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Variations in sialic acid content of gamma-glutamyltransferase: a consequence for immunochemical determinations?
21
Clinica Chimica Acta, 148 (1985) 21-30
Elsevier CCA 03160
Variations in sialic acid content of gamma-glutamyltransferase: a consequence for immunochemical determinations? Maria Wellman-Bednawska
a, Yves Artur a and Gerard
Siest a*b
a Centre du Midicament, UA CNRS No 597, 30 Rue Lionnois, 54000 Nancy and b Centre de Mkdecine Prkventive (Dir. Pr. Senault), 2 Avenue du Doyen Jacques Parisot, 54500 Vandoeuvre- les - Nancy (France) (Received July 17th, 1984; revision January 15th, 1985) Key words: gamma
-Glutamyltransferase,
Human analysis; Sialic aciak analysis; Immunochemistry
analysis
Summary Using specific antibodies against the human kidney enzyme, gamma-glutamyltransferase (GGT, EC 2.3.2.2) was assayed from human kidney and serum by electroimmunodiffusion. Determination of the enzyme by such a method was highly influenced by the sialic acid content of the molecule. The peaks corresponding to the sialylated GGT were higher than those corresponding to the neuraminidase-treated enzyme. In contrast, sialylation of the protein had no influence on the results observed when measuring the enzyme by radial immunodiffusion. Moreover, immunoprecipitation curves of both sialylated and neuraminidase-treated samples were identical. The varying degrees of sialylation of GGT occurring under physiological or pathological conditions are known to be partly responsible for the heterogeneity of the enzyme in organs and biological fluids. Therefore, determination of the enzyme by electroimmunodiffusion may be hazardous.
Introduction gamma-Glutamyltransferase (GGT, EC 2.3.2.2) is a glycoprotein found in many tissues and biological fluids. According to Huseby [l], GGT is present in human organs as an amphiphilic membrane-bound enzyme. Detergents may solubilize the enzyme by binding to the hydrophobic, membrane-binding domain of the enzyme, forming a micellar-like complex, the ‘heavy’ or amphiphilic form of the enzyme. Treatment with proteases such as papain or trypsin removes the hydrophobic domain, thus forming a soluble, hydrophilic ‘light’ form of the enzyme. Both the heavy and the light forms are present in serum [2]. 0009-8981/85/$03.30
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
22
GGT activity is markedly increased in the sera of patients with hepatobiliary disorders [3]. Many chromogenic or fluorogenic substrates have been proposed for determining this activity (see short review in [4]). However, much less work has been done on the immunochemical measurement of the GGT protein. Rocket electroimmunoassays were performed by Scherberich et al for the urine enzyme [5], and by Huseby for a partially purified liver GGT [l], but without quantitative application. A radioimmunoassay for human serum and pancreatic GGT [6] and an enzyme-linked immunosorbent assay (ELISA) for the enzyme from some bovine body fluids and tissue preparations [7] have recently been proposed. The present paper displays the results we obtained when determining the enzyme by electroimmunodiffusion on cellulose acetate plates. We placed special emphasis on the study of the involvement of sialic acid residues in electroimmunodiffusion of GGT: indeed GGT is a sialoglycoprotein, and the degree of sialylation has been previously shown to have an effect upon the height of the rockets in the case of alpha-l-acid glycoprotein [8] and transferrin [9]; moreover, the varying degrees of sialylation occurring under physiological or pathological conditions seem to contribute to the heterogeneity of the enzyme in serum [lo-121, kidney [13] and liver [14,15]. Materials and methods Purification of human GGTs The light form of human kidney GGT was purified from cortical slides, using a procedure similar to that used by Tate and Meister [16]. The light form of human liver GGT was prepared by the technique proposed by Huseby [17]. Specific activities of the liver and kidney GGT preparations were 110 and 210 U/mg of protein, respectively. The purified kidney GGT contained 20 nmol sialic acid/mg protein. Liver and kidney were obtained at autopsy within 48 h of death. Preparation of the antiserum The antiserum against light GGT was raised in rabbits (Fauves de Bourgogne, France). The animals were inoculated subcutaneously with 100 pg of kidney GGT preparation mixed with 0.5 ml of Freund’s complete adjuvant. Similar booster injections, but using Freund’s incomplete adjuvant, were given at 2-wk intervals. Each rabbit was bled from the ear vein 8 days after the third booster challenge. Human serum samples Sera were obtained from 7 patients with various liver diseases: four with alcoholic cirrhosis (GGT activities: 592,692,989 and 1,212 U/l) and one each with congestive heart failure (1,030 U/l), liver metastasis (344 U/l) and nutritional steatosis (776 U/l). Neuraminidase treatment The purified kidney GGT was incubated with 2 U neuraminidase/mg protein in a 100 mmol/l acetate buffer, pH 5.5, containing 10 mmol/l CaCl,, at 37°C for
23
