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Human amniotic fluid α-glucosidase
53
Clinica Chimicu Actu, I I7 (1981) 53-62 Elsevier/North-Holland Biomedical Press
CCA
1947
Human amniotic fluid ar-glucosidase Livia
Poenaru,
Marie-Claude
Institut de Pathologic Molkdaire
Vinet
and Jean-Claude
Dreyfus
**
*, CHU Cochin, 24 rue du Fauhourg St. Jacques, 75674 Paris Ckdex I4 (France) (Received
April 20th. 1981)
Amniotic fluid in midpregnancy contains significant a-glucosidase activity. This enzyme is distinguishable from the lysosomal acid a-glucosidase, deficiency of which is associated with Pompe’s disease. The two enzymes differ in optimum pH, thermal stability, electrophoretic migration, isoelectric point, molecular mass, and immunological response. Amniotic a-glucosidase is also different from the classical neutral form. Immuno-cross reactions suggest that the amniotic fluid enzyme has a double fetal origin: renal and intestinal. It seems that a-glucosidase in amniotic fluid is linked to lipids.
Introduction Two forms of cu-1,4glucosidase (EC 3.2.1.20) are generally described in human tissues [l-3]: one with maximum activity at acidic pH and a second with a maximum at neutral pH. A special form, immunologically independent, has been demonstrated in kidney [4,5] and in leucocytes [5]. Type II glycogenosis (Pompe’s disease) is a fatal autosomal recessive disorder characterized by the accumulation of glycogen in the heart, skeletal muscle, brain, liver and kidney [6]. Deficiency of a-1,Cglucosidase in liver [7], heart [8], skeletal muscle [9], brain [9], pancreas [9], cultivated skin fibroblasts [lO,l l] and leucocytes [ 12,131 of patients with this disorder has been reported. Pompe’s disease has been detected in utero on the basis of a deficiency of a- 1,Cglucosidase in cultured amniotic cells [ 14- 161.
* Universite Paris, Unite 129 de 1’Institut National de la Sante et de la Recherche Medicale. Laboratoire Associe 85 du Centre National de la Recherche Scientifique. ** Correspondence should be addressed to J.-C. Dreyfus. Institut de Pathologie Moleculaire, 24 rue du Faubourg St. Jacques, 75014 Paris, France.
0009-898
I /81 /OOOO-0000/$02.75
$1 I98
I Elsevier/North-Holland
Biomedical
Press
54
A different cr-glucosidase activity has been reported in amniotic fluid in midpregnancy [ 14- 18). This enzyme is not implicated in Pompe’s disease [ 15,17 and our unpublished observations] and it disappears between the 20th and 26th weeks of pregnancy [ 171. The purpose of this study was to characterize the a-glucosidase in amniotic fluid and compare it with that in urine, placenta, kidney, intestine, fibroblasts and cultivated amniotic cells in an attempt to determine the origin of the enzyme in amniotic fluid. Materials and methods Amniotic fluid was obtained by transabdominal amniocentesis during the 17th week of pregnancy from women whose pregnancies were being monitored for a variety of fetal disorders. The amniotic fluid was centrifuged at 800 X g and the supematant was assayed immediately, or stored at -20°C. Fibroblasts and cultured amniotic cells were a gift from Dr. J. Boue (Chateau de Longchamp, Paris, France). Serum, urine and leucocytes prepared by sedimentation in dextran followed by lysis of red cells by 0.17 mol/l ammonium chloride were used immediately or stored at -20°C. Placenta, fetal liver, kidney and intestine were obtained after therapeutic abortion and stored at -80°C. Tissues were extracted by 0.1% Triton X-100 with a Potter-Elvehjem apparatus. Enzyme determination a-Glucosidase activity was determined at several pHs using as substrate the fluorogenic compound 4-methylumbelliferyl-cy-glucoside (Koch-Light) as described previously [ 191. The sucrase and maltase activity was also determined by calorimetric methods [20] using sucrose and maltose as substrates. Thermal stability Amniotic fluid thermal stability at 40°C in phosphate buffer, pH 6.9, was compared with thermostability of the fraction not retained by Concanavalin A Sepharose, containing the neutral a-glucosidase activity from liver, placenta, kidney and intestine. Electrophoresis and electrofocusing Electrophoresis was carried out on cellulose acetate (Cellogel-Chemetron) [21], and electrofocusing was performed on horizontal ampholine polyacrylamide gel plates (LKB), pH range 3.5-9.5. Enzymes were stained with the same fluorogenic substrate. Molecular mass The molecular
mass was determined
in 5520% (w/v)
sucrose linear
gradients
in
55
water or in 0.1% Triton X- 100. Then, 0.5 ml of ten times concentrated amniotic fluid were centrifuged in each gradient for 3 h at 236500 X g (using the vertical rotor TY-850 of a DuPont ultracentrifuge). 32 fractions of 1.2 ml each were collected and assayed for a-glucosidase activity at pH 4.0, 5.5 and 6.5. Sephadex G-100 equilibrated in 25 mmol/l NaCl, EDTA 1 mmol/l and Concanavalin A Sepharose 4-B equilibrated in 10 mmol/l phosphate buffer, pH 6.8, containing 0.5 mol/l NaCl, 0.1 mol/l MgCl,, 0.1 mol/l CaCl, were used to test a-glucosidase affinity for these gels in batch and in microcolumns (1 ml) at 4’C.
