- Email: [email protected]
On the isoenzymes of human red cell acid phosphatase
BIOCHEMICAL
MEDICINE
16, 173-176
(1976)
On the lsoenzymes of Human Red Cell Acid Phosphatase M. JAN KACZMAREK Medizinisch-Chemisches Zentrallaboratorium, Raemistrasse 100, 8006 Zurich, Received
Kantonsspital
Zurich,
Switzerland
July 7, 1976
The presence of acid phosphatase (acid phosphomonoesterase, EC 3.1.3.2) in human red cells was recognized in 1934 (1) and the properties of this enzyme had been studied for almost 30 years (2-7) before its ,?ole in human genetics was revealed by Hopkinson et al. (8). The authors uxamined acid phosphatase from red cell hemolysates by starch-gel electrophoresis. They found two to four active zones, and they distinguished five different red cell acid phosphatase patterns that appeared to be characteristic for the individual and to be genetically determined. In the present work human erythrocytic acid phosphatase was separated into isoenzymes by means of electrofocusing. The isoenzymes were shown to contain increasing numbers of neuraminic acid residues. No genetic variations were observed. Molecular weights of the isoenzymes and their isoelectric points were estimated. METHODS 1. Purijcation of human red cell acid phosphatase. Fifty-milliliter blood samples from 15 individuals were collected. Red cells from these samples were isolated, washed with 0.9% saline, and hemolyzed with four volumes of distilled water. Acid phosphatase was separated from hemolysates by the method described by Ito et al. (9). Purified samples were dialyzed against 0.05 M citrateacetate buffer, pH 4.8, and diluted with the same buffer to yield a final activity of 700 IU. 2. Thin-layer electrofocusing. Thin-layer electrofocusing was performed on a 2117 Multiphor apparatus (LKB, S-16125 Bromma, Sweden) at 1°C. Polyacrylamide-gel slabs were prepared as described in the Application Note (10) with the following modifications: Gel thickness was reduced to 1 mm. pH gradient was formed by 3% Ampholine solution consisting of Ampholine pH 3.5-5 (LKB) and Ampholine pH 5-7 mixed in the proportions of 1:l. Three percent Ampholine pH 7-9 was used as the cathodic electrode solution and 3% Ampholine pH 2-4 as the anodic electrode solution. 173 Copyright All rights
@ 1976 by Academic Press, Inc. of reproduction in any form reserved.
174
M. JAN
KACZMAREK
The run was started with 400 V. Voltage was increased in increments of 100 every 10 min up to 800 V. One hour and twenty minutes was found to be an optimal time of electrofocusing. After completing the run, isoenzymes were demonstrated by the method described by Pearse (11). 3. Preparative electrofocusing. Preparative electrofocusing was performed on a 7900 Uniphor apparatus (LKB) using a 220-ml glass column. A density gradient was formed by 60-30% sucrose with addition of 3% Ampholine solution pH 3.5-7 that established the pH gradient. Electrode solutions were the same as for thin-layer electrofocusing. Experiments were run for 72 hr at 1°C with a constant voltage of 1200 V. After 72 hr, l-ml fractions were collected and the activity was estimated by the p-nitrophenyl phosphate method of Hudson et al. ( 12). 4. Neuraminidase treatment. One-hundred microliters of purified enzyme solution with an activity of 700 IU was mixed with 1 ml of 0.05 M citrate-acetate buffer, pH 6.0, and 50 ~1 of Neuraminidase (Calbiochem, San Diego, California), known to hydrolyze glycosidic bonds between acidic sugar moieties, was added. The isoenzymes isolated were diluted with citrate-acetate buffer, pH 6.0, or concentrated by freeze-drying to a final activity of 70 IU. One milliliter of isoenzyme solution was mixed with 50 ~1 of neuraminidase and incubated at 35°C for increasing periods of time. After each hour of incubation samples were examined by thinlayer electrofocusing. Isoenzymes with isoelectric points of pH 5.8-5.75 achieved their final isoelectric point within 1 hr. Isoenzymes with lower isoelectric points needed successively longer periods of incubation in order to achieve the final isoelectric point. After 10 hr of incubation, purified enzyme and isoenzymes with the lowest isoelectric points established their definitive isoelectric point. 5. Molecular weight estimation. Molecular weights of the purified enzyme and isoenzymes isolated were estimated by Sephadex gel filtration. Sephadex G-75 was equilibrated with 0.05 M citrate-acetate buffers of pH corresponding to the isoelectric point of the single isoenzyme and of pH 5.8 in the case of purified enzyme. RESULTS
AND DISCUSSION
Red cell acid phosphatase has been separated by electrofocusing into 2 1 isoenzymes (Fig. 1). The isoenzymes have been found to be sialoproteins carrying increasing numbers of neuraminic acid residues. After incubation with neuraminidase for 10 hr, only a single activity peak with the isoelectric point of pH 5.8 (Fig. 2) was visible. Isoelectric points of the isoenzymes lie between pH 4.2 and 5.8 (Fig. 1). The molecular weight of the isoenzymes having isoelectric points between pH 4.2 and 5.2 was found to be 21,000. Isoenzymes with isoelectric
175
M. JAN KACZMAREK
100
2
25
20
FIG. 1. Zymogram electrofocusing.