0.5-48 h. Neuraminidase (100 U/117 mg protein) was from Sigma Chemical Co. (Saint Louis, MO, USA). For serum, desialylation was carried out in the same way, but using 5 U neuraminidase/rnl serum. Incubation with neuraminidase had no effect upon the enzyme activity in these conditions. Radial immunodifftkon and double immunodiffusion tests In radial immunodiffusion and double irnmunodiffusion tests, we used Indubiose (Industrie Biologique Francaise, Clichy, France) at a concentration of 10 g/l in a 50 mmol/l barbital-sodium barbital buffer, pH 8.6. The concentration of antiserum in the gels of radial immunodiffusion was 20 ml of antiserum/l of gel, and 8 ~1 portions of the various samples were applied. In both cases, diffusion was allowed to take place for 48 h. Unprecipitated proteins were removed by washing the gels in 9 g/l NaCl and water. As in electroimmunodiffusion tests, the precipitates were observed after enzyme specific staining (see below). Electroimmunodiffusion tests We used Titan III Zip Zone cellulose acetate plates (Helena Labs., Beaumont, TX, USA). The plates were wetted in a 25 nunol/l barbital-sodium barbital buffer, pH 8.6, containing 40 ml of antiserum/l of buffer. We applied 0.6 ~1 of the various enzyme preparations, or 1.2 ~1 of serum. The strips were then electrophoresed for 45 min with a constant 180 V. Unprecipitated proteins were removed in a 60-min buffer wash, using a magnetic stirrer. Enzyme specific staining of the peaks was realized using the substrate gamma-L-glutamyl-alpha-naphthylamide (Sigma Chemical Co, Saint Louis, MO, USA). Glycylglycine and Tris were from Merck (Darmstadt, FRG). The plates were incubated at 37°C for 150 min with the substrate solution (per liter, 5 nun01 of gamma-rglutamyl-alpha-naphthylamide, and 100 mm01 each of Tris and glycylglycine, pH 8.25). After quickly washing the plates with water, we detected the released alpha-naphthylamine by applying a freshly prepared 1 g/l aqueous solution of Fast Garnet GBC (Serva, Heidelberg, FRG), and fixed the resulting red peaks with a 100 ml/l aqueous solution of acetic acid. Sodium deoxycholate was from Sigma Chemical Co. Immunoprecipitation tests Immunoprecipitation tests were carried out as previously described [18], incubating increasing volumes of antiserum with constant samples of GGT preparations or serum. Other analytical methodr We measured GGT activities at 30°C using a Cary 219 spectrophotometer (Varian Associates, Palo Alto, CA, USA) as previously described [19]. The substrate gamma-L-glutamyl-3-carboxy-4nitroanilide was from Boehringer (Mannheim, FRG). Sialic acid was determined by Warren’s method [20], using N-acetyl neuraminic acid (Sigma Chemical Co.) as standard.
24
Results
By double immunodiffusion in agarose gels, an apparent identity reaction was seen between the kidney and the liver GGT: indeed, the single precipitation lines fuse smoothly with one another (Fig. 1). As previously described [19], immunoprecipitates retained enzyme activity, allowing enzyme-specific staining of the arcs. A simple and rapid method for determining the enzyme by el~troi~unodiffusion was developed (see ‘Materials and Methods’). Using the purified light form of kidney GGT as standard, we observed a linear relationship between the peak height and the logarithm of the protein concentration (Fig. 2). The corresponding rockets are shown in Fig. 3A. Amounts of GGT as low as 20 ng were detected and the calibration curve was linear up to 0.6 pg at least. Applying identical amounts of native and desialylated GGT, we observed that the neuraminidase treatment of the enzyme led to an important decrease in the peak height. Figure 3B shows the rockets corresponding to the native sialylated GGT and the desialylated enzyme at various times of neuraminidase treatment. After 60 min of incubation with neuraminidase, 20 nmol of sialic acid were liberated per mg of protein. A longer incubation did not change sialic acid liberated or the height of the peaks. The maximum difference in peak height we found here was about 208, and
Fig. 1. Double immunodiffusion: reactivity of kidney and liver GGT towards rabbit antiserum produced against purified human kidney GGT. The center well [7] contains antiserum. Peripheral wells contain: 1, 3, 5: liver GGT, 2, 4, 6: kidney GGT. The precipitate was stained for GGT activity (see ‘Materials and Methods’).
25 .paak
haight
(cm)
.
‘L 20
50
loo
500
kiirmy
GGT
Fig. 2. Electroimmunodiffusion of various amounts of native sialylated GGT from human kidney. The logarithm of the amount of GGT applied on the strip is plotted against the peak height. The corresponding rockets are shown in Fig. 3A. The amounts of GGT applied were: 18.7, 37.5, 75, 150, 300 and 600 ng.
Fig. 3. Electroimmunodiffusion of kidney GGT. Conditions for electrophoresis are described in ‘Materials and Methods’. The rockets are stained for GGT activity as shown in the text. A. Decreasing amounts of native sialylated GGT: 1, 600 ng; 2, 300 ng; 3, 150 ng; 4, 75 ng; 5, 37.5 ng; 6, 18.7 ng. B. Native sialylated GGT: 7, 300 ng; 8, 75 ng; 9, 10, 11, 12, neuraminidase&eated samples (300 ng of GGT for each) after incubation with neuraminidase for 0.5,3,22 and 48 h, respectively. The specific activity of the undiluted GGT preparation used was 210 U/mg of protein. Its protein concentration was 1 mg/ml. Bar shows point of application.
