303
Clinica Chimica Acta, 64 (1975) 303-306 @ Etsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7330
SIALIC ACID CONTAINING
ABNORMAL
AMYLASES
IN HUMAN SERA
KAYOKO SUDO and TAKASHI KANNO Division of Clinical Biochemistry, Clinical Laboratories, Keio University Hospital, School of Medicine, Keio University, Shinanomachi-35, Shinjuku, Tokyo 160 (Japan} (Received May 27, 1975)
Summary The properties of abnormal amylases with unusually fast electrophoretic mobility from five patients were studied. These abnormal amylases , showed markedly reduced electrophoretic mi~ation to the cathodic side by digestion with neur~inid~e. It was suggested that these five abnormal amylases contained sialic acid in their molecules, and the increased electrophoretic mobility resulted from sialic acid, which is not present in normal human amylases.
Recently, the heterogeneity of human serum amylases has been extensively studied and the serum amylases were electrophoretically separated into two or more activity bands in fast-gamma and pre-gamma regions. It has been postulated that the fast-gamma portion is derived from pancreas and the pre-gamma portion from the salivary glands. However, several investigators reported ectopically produced amylases in patients with carcinoma of the lung [ 1,2] and demonstrated that these amylases show different electrophoretic mobilities compared with normal amylases. In the same way, many cases of amylases linked to immunoglobulin have been reported in which one type showed a different mobility on electrophoresis ]3,4] In 1970, Finnegan and Hope [ 5] reported that the ~eatment of marsupial mouse amylases with neuraminidase reduced the electrophoretic mobility of the isoenzymes towards the cathodic side and that the amylases of this mouse were characterized by the presence of sialic acid residues. In human amylases, such changes in mobilities were not observed on treatment with neuraminidase, and it was considered that residues of sialic acid were not present in human amylases.
In this report, abnormal sialic acid-containing human serum amyiases with electrophoretic mobilities different from normal amylases are investigated. Amylase activity was measured by the insoluble chromogenic Blue-starch method described by Ceska [6] and the enzyme activities were expressed as mI.U./ml. Analysis of the amylase isoenzymes was based on electrophoresis on acetate gel (CeIlogeI) with the discontinous buffer system described by Davis [ 71. The amylase activity bands were visu~ized as follows: At the conclusion of electrophoresis, the acetate gel was placed face down on the pasted Bluestarch reagents and incubated at 37°C for 30 min [S]. By this method, the heterogeneity of the serum amylases was clearly demonstrated. The molecular size of amylases was roughly evaluated by thin-layer gel filtration using Sephadex G-200 superfine. The migrated proteins were absorbed on Whatman 3 MM filter paper and amylase activity was demonstrated by the same technique as described for the electrophoresis. Incubation of patient’s sera with neur~inidase was performed with 0.2 unit of neuram~nidase added to 1 ml of serum at 37°C for 20 to 48 h in phosphate buffer of pH 7.2. Binding of amylases to immunoglobulin was demonstrated as follows: immunoelectrophoresis was performed and precipitin lines for specific antisera (e.g. anti-IgA or anti-light chain lambda) were stained by the technique for assessing amylase activity as described above [ 8 J . Changes in electrophoretic mobility were observed in 5 patients after neuraminidase treatment of the sera; three were patients with macroamylasemia, linked to immunoglobulin G and A, one patient (M.U.) was a 45-year-old male with chronic pancreatitis and carcinoma of the pancreas, and the other (H.H.) was a 6%year-old male with luiig cancer and hyperamylasemia. Examples of the changes of mobilities are shown in Fig. 1. Normal serum with 352 mI.U./ml of amylase activity, pancreatic juice (925 mI.U./ml), and salivary juice (925 mIU/ml) did not show mobility changes on neuraminidase treatment. The abnormal amylases of patient S.T. with immunoglobulin-linked amylase, M.U. with chronic pancreatitis with 647 mI.U./ml serum amylase activity, and H.H. with lung cancer with 1690 mI.U./ml of amyfase activity showed faster electrophoretic mobility than salivary gland amylase and were clearly distinct from normal subjects. These abnormal amylases thus show reduced electrophoretic mobility to the cathodic side after neuraminidase treatment. The molecular sizes of the amylases from patients M.U. and H.H. were normal on gel-filtration, and binding to serum proteins was not observed on immunoelectrophoresis followed by amylase staining. After treatment with r~euraminidase, the molecular sizes in these two patients were not reduced. In the cases of ma~roamylasemia, immunoele~trophoresis followed by amylase staining was employed to show the binding of immunoglobulins to the enzyme. The classes of binding immunoglobulin were IgG in patient S.T. and S-N., and IgA in K.I. Neuraminidase treatment of these immunoglobulin-linked amylases showed reduction of electrophoretic mobility to the cathodic side, but the molecular size of the neuraminidase-treated amylase was not changed on Sephadex G-200 gel-filtration. The percentage of residual activity after treatment at 56°C for 2 h was
305
Fig. 1. Changes of eleetro~horetic mobilities on neuraminidase treatment. The abnormal amylase isoenzyme bands of patients S.T. and KU. show the elect~ophor~~c&lIy reduced mobility to the cathodic side on treatment with neuraminidase. (-_) show the enzymograms without neuraminidase treatment and f+) that with neuraminidase treatment. The amylases obtained from pancreas and salivary juice, and normal serum did not show changes of eleetrophoretic mobilities on neuraminidase treatment.
