cellular localization of the hPXR proteins was analyzed. All four variants as well as the wild-type hPXR localized exclusively to the nucleus, regardless of the presence or absence of rifampicin. These data suggest that the R98C, R381W, and I403V hPXR variants, especially R98C, may influence the expression of drug-metabolizing enzymes and transporters, which are transactivated by PXR.

Amino Acid Sequence↗

PubMed1970

Penicillinase Studies on L-Phase Variants, G-Phase Variants, and Reverted Strains of Staphylococcus aureus.

L-phase variants and small colony (G-phase) variants derived from penicillinase-producing Staphylococcus aureus strains were tested for penicillinase (beta lactamase) production. A refined variation of the modified Gots test for penicillinase was used to demonstrate penicillinase synthesis. Penicillinase synthesis was reduced in L-phase variants and G-phase variants when compared to parental strains. After reversion of variants to vegetative stages had been induced, revertants were tested for production of penicillinase, coagulase, and alpha hemolysin, mannitol fermentation, and pigment production, and comparisons were made between parent and reverted vegetative forms. All revertants of G-phase variants retained penicillinase activity. Most revertants of L-phase variants showed reduction or loss of penicillinase activity. Retention of coagulase activity, alpha hemolysin production, mannitol fermentation, pigmentation, and phage type varied among revertants.

Journal Article↗

PubMed1991

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.

Alleles↗

PubMedDate not supplied

Papillary thyroid carcinoma and its variants in fine needle aspiration smears. A cytomorphologic study with special reference to tall cell variant.

OBJECTIVE: To study the fine needle aspiration (FNA) cytologic features of papillary thyroid carcinoma (PTC) with special reference to its tall cell variant (TCV), which is the most aggressive of the variants. STUDY DESIGN: Fifty-four PTC cases were classified into variants, and the frequency of well-known morphologic criteria was determined. Four parameters were quantitatively analyzed based on a study of 200 consecutive neoplastic follicular cells: shape of cells, color of cytoplasm, intranuclear cytoplasmic inclusion (INCI) and nuclear grooves. RESULTS: The PTC cases included 6 TCV (> or = 30% tall cells), 8 cases with a significant tall cell component (sig. TCC) having 10-29% tall cells, 17 usual variant (UV), 17 follicular variant (FV) and 6 miscellaneous variants. TCV differed significantly from UV and FV in having a higher tall cell count, higher count of cells with reddish cytoplasm and INCI, and higher frequency of cases with lymphocytic infiltration. PTC (with significant tall cell component [TCC]) differed significantly from TCV with regard to tall cell count and lymphocytic infiltration, from UV with respect to tall cell count and monolayered sheets, and from FV with respect to tall cells, INCI, grooved nuclei, acinar formation, fire-flare appearance and giant cells. CONCLUSION: TCV was cytologically distinct from other variants. The biologic behavior of PTC cases with significant TCC, which morphologically seem to be a group intermediate between TCV on the one hand and UV and FV on the other, however, needs to be carefully monitored.

Adolescent↗

PubMed2004

Hemoglobin C and hemoglobin O-Arab variants can be diagnosed using the Bio-Rad Variant II high-performance liquid chromatography system without further confirmatory tests.

CONTEXT: Current standards for laboratory accreditation from the College of American Pathologists state that when high-performance liquid chromatography (HPLC) is used as a screening test, all non-A, non-S abnormal hemoglobin (Hb) variants must be confirmed by an alternative method, including alkaline and acid electrophoresis. OBJECTIVE: To determine whether confirmation of Hb C and Hb O-Arab variants by an alternative method is required when using the Bio-Rad Variant II HPLC system. DESIGN: We reviewed 48 478 consecutive hemoglobin identification test results performed on the Bio-Rad Variant II HPLC system during the period November 15, 2000 to January 15, 2003. SETTING: Special Hematology Laboratory, Department of Pathology, Bellevue Hospital Center, New York, NY. MAIN OUTCOME MEASURES: The chromatogram patterns and retention times (RTs) for specimens containing Hb C and Hb O-Arab were analyzed. We compared the results by the HPLC method with those by the confirmatory tests (alkaline and acid electrophoresis) for both variants. RESULTS: We identified 3668 cases of abnormal hemoglobin variants, including 660 cases of Hb C trait (17%), 5 cases of Hb O-Arab trait (0.1%), and 1 case of Hb SO-Arab (0.03%). A unique pattern of separation on the chromatogram for Hb O-Arab was revealed, presenting as 2 distinct peaks in 2 different manufacturer-defined RT windows, namely, D and C. The chromatogram for Hb C did not show the D window in any of the reviewed cases. The RT in the C window (C-RT) revealed a statistically significant difference for Hb C and Hb O-Arab (5.18 +/- 0.01 minutes and 4.91 +/- 0.01 minutes, respectively; P <.001). CONCLUSION: According to our review, the identification of Hb C and Hb O-Arab is accurate using HPLC methodology, as performed by the Bio-Rad Variant II HPLC system. This method can be both confirmatory and diagnostic at the same time.

