Search PubMed⌕ Search

Biomedical subjects

O Lockridge

Publications and source records attributed to O Lockridge.

At least 55 records · Page 3Linked to original sources
PubMed1994

Promoter and transcription start site of human and rabbit butyrylcholinesterase genes.

Two kilobase segments of the 5'-untranslated regions of the human and rabbit butyrylcholinesterase (BCHE) genes were characterized. The sequences shared extensive identity except for a 333-base pair (bp) Alu repeat present only in human BCHE. One single transcription start site was found in both genes with the techniques of primer extension, amplification of the 5'-end of mRNA, and RNase protection. Cap sites in human and rabbit BCHE genes were found in strictly homologous positions. In human BCHE, the transcription start site was found 157 bp upstream of Met-28, the translation start site. Potential regulatory elements in both promoters included one AP1 site and multiple sites for topoisomerase, Oct-1 and PEA-3. Transient expression of BCHE-reporter gene constructs showed that a 194-bp fragment of the 5'-flanking region of human BCHE and a 570-bp fragment of rabbit BCHE were sufficient for promoting chloramphenicol acetyltransferase activity in HeLa cells. No consensus TATA and CAAT boxes were found. However, the sequence around the transcription start site exhibited homology with initiator elements found in other TATA-less promoters in developmentally regulated genes.

Amino Acid Sequence↗
PubMed1994

Mutation His322Asn in human acetylcholinesterase does not alter electrophoretic and catalytic properties of the erythrocyte enzyme.

The YT blood group antigen is located on human red blood cell (RBC) acetylcholinesterase. Wild-type acetylcholinesterase, YT1, has histidine at codon 322, whereas the genetic variant of acetylcholinesterase, YT2, has asparagine. This mutation is located within exon 2 of the ACHE gene, an exon that is present in all alternatively spliced forms of acetylcholinesterase. Therefore, acetylcholinesterase in brain and muscle has the same mutation as RBC acetylcholinesterase. We compared the electrophoretic and kinetic properties of RBC acetylcholinesterases having His 322 or Asn 322. We found no differences in the isoelectric point, mobility on non-denaturing gel electrophoresis, affinity for acetylthiocholine, activity per milligram of RBC ghost protein, substrate inhibition constants, binding to the peripheral site ligand (propidium), and binding to active site ligands (tetrahydroaminoacridine and edrophonium). Thus, although the point mutation elicits antibody production in nonmatching blood transfusion recipients, it has no effect on the enzymatic properties of acetylcholinesterase.

Acetylcholinesterase↗
PubMed1994

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13.

Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.

Acetylcholinesterase↗
PubMed1994

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA.

Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetylcholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

Acetylcholinesterase↗
PubMed1994

Bacterial expression and in vitro folding of the beta-subunit of human chorionic gonadotropin (hCG beta) and functional assembly of recombinant hCG beta with hCG alpha.

A bacterial expression system for the beta-subunit of hCG (hCG beta) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG beta in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG beta complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG beta (rhCG beta) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG beta were recovered from 1 liter culture, rhCG beta was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCG alpha and the purified rhCG beta/urinary alpha dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG beta/urinary alpha dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG beta. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG beta are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG beta in bacteria and of folding it in vitro implies that the beta-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

Animals↗
PubMed1993

Protein folding and assembly in vitro parallel intracellular folding and assembly. Catalysis of folding and assembly of the human chorionic gonadotropin alpha beta dimer by protein disulfide isomerase.

Although purified proteins will refold and assemble in vitro, it is not known if cellular factors change the mechanisms of these processes. Based on the gel migration of folding intermediates, the kinetic relationships between these intermediates, and on the order of formation of six disulfide bonds, we have found that the in vitro folding pathway of the human chorionic gonadotropin beta subunit (hCG-beta) is indistinguishable from the intracellular folding pathway. The same rate-limiting event was found in both folding environments; however, the t1/2 for this step in a cell is 4 min, whereas in vitro the t1/2 was > or = 80 min. Protein disulfide isomerase (PDI) increased the in vitro rate of this event (t1/2 = 25 min) without changing the order of disulfide bond formation. PDI also catalyzed the in vitro rate of assembly of hCG subunits. In intact cells, assembly of the alpha beta heterodimer occurs before all of the intramolecular disulfide bonds of beta are formed. In vitro, assembly was increased after reduction of two of the carboxyl-terminal disulfide bonds of hCG-beta by PDI. These results strongly suggest that both in intact cells and in vitro, partially unfolded hCG-beta is more assembly-competent than is fully folded hCG-beta. The comparison of in vitro and intracellular hCG-beta folding and hCG subunit assembly which is shown in this report indicates that the assisted folding and assembly pathway that occurs in cells, where proteins such as PDI play a role, differs only in rate but not in the order of disulfide bond formation or in the precursor-product relationships among the folding intermediates.