80.0 in patient H.H., 77.0 in S.T., 54.2 in M.U., 86.2 in a patient with mumps, and 23.4 in a patient with acute pancreatitis. Thus, heat stability of these neur~in~d~e-sensitive amylases was higher than those of normal and pancreatic amylases, This higher stability may indicate that these abnormal amylases contained many more polysaccharide residues than normal amylases. Muus and Vnechak [9] reported that human salivary amylases have three
306
or more heterogeneities in acrylamide gel electrophoresis, and Finnegan and Hope [5] suggested that sialic acid in amylase molecules may contribute to the faster mobility of the amylase heterogeneities on electrophoretic analysis. On the other hand, Sudo et al. [lO,ll] reported that serum amylase treated by incubation at 37°C showed faster electrophoretic migration and increasing activity bands on the faster, anodic side. This phenomenon was proved to be due to digestive modification of serum amylases by plasmin-like proteolytic activity of human sera, and the electrophoretic mobilities of these modified faster activity bands were not reduced by treatment with neuraminidase or antiplasmin reagents. These mobility changes were clearly prevented by the addition of several plasmin inhibitors, such as trans-4-aminoethylcyclohexane carboxylic acid and epsilon-aminocaproic acid, while trypsin inhibitors and SH-inhibitors had no effect. In the five electrophoretically abnormal isoenzymes described above, the faster electrophoretic mobility was clearly reduced by neuraminidase digestion. Therefore, this increased was not due to the plasmin-like proteolytic activity but to the sialic acid residues in the amylase molecule or to immunoglobulin linked to amylase. In the case of immunoglobulin-linked amylases, that of patient S.T., it could be postulated that sialic acid may be contained in the moieties of the heavy chains of immunoglobulin. However, the molecular size of these immunoglobulin-linked amylases was markedly reduced by papain digestion, and the electrophoretic mobility of digestive macroamylases was similarly affected by the neuraminidase treatment. These results suggest that other glycoproteins may be involved in the amylase IgG complexes. On the other hand the electrophoretic mobilities of the abnormal amylases from the two patients M.U. and H.H. resembled those reported by Harada and Kitamura [ 11, and it may be postulated that these amylases contain sialic acid. The higher heat stability of the amylases of these two patients also supports the possibility of an increase in polysaccharides or other polysaccharide residues in their molecules which were produced ectopically by the tumor or some other tissues. Acknowledgment The authors thank Dr Shoji Kasagi and Dr Teiichi Sasaki for supplying blood sample of H.H. References 1
Harada.
2
Ammann,
K. and
Kitamura,
R.W..
M. (1972)
et al. (1973)
3
Koami.
E.,
4
Hai-ada,
K.,
5
Finnegan,
D.J.
6
Ceska.
Birath,
K. and
(1972)
J. Clin.
M.,
et al. (1972)
Jap.
et al. (1973) and
7
Davis,
T.J.
8
Sudo.
K.,
9
Muus,
J. and
10
Sudo,
K..
Kane,
11
Sudo.
K.,
Kanno.
Vnechak. S. and
Biol. Med.
Chem. (1964)
25,
78.
Phys.
Aust.
82,
155-158
521-525 Pathol.
J. EXP.
B. (1969)
Pathol.
(Japan)
1. 192-197
Chem.
(1970)
Brown.
Phys.
Chem.
Symp.
R.M.
J.M.
Med. Intern.
J. Clin.
Proc.
Hope.
et al. (1975)
Ann.
Clin.
Biol.
Chim.
(Japan) Med.
Acta
26,
13,136
Sci.
48,
237-240
437444
266-267 Biol.
Nature
(Japan)
20.
204.
283-285
Kanno.
T. (1974)
Phys.
T. and Kane,
S. (1974)
Jap.
Chem. J. Clin.
in press Biol. Chem.
