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O Lockridge

Publications and source records attributed to O Lockridge.

At least 73 records · Page 4Linked to original sources
PubMed1990

Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine.

People with genetic variants of cholinesterase respond abnormally to succinylcholine, experiencing substantial prolongation of muscle paralysis with apnea rather than the usual 2-6 min. The structure of usual cholinesterase has been determined including the complete amino acid and nucleotide sequence. This has allowed identification of altered amino acids and nucleotides. The variant most frequently found in patients who respond abnormally to succinylcholine is atypical cholinesterase, which occurs in homozygous form in 1 out of 3500 Caucasians. Atypical cholinesterase has a single substitution at nucleotide 209 which changes aspartic acid 70 to glycine. This suggests that Asp 70 is part of the anionic site, and that the absence of this negatively charged amino acid explains the reduced affinity of atypical cholinesterase for positively charged substrates and inhibitors. The clinical consequence of reduced affinity for succinylcholine is that none of the succinylcholine is hydrolyzed in blood and a large overdose reaches the nerve-muscle junction where it causes prolonged muscle paralysis. Silent cholinesterase has a frame shift mutation at glycine 117 which prematurely terminates protein synthesis and yields no active enzyme. The K variant, named in honor of W. Kalow, has threonine in place of alanine 539. The K variant is associated with 33% lower activity. All variants arise from a single locus as there is only one gene for human cholinesterase (EC 3.1.1.8). Comparison of amino acid sequences of esterases and proteases shows that cholinesterase belongs to a new family of serine esterases which is different from the serine proteases.

Amino Acid Sequence↗
PubMed1990

Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT----GGAG).

A frameshift mutation that causes a silent phenotype for human serum cholinesterase was identified in the DNA of seven individuals of two unrelated families. The mutation, identified using the polymerase chain reaction, causes a shift in the reading frame from Gly 117, where GGT (Gly)----GGAG (Gly+ 1 base) to a new stop codon created at position 129. This alteration is upstream of the active site (Ser 198), and, if any protein were made, it would represent only 22% of the mature enzyme found in normal serum. Results of analysis of the enzymatic activities in serum agreed with the genotypes inferred from the nucleotide sequence. Rocket immunoelectrophoresis using alpha-naphthyl acetate to detect enzymatic activity showed an absence of cross-reactive material, as expected. One additional individual with a silent phenotype did not show the same frameshift mutation. This was not unexpected, since there must be considerable molecular heterogeneity involved in causes for the silent cholinesterase phenotype. This is the first report of a molecular mechanism underlying the silent phenotype for serum cholinesterase. The analytical approach used was similar to the one we recently employed to identify the mutation that causes the atypical cholinesterase variant.

Amino Acid Sequence↗
PubMed1989

Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.

A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.

Base Sequence↗
PubMed1987

Location of disulfide bonds within the sequence of human serum cholinesterase.

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.

Acetylcholinesterase↗
PubMed1987

Complete amino acid sequence of human serum cholinesterase.

The complete amino acid sequence of human serum cholinesterase (choline esterase II (unspecific), EC 3.1.1.8) was determined by Edman degradation of purified peptides. The protein contains 574 amino acids per subunit and nine carbohydrate chains attached to 9 asparagines. The four subunits of cholinesterase appear to be identical. The active site serine is the 198th residue from the amino terminus. The sequence of human serum cholinesterase is 53.8% identical with the sequence of acetylcholinesterase from Torpedo californica and 28% identical with the carboxyl-terminal portion of bovine thyroglobulin.

Amino Acid Sequence↗
PubMed1987

Brain cDNA clone for human cholinesterase.

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase (EC 3.1.1.8). Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase (EC 3.1.1.8) rather than acetylcholinesterase (EC 3.1.1.7). It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes, coded for cholinesterase.

Amino Acid Sequence↗
PubMed1986

Amino acid sequence of the active site of human serum cholinesterase from usual, atypical, and atypical-silent genotypes.

Active-site tryptic peptides were isolated from three genetic types of human serum cholinesterase. The active-site peptide was identified by labeling the active-site serine with [3H]diisopropylfluorophosphate. Peptides were purified by high-performance liquid chromatography. Amino acid composition and sequence analysis showed that the peptide from the usual genotype contained 29 residues with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-His-Leu- Leu-Ser-Pro-Gly-Ser-His-Ser-Leu-Phe-Thr-Arg. The active-site serine was the eighth residue from the N-terminal. The peptide containing the active-site serine from the atypical genotype contained 22 residues with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-His-Leu- Leu-Ser-Pro-Gly. The peptide from the atypical-silent genotype contained eight residues with the sequence Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser. Thus, the sequences of the atypical and atypical-silent active-site peptides were identical to the corresponding portions of the usual peptide.