Immunochemical studies Three antisera raised in rabbits against human enzymes were used: (a) an anti-acid maltase, prepared against acid a-glucosidase from placenta; purified by two steps: 1. Concanavalin A Sepharose 4-B (Pharmacia), chromatography involving a selective elution with 0.5 mol/l methyl-a-D-mannoside (Koch-Light). 2. Sephadex G-100 specific chromatography eluted by 0.25% maltose. This antiserum was used in the form of purified globulins; (b) an antiserum prepared against pure human renal maltase [4] kept as whole serum, was kindly donated by Drs. de Burlet and Sudaka (Faculte de Medecine, Nice, France); (c) an antiserum anti-amniotic fluid a-glucosidase was obtained by injection into rabbits of the sucrose gradient fraction containing a-glucosidase. We used the immunoprecipitation technique; extracts were incubated with antisera or normal serum overnight. Complete precipitation of the antigen-antibody complex was ensured by addition of polyethylenglycol (PEG) to a final concentration of 5%. After 15 min centrifugation at 10000 X g the supernatant was used to test the non-precipitated enzyme. Detection and identification of total fatty acids from the top fraction of sucrose gradient, containing a-glucosidase activity, was performed by gas liquid chromatography according to the method of Klenk and Kahlke [22]. Results 40 amniotic fluids between 17 and 19 weeks of gestation were tested (Fig. 1). The average of a-glucosidase activity at pH 5.5, expressed in mmol .l~ ’ . h- I, was 360 with SD 167.
pH activity curves The profile of amniotic
fluid a-glucosidase activity as a function of pH is represented in Fig. 2 and compared with other tissues. The maximum of amniotic fluid activity is situated at pH 5.5 as for intestinal enzyme. Two peaks of activity, one at pH 4.0-4.5 and a second at pH 6.0-6.5, could be observed for fibroblasts, kidney, liver and placenta.
Thermal stability Activity
curves
after
incubation
at 40°C
indicate
that only
the amniotic
fluid
56
mmo,. I-‘.h-’
Fig. 1. Amniotic
fluid cr-glucosidase
activity
tested at pH 5.5 from
17 and 19 weeks of gestation.
too. 90.
80 _
70 _ 2 :z
60.
2 Lc 50. 0 s
40.
30. I
20.
/ /
10. /
L 0
/
,
3.5
4
4.5
5
5.5
6
6.5
7
75 PH
Fig. 2. pH v -V,
activity kidney;
curves 0 -
of a-glucosidase from: A -A, fibroblasts; 0, amniotic fluid; W n , placenta; 0 -
l0, 0, liver.
intestine:
57
enzyme is completely inactivated at this temperature. This apparent thermal lability, as already observed at 55°C 1171 is probably due to a low concentration of proteins since the cw-glucosidase from amniotic fluid concentrated 20 times is inactivated only 50% in 50 min at 40°C.
Eiectrophoresis Fig. 3 shows the electrophoretic pattern of a-glucasidase from different tissues stained at pH 5.5-5.6. The most important part of amniotic fluid enzyme (line 6) migrates in a position intermediate between the fast band (neutral cr-glucosidase) and the slower one (acid ~-~ucosidase). The neutral activity of some tissues (liver, intestine) is not visible on electrophoresis (line 1 and 3) since the proportion of this form is not high enough. Isoelectric focusing
Isoelectric foeusing of Lu-glucosidase revealed an isoelectric point (~1) between 3.5-3.X for amniotic fluid, 3.8-4 for specific renal forms, 4.2-4.6 for intestine and acidic forms from other organs, and 5-5S for neutral forms.
+
1
2
3
4
5
6
7
Fig. 3. Cellulose acetate electrophoresis of or-glucosidase; pH of rue: 6.5, pH of vizuaiization: 5.5 (both
acid and neutralforms are visualized at this pH). 1, liver; 2, fibroblasts; 3, intestine; 4, kidney; 5, urine concentrated 20 times; 6, amniotic fluid concentrated IO times; 7, placenta.