Blood Protein Electrophoresis↗

PubMed1988

Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling.

Studies are reported on the characterization of a new isolate within a novel class of variants of the L1210 cell exhibiting markedly increased transport inward of folate analogues. This variant (L1210/R83), which was selected in the presence of the antifolate metoprine, exhibited a 40-fold increase in [3H]aminopterin influx compared to parental cells and a modest (4-5-fold) increase in [3H]aminopterin efflux. The increase in influx was associated with a comparable increase in influx Vmax for the one-carbon, reduced folate transport system and the same increase in the amount of specific binding of [3H]aminopterin on the cell surface. Values for influx Km for [3H]aminopterin and specificity for various folate structures were unchanged. The alteration in influx Vmax and more rapid efflux accounted for the different level of intracellular exchangeable level of drug at steady state in this variant compared with parental L1210 cells. Otherwise, membrane potential was unchanged. The N-hydroxysuccinimide ester of [3H]aminopterin was used to covalently label the specific binding protein for folate compounds in the plasma membrane of variant and parental L1210 cells. Incorporation of label into this protein was stable under a variety of conditions and accounted for 97 and 52% of total cellular labeling, respectively, for membrane derived from R83 and parental L1210 cells at a reagent concentration of 20 nM. Specific affinity labeling on the surface of parental and variant cells was decreased in the presence of aminopterin, methotrexate, or 5-formyltetrahydrofolate, but not in the presence of folic acid. Also, [3H]aminopterin influx in these cells was inhibited by the N-hydroxysuccinimide ester of aminopterin or methotrexate, but not the N-hydroxysuccinimide ester of folic acid. These findings, in addition to the increased affinity labeling of this variant, which corresponds to the increase in influx of [3H] aminopterin also seen, appears to identify the affinity labeled protein as a component of the "classical" one-carbon, reduced folate transport system in these cells. The affinity labeled protein from each cell type was solubilized in sodium dodecyl sulfate or extracted in detergent in the presence of proteinase inhibitors and was found to elute from Sephacryl S-300 and migrate during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single peak of Mr = 45,000-48,000. Recovery of labeled binding protein in these fractions from R83 variant cells was approximately 40 times greater than that from parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗

PubMed1988

Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolytic characterization of genetic variants lacking the fibronectin finger-like and/or the epidermal growth factor domains.

Tissue-type plasminogen activator (t-pa) is a serine protease comprising four different putative structural domains with homologies to fibronectin finger-like structures (finger), epidermal growth factor, kringle structures, and the active site of serine proteases. Only the finger and epidermal growth factor domain are each entirely encoded by unique single exons. We assessed the functional contribution of these two structural domains by making mutants precisely deleted for one or both of the relevant exons. The three mutant genes were expressed in monkey cells, and the variant proteins, purified from the culture medium, were characterized for their fibrinolytic activity, fibrinogenolytic potential, and affinity for fibrin. No significant difference in any biochemical property was observed among the variants. All three variants retained a catalytic dependence on cyanogen bromide fragments of fibrinogen which could not be distinguished from the wild-type enzyme. The activities of the variants were also very similar to that of wild-type t-pa, showing no detectable fibrinogenolytic potential in human plasma at activator concentrations of 500 IU/ml, or when their fibrinolytic activity was tested in human plasma using the 125I-labeled fibrin clot lysis assay at activator concentrations of 150 IU/ml or greater. However, the variants were markedly defective in fibrinolysis at low activator concentrations such that essentially no fibrinolysis was detected at 15 IU/ml. Measurement of fibrin binding showed that the variants lacked the high fibrin binding characteristic of wild-type t-pa. These results demonstrate that the fibrin specificity and fibrin-dependent activity of t-pa are independent of the protein's high affinity for fibrin. The implication of these results is that the t-pa variants would be ineffective activators at a physiological concentration of approximately 2 IU/ml but would be expected to behave similarly to wild-type t-pa at the steady-state plasma concentrations of 0.75-1.25 micrograms/ml (approximately 500 IU/ml) currently required for coronary reperfusion in patients receiving t-pa for acute myocardial infarction (Garabedian, H.D., Gold, H.K., Leinbach, R.C., Yasuda, T., Johns, J.A., and Collen, D. (1986) Am. J. Cardiol. 58, 673-679).