26
the height of the lowest rocket obtained with 300 ng of desialylated enzyme corresponded approximately to the height of the rocket observed with 75 ng of native sialylated GGT. The presence of detergent (sodium deoxycholate at the concentration of 5 g/l both in the sample and in the migrating buffer) did not influence the peak height. We then submitted serum samples to electroimmunodiffusion. In the absence of detergent, we obtained unsatisfactory pictures: for each serum, we observed two superimposed rockets, but these rockets formed trails, and a fraction of the enzyme did not migrate. This phenomenon may be partly attributed to the binding of the enzyme to lipids. Indeed, we previously observed the binding of GGT to lipoproteins in serum of patients with hepatobiliary diseases [18,21]. The fraction which did not migrate might be a GGT fraction associated with chylomicrons as assumed by Von Freise et al [22]. Nevertheless, the trails corresponding to the untreated sera were higher than those corresponding to the neuraminidase-treated samples (data not shown). Figure 4 represents an example of the pictures we observed when sodium deoxycholate was added to the samples and to the migrating buffer. For each serum, two superimposed rockets with GGT activity were obtained, but no GGT activity
Fig. 4. Electroimmunodiffusion of two sera before (1, 2) and after (lN, 2N) neuraminidase treatment. Desialylation was carried out as described in the text, incubating 5 U neuraminidase/mf serum for 16 h at 37’C. Electrophoresis was performed as described in the text, with sodium deoxycholate present in the samples and in the migrating buffer (5 g/l). The peaks were stained for GGT activity (see ‘Materials and Methods’). Both sera were- from patients with alcoholic cirrhosis. GGT activities: 1,989 U/l; 2,1212 U/l. Bar shows point of application.
21
Fig. 5. Radial immunodiffusion of native sialylated and neuraminidase-treated human kidney GGT. Conditions for neuraminidase treatment, diffusion and specific staining of the precipitates are described in ‘Materials and Methods’. Decreasing amounts of native sialylated GGT: 1, 8 pg; 2, 6 pg; 3, 5 gg, 4, 4 gg and 5, 2 pg. Five pg of neuraminidase-treated GGT, incubated at 37°C for: 6, 0.5 h; 7, 1 h; 8, 5 h; 9, 16 h; lo,24 h and 11,24 h.
was detected at the point of application. The neuraminidase treatment of the sera led to a decrease in the height of both of the superimposed rockets. We then studied the possible influence of desialylation on the behavior of the enzyme when performing other immunochemical tests. Thus, the radial immunodiffusion technique was applied to the determination of the purified enzyme (Fig. 5). The calibration curve, where areas of the circular immunoprecipitates, expressed as the squared diameters, were plotted as a function of GGT concentration, was linear
% activity in supernates
100
I
1
2
j
4
antiserumadded (p/mu. GGT
)
Fig. 6. Immunoprecipitation of GGT by antiserum against kidney GGT: purified kidney GGT before and after (o.-.-.o) serum GGT before (0 ......O) (O------Cl) and after desialylation (m-m); desialylation (patient with hepatic metastasis, GGT activity: 344 U/I). Immunoprecipitation was realized as previously described [18]. DesiaIylation of the serum was performed as mentioned in the legend of Fig. 4. For neuraminidase treatment of purified GGT, see text.
28
up to 8 pg of protein (data not shown). Figure 5 clearly demonstrates that desialylation of GGT has no effect on the area of the circular immunoprecipitates. Moreover, as shown in Fig. 6, neuraminidase treatment did not induce any modification in immunoprecipitation curves of serum or purified kidney enzyme. In all cases, 80-90% of GGT activity was precipitated by the anti-GGT antiserum. The consistency of the results obtained by radial immunodiffusion or by immunoprecipitation tests indicates that the phenomenon observed in electroimmunodiffusion studies was not due to a change in GGT antigenic properties, but was rather a consequence of the modification in the charge of the molecule induced by the neuraminidase treatment. Discussion
We have investigated the immunological identity of the light GGTs from liver and kidney by double immunodiffusion. In a previous study [19], we demonstrated the immunological identity of the light and the heavy forms of the liver enzyme. As reported by many authors, specific activity of GGT is higher in human kidney than in liver. For this reason, the yield of purification is better when the enzyme is extracted from the kidney. Therefore, we used the light form of human kidney GGT and the corresponding antiserum for the present study, although serum GGT is generally thought to originate from the liver. Removing sialic acid residues of GGT by neuraminidase treatment led to a decrease in the peak height in electroimmunodiffusion. However, we did not observe any modification induced by the desialylation when measuring the enzyme by radial immunodiffusion or when studying immunoprecipitation of the protein by specific antibodies. Such a phenomenon was previously observed for transferrin by Van Eijk et al [9] and for alpha-l-acid glycoprotein by Bordas et al [8], who assumed that in electroimmunodiffusion the shape of the rocket was dependent on lateral diffusion as well as on electrophoretic mobility. Desialylation of the glycoprotein led to a decrease in mobility and as a consequence diffusion was promoted and the peak height was reduced. Heterogeneity of serum GGT apparently results from three types of phenomena occurring simultaneously: binding of the enzyme to such other serum components as