5x
cY-Glucosidase affinity for Sephadex G 100 and Con A Sepharose Amniotic fluid a-glucosidase was not retarded by Sephadex G 100. The activity was eluted with the bulk of the proteins. By contrast, most of the placental acid a-glucosidase was strongly retarded on dextran gel and eluted after the peak of the proteins. Absorbtion on Concanavalin A Sepharose was very small: 10 to 20% of activity was retained either in batch or in column whereas all the acid a-glucosidase was retained. Molecular mass Since some a-glucosidase isozymes are specifically retained by different gels (Sepharose G 100, Sephacryl S 200, unpublished results), we adopted centrifugation in sucrose linear gradient to determine the molecular mass of a-glucosidase. The results from the experiments made on a sucrose gradient in water (Fig. 4) show an acid a-glucosidase activity peak from liver, kidney and intestine in the 25th fraction; the neutral intestinal and renal activity peak is in the 17th fraction. For amniotic fluid all the activity remained on the top and was found in the 31st and 32nd fractions (Fig. 4, peak A).
A
h
AMNIOTIC
Fig. 4. Centrifugation on a 5 to 20% sucrose linear gradient. The pattern of liver, kidney and intestine is represented as the a-glucosidase activity at pH 6.5 by empty symbols and at pH 4 by full symbols. The pattern was identical in Triton and in water. Amniotic fluid cr-glucosidase activity was tested at pH 5.5. The empty symbols represent separation in Triton and the full symbols in water.
pl
of antiserum
Fig. 5. Immunoprecipitation of amniotic fluid cr-glucosidase glucosidase; I 8, anti-renal tu-glucosidase; A activity was tested at $3 5.5 and 6.5 with identical results.
using antisera: A, anti-amniotic
l0, anti-acid afluid a-glucosidase. The
fiperiments performed on a sucrose gradient in Triton show an unchanged pattern for all tissues except for amniotic fluid enzyme, the position of which was changed and the maximal activity was found in the 17th fraction (Fig. 4, peak B). After centrifugation on a sucrose gradient in water the amniotic fluid fraction containing cu-glucosidase activity was analysed by gas liquid c~omato~aphy for the presence of lipids. A small peak (less than 5 mg/l of fatty acids) was observed in the C ,8 zone.
The action of three antisera: anti-acid maltase, anti-renal maltase and antiamniotic maltase, on amniotic fluid enzyme tested by immunoprecipitation is represented in Fig. 5. The activity is not modified by anti-acid maltase antiserum, is partially precipitated (75%) by anti-renal maltase antiserum and completely precipitated by anti-amniotic fluid antiserum.
pl Fig. 6. The action intestine; +
of anti-amniotic +, amniotic
Of antiserum
fluid n-glucosidase fluid. a-Glucosidase
antiserum: AA, kidney; n -m, activity was tested at pH 5.5 and 6.5.
60
. 20 pl
of antiserum
intestine; Fig. 7. The action of anti-renal n-glucosidase antiserum: 0 ---0, fluid; AA, kidney. a-Glucosidase activity was tested at pH 5.5 and 6.5.
W-
W, amniotic
Anti-amniotic fluid antiserum incubated with kidney extract, intestinal extract and amniotic fluid (Fig. 6) precipitates the a-glucosidase from all these origins but in different proportions; 50% 90% and 100% respectively. No effect of this antiserum was observed on placenta, liver, leukocytes, urine and fibroblasts. Anti-renal maltase antiserum acts in the same way on intestine, amniotic fluid and kidney enzyme (Fig. 7) precipitating respectively 65%, 75% and 80% of activity tested at pH 6.5. This antiserum also precipitates part of leukocyte a-glucosidase activity [5] but no action was observed on placenta, liver, urine and fibroblasts. It is known that in intestinal extracts and in amniotic fluid there exist several neutral disaccharidases acting on various a-glucosides. We, therefore, assayed our antisera on two disaccharidase activities, i.e. maltase and sucrase. Anti-renal maltase antiserum inhibited only maltase activity. By contrast, anti-amniotic fluid maltase antiserum precipitated completely both maltase and sucrase. Discussion The presence of a-glucosidase activity in amniotic fluid from the second trimester of pregnancy has been reported several times but the source of this activity has not been identified. The properties described in this paper provide evidence for a distinction between amniotic fluid a-glucosidase and acid and neutral classical forms, including the neutral cu-glucosidase C recently described by R. Hirschhorn and colleagues [23,24]. Unlike the acid form, amniotic a-glucosidase is not affected in Pompe’s disease, it does not react with anti acid qglucosidase antiserum [ 181, its pH optimum is not 4 but 5.5 and it has no affinity either for Concanavalin A or for Sephadex G-100. On electrophoresis and isoelectric focusing amniotic enzyme migrates in a different position from acid and neutral maltase. On centrifugation on a sucrose gradient the molecular mass is 142000 for amniotic fluid, 90000-100000 for acid forms and 177 000 for the neutral form from various tissues.
