L-Glutamic acid γ-(β-naphthylamide)

Background: Measurement of cholinesterase activity in serum is important to identify substantial liver disease and damage by pesticides, and to assess the degree of development of fatty liver and preoperative risk. Many procedures using various artificial substrates have been developed but suffer from problems with substrate specificity and interference by endogenous substances. Methods: An assay pseudocholinesterase (ChE, EC 3.1.1.8 acylcholine acylhydrolase) activity was developed using a stable substrate specific to ChE, benzoylthiocholine iodide (BZTC). The thiocholine generated by hydrolysis of BZTC by ChE activity reacts with 2, 2'-dipyridildisulfide (2-PDS) to produce 2-thiopyridine (2-TP), which is measured at 340 nm. Optimum pH, buffer types and concentrations, substrate concentrations, and optimum conditions of the color reaction were investigated. The substrate specificity, test interferences, correlation with other measurement methods, and reference interval were evaluated. Results: The optimum pH of this method was 7.8, and 3-[4-(2-hydroxyethyl)-1-piperazinyl] propanesulfonic acid (EPPS) buffer solution was selected. Constant activity was shown at buffer concentrations >200 mmol/l, and the maximum activity was shown at a substrate concentration of 0.2 mmol/l. When a Hill plot was utilized, the Hill number was 1.08 and 1.09. The reaction velocity at this substrate concentration was 94% of V(max). The K(m) of ChE to BZTC was between 1.2 x 10(-2) and 1.3 x 10(-2) mmol/l. The range was 0-300 U/l. The coefficients of variation (CV) for 20 measurements of serum containing 53.1, 96.6, and 270.7 U/l of ChE were 0.82%, 0.76%, and 0.54%, respectively. The relative reactivity of acetylcholinesterase (AChE) to this substrate was 2%. The correlation factors of this method to three other methods were between 0.993 and 0.998. Conclusions: This method provides excellent specificity, reproducibility, a wide measurement range, and minimal interference from endogenous substances to common serum analytes. Correlation of this method with conventional methods was good. Because the reagents are stable after preparation, this assay is useful for routine analysis.

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.

Alanine aminopeptidase was partially purified from cultured human melanoma cells (Bowes) by gel filtration on Sephadex G-200 and DEAE Sepharose column chromatography. The molecular weight of the enzyme was about 52,000 as determined by gel filtration on Sephadex G-100. The enzyme hydrolyzed L-alanine beta-naphthylamide (NA), but not or slightly L-methionine-NA, L-leucine-NA, and L-arginine-NA. The Km value for L-alanine-NA was 0.17 mmol/l, pH optimum was 7.4. The enzyme was stable at 50 degrees C for 20 min, but lost about 50% of its activity at 60 degrees C within 20 min. It was markedly stimulated by chloride ions, and was inhibited by sulfhydryl blocking agents and EDTA. The activity was restored by the addition of Co2+ or Zn2+ after EDTA treatment. The enzyme is a metallo- and thiol-dependent and chloride-activated, low-molecular weight aminopeptidase.