lipoproteins or immunoglobulins [l&21,22], proteolytic modification of the enzyme occurring in vivo or during the storage of the samples [2], and variation in the degree of sialylation of the molecule. Thus, Kiittgen and Gerok observed that, in the case of alcoholic hepatitis, serum GGT lost its concanavalin A affinity because of its increased sialylation [ll]. Fractionating serum GGT of patients with hepatic cancer by polyacrylamide gradient gel slab electrophoresis, Kojima et al demonstrated the existence of 3 hepatoma-related fractions and assumed that there was a possibility that one of them was a sialic acid-rich fetal type [lo]. Indeed GGT from rat fetal liver [14], human hepatoma [15] and human renal carcinoma [13] were found to be more sialylated than the corresponding normal adult liver or kidney enzymes. This variability in sialylation of tissue or serum GGT and our present results show that electroimmunodiffusion _is not directly suitable for determining the
29
enzyme. Perhaps this technique could be applicable after a previous systematic treatment of the sample by neuraminidase, and using a desialylated purified enzyme as standard. In addition, electroimmunodiffusion of serum GGT led to our obtaining two superimposed rockets, even in the presence of a detergent. Explanations for this phenomenon need further investigation. In the same way, the development of radioimmunoassay or enzyme-linked immunosorbent assay of GGT seems to be hindered by some differences in immunoreactivity of the various forms of the enzyme: to some degree, this immunoreactivity might depend on such parameters as molecular mass [6,7]. Acknowledgements
We thank Mrs. M. Chaussard for her excellent technical assistance, Dr. F. Schiele for stimulating discussions and Miss D. Aguillon for her help. References 1 Huseby NE. Separation and characterization of human gamma-glutamyltransferase. Chn Chim Acta 1981; 111: 39-45. 2 Huseby NE. Hydrophylic form of gamma-glutamyltransferase: proteolytic formation in liver homogenates and its estimation in serum. Clin Chim Acta 1982; 124: 113-121. 3 Goldberg DM. Structural, functional, and clinical aspects of gamma-ghttamyltransferase. Chn Crit Rev Clin Lab Sci 1980; 12: l-58. 4 GaIteau MM, Schiele F. Measurement of plasma GGT activities and standardization. In: Siest G, Heusghem C, eds. gamma-Glutamyltransferases: advances in biochemical pharmacology, 3rd ed. Paris: Masson Publ., 1982: 127-132. 5 Scherberich JE, KIeeman B, Mondorf W. Isolation of kidney brush border gamma-glutamyltranspeptidase from urine by specific antibody gel chromatography. Chn Chim Acta 1979; 93: 35-41. 6 Masuike M, Ogawa M, Kitahara T, Murata A, Matsuda K, Kosaki G. Development of radioimmunoassay for gamma-glutamyltransferase using pancreatic enzyme. Ann Clin Biochem 1983; 20: 247-250. 7 Szewczuk A, Kuropatwa M, Lang D. Enzyme-linked immunosorbent assay (ELISA) and calorimetric determination of cow gamma-ghttamyltransferase. Arch Immunol Ther Exp 1983; 31: 121-125. 8 Bordas MC, Biou DR, Feger JM, Durand GM, Joziasse DH, Van Den Eijnden DH. Involvement of sialic acid residues in electroimmunodiffusion of alpha l-acid glycoprotein. A method for determining the degree of sialylation of serum glycoproteins. Chn Chim Acta 1981; 116: 17-24. 9 Van Eijk HG, Van Noort WL, Van der Heul C. Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations? Clin Chim Acta 1982; 126: 193-195. 10 Kojima J, Kanatani M, Nakamura N, Kashiwagi T, Tohjoh F, Akiyama M. Electrophoretic fractionation of serum gamma-ghttamyltranspeptidase in human hepatic cancer. Clin Chim Acta 1980; 106: 165-172. 11 Kottgen E, Gerok W. Isoenzymedifferenzierung der gamma-Glutamyltransferase mit Concanavahn A und Con-A Sepharose. Khn Wochenschr 1976; 54: 439-444. 12 Tsuchida S, Imai F, Sato K. Immunological characterization of gamma-glutamyltransferase in human serum. J Biochem 1981; 89: 775-782. 13 Hada T, Higashino K, Yamamoto H, Okochi T, Sumikawa K, Yamamura Y. Further investigations on a novel gamma-ghttamyltranspeptidase in human renal carcinoma. Clin Chim Acta 1981; 112: 135-140. 14 Kottgen E, Reutter W, Gerok W. Two different gamma-glutamyltransferases during development of liver and small intestine: a fetal (sialo-) and an adult (asialo-) glycoprotein. Biochem Biophys Res Commun 1976; 72: 61-66.
30
15 Y~~oto H, Sumikawa K, Hada T, Higashino K, Yamamura Y. g~a-Glut~yl~~sfera~ from human hepatoma tissue in comparison with normal liver enzyme. Chn Chim Acta 1981; 111: 229-237. 16 Tale SS, Meister H. Identity of mafeate-stimulated ghrtaminase with gamma-ghttamyhranspeptidase in rat kidney. J Biol Chem 1975; 250: 4619-4627. 17 Huseby NE. Purification and some properties of gamma-ghttamyltransferase from human liver. Biocbim Biophys Acta 1977; 483: 46-56. 18 Artur Y, WeBman-Bednawska M, Jacquier A, Siest G. Complexes of serum gamma-ghttamyltr~sfera~ with apo~~proteins and i~~~obu~n A. Clin Chem 1984, 30: 631-633. 19 Artur Y, W~rn~-~nawska M, Siest G. Immunological studies of human liver pmma-glutamyltransferases. In: Siest G, Heusghem C. eds. gamma-Glutamyltransferases: advances in biochemical pharmacology, 3rd ed. Paris: Masson Publ. 1982: 61-67. 20 Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959; 234: 1971-1975. 21 Artur Y, Wellman-Bednawska M, Jacquier A, Siest G. Associations between serum gamma-glutamyhransferase and apolipoproteins: relationships with hepatobiliary diseases. Clin Chem 1984; 30: 1318-1321. 22 Von F&se J, Magerstedt P, Schmidt E. Das elektrophoretische Muster der gamma-Glutamyltransferase in Serum und seine Anderung durch Chylomikronen. J Clin Chem Clin Biochem 1976; 14: 589-594.