61
The total precipitation of amniotic fluid a-glucosidase by anti-amniotic fluid antiserum and the total absence of reaction of this antiserum with acid and neutral enzyme from different tissues constitute additional arguments for the dissimilarity between these enzymes. Immunological results obtained by incubation of anti-renal maltase anti-serum and anti-amniotic fluid antiserum with kidney extract, intestinal extract and amniotic fluid, assayed for a-glucosidase at pH 5.5, show by cross reaction certain immunological similarities between the three enzymes. In this case, it is difficult to understand the absence of recognition of the renal form of cr-glucosidase present in leukocytes [5] by anti-amniotic fluid antiserum. The centrifugation on a sucrose gradient in water reveals that a-glucosidase from amniotic fluid remains in the top fraction. Our hypothesis to explain this behaviour is that the enzyme in amniotic fluid is linked to the lipids, the low density and insolubility in water of which prevent the enzyme from entering the gradient. This hypothesis was confirmed by the experiments made in Triton which, probably by destroying the association protein-lipids, allowed a-glucosidase to migrate as far as the 17th fraction. The presence of fatty acids demonstrated by gas liquid chromatography also supports this hypothesis. In conclusion, it seems that amniotic fluid a-glucosidase has a double fetal origin, renal and intestinal. The complete absence of identity of the amniotic fluid enzyme with acid maltase constitutes a formal counter-indication for the utilisation of amniotic fluid in prenatal diagnosis of Pompe’s disease. The amniotic fluid fraction coming from kidney is immunologically not identical with the renal type existing in leukocytes. At least part of the fraction coming from intestine consists of sucrase [25,26] which is precipitated by anti-amniotic fluid antiserum. The comparison of properties of a-glucosidase from kidney, intestine and amniotic fluid reveals similarities but also differences. It is very likely that amniotic fluid a-glucosidase is derived from the intestinal, and perhaps partly from the renal enzyme. But it is also apparent that within the amniotic fluid, the enzyme undergoes modifications which cause changes in some of its properties. The mechanism by which a-glucosidase activity disappears in late pregnancy remains to be explained; intestinal and renal specific a-glucosidase exist in these tissues at birth and in adult tissues. Acknowledgements We thank Dr. Papa for the gift of amniotic fluids and Dr. M.H. Laudat detection of fatty acids. This work was supported by Grant INSERM CRL 79.5.093.1.
for the
62
References I Hers HG. Van Hoof F. Lysosomal u-glucosidase. In: Dickens F. Randle PJ. Whelan WJ, eds. Carbohydrate metabolism and its disorders. Academic Press, 15 I - 160, New York. 2 Koster JF. Slee RG, Hulsmann WC. NiermetJer MF. Electrophoretic pattern and activities of acidic and neutral maltase of cultivated fibroblasts and amniotic fluid cells from controls and patients with the variant of glycogen storage disease type II. Clin Chim Acta 1972; 40: 294-297. 3 Shin-Buering YS. Drefers M, Kronner H. Osang M. Schaub J. Separation of acid and neutral a-glucoaidase isoenzymes from fetal and adult tissues. cultivated fibroblasts and amniotic fluid cells by DEAE-cellulose and Sephadex G- 100 column chromatography. Clin Chim Acta 197X: X9: 393-404. 4 De Burlet G. Sudaka P. Purification dc la maltaac ncutre r&tale humaine. Biochimic 1976: 5X: 62 I-623. 5 Dreyfus JC, Poenaru L. Alpha-glucosidases in white blood cells. with rcfcrence to the detection of acid-a- I .4-glucosidase deficiency Biochem Biophya Rcs Commun 197X; X5: 6 155622. 6 Hug G. Schubert WK. Glycogenosis type II glycogcn distribution in tissues. Arch Pathol 1967: X4: 141-152. 7 Hers HG. a-Glucosidase deficiency in generalized glycogen storage disease (Pompc’s disease). Biothem J 1963: X6: 1I-16. X Smith HL. Amick LD. Sidbury JB. Type II glycogenoais. Am J Dis Child 1966: I 1I : 4755479. 9 Steinitz K. Rutenberg A. Tissue a-glucosidase activity and glycogen content in patients with generalized glycogencais. Isr J Med Sci 1967; 3: 41 I-415. IO Nitowsky HM, Grunfeld A. Lysosomal glucosidase in type II glycogcneais: activity in leukocytes and cell cultured in relation to genotype. J Lab Clin Med 1967: 69: 472-482. I I Dan& J, Hultzer, J. Lynficld J. Cox RP. Absence of acid maltaae in glycogenesia type 2 (Pompc’s disease) in tissue culture. Am J Dis Child 1969; I 17: IOX- 112. I2 Williams HE. a-Glucosidase activity in human lcucocytcs. Biochim Biophya Acta 1966: 124: 34-Xx. I3 Dreyfus JC, Poenaru L. White blood cells and the diagnosis of n-glucoaidaae deficiency. Pediatr Res 19X0; 14: 342-344. 