Animals↗
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↗
PubMed1993

Mutation at codon 322 in the human acetylcholinesterase (ACHE) gene accounts for YT blood group polymorphism.

Acetylcholinesterase is present in innervated tissues, where its function is to terminate nerve impulse transmission. It is also found in the red blood cell membrane, where its function is unknown. We report the first genetic variant of human acetylcholinesterase and support the identity of acetylcholinesterase as the YT blood group antigen. DNA sequencing shows that the wild-type sequence of acetylcholinesterase with His322 (CAC) is the YT1 blood group antigen and that the rare variant of acetylcholinesterase with Asn322 (AAC) is the YT2 blood group antigen. Two additional point mutations in the acetylcholinesterase gene do not affect the amino acid sequence of the mature enzyme.

Acetylcholinesterase↗
PubMed1992

Identification of two different point mutations associated with the fluoride-resistant phenotype for human butyrylcholinesterase.

The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier we reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work we have identified two different point mutations associated with the fluoride-resistant phenotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members.

Amino Acid Sequence↗
PubMed1992

DNA mutation associated with the human butyrylcholinesterase K-variant and its linkage to the atypical variant mutation and other polymorphic sites.

Genomic DNA from two families exhibiting the K-variant phenotype of serum butyrylcholinesterase was amplified by PCR and sequenced to determine the molecular basis of this variant. The K-variant phenotype was found to be associated with a DNA transition from guanine to adenine at nucleotide 1615, which caused an amino acid change from alanine 539 to threonine (GCA----ACA; Ala539----Thr). There was a 30% reduction of serum butyrylcholinesterase activity associated with this mutation. Amplification and sequencing of DNA from a random sample of 47 unrelated people gave a frequency of .128 for the K-variant allele. Thus, 1 person in 63 should be homozygous for the K-variant, making the K-variant the most common butyrylcholinesterase variant. The K-variant mutation was also found to be present in 17 (89%) of 19 butyrylcholinesterase genes containing the point mutation which causes the atypical phenotype of butyrylcholinesterase (GAT----GGT; Asp70----Gly). The presence of the K-variant in the same molecule as the atypical variant does not contribute to the qualitative change in the atypical enzyme, but it most likely accounts for the approximately one-third reduction in Vmax of butyrylcholinesterase activity in atypical serum. Two additional point mutations located in noncoding regions of the gene were also observed to be in linkage disequilibrium with the K-variant mutation. As many as four different point mutations have been identified within a single butyrylcholinesterase gene. Inhibition tests of the enzyme in plasma are usually used to distinguish the K-variant from the usual enzyme when the former is present with the heterozygous atypical variant (AK phenotype vs. UA phenotype). Inhibition tests were performed on plasma enzyme from the four possible genotypic combinations of the heterozygous atypical mutation with or without the K-variant mutation on either allele; we found that the AK phenotype was caused by three genotypes (A/K, AK/K, and U/A) and that the UA phenotype was caused by two genotypes (U/A and U/AK).

Amino Acid Sequence↗
PubMed1991

The cloning and expression of a gene encoding Old Yellow Enzyme from Saccharomyces carlsbergensis.

We have identified a gene that encodes Old Yellow Enzyme in brewer's bottom yeast. The open reading frame encodes a polypeptide of 400 amino acids with Mr = 45,021. Using the T7 RNA polymerase system, recombinant enzyme was expressed in Escherichia coli. 17 mg of Old Yellow Enzyme was obtained from a 3-liter cell culture, and the recombinant enzyme had NADPH oxidase activity. On fast protein liquid chromatography separation, the recombinant enzyme showed a single large peak, while native enzyme from brewer's bottom yeast separated into five fractions on fast protein liquid chromatography. Southern blot analysis showed that there are at least two Old Yellow Enzyme genes in brewer's bottom yeast genomic DNA. These results suggest that the heterogeneity of Old Yellow Enzyme in Saccharomyces carlsbergensis is due to the presence of multiple genes.

Amino Acid Sequence↗
PubMed1991

Use of the polymerase chain reaction for homology probing of butyrylcholinesterase from several vertebrates.

Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.

Amino Acid Sequence↗
PubMed1991

Proposed nomenclature for human butyrylcholinesterase genetic variants identified by DNA sequencing.