Clinica Chimica Acta, 148 (1985) 21-30
Elsevier CCA 03160
Variations in sialic acid content of gamma-glutamyltransferase: a consequence for immunochemical determinations? Maria Wellman-Bednawska
a, Yves Artur a and Gerard
Siest a*b
a Centre du Midicament, UA CNRS No 597, 30 Rue Lionnois, 54000 Nancy and b Centre de Mkdecine Prkventive (Dir. Pr. Senault), 2 Avenue du Doyen Jacques Parisot, 54500 Vandoeuvre- les - Nancy (France) (Received July 17th, 1984; revision January 15th, 1985) Key words: gamma
-Glutamyltransferase,
Human analysis; Sialic aciak analysis; Immunochemistry
analysis
Summary Using specific antibodies against the human kidney enzyme, gamma-glutamyltransferase (GGT, EC 2.3.2.2) was assayed from human kidney and serum by electroimmunodiffusion. Determination of the enzyme by such a method was highly influenced by the sialic acid content of the molecule. The peaks corresponding to the sialylated GGT were higher than those corresponding to the neuraminidase-treated enzyme. In contrast, sialylation of the protein had no influence on the results observed when measuring the enzyme by radial immunodiffusion. Moreover, immunoprecipitation curves of both sialylated and neuraminidase-treated samples were identical. The varying degrees of sialylation of GGT occurring under physiological or pathological conditions are known to be partly responsible for the heterogeneity of the enzyme in organs and biological fluids. Therefore, determination of the enzyme by electroimmunodiffusion may be hazardous.
Introduction gamma-Glutamyltransferase (GGT, EC 2.3.2.2) is a glycoprotein found in many tissues and biological fluids. According to Huseby [l], GGT is present in human organs as an amphiphilic membrane-bound enzyme. Detergents may solubilize the enzyme by binding to the hydrophobic, membrane-binding domain of the enzyme, forming a micellar-like complex, the ‘heavy’ or amphiphilic form of the enzyme. Treatment with proteases such as papain or trypsin removes the hydrophobic domain, thus forming a soluble, hydrophilic ‘light’ form of the enzyme. Both the heavy and the light forms are present in serum [2]. 0009-8981/85/$03.30
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
22
GGT activity is markedly increased in the sera of patients with hepatobiliary disorders [3]. Many chromogenic or fluorogenic substrates have been proposed for determining this activity (see short review in [4]). However, much less work has been done on the immunochemical measurement of the GGT protein. Rocket electroimmunoassays were performed by Scherberich et al for the urine enzyme [5], and by Huseby for a partially purified liver GGT [l], but without quantitative application. A radioimmunoassay for human serum and pancreatic GGT [6] and an enzyme-linked immunosorbent assay (ELISA) for the enzyme from some bovine body fluids and tissue preparations [7] have recently been proposed. The present paper displays the results we obtained when determining the enzyme by electroimmunodiffusion on cellulose acetate plates. We placed special emphasis on the study of the involvement of sialic acid residues in electroimmunodiffusion of GGT: indeed GGT is a sialoglycoprotein, and the degree of sialylation has been previously shown to have an effect upon the height of the rockets in the case of alpha-l-acid glycoprotein [8] and transferrin [9]; moreover, the varying degrees of sialylation occurring under physiological or pathological conditions seem to contribute to the heterogeneity of the enzyme in serum [lo-121, kidney [13] and liver [14,15]. Materials and methods Purification of human GGTs The light form of human kidney GGT was purified from cortical slides, using a procedure similar to that used by Tate and Meister [16]. The light form of human liver GGT was prepared by the technique proposed by Huseby [17]. Specific activities of the liver and kidney GGT preparations were 110 and 210 U/mg of protein, respectively. The purified kidney GGT contained 20 nmol sialic acid/mg protein. Liver and kidney were obtained at autopsy within 48 h of death. Preparation of the antiserum The antiserum against light GGT was raised in rabbits (Fauves de Bourgogne, France). The animals were inoculated subcutaneously with 100 pg of kidney GGT preparation mixed with 0.5 ml of Freund’s complete adjuvant. Similar booster injections, but using Freund’s incomplete adjuvant, were given at 2-wk intervals. Each rabbit was bled from the ear vein 8 days after the third booster challenge. Human serum samples Sera were obtained from 7 patients with various liver diseases: four with alcoholic cirrhosis (GGT activities: 592,692,989 and 1,212 U/l) and one each with congestive heart failure (1,030 U/l), liver metastasis (344 U/l) and nutritional steatosis (776 U/l). Neuraminidase treatment The purified kidney GGT was incubated with 2 U neuraminidase/mg protein in a 100 mmol/l acetate buffer, pH 5.5, containing 10 mmol/l CaCl,, at 37°C for
23