(Japan)
18,
3, 209-216
205-206
the
Clinica Chimica Acta, 64 (1975) 303-306 @ Etsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7330
SIALIC ACID CONTAINING
ABNORMAL
AMYLASES
IN HUMAN SERA
KAYOKO SUDO and TAKASHI KANNO Division of Clinical Biochemistry, Clinical Laboratories, Keio University Hospital, School of Medicine, Keio University, Shinanomachi-35, Shinjuku, Tokyo 160 (Japan} (Received May 27, 1975)
Summary The properties of abnormal amylases with unusually fast electrophoretic mobility from five patients were studied. These abnormal amylases , showed markedly reduced electrophoretic mi~ation to the cathodic side by digestion with neur~inid~e. It was suggested that these five abnormal amylases contained sialic acid in their molecules, and the increased electrophoretic mobility resulted from sialic acid, which is not present in normal human amylases.
Recently, the heterogeneity of human serum amylases has been extensively studied and the serum amylases were electrophoretically separated into two or more activity bands in fast-gamma and pre-gamma regions. It has been postulated that the fast-gamma portion is derived from pancreas and the pre-gamma portion from the salivary glands. However, several investigators reported ectopically produced amylases in patients with carcinoma of the lung [ 1,2] and demonstrated that these amylases show different electrophoretic mobilities compared with normal amylases. In the same way, many cases of amylases linked to immunoglobulin have been reported in which one type showed a different mobility on electrophoresis ]3,4] In 1970, Finnegan and Hope [ 5] reported that the ~eatment of marsupial mouse amylases with neuraminidase reduced the electrophoretic mobility of the isoenzymes towards the cathodic side and that the amylases of this mouse were characterized by the presence of sialic acid residues. In human amylases, such changes in mobilities were not observed on treatment with neuraminidase, and it was considered that residues of sialic acid were not present in human amylases.
In this report, abnormal sialic acid-containing human serum amyiases with electrophoretic mobilities different from normal amylases are investigated. Amylase activity was measured by the insoluble chromogenic Blue-starch method described by Ceska [6] and the enzyme activities were expressed as mI.U./ml. Analysis of the amylase isoenzymes was based on electrophoresis on acetate gel (CeIlogeI) with the discontinous buffer system described by Davis [ 71. The amylase activity bands were visu~ized as follows: At the conclusion of electrophoresis, the acetate gel was placed face down on the pasted Bluestarch reagents and incubated at 37°C for 30 min [S]. By this method, the heterogeneity of the serum amylases was clearly demonstrated. The molecular size of amylases was roughly evaluated by thin-layer gel filtration using Sephadex G-200 superfine. The migrated proteins were absorbed on Whatman 3 MM filter paper and amylase activity was demonstrated by the same technique as described for the electrophoresis. Incubation of patient’s sera with neur~inidase was performed with 0.2 unit of neuram~nidase added to 1 ml of serum at 37°C for 20 to 48 h in phosphate buffer of pH 7.2. Binding of amylases to immunoglobulin was demonstrated as follows: immunoelectrophoresis was performed and precipitin lines for specific antisera (e.g. anti-IgA or anti-light chain lambda) were stained by the technique for assessing amylase activity as described above [ 8 J . Changes in electrophoretic mobility were observed in 5 patients after neuraminidase treatment of the sera; three were patients with macroamylasemia, linked to immunoglobulin G and A, one patient (M.U.) was a 45-year-old male with chronic pancreatitis and carcinoma of the pancreas, and the other (H.H.) was a 6%year-old male with luiig cancer and hyperamylasemia. Examples of the changes of mobilities are shown in Fig. 1. Normal serum with 352 mI.U./ml of amylase activity, pancreatic juice (925 mI.U./ml), and salivary juice (925 mIU/ml) did not show mobility changes on neuraminidase treatment. The abnormal amylases of patient S.T. with immunoglobulin-linked amylase, M.U. with chronic pancreatitis with 647 mI.U./ml serum amylase activity, and H.H. with lung cancer with 1690 mI.U./ml of amyfase activity showed faster electrophoretic mobility than salivary gland amylase and were clearly distinct from normal subjects. These abnormal amylases thus show reduced electrophoretic mobility to the cathodic side after neuraminidase treatment. The molecular sizes of the amylases from patients M.U. and H.H. were normal on gel-filtration, and binding to serum proteins was not observed on immunoelectrophoresis followed by amylase staining. After treatment with r~euraminidase, the molecular sizes in these two patients were not reduced. In the cases of ma~roamylasemia, immunoele~trophoresis followed by amylase staining was employed to show the binding of immunoglobulins to the enzyme. The classes of binding immunoglobulin were IgG in patient S.T. and S-N., and IgA in K.I. Neuraminidase treatment of these immunoglobulin-linked amylases showed reduction of electrophoretic mobility to the cathodic side, but the molecular size of the neuraminidase-treated amylase was not changed on Sephadex G-200 gel-filtration. The percentage of residual activity after treatment at 56°C for 2 h was
305
Fig. 1. Changes of eleetro~horetic mobilities on neuraminidase treatment. The abnormal amylase isoenzyme bands of patients S.T. and KU. show the elect~ophor~~c&lIy reduced mobility to the cathodic side on treatment with neuraminidase. (-_) show the enzymograms without neuraminidase treatment and f+) that with neuraminidase treatment. The amylases obtained from pancreas and salivary juice, and normal serum did not show changes of eleetrophoretic mobilities on neuraminidase treatment.