14 Nadler HL, Messina AM. In utero detection of type II glycogencsis (Pompc’s discasc). Lancet 1969: 2: 1277. 15 Cox RP, Duglas G. Hutzler J. Lynfield J. Dan& J. In utero detection of Pompe’s disease. Lancet lY70: I: x93. 16 Nadler HL. Bigley RH, Hug G. Prenatal detection of Pompc’s disease. Lancct 1970; 2: 369. 17 Fluharty AL, Scott ML, Porter MT, Kihara H, Wilson MC, Towner JW. Acid n-glucoaidaac in amniotic fluid. Biochem Mcd 1973: 7: 39-51. IX Poenaru L, Dreyfus JC. Re-evaluation of enzymatic assays of amniotic fluid and fresh amniotic cells. In: Murken JD. Stengal-Rutkowski S, Schwinger E. eds. Prenatal diagnosis. Stuttgart: Enke. F. Publ.. 1079: 2X4-288. 19 Dreyfus JC. Poenaru L. Le diagnostic enzymatique dea sphingolipidosea. Arch Franc Pediatr 1075: 32: 503-514. 20 Dahlqvist A. Method for assay of intestinal disaccharidascs. Anal Biochem 1964; 7: I X-25. 21 Poenaru L. Drcyfua JC. Electrophorctic heterogeneity of human a-mannosidaac. Biochim Biophya Acta 1973: 303: 171-174. 22 Klenk E. Kahlke W. Uebcr das Vorkommcn der 3.7.1 I,15 Tctramcthyl Hexo-Dccansatirc (Phytansaurc) in den Cholesterinestcrn und andern Lipoid-Fraktionen der Organe bci cinem Krankhcitsfall unnbckannter Geneac. Verdacht auf Heredopathia atactica Polyneuritiformis (Refsum Syndrom) Hoppe Seylera Z Physiol Chem 1963: 333: l33- 139. 23 Swallow DM. Corney G. Harris H, Hirschhorn R. Acid a-glucosidase: a new polymorphism in man demonstrable by affinity electrophoresis. Ann Hum Genet 1975: 3X: 39 I-406. 24 Martiniuk F. Hirschhorn R. Characterization of neutral isozymea of human n-glucosidasc. Differences in substrate specificity molecular weight and clectrophoretic mobility. Biochim Biophys Acta 19X1: 65X: 24X-26 1. 25 Poticr M. Melancon SB, Da&tire ‘L. Fetal intestinal disaccharidasea in human amniotic fluid. Biomedicine 1’376; 25: l67- 16Y. 26 Antonowicz I. Milunsky A. Lobcnthal E. Shwachman H. Disaccharidasc and lyaosomal enzyme activities in amniotic fluid, intestinal mucosa and meconium. Biol Nconat 1977: 32: 2X0-2X9.
Clinica Chimicu Actu, I I7 (1981) 53-62 Elsevier/North-Holland Biomedical Press
CCA
1947
Human amniotic fluid ar-glucosidase Livia
Poenaru,
Marie-Claude
Institut de Pathologic Molkdaire
Vinet
and Jean-Claude
Dreyfus
**
*, CHU Cochin, 24 rue du Fauhourg St. Jacques, 75674 Paris Ckdex I4 (France) (Received
April 20th. 1981)
Amniotic fluid in midpregnancy contains significant a-glucosidase activity. This enzyme is distinguishable from the lysosomal acid a-glucosidase, deficiency of which is associated with Pompe’s disease. The two enzymes differ in optimum pH, thermal stability, electrophoretic migration, isoelectric point, molecular mass, and immunological response. Amniotic a-glucosidase is also different from the classical neutral form. Immuno-cross reactions suggest that the amniotic fluid enzyme has a double fetal origin: renal and intestinal. It seems that a-glucosidase in amniotic fluid is linked to lipids.
Introduction Two forms of cu-1,4glucosidase (EC 3.2.1.20) are generally described in human tissues [l-3]: one with maximum activity at acidic pH and a second with a maximum at neutral pH. A special form, immunologically independent, has been demonstrated in kidney [4,5] and in leucocytes [5]. Type II glycogenosis (Pompe’s disease) is a fatal autosomal recessive disorder characterized by the accumulation of glycogen in the heart, skeletal muscle, brain, liver and kidney [6]. Deficiency of a-1,Cglucosidase in liver [7], heart [8], skeletal muscle [9], brain [9], pancreas [9], cultivated skin fibroblasts [lO,l l] and leucocytes [ 12,131 of patients with this disorder has been reported. Pompe’s disease has been detected in utero on the basis of a deficiency of a- 1,Cglucosidase in cultured amniotic cells [ 14- 161.
* Universite Paris, Unite 129 de 1’Institut National de la Sante et de la Recherche Medicale. Laboratoire Associe 85 du Centre National de la Recherche Scientifique. ** Correspondence should be addressed to J.-C. Dreyfus. Institut de Pathologie Moleculaire, 24 rue du Faubourg St. Jacques, 75014 Paris, France.