1. New information identifying nucleotide alterations of human butyrylcholinesterase allows the use of more specific nomenclature for the variants commonly known as atypical, fluoride, silent, and K variant. 2. In addition to suggesting a system of trivial names and abbreviations, we provide a list of formal names that follow the guidelines of the Committee for Human Gene Nomenclature. 3. It is suggested that formal names be included in publications whenever possible.

Amino Acid Sequence↗
PubMed1991

The cloned butyrylcholinesterase (BCHE) gene maps to a single chromosome site, 3q26.

Human tissues have two distinct cholinesterase activities: acetylcholinesterase and butyrylcholinesterase. Acetylcholinesterase functions in the transmission of nerve impulses, whereas the physiological function of butyryl-cholinesterase remains unknown. An atypical form of butyrylcholinesterase or the absence of its activity leads to prolonged apnea following administration of the muscle relaxant suxamethonium. Inheritance of these butyrylcholinesterase variants is consistent with the enzyme activity being encoded in a single autosomal locus, BCHE (formerly CHE1 and E1), which has been assigned to chromosome 3. Previous in situ hybridization of a BCHE cDNA probe gave evidence of homologous sequences at 3q26 and 16q11-q23, raising the possibility of more than one locus coding for butyrylcholinesterase [H. Soreq, R. Zamir, D. Zevin-Sonkin, and H. Zakut (1987) Hum. Genet. 77: 325-328]. Using a different cDNA probe hybridized in situ to 46,XX,inv(3)(p25q21) metaphase chromosomes, we report here the localization of BCHE to a single autosomal location: 3q26.

Autoradiography↗
PubMed1990

Evidence for a single butyrylcholinesterase gene in individuals carrying the C5 plasma cholinesterase variant (CHE2).

DNA of 3 unrelated individuals carrying the human plasma butyrylcholinesterase C5 variant (CHE2) was isolated from white blood cells. Southern blot patterns of DNA restriction fragments probed with each of the 4 butyrylcholinesterase exons provided evidence that the production of C5 is not directed by a second butyrylcholinesterase gene. This finding supports the suggestion that the C5 variant is a hybrid enzyme resulting from the association of butyrylcholinesterase subunits with a non-cholinesterase protein.

Butyrylcholinesterase↗
PubMed1990

Structure of the gene for human butyrylcholinesterase. Evidence for a single copy.

We have isolated five genomic clones for human butyrylcholinesterase (BChE), using cDNA probes encoding the catalytic subunit of the hydrophilic tetramer [McTiernan et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6682-6686]. The BChE gene is at least 73 kb long and contains four exons. Exon 1 contains untranslated sequences and two potential translation initiation sites at codons -69 and -47. Exon 2 (1525 bp) contains 83% of the coding sequence for the mature protein, including the N-terminal and the active-site serine, and a third possible translation initiation site (likely functional), at codon -28. Exon 3 is 167 nucleotides long. Exon 4 (604 bp) codes for the C-terminus of the protein and the 3' untranslated region where two polyadenylation signals were identified. Intron 1 is 6.5 kb long, and the minimal sizes of introns 2 and 3 are estimated to be 32 kb each. Southern blot analysis of total human genomic DNA is in complete agreement with the gene structure established by restriction endonuclease mapping of the genomic clones: this strongly suggests that the BChE gene is present in a single copy.

Base Sequence↗
PubMed1990

Phenotypic and molecular biological analysis of human butyrylcholinesterase variants.

Our laboratory has recently shown that several variant forms of human butyrylcholinesterase, associated with unusual sensitivity to succinylcholine, are caused by specific mutations within the structural DNA coding for this enzyme. Atypical (dibucaine-resistant) butyrylcholinesterase is caused by a point mutation at nucleotide position 209(GAT-- greater than GGT), which changes aspartate 70 to glycine. One fluoride-resistant variant family has a point mutation at nucleotide 728(ACG-- greater than ATG), which changes threonine 243 to methionine. Another type of fluoride-resistant variant has a point mutation at nucleotide 1169(GGT-- greater than GTT), which changes glycine 390 to valine. One type of silent phenotype is due to a frame-shift mutation at nucleotide position 351(GGT-- greater than GGAG). A polymorphic site at nucleotide position 1615 (GCA/ACA), coding for Ala/Thr, accounts for the quantitative K-variant, which causes an approximate one-third reduction of activity, if Thr occupies that position at codon 539. Examples are given to illustrate the advantages of using a combination of the new DNA analytical techniques, including: the use of allele-specific probes, with the standard serum cholinesterase phenotyping methods. More accurate typing of patients with certain variants is now possible; pedigree analysis will be aided by the improved methodology.

Alleles↗