Animals↗

PubMed1976

Genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase. Kinetic and thermodynamic parameters of variants A, B, and A- in relation to quaternary structure.

The values of Vmax and Km for the three genetic variants A, B, and A- of erythrocyte glucose-6-phosphate dehydrogenase have been determined at 10 different pH values in the range from 5.5 to 9.5, and at four different temperatures in the range from 18.5-40.0 degrees. The log Vmax versus pH curve for each of the enzymes shows a monotonic increase between pH 5.5 and 7, and a plateau from pH 7.5 upwards. These curves, and their temperature dependence, are compatible with the presence of a single ionizable group which, in its conjugate acid form, renders the enzyme-substrate complex inactive. The pK of this group is 6.94 at 18.5 degrees, and its enthalpy of ionization is 7.0 kcal mol-1. The log Km versus pH curves show a broad plateau between pH 6.2 and 8.2, interrupted by a sharp minimum at pH 7.2 for variant B, while variants A and A- show sharp maxima at pH 7.2 and 7.45, respectively. It is proposed that this unusual behavior depends on the dissociation of the tetrameric enzyme to dimers in this pH region. Specifically, it is shown that a sharp maximum or minimum of Km can arise if cooperative uptake or release of protons is linked to dimer formation, and if the degree of cooperativity is different for the free enzyme compared to the enzyme-substrate complex. The pH dependence of the equilibrium between the tetrameric and the dimeric form of the enzyme has been determined by gel filtration for the same three genetic variants B, A, and A-. In agreement with previous ultracentrifugal data, the enzyme is a tetramer in acid solution and a dimer in alkaline solution. The pH at which half of the enzyme is in dimeric form, under our experimental conditions, is 7.15 +/- 0.05 for variants A and B, and 7.35 +/- 0.05 for variant A-. These pH values correspond closely, for all three variants, to the sharp extrema in the pH dependence of their Km values for glucose 6-phosphate. From the measured dissociation equilibria, it can be inferred that the tetramer-dimer transition entails cooperative release of protons. The degree of cooperativity estimated from these data agrees closely with the independent estimate based on the pH dependence of Km.

Erythrocytes↗

PubMed1982

Histone variants in plants. Evidence for primary structure variants differing in molecular weight.

Variants of H2a and H2b histones from wheat embryos and pea shoots have been isolated and characterized by two-dimensional gel electrophoresis, amino acid content, and peptide mapping. In striking contrast to the animal H2a or H2b variants which co-migrate on sodium dodecyl sulfate polyacrylamide gels, their plant counterparts have differential electrophoretic mobilities. The estimated molecular weights of the wheat H2a variants range from 16,600 to 19,000. The H2b variants range from 15,300 to 19,000. Correlating with their electrophoretic mobilities, the plant histone variants elute differentially from gel exclusion columns in a manner consistent with the molecular weight differences inferred from the sodium dodecyl sulfate gels. The differences in peptide maps of the wheat H2a variants are extensive and indicate amino acid substitutions throughout the molecules. The maps of the plant H2a histones are also distinct from those of the major calf thymus H2a histones. The peptide maps of the plant H2b variants are very similar to each other but are markedly different from those of calf thymus H2b histones.

Amino Acid Sequence↗

PubMed1993

Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant.

Kinetics of recombinant fluoride-2 variant of human butyrylcholinesterase (Gly390 Val) secreted by Chinese hamster ovary cells were compared to recombinant usual and to usual butyrylcholinesterase purified from human plasma. The usual and fluoride-2 variant were indistinguishable with regard to hydrolysis of benzoylcholine (Km = 5 microM), neutral esters, and at high concentrations of acetylthiocholine, propionylthiocholine, and butyrylthiocholine. However, at low substrate concentrations Km values for acetylthiocholine and succinyldithiocholine were 2-6-fold higher for the fluoride-2 variant. pH rate profiles revealed small differences in pKa that could be attributed to changes in the active site histidine environment. On the other hand, Arrhenius plot analysis of o-nitrophenylbutyrate hydrolysis at pH 7.5 showed no difference in activation energy between fluoride-2 and usual butyrylcholinesterases. Both exhibited an anomalous temperature dependence with a wavelike change in activation energy around 18 degrees C. Affinity of the fluoride-2 variant for sodium fluoride, tacrine, dibucaine, amodiaquin, and succinyldicholine was lower than for usual enzyme. Apparent Ki for succinyldicholine was 125 microM for the fluoride-2 variant and 20 microM for the usual enzyme. Organophosphate inhibition showed equivalent reactivity, indicating that the point mutation altered only the binding properties of the variant. Thus, Km and Ki changes explain the succinyldicholine sensitivity of people carrying the fluoride-2 variant.

Amino Acid Sequence↗