80.0 in patient H.H., 77.0 in S.T., 54.2 in M.U., 86.2 in a patient with mumps, and 23.4 in a patient with acute pancreatitis. Thus, heat stability of these neur~in~d~e-sensitive amylases was higher than those of normal and pancreatic amylases, This higher stability may indicate that these abnormal amylases contained many more polysaccharide residues than normal amylases. Muus and Vnechak [9] reported that human salivary amylases have three
306
or more heterogeneities in acrylamide gel electrophoresis, and Finnegan and Hope [5] suggested that sialic acid in amylase molecules may contribute to the faster mobility of the amylase heterogeneities on electrophoretic analysis. On the other hand, Sudo et al. [lO,ll] reported that serum amylase treated by incubation at 37°C showed faster electrophoretic migration and increasing activity bands on the faster, anodic side. This phenomenon was proved to be due to digestive modification of serum amylases by plasmin-like proteolytic activity of human sera, and the electrophoretic mobilities of these modified faster activity bands were not reduced by treatment with neuraminidase or antiplasmin reagents. These mobility changes were clearly prevented by the addition of several plasmin inhibitors, such as trans-4-aminoethylcyclohexane carboxylic acid and epsilon-aminocaproic acid, while trypsin inhibitors and SH-inhibitors had no effect. In the five electrophoretically abnormal isoenzymes described above, the faster electrophoretic mobility was clearly reduced by neuraminidase digestion. Therefore, this increased was not due to the plasmin-like proteolytic activity but to the sialic acid residues in the amylase molecule or to immunoglobulin linked to amylase. In the case of immunoglobulin-linked amylases, that of patient S.T., it could be postulated that sialic acid may be contained in the moieties of the heavy chains of immunoglobulin. However, the molecular size of these immunoglobulin-linked amylases was markedly reduced by papain digestion, and the electrophoretic mobility of digestive macroamylases was similarly affected by the neuraminidase treatment. These results suggest that other glycoproteins may be involved in the amylase IgG complexes. On the other hand the electrophoretic mobilities of the abnormal amylases from the two patients M.U. and H.H. resembled those reported by Harada and Kitamura [ 11, and it may be postulated that these amylases contain sialic acid. The higher heat stability of the amylases of these two patients also supports the possibility of an increase in polysaccharides or other polysaccharide residues in their molecules which were produced ectopically by the tumor or some other tissues. Acknowledgment The authors thank Dr Shoji Kasagi and Dr Teiichi Sasaki for supplying blood sample of H.H. References 1
Harada.
2
Ammann,
K. and
Kitamura,
R.W..
M. (1972)
et al. (1973)
3
Koami.
E.,
4
Hai-ada,
K.,
5
Finnegan,
D.J.
6
Ceska.
Birath,
K. and
(1972)
J. Clin.
M.,
et al. (1972)
Jap.
et al. (1973) and
7
Davis,
T.J.
8
Sudo.
K.,
9
Muus,
J. and
10
Sudo,
K..
Kane,
11
Sudo.
K.,
Kanno.
Vnechak. S. and
Biol. Med.
Chem. (1964)
25,
78.
Phys.
Aust.
82,
155-158
521-525 Pathol.
J. EXP.
B. (1969)
Pathol.
(Japan)
1. 192-197
Chem.
(1970)
Brown.
Phys.
Chem.
Symp.
R.M.
J.M.
Med. Intern.
J. Clin.
Proc.
Hope.
et al. (1975)
Ann.
Clin.
Biol.
Chim.
(Japan) Med.
Acta
26,
13,136
Sci.
48,
237-240
437444
266-267 Biol.
Nature
(Japan)
20.
204.
283-285
Kanno.
T. (1974)
Phys.
T. and Kane,
S. (1974)
Jap.
Chem. J. Clin.
in press Biol. Chem.
(Japan)
18,
3, 209-216
205-206
the
