Dibucaine inhibition of serum cholinesterase

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an IC(50) of 5.3 microM with BuTch or 3.8 microM with AcTch. The inhibition was competitive with respect to BuTch with a K(i) of 1.3 microM and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, K(i) and K(I) of 0.66 and 2.5 microM, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.     

Structure of human serum cholinesterase

Human cholinesterase has recently been sequenced and cloned. It is a glycoprotein of 4 identical subunits, each subunit containing 9 carbohydrate chains and 3.5 disulfide bonds. Protein folding is likely to be very similar in human cholinesterase and Torpedo acetylcholinesterase. The cholinesterases have no significant sequence homology with the serine proteases and seem to belong to a separate serine esterase family.     

Variations in the level of human serum albumin during glucose tolerance test

The serum protein level in patients taking the glucose tolerance test was found to vary. This variation was accounted for by changes in the level of serum albumin which dropped an average of 17% one hour after the administration of glucose. The serum albumin level returned to its normal value when blood glucose and non-esterified fatty acid levels returned to their initial levels. It is suggested that this variation is the result of a shift of albumin between the intravascular and extravascular compartments.     

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.     

Active site kinetics of horse serum cholinesterase

Horse serum Cholinesterase hydrolyzes choline and p-nitrophenol esters at different rates. Deacylation and acylation seem to be the rate-limiting step for charged and uncharged substrates respectively. Activation energy is similar for the acetic, propionic, and butyric esters of thiocholine, but it is higher for p-nitrophenylpropionate. Inhibition by the tetramethyl-ammonium ion is competitive. Tetraethyl-, tetrapropyl-, and tetrabutyl-ammonium ions are mixed-type inhibitors. The pH studies demonstrated the existence of a residue, pK = 6.33, involved in catalysis.