0009-898
I /81 /OOOO-0000/$02.75
$1 I98
I Elsevier/North-Holland
Biomedical
Press
54
A different cr-glucosidase activity has been reported in amniotic fluid in midpregnancy [ 14- 18). This enzyme is not implicated in Pompe’s disease [ 15,17 and our unpublished observations] and it disappears between the 20th and 26th weeks of pregnancy [ 171. The purpose of this study was to characterize the a-glucosidase in amniotic fluid and compare it with that in urine, placenta, kidney, intestine, fibroblasts and cultivated amniotic cells in an attempt to determine the origin of the enzyme in amniotic fluid. Materials and methods Amniotic fluid was obtained by transabdominal amniocentesis during the 17th week of pregnancy from women whose pregnancies were being monitored for a variety of fetal disorders. The amniotic fluid was centrifuged at 800 X g and the supematant was assayed immediately, or stored at -20°C. Fibroblasts and cultured amniotic cells were a gift from Dr. J. Boue (Chateau de Longchamp, Paris, France). Serum, urine and leucocytes prepared by sedimentation in dextran followed by lysis of red cells by 0.17 mol/l ammonium chloride were used immediately or stored at -20°C. Placenta, fetal liver, kidney and intestine were obtained after therapeutic abortion and stored at -80°C. Tissues were extracted by 0.1% Triton X-100 with a Potter-Elvehjem apparatus. Enzyme determination a-Glucosidase activity was determined at several pHs using as substrate the fluorogenic compound 4-methylumbelliferyl-cy-glucoside (Koch-Light) as described previously [ 191. The sucrase and maltase activity was also determined by calorimetric methods [20] using sucrose and maltose as substrates. Thermal stability Amniotic fluid thermal stability at 40°C in phosphate buffer, pH 6.9, was compared with thermostability of the fraction not retained by Concanavalin A Sepharose, containing the neutral a-glucosidase activity from liver, placenta, kidney and intestine. Electrophoresis and electrofocusing Electrophoresis was carried out on cellulose acetate (Cellogel-Chemetron) [21], and electrofocusing was performed on horizontal ampholine polyacrylamide gel plates (LKB), pH range 3.5-9.5. Enzymes were stained with the same fluorogenic substrate. Molecular mass The molecular
mass was determined
in 5520% (w/v)
sucrose linear
gradients
in
55
water or in 0.1% Triton X- 100. Then, 0.5 ml of ten times concentrated amniotic fluid were centrifuged in each gradient for 3 h at 236500 X g (using the vertical rotor TY-850 of a DuPont ultracentrifuge). 32 fractions of 1.2 ml each were collected and assayed for a-glucosidase activity at pH 4.0, 5.5 and 6.5. Sephadex G-100 equilibrated in 25 mmol/l NaCl, EDTA 1 mmol/l and Concanavalin A Sepharose 4-B equilibrated in 10 mmol/l phosphate buffer, pH 6.8, containing 0.5 mol/l NaCl, 0.1 mol/l MgCl,, 0.1 mol/l CaCl, were used to test a-glucosidase affinity for these gels in batch and in microcolumns (1 ml) at 4’C.
Immunochemical studies Three antisera raised in rabbits against human enzymes were used: (a) an anti-acid maltase, prepared against acid a-glucosidase from placenta; purified by two steps: 1. Concanavalin A Sepharose 4-B (Pharmacia), chromatography involving a selective elution with 0.5 mol/l methyl-a-D-mannoside (Koch-Light). 2. Sephadex G-100 specific chromatography eluted by 0.25% maltose. This antiserum was used in the form of purified globulins; (b) an antiserum prepared against pure human renal maltase [4] kept as whole serum, was kindly donated by Drs. de Burlet and Sudaka (Faculte de Medecine, Nice, France); (c) an antiserum anti-amniotic fluid a-glucosidase was obtained by injection into rabbits of the sucrose gradient fraction containing a-glucosidase. We used the immunoprecipitation technique; extracts were incubated with antisera or normal serum overnight. Complete precipitation of the antigen-antibody complex was ensured by addition of polyethylenglycol (PEG) to a final concentration of 5%. After 15 min centrifugation at 10000 X g the supernatant was used to test the non-precipitated enzyme. Detection and identification of total fatty acids from the top fraction of sucrose gradient, containing a-glucosidase activity, was performed by gas liquid chromatography according to the method of Klenk and Kahlke [22]. Results 40 amniotic fluids between 17 and 19 weeks of gestation were tested (Fig. 1). The average of a-glucosidase activity at pH 5.5, expressed in mmol .l~ ’ . h- I, was 360 with SD 167.
pH activity curves The profile of amniotic
fluid a-glucosidase activity as a function of pH is represented in Fig. 2 and compared with other tissues. The maximum of amniotic fluid activity is situated at pH 5.5 as for intestinal enzyme. Two peaks of activity, one at pH 4.0-4.5 and a second at pH 6.0-6.5, could be observed for fibroblasts, kidney, liver and placenta.