40
60
60
100
120
l&
180 180 FRACTION No.
of red cell acid phosphatase obtained by means of preparative
points of pH 5.2-5.8 were found to have a molecular weight of 12,000. The molecular weight of purified enzyme was found to be 44,000. Molecular weight differences between isoenzymes and the purified enzyme suggest that the enzyme is a dimer or tetramer. All of the 15 samples investigated showed the same zymogram as in Fig. 1. No genetic variations were observed. Most probably, results obtained by Hopkinson et al. (8) are due to the relatively low resolving power of starch-gel electrophoresis. The authors applied blood hemolysates without purifying the enzyme, so that the possibility of association with other erythrocytic proteins existed. 5.a
54
5.0
46
ial-
4.2 PH
20
40
60
80
loo
120
140 IW FRACTION
WI No
FIG. 2. Zymogram of red cell acid phosphatase incubated for 10 hr with neuraminidase, obtained by means of preparative electrofocusing.
176
M. JAN KACZMAREK
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Davies, D. R., Biuchcm. J. 28, 525 (1934). Abul-Fad], M. A. M., and King. E. J.. Bioch~m. J. 45, 51 (1948). King, E. J., Wood, E. J., and Delory, G. E., Biochem. .I. 39, 24 (1945). Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. Biophys. 43, 339 (1953). Tsuboi, K. K., and Hudson, P. B.. Arch. Biochem. Biophys. 53, 341 (1954). Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. Biophys. 55, 206 (1955). Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. Biophys. 61, 197 (1956). Hopkinson, D. A., Spencer, N., and Harris, H., Nufure (London) 199, 968 (1963). Ito, M., Hashimoto, T., and Yoshikawa, H., J. Biochem. (Tokyo) 55, 321 (1964). LKB Application Note No. 75, LKB, 16125 Bromma, Sweden Pearse, A. G. E.. in “Histochemie,” p. 148, Springer-Verlag, BerliniHeidelberglNew York. 12. Hudson, P. B., Brendler, H.. and Scott, W. W., J. Ural. 58, 89 (1947).
MEDICINE
16, 173-176
(1976)
On the lsoenzymes of Human Red Cell Acid Phosphatase M. JAN KACZMAREK Medizinisch-Chemisches Zentrallaboratorium, Raemistrasse 100, 8006 Zurich, Received
Kantonsspital
Zurich,
Switzerland
July 7, 1976
The presence of acid phosphatase (acid phosphomonoesterase, EC 3.1.3.2) in human red cells was recognized in 1934 (1) and the properties of this enzyme had been studied for almost 30 years (2-7) before its ,?ole in human genetics was revealed by Hopkinson et al. (8). The authors uxamined acid phosphatase from red cell hemolysates by starch-gel electrophoresis. They found two to four active zones, and they distinguished five different red cell acid phosphatase patterns that appeared to be characteristic for the individual and to be genetically determined. In the present work human erythrocytic acid phosphatase was separated into isoenzymes by means of electrofocusing. The isoenzymes were shown to contain increasing numbers of neuraminic acid residues. No genetic variations were observed. Molecular weights of the isoenzymes and their isoelectric points were estimated. METHODS 1. Purijcation of human red cell acid phosphatase. Fifty-milliliter blood samples from 15 individuals were collected. Red cells from these samples were isolated, washed with 0.9% saline, and hemolyzed with four volumes of distilled water. Acid phosphatase was separated from hemolysates by the method described by Ito et al. (9). Purified samples were dialyzed against 0.05 M citrateacetate buffer, pH 4.8, and diluted with the same buffer to yield a final activity of 700 IU. 2. Thin-layer electrofocusing. Thin-layer electrofocusing was performed on a 2117 Multiphor apparatus (LKB, S-16125 Bromma, Sweden) at 1°C. Polyacrylamide-gel slabs were prepared as described in the Application Note (10) with the following modifications: Gel thickness was reduced to 1 mm. pH gradient was formed by 3% Ampholine solution consisting of Ampholine pH 3.5-5 (LKB) and Ampholine pH 5-7 mixed in the proportions of 1:l. Three percent Ampholine pH 7-9 was used as the cathodic electrode solution and 3% Ampholine pH 2-4 as the anodic electrode solution. 173 Copyright All rights
@ 1976 by Academic Press, Inc. of reproduction in any form reserved.
