Apolinary Szewczuk

Determination of γ-glutamyl transpeptidase activity in human serum and urine

Abstract A simple and improved method for the synthesis of γ- l -glutamyl-α- and β-naphthylamide is described. A procedure for the determination of γ-glutamyl transpeptidase activity based on the colorimetric determination of α-naphthylamine liberated from its γ-glutamyl derivative is given.

The use of α-(N-γ-dl-glutamyl)-aminonitriles for the colorimetric determination of a specific peptidase in blood serum

Abstract The occurrence of a specific peptidase in human serum is described. The enzyme catalyses the hydrolysis of α-(N-γ- dl -glutamyl)-aminopropionitrile as well as of γ-glutamyl derivatives of other α-aminonitriles. A method is described for assay of the enzyme activity, based on the colorimetric determination of α-aminopropionitrile liberated during the enzymatic incubation ; as little as 0.2 μg of α-aminopropionitrile can be estimated. Some properties of the enzyme were investigated and its specificity is discussed. The activity in sera of normal human subjects was 10.9 units (1 unit = 1 μmole α-aminopropionitrile liberated from the substrate per 100 ml serum after a 2-h incubation at pH 8.6 and 37°). Changes in the activity of the peptidase were found in some pathological conditions. Besides human serum, the enzyme activity was found in sera of many animals, as well as in human urine and pathological exudations. No activity was found in human red blood cells.

Colorimetric assay of penicillin amidase activity using phenylacetyl-aminobenzoic acid as substrate.

Abstract All three isomers of phenylacetyl-aminobenzoic acid are stable in buffer solutions and are suitable as substrates for the simple and sensitive assay of penicillin amidase activity in purified preparations, in bacterial cells as well as in immobilized preparations. By titration of the active center with phenylmethylsulfonyl fluoride it was demonstrated that phenylacetyl-4-aminobenzoic acid as well as benzylpenicillin are hydrolyzed by the same enzyme. The new substrate was also used for rapid assay of the amidase activity in bacterial colonies. Both phenylacetyl-naphthylamides are very slowly hydrolyzed by the amidase.

Immunofluorescent localization of γ-glutamyl transferase in rat and bovine tissues

Summary A distribution of the enzyme in tissue sections was studied by direct fluorescent technique using immunoglobulins from rabbits immunized with the rat and bovine kidney enzyme. For studies rat and bovine sections of kidneys, livers, pancreas, and small intestine were used. Cell distribution of the enzyme by immunofluorescent method was the same as that by the classical histoehemical method. No differences in the enzyme distribution were noted when either immunoglobulins anti-heavy form or immunoglobulins anti light form of the enzyme were used. Bovine kidney enzyme coupled to diazotized p-aminobenzoyl-hexamethylenediamine-sepha-rose is a effective sorbant for affinity chromatography of antibody. Precipitates formed between antibodies and purified rat or bovine γ -glutamyl transferases preserves over 60% of the initial enzyme activity.

A Note on the occurrence of γ-glutamyl transpeptidase in human serum

Abstract The results presented support the view that the specific peptidase in blood serum is γ-glutamyl transpeptidase. Thus our method previously described and based on the colorimetric determination of α-aminopropionitrile liberated from its γ-glutamyl derivate during the enzymatic reaction, determines γ-glutamyl transpeptidase activity. For the same purpose we used recently a new synthetic substrate γ- l -glutamyl-α-naphihylamide6.

The use of γ-glutamyl-p-aminobenzoic acid as the substrate for determination of γ-glutamyltranspeptidase activity in blood serum

Abstract By the phthaloyl method less toxic and readily soluble γ- l -glutamyl- p -aminobenzoic acid was synthesized. This substance was used as a substrate for γ-glutamyltranspeptidase activity assay in blood serum and urine. Close correlation was shown between the results obtained with the new method and with the old one which used γ- l -glutamyl- p -nitroanilide.

A new fluorimetric method for the determination of 7-glutamyltransferase activity in blood serum

Using 7-(gamma-L-glutamyl)-4-methyl-coumarylamide as the fluorogenic substrate a simple and sensitive method for the assay of transferase activity of gamma-glutamyl-transferase in human blood serum and some other biological fluids is described. The substrate was synthesized with a good yield by the King and Kidd method. Close correlation and good agreement was noted between activities measured by the fluorimetric method and by the old colorimetric one in which gamma-L-glutamyl-p-nitroanilide was used.

Cobalt-activated acylase in human serum. Occurrence in hepatobiliary diseases

Abstract A simple colorimetric method for the determination of acylase activity in serum using N-chloroacetyl-γ- l -glutamyl-β-naphthylamide as substrate has been elaborated. The enzyme is strongly activated by Co2+ and inhibited by EDTA. The acylase activity was assayed in the sera of 60 healthy subjects and in 260 patients with various internal diseases. A distinct enzyme activity was observed in all cases of viral hepatitis studied early in the icteric phase of the disease. On the contrary, only traces of the activity were found in sera of patients with some hepatobiliary disorders. No activity could be demonstrated in patients with other diseases or in healthy subjects. The results obtained indicate that serum acylase determination may be of use in the differential diagnosis of some hepatobiliary diseases.

Colorimetric method for assay of serum γ-glutamyltransferase activity with some L-γ-glutamyl-carboxyanilides

Abstract A simple method for the assay of serum γ-glutamyltransferase activity with soluble l -γ-glutamyl-carboxyanilides is presented. It is based on transformation of aminobenzoic acid liberated by the enzyme into colour complex with 4-dimethyl-aminobenzaldehyde or with 4-dimethylaminocinnamaldehyde in acid medium. Among three carboxyanilides studied the highest enzyme activity was noted with l -γ-glutamyl-3-carboxyanilide and no activity with l -γ-glutamyl-2-carboxyanilide. A good correlation was demonstrated for serum γ-glutamyltransferase activity determined by the new colorimetric method and by the standard one with l -γ-glutamyl-3-carboxy-4-nitroanilide.

Acylases in plasma of hamsters with experimental hepatitis.

After the administration of D-galactosamine to golden Syrian hamsters, a rapid but short-lived increase in acylase I and cobalt-activated (AA-Co) activity was noted in the blood plasma, but not in the liver. In the plasma of control hamsters, only form 2 AA-Co was identified, but during experimental hepatitis, the appearance of form 1 was observed. Only form 2 hamster enzyme is strongly inhibited by alpha-hydroxyisocaproil-tyrosine and reacts with antihuman form 2 antibody.