Thermal stability Activity
curves
after
incubation
at 40°C
indicate
that only
the amniotic
fluid
56
mmo,. I-‘.h-’
Fig. 1. Amniotic
fluid cr-glucosidase
activity
tested at pH 5.5 from
17 and 19 weeks of gestation.
too. 90.
80 _
70 _ 2 :z
60.
2 Lc 50. 0 s
40.
30. I
20.
/ /
10. /
L 0
/
,
3.5
4
4.5
5
5.5
6
6.5
7
75 PH
Fig. 2. pH v -V,
activity kidney;
curves 0 -
of a-glucosidase from: A -A, fibroblasts; 0, amniotic fluid; W n , placenta; 0 -
l0, 0, liver.
intestine:
57
enzyme is completely inactivated at this temperature. This apparent thermal lability, as already observed at 55°C 1171 is probably due to a low concentration of proteins since the cw-glucosidase from amniotic fluid concentrated 20 times is inactivated only 50% in 50 min at 40°C.
Eiectrophoresis Fig. 3 shows the electrophoretic pattern of a-glucasidase from different tissues stained at pH 5.5-5.6. The most important part of amniotic fluid enzyme (line 6) migrates in a position intermediate between the fast band (neutral cr-glucosidase) and the slower one (acid ~-~ucosidase). The neutral activity of some tissues (liver, intestine) is not visible on electrophoresis (line 1 and 3) since the proportion of this form is not high enough. Isoelectric focusing
Isoelectric foeusing of Lu-glucosidase revealed an isoelectric point (~1) between 3.5-3.X for amniotic fluid, 3.8-4 for specific renal forms, 4.2-4.6 for intestine and acidic forms from other organs, and 5-5S for neutral forms.
+
1
2
3
4
5
6
7
Fig. 3. Cellulose acetate electrophoresis of or-glucosidase; pH of rue: 6.5, pH of vizuaiization: 5.5 (both
acid and neutralforms are visualized at this pH). 1, liver; 2, fibroblasts; 3, intestine; 4, kidney; 5, urine concentrated 20 times; 6, amniotic fluid concentrated IO times; 7, placenta.
5x
cY-Glucosidase affinity for Sephadex G 100 and Con A Sepharose Amniotic fluid a-glucosidase was not retarded by Sephadex G 100. The activity was eluted with the bulk of the proteins. By contrast, most of the placental acid a-glucosidase was strongly retarded on dextran gel and eluted after the peak of the proteins. Absorbtion on Concanavalin A Sepharose was very small: 10 to 20% of activity was retained either in batch or in column whereas all the acid a-glucosidase was retained. Molecular mass Since some a-glucosidase isozymes are specifically retained by different gels (Sepharose G 100, Sephacryl S 200, unpublished results), we adopted centrifugation in sucrose linear gradient to determine the molecular mass of a-glucosidase. The results from the experiments made on a sucrose gradient in water (Fig. 4) show an acid a-glucosidase activity peak from liver, kidney and intestine in the 25th fraction; the neutral intestinal and renal activity peak is in the 17th fraction. For amniotic fluid all the activity remained on the top and was found in the 31st and 32nd fractions (Fig. 4, peak A).
A
h
AMNIOTIC
Fig. 4. Centrifugation on a 5 to 20% sucrose linear gradient. The pattern of liver, kidney and intestine is represented as the a-glucosidase activity at pH 6.5 by empty symbols and at pH 4 by full symbols. The pattern was identical in Triton and in water. Amniotic fluid cr-glucosidase activity was tested at pH 5.5. The empty symbols represent separation in Triton and the full symbols in water.
pl
of antiserum
Fig. 5. Immunoprecipitation of amniotic fluid cr-glucosidase glucosidase; I 8, anti-renal tu-glucosidase; A activity was tested at $3 5.5 and 6.5 with identical results.
using antisera: A, anti-amniotic
l0, anti-acid afluid a-glucosidase. The
fiperiments performed on a sucrose gradient in Triton show an unchanged pattern for all tissues except for amniotic fluid enzyme, the position of which was changed and the maximal activity was found in the 17th fraction (Fig. 4, peak B). After centrifugation on a sucrose gradient in water the amniotic fluid fraction containing cu-glucosidase activity was analysed by gas liquid c~omato~aphy for the presence of lipids. A small peak (less than 5 mg/l of fatty acids) was observed in the C ,8 zone.