0.5-48 h. Neuraminidase (100 U/117 mg protein) was from Sigma Chemical Co. (Saint Louis, MO, USA). For serum, desialylation was carried out in the same way, but using 5 U neuraminidase/rnl serum. Incubation with neuraminidase had no effect upon the enzyme activity in these conditions. Radial immunodifftkon and double immunodiffusion tests In radial immunodiffusion and double irnmunodiffusion tests, we used Indubiose (Industrie Biologique Francaise, Clichy, France) at a concentration of 10 g/l in a 50 mmol/l barbital-sodium barbital buffer, pH 8.6. The concentration of antiserum in the gels of radial immunodiffusion was 20 ml of antiserum/l of gel, and 8 ~1 portions of the various samples were applied. In both cases, diffusion was allowed to take place for 48 h. Unprecipitated proteins were removed by washing the gels in 9 g/l NaCl and water. As in electroimmunodiffusion tests, the precipitates were observed after enzyme specific staining (see below). Electroimmunodiffusion tests We used Titan III Zip Zone cellulose acetate plates (Helena Labs., Beaumont, TX, USA). The plates were wetted in a 25 nunol/l barbital-sodium barbital buffer, pH 8.6, containing 40 ml of antiserum/l of buffer. We applied 0.6 ~1 of the various enzyme preparations, or 1.2 ~1 of serum. The strips were then electrophoresed for 45 min with a constant 180 V. Unprecipitated proteins were removed in a 60-min buffer wash, using a magnetic stirrer. Enzyme specific staining of the peaks was realized using the substrate gamma-L-glutamyl-alpha-naphthylamide (Sigma Chemical Co, Saint Louis, MO, USA). Glycylglycine and Tris were from Merck (Darmstadt, FRG). The plates were incubated at 37°C for 150 min with the substrate solution (per liter, 5 nun01 of gamma-rglutamyl-alpha-naphthylamide, and 100 mm01 each of Tris and glycylglycine, pH 8.25). After quickly washing the plates with water, we detected the released alpha-naphthylamine by applying a freshly prepared 1 g/l aqueous solution of Fast Garnet GBC (Serva, Heidelberg, FRG), and fixed the resulting red peaks with a 100 ml/l aqueous solution of acetic acid. Sodium deoxycholate was from Sigma Chemical Co. Immunoprecipitation tests Immunoprecipitation tests were carried out as previously described [18], incubating increasing volumes of antiserum with constant samples of GGT preparations or serum. Other analytical methodr We measured GGT activities at 30°C using a Cary 219 spectrophotometer (Varian Associates, Palo Alto, CA, USA) as previously described [19]. The substrate gamma-L-glutamyl-3-carboxy-4nitroanilide was from Boehringer (Mannheim, FRG). Sialic acid was determined by Warren’s method [20], using N-acetyl neuraminic acid (Sigma Chemical Co.) as standard.
24
Results
By double immunodiffusion in agarose gels, an apparent identity reaction was seen between the kidney and the liver GGT: indeed, the single precipitation lines fuse smoothly with one another (Fig. 1). As previously described [19], immunoprecipitates retained enzyme activity, allowing enzyme-specific staining of the arcs. A simple and rapid method for determining the enzyme by el~troi~unodiffusion was developed (see ‘Materials and Methods’). Using the purified light form of kidney GGT as standard, we observed a linear relationship between the peak height and the logarithm of the protein concentration (Fig. 2). The corresponding rockets are shown in Fig. 3A. Amounts of GGT as low as 20 ng were detected and the calibration curve was linear up to 0.6 pg at least. Applying identical amounts of native and desialylated GGT, we observed that the neuraminidase treatment of the enzyme led to an important decrease in the peak height. Figure 3B shows the rockets corresponding to the native sialylated GGT and the desialylated enzyme at various times of neuraminidase treatment. After 60 min of incubation with neuraminidase, 20 nmol of sialic acid were liberated per mg of protein. A longer incubation did not change sialic acid liberated or the height of the peaks. The maximum difference in peak height we found here was about 208, and
Fig. 1. Double immunodiffusion: reactivity of kidney and liver GGT towards rabbit antiserum produced against purified human kidney GGT. The center well [7] contains antiserum. Peripheral wells contain: 1, 3, 5: liver GGT, 2, 4, 6: kidney GGT. The precipitate was stained for GGT activity (see ‘Materials and Methods’).
25 .paak
haight
(cm)
.
‘L 20
50
loo
500
kiirmy
GGT
Fig. 2. Electroimmunodiffusion of various amounts of native sialylated GGT from human kidney. The logarithm of the amount of GGT applied on the strip is plotted against the peak height. The corresponding rockets are shown in Fig. 3A. The amounts of GGT applied were: 18.7, 37.5, 75, 150, 300 and 600 ng.
Fig. 3. Electroimmunodiffusion of kidney GGT. Conditions for electrophoresis are described in ‘Materials and Methods’. The rockets are stained for GGT activity as shown in the text. A. Decreasing amounts of native sialylated GGT: 1, 600 ng; 2, 300 ng; 3, 150 ng; 4, 75 ng; 5, 37.5 ng; 6, 18.7 ng. B. Native sialylated GGT: 7, 300 ng; 8, 75 ng; 9, 10, 11, 12, neuraminidase&eated samples (300 ng of GGT for each) after incubation with neuraminidase for 0.5,3,22 and 48 h, respectively. The specific activity of the undiluted GGT preparation used was 210 U/mg of protein. Its protein concentration was 1 mg/ml. Bar shows point of application.