Amino Acid Sequence↗
PubMed1986

Molecular biology of human serum cholinesterase.

More than 90% of the amino acid sequence of purified human serum cholinesterase has been determined in our laboratory. Purified enzyme was digested with several proteolytic enzymes; the resulting polypeptides were then separated, purified, and sequenced. Optimal sequence regions were identified and used as the basis for the synthesis of three 17-mer oligonucleotide probes. In addition, one long peptide of 58 amino acid residues was selected for construction of two unique sequence oligonucleotide probes of 39-mer and 53-mer; the peptide regions corresponding to the latter are six amino acids apart. The probes have been used to screen a human liver cDNA library and a human genomic library. Several positive clones to both types of probes have been identified. These are being characterized, and some of them have been or are now being sequenced. A high degree of homology in the amino acid sequence of the active center of human serum cholinesterase and that of acetylcholinesterase from the Torpedo fish has been noted. It appears that this region of cholinesterases has been conserved during evolution, and there may be an important, still unrecognized role for serum nonspecific cholinesterase in mammalian metabolism.

Amino Acid Sequence↗
PubMed1983

Immunological comparison of the usual and atypical human serum cholinesterase phenotypes.

Antiserum prepared against highly purified usual human serum cholinesterase (the most common phenotype) cross-reacted identically with the atypical serum cholinesterase. The level of circulating atypical enzyme protein, determined immunologically, was about 30% lower when the enzyme came from an atypical rather than a usual phenotype, and the level of enzyme activity measured enzymatically at Vmax with either o-nitrophenylbutyrate or benzoylcholine as substrate showed approximately the same degree of reduction. The average specific activity (activity at Vmax per microgram of enzyme protein) in sera from 28 usual and 20 atypical individuals did not differ significantly. These findings suggest that the atypical enzyme not only has altered catalytic properties (Km) but also might be synthesized more slowly, or cleared in vivo more rapidly, than the usual enzyme.

Antigen-Antibody Complex↗
PubMed1982

Substance P hydrolysis by human serum cholinesterase.

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.

Amino Acids↗
PubMed1980

Hydrolysis of diacetylmorphine (heroin) by human serum cholinesterase.

The enzyme in human serum that rapidly hydrolyzes diacetylmorphine (heroin) to 6-acetylmorphine is identified in this report as serum cholinesterase (EC 3.1.1.8, acylcholine acylhydrolase; also called pseudocholinesterase or butyrylcholine esterase). The rate of heroin hydrolysis was measured spectrophotometrically at 245 nm using highly purified serum cholinesterase. The turnover number was 500 mumol of heroin hydrolyzed per min per mumol active site. The product was identified spectrophotometrically and by thin-layer chromatography to be 6-acetylmorphine. There appeared to be marked product inhibition of heroin hydrolysis, as 6-acetylmorphine (Ki = 0.015 mM) bound 7 times more tightly than heroin (Ki = 0.11 mM). Purified human serum arylesterase did not hydrolyze heroin. Purified serum cholinesterase accounted for all the observed heroin hydrolysis by whole serum. The genetic variants of human serum cholinesterase, silent and atypical cholinesterase, were also tested. Serum from a person identified as having silent cholinesterase did not hydrolyze heroin. Purified atypical cholinestearase hydrolyzed heroin, but the binding was less tight (Km = 0.45 mM) than with usual cholinesterase (Km = 0.11 mM). The possibility that heroin potency may be influenced by serum cholinesterase genotype or activity level remains to be investigated.

Cholinesterase Inhibitors↗
PubMed1978

Comparison of atypical and usual human serum cholinesterase. Purification, number of active sites, substrate affinity, and turnover number.

Atypical and usual human serum cholinesterases were purified and studied with the fluorescent probe, N-methyl-(7-dimethylcarbamoxy)quinolinium iodide. Four active sites per tetramer were found in each enzyme. The turnover numbers of usual and atypical cholinesterases were the same: 15,000 mumol of benzoylcholine hydrolyzed/min/mumol of active site; 48,000 min-1 for o-nitrophenylbutyrate; and 0.0025 min-1 for N-methyl-(7-dimethylcarbamoxy)quinolinium iodide. They had identical rate constants for carbamylation, (5.0 min-1) and for decarbamylation (0.15 h-1). The major difference between the two genetically determined forms of the enzyme was substrate affinity, KD being 0.16 mM for usual and 5.4 mM for atypical cholinesterase, for the fluorescent probe substrate. Km for the uncharged ester, o-nitrophenylbutyrate, was 0.14 mM for both enzymes, whereas Km for benzoylcholine was 0.005 mM for usual and 0.024 mM for atypical cholinesterase. We interpret these data to mean that the two enzymes differ only in the structure of their anionic site.

Binding Sites↗