The action of three antisera: anti-acid maltase, anti-renal maltase and antiamniotic maltase, on amniotic fluid enzyme tested by immunoprecipitation is represented in Fig. 5. The activity is not modified by anti-acid maltase antiserum, is partially precipitated (75%) by anti-renal maltase antiserum and completely precipitated by anti-amniotic fluid antiserum.
pl Fig. 6. The action intestine; +
of anti-amniotic +, amniotic
Of antiserum
fluid n-glucosidase fluid. a-Glucosidase
antiserum: AA, kidney; n -m, activity was tested at pH 5.5 and 6.5.
60
. 20 pl
of antiserum
intestine; Fig. 7. The action of anti-renal n-glucosidase antiserum: 0 ---0, fluid; AA, kidney. a-Glucosidase activity was tested at pH 5.5 and 6.5.
W-
W, amniotic
Anti-amniotic fluid antiserum incubated with kidney extract, intestinal extract and amniotic fluid (Fig. 6) precipitates the a-glucosidase from all these origins but in different proportions; 50% 90% and 100% respectively. No effect of this antiserum was observed on placenta, liver, leukocytes, urine and fibroblasts. Anti-renal maltase antiserum acts in the same way on intestine, amniotic fluid and kidney enzyme (Fig. 7) precipitating respectively 65%, 75% and 80% of activity tested at pH 6.5. This antiserum also precipitates part of leukocyte a-glucosidase activity [5] but no action was observed on placenta, liver, urine and fibroblasts. It is known that in intestinal extracts and in amniotic fluid there exist several neutral disaccharidases acting on various a-glucosides. We, therefore, assayed our antisera on two disaccharidase activities, i.e. maltase and sucrase. Anti-renal maltase antiserum inhibited only maltase activity. By contrast, anti-amniotic fluid maltase antiserum precipitated completely both maltase and sucrase. Discussion The presence of a-glucosidase activity in amniotic fluid from the second trimester of pregnancy has been reported several times but the source of this activity has not been identified. The properties described in this paper provide evidence for a distinction between amniotic fluid a-glucosidase and acid and neutral classical forms, including the neutral cu-glucosidase C recently described by R. Hirschhorn and colleagues [23,24]. Unlike the acid form, amniotic a-glucosidase is not affected in Pompe’s disease, it does not react with anti acid qglucosidase antiserum [ 181, its pH optimum is not 4 but 5.5 and it has no affinity either for Concanavalin A or for Sephadex G-100. On electrophoresis and isoelectric focusing amniotic enzyme migrates in a different position from acid and neutral maltase. On centrifugation on a sucrose gradient the molecular mass is 142000 for amniotic fluid, 90000-100000 for acid forms and 177 000 for the neutral form from various tissues.
61
The total precipitation of amniotic fluid a-glucosidase by anti-amniotic fluid antiserum and the total absence of reaction of this antiserum with acid and neutral enzyme from different tissues constitute additional arguments for the dissimilarity between these enzymes. Immunological results obtained by incubation of anti-renal maltase anti-serum and anti-amniotic fluid antiserum with kidney extract, intestinal extract and amniotic fluid, assayed for a-glucosidase at pH 5.5, show by cross reaction certain immunological similarities between the three enzymes. In this case, it is difficult to understand the absence of recognition of the renal form of cr-glucosidase present in leukocytes [5] by anti-amniotic fluid antiserum. The centrifugation on a sucrose gradient in water reveals that a-glucosidase from amniotic fluid remains in the top fraction. Our hypothesis to explain this behaviour is that the enzyme in amniotic fluid is linked to the lipids, the low density and insolubility in water of which prevent the enzyme from entering the gradient. This hypothesis was confirmed by the experiments made in Triton which, probably by destroying the association protein-lipids, allowed a-glucosidase to migrate as far as the 17th fraction. The presence of fatty acids demonstrated by gas liquid chromatography also supports this hypothesis. In conclusion, it seems that amniotic fluid a-glucosidase has a double fetal origin, renal and intestinal. The complete absence of identity of the amniotic fluid enzyme with acid maltase constitutes a formal counter-indication for the utilisation of amniotic fluid in prenatal diagnosis of Pompe’s disease. The amniotic fluid fraction coming from kidney is immunologically not identical with the renal type existing in leukocytes. At least part of the fraction coming from intestine consists of sucrase [25,26] which is precipitated by anti-amniotic fluid antiserum. The comparison of properties of a-glucosidase from kidney, intestine and amniotic fluid reveals similarities but also differences. It is very likely that amniotic fluid a-glucosidase is derived from the intestinal, and perhaps partly from the renal enzyme. But it is also apparent that within the amniotic fluid, the enzyme undergoes modifications which cause changes in some of its properties. The mechanism by which a-glucosidase activity disappears in late pregnancy remains to be explained; intestinal and renal specific a-glucosidase exist in these tissues at birth and in adult tissues. Acknowledgements We thank Dr. Papa for the gift of amniotic fluids and Dr. M.H. Laudat detection of fatty acids. This work was supported by Grant INSERM CRL 79.5.093.1.
for the
62
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