26
the height of the lowest rocket obtained with 300 ng of desialylated enzyme corresponded approximately to the height of the rocket observed with 75 ng of native sialylated GGT. The presence of detergent (sodium deoxycholate at the concentration of 5 g/l both in the sample and in the migrating buffer) did not influence the peak height. We then submitted serum samples to electroimmunodiffusion. In the absence of detergent, we obtained unsatisfactory pictures: for each serum, we observed two superimposed rockets, but these rockets formed trails, and a fraction of the enzyme did not migrate. This phenomenon may be partly attributed to the binding of the enzyme to lipids. Indeed, we previously observed the binding of GGT to lipoproteins in serum of patients with hepatobiliary diseases [18,21]. The fraction which did not migrate might be a GGT fraction associated with chylomicrons as assumed by Von Freise et al [22]. Nevertheless, the trails corresponding to the untreated sera were higher than those corresponding to the neuraminidase-treated samples (data not shown). Figure 4 represents an example of the pictures we observed when sodium deoxycholate was added to the samples and to the migrating buffer. For each serum, two superimposed rockets with GGT activity were obtained, but no GGT activity
Fig. 4. Electroimmunodiffusion of two sera before (1, 2) and after (lN, 2N) neuraminidase treatment. Desialylation was carried out as described in the text, incubating 5 U neuraminidase/mf serum for 16 h at 37’C. Electrophoresis was performed as described in the text, with sodium deoxycholate present in the samples and in the migrating buffer (5 g/l). The peaks were stained for GGT activity (see ‘Materials and Methods’). Both sera were- from patients with alcoholic cirrhosis. GGT activities: 1,989 U/l; 2,1212 U/l. Bar shows point of application.
21
Fig. 5. Radial immunodiffusion of native sialylated and neuraminidase-treated human kidney GGT. Conditions for neuraminidase treatment, diffusion and specific staining of the precipitates are described in ‘Materials and Methods’. Decreasing amounts of native sialylated GGT: 1, 8 pg; 2, 6 pg; 3, 5 gg, 4, 4 gg and 5, 2 pg. Five pg of neuraminidase-treated GGT, incubated at 37°C for: 6, 0.5 h; 7, 1 h; 8, 5 h; 9, 16 h; lo,24 h and 11,24 h.
was detected at the point of application. The neuraminidase treatment of the sera led to a decrease in the height of both of the superimposed rockets. We then studied the possible influence of desialylation on the behavior of the enzyme when performing other immunochemical tests. Thus, the radial immunodiffusion technique was applied to the determination of the purified enzyme (Fig. 5). The calibration curve, where areas of the circular immunoprecipitates, expressed as the squared diameters, were plotted as a function of GGT concentration, was linear
% activity in supernates
100
I
1
2
j
4
antiserumadded (p/mu. GGT
)
Fig. 6. Immunoprecipitation of GGT by antiserum against kidney GGT: purified kidney GGT before and after (o.-.-.o) serum GGT before (0 ......O) (O------Cl) and after desialylation (m-m); desialylation (patient with hepatic metastasis, GGT activity: 344 U/I). Immunoprecipitation was realized as previously described [18]. DesiaIylation of the serum was performed as mentioned in the legend of Fig. 4. For neuraminidase treatment of purified GGT, see text.
28
up to 8 pg of protein (data not shown). Figure 5 clearly demonstrates that desialylation of GGT has no effect on the area of the circular immunoprecipitates. Moreover, as shown in Fig. 6, neuraminidase treatment did not induce any modification in immunoprecipitation curves of serum or purified kidney enzyme. In all cases, 80-90% of GGT activity was precipitated by the anti-GGT antiserum. The consistency of the results obtained by radial immunodiffusion or by immunoprecipitation tests indicates that the phenomenon observed in electroimmunodiffusion studies was not due to a change in GGT antigenic properties, but was rather a consequence of the modification in the charge of the molecule induced by the neuraminidase treatment. Discussion
We have investigated the immunological identity of the light GGTs from liver and kidney by double immunodiffusion. In a previous study [19], we demonstrated the immunological identity of the light and the heavy forms of the liver enzyme. As reported by many authors, specific activity of GGT is higher in human kidney than in liver. For this reason, the yield of purification is better when the enzyme is extracted from the kidney. Therefore, we used the light form of human kidney GGT and the corresponding antiserum for the present study, although serum GGT is generally thought to originate from the liver. Removing sialic acid residues of GGT by neuraminidase treatment led to a decrease in the peak height in electroimmunodiffusion. However, we did not observe any modification induced by the desialylation when measuring the enzyme by radial immunodiffusion or when studying immunoprecipitation of the protein by specific antibodies. Such a phenomenon was previously observed for transferrin by Van Eijk et al [9] and for alpha-l-acid glycoprotein by Bordas et al [8], who assumed that in electroimmunodiffusion the shape of the rocket was dependent on lateral diffusion as well as on electrophoretic mobility. Desialylation of the glycoprotein led to a decrease in mobility and as a consequence diffusion was promoted and the peak height was reduced. Heterogeneity of serum GGT apparently results from three types of phenomena occurring simultaneously: binding of the enzyme to such other serum components as lipoproteins or immunoglobulins [l&21,22], proteolytic