174
M. JAN
KACZMAREK
The run was started with 400 V. Voltage was increased in increments of 100 every 10 min up to 800 V. One hour and twenty minutes was found to be an optimal time of electrofocusing. After completing the run, isoenzymes were demonstrated by the method described by Pearse (11). 3. Preparative electrofocusing. Preparative electrofocusing was performed on a 7900 Uniphor apparatus (LKB) using a 220-ml glass column. A density gradient was formed by 60-30% sucrose with addition of 3% Ampholine solution pH 3.5-7 that established the pH gradient. Electrode solutions were the same as for thin-layer electrofocusing. Experiments were run for 72 hr at 1°C with a constant voltage of 1200 V. After 72 hr, l-ml fractions were collected and the activity was estimated by the p-nitrophenyl phosphate method of Hudson et al. ( 12). 4. Neuraminidase treatment. One-hundred microliters of purified enzyme solution with an activity of 700 IU was mixed with 1 ml of 0.05 M citrate-acetate buffer, pH 6.0, and 50 ~1 of Neuraminidase (Calbiochem, San Diego, California), known to hydrolyze glycosidic bonds between acidic sugar moieties, was added. The isoenzymes isolated were diluted with citrate-acetate buffer, pH 6.0, or concentrated by freeze-drying to a final activity of 70 IU. One milliliter of isoenzyme solution was mixed with 50 ~1 of neuraminidase and incubated at 35°C for increasing periods of time. After each hour of incubation samples were examined by thinlayer electrofocusing. Isoenzymes with isoelectric points of pH 5.8-5.75 achieved their final isoelectric point within 1 hr. Isoenzymes with lower isoelectric points needed successively longer periods of incubation in order to achieve the final isoelectric point. After 10 hr of incubation, purified enzyme and isoenzymes with the lowest isoelectric points established their definitive isoelectric point. 5. Molecular weight estimation. Molecular weights of the purified enzyme and isoenzymes isolated were estimated by Sephadex gel filtration. Sephadex G-75 was equilibrated with 0.05 M citrate-acetate buffers of pH corresponding to the isoelectric point of the single isoenzyme and of pH 5.8 in the case of purified enzyme. RESULTS
AND DISCUSSION
Red cell acid phosphatase has been separated by electrofocusing into 2 1 isoenzymes (Fig. 1). The isoenzymes have been found to be sialoproteins carrying increasing numbers of neuraminic acid residues. After incubation with neuraminidase for 10 hr, only a single activity peak with the isoelectric point of pH 5.8 (Fig. 2) was visible. Isoelectric points of the isoenzymes lie between pH 4.2 and 5.8 (Fig. 1). The molecular weight of the isoenzymes having isoelectric points between pH 4.2 and 5.2 was found to be 21,000. Isoenzymes with isoelectric
175
M. JAN KACZMAREK
100
2
25
20
FIG. 1. Zymogram electrofocusing.
40
60
60
100
120
l&
180 180 FRACTION No.
of red cell acid phosphatase obtained by means of preparative
points of pH 5.2-5.8 were found to have a molecular weight of 12,000. The molecular weight of purified enzyme was found to be 44,000. Molecular weight differences between isoenzymes and the purified enzyme suggest that the enzyme is a dimer or tetramer. All of the 15 samples investigated showed the same zymogram as in Fig. 1. No genetic variations were observed. Most probably, results obtained by Hopkinson et al. (8) are due to the relatively low resolving power of starch-gel electrophoresis. The authors applied blood hemolysates without purifying the enzyme, so that the possibility of association with other erythrocytic proteins existed. 5.a
54
5.0
46
ial-
4.2 PH
20
40
60
80
loo
120
140 IW FRACTION
WI No
FIG. 2. Zymogram of red cell acid phosphatase incubated for 10 hr with neuraminidase, obtained by means of preparative electrofocusing.
176
M. JAN KACZMAREK
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Davies, D. R., Biuchcm. J. 28, 525 (1934). Abul-Fad], M. A. M., and King. E. J.. Bioch~m. J. 45, 51 (1948). King, E. J., Wood, E. J., and Delory, G. E., Biochem. .I. 39, 24 (1945). Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. Biophys. 43, 339 (1953). Tsuboi, K. K., and Hudson, P. B.. Arch. Biochem. Biophys. 53, 341 (1954). Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. Biophys. 55, 206 (1955). Tsuboi, K. K., and Hudson, P. B., Arch. Biochem. Biophys. 61, 197 (1956). Hopkinson, D. A., Spencer, N., and Harris, H., Nufure (London) 199, 968 (1963). Ito, M., Hashimoto, T., and Yoshikawa, H., J. Biochem. (Tokyo) 55, 321 (1964). LKB Application Note No. 75, LKB, 16125 Bromma, Sweden Pearse, A. G. E.. in “Histochemie,” p. 148, Springer-Verlag, BerliniHeidelberglNew York. 12. Hudson, P. B., Brendler, H.. and Scott, W. W., J. Ural. 58, 89 (1947).