modification of the enzyme occurring in vivo or during the storage of the samples [2], and variation in the degree of sialylation of the molecule. Thus, Kiittgen and Gerok observed that, in the case of alcoholic hepatitis, serum GGT lost its concanavalin A affinity because of its increased sialylation [ll]. Fractionating serum GGT of patients with hepatic cancer by polyacrylamide gradient gel slab electrophoresis, Kojima et al demonstrated the existence of 3 hepatoma-related fractions and assumed that there was a possibility that one of them was a sialic acid-rich fetal type [lo]. Indeed GGT from rat fetal liver [14], human hepatoma [15] and human renal carcinoma [13] were found to be more sialylated than the corresponding normal adult liver or kidney enzymes. This variability in sialylation of tissue or serum GGT and our present results show that electroimmunodiffusion _is not directly suitable for determining the
29
enzyme. Perhaps this technique could be applicable after a previous systematic treatment of the sample by neuraminidase, and using a desialylated purified enzyme as standard. In addition, electroimmunodiffusion of serum GGT led to our obtaining two superimposed rockets, even in the presence of a detergent. Explanations for this phenomenon need further investigation. In the same way, the development of radioimmunoassay or enzyme-linked immunosorbent assay of GGT seems to be hindered by some differences in immunoreactivity of the various forms of the enzyme: to some degree, this immunoreactivity might depend on such parameters as molecular mass [6,7]. Acknowledgements
We thank Mrs. M. Chaussard for her excellent technical assistance, Dr. F. Schiele for stimulating discussions and Miss D. Aguillon for her help. References 1 Huseby NE. Separation and characterization of human gamma-glutamyltransferase. Chn Chim Acta 1981; 111: 39-45. 2 Huseby NE. Hydrophylic form of gamma-glutamyltransferase: proteolytic formation in liver homogenates and its estimation in serum. Clin Chim Acta 1982; 124: 113-121. 3 Goldberg DM. Structural, functional, and clinical aspects of gamma-ghttamyltransferase. Chn Crit Rev Clin Lab Sci 1980; 12: l-58. 4 GaIteau MM, Schiele F. Measurement of plasma GGT activities and standardization. In: Siest G, Heusghem C, eds. gamma-Glutamyltransferases: advances in biochemical pharmacology, 3rd ed. Paris: Masson Publ., 1982: 127-132. 5 Scherberich JE, KIeeman B, Mondorf W. Isolation of kidney brush border gamma-glutamyltranspeptidase from urine by specific antibody gel chromatography. Chn Chim Acta 1979; 93: 35-41. 6 Masuike M, Ogawa M, Kitahara T, Murata A, Matsuda K, Kosaki G. Development of radioimmunoassay for gamma-glutamyltransferase using pancreatic enzyme. Ann Clin Biochem 1983; 20: 247-250. 7 Szewczuk A, Kuropatwa M, Lang D. Enzyme-linked immunosorbent assay (ELISA) and calorimetric determination of cow gamma-ghttamyltransferase. Arch Immunol Ther Exp 1983; 31: 121-125. 8 Bordas MC, Biou DR, Feger JM, Durand GM, Joziasse DH, Van Den Eijnden DH. Involvement of sialic acid residues in electroimmunodiffusion of alpha l-acid glycoprotein. A method for determining the degree of sialylation of serum glycoproteins. Chn Chim Acta 1981; 116: 17-24. 9 Van Eijk HG, Van Noort WL, Van der Heul C. Microheterogeneity of human serum transferrins: a consequence for immunochemical determinations? Clin Chim Acta 1982; 126: 193-195. 10 Kojima J, Kanatani M, Nakamura N, Kashiwagi T, Tohjoh F, Akiyama M. Electrophoretic fractionation of serum gamma-ghttamyltranspeptidase in human hepatic cancer. Clin Chim Acta 1980; 106: 165-172. 11 Kottgen E, Gerok W. Isoenzymedifferenzierung der gamma-Glutamyltransferase mit Concanavahn A und Con-A Sepharose. Khn Wochenschr 1976; 54: 439-444. 12 Tsuchida S, Imai F, Sato K. Immunological characterization of gamma-glutamyltransferase in human serum. J Biochem 1981; 89: 775-782. 13 Hada T, Higashino K, Yamamoto H, Okochi T, Sumikawa K, Yamamura Y. Further investigations on a novel gamma-ghttamyltranspeptidase in human renal carcinoma. Clin Chim Acta 1981; 112: 135-140. 14 Kottgen E, Reutter W, Gerok W. Two different gamma-glutamyltransferases during development of liver and small intestine: a fetal (sialo-) and an adult (asialo-) glycoprotein. Biochem Biophys Res Commun 1976; 72: 61-66.
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15 Y~~oto H, Sumikawa K, Hada T, Higashino K, Yamamura Y. g~a-Glut~yl~~sfera~ from human hepatoma tissue in comparison with normal liver enzyme. Chn Chim Acta 1981; 111: 229-237. 16 Tale SS, Meister H. Identity of mafeate-stimulated ghrtaminase with gamma-ghttamyhranspeptidase in rat kidney. J Biol Chem 1975; 250: 4619-4627. 17 Huseby NE. Purification and some properties of gamma-ghttamyltransferase from human liver. Biocbim Biophys Acta 1977; 483: 46-56. 18 Artur Y, WeBman-Bednawska M, Jacquier A, Siest G. Complexes of serum gamma-ghttamyltr~sfera~ with apo~~proteins and i~~~obu~n A. Clin Chem 1984, 30: 631-633. 19 Artur Y, W~rn~-~nawska M, Siest G. Immunological studies of human liver pmma-glutamyltransferases. In: Siest G, Heusghem C. eds. gamma-Glutamyltransferases: advances in biochemical pharmacology, 3rd ed. Paris: Masson Publ. 1982: 61-67. 20 Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959; 234: 1971-1975. 21 Artur Y, Wellman-Bednawska M, Jacquier A, Siest G. Associations between serum gamma-glutamyhransferase and apolipoproteins: relationships with hepatobiliary diseases. Clin Chem 1984; 30: 1318-1321. 22 Von F&se J, Magerstedt P, Schmidt E. Das elektrophoretische Muster der gamma-Glutamyltransferase in Serum und seine Anderung durch Chylomikronen. J Clin Chem Clin Biochem 1976; 14: 589-594.