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Acid phosphatases from different cell types in rat liver
218
SHORT COMMUNICATIONS
the rate of acetate utilisation by the placenta is low, particularly as adult ruminant liver, which has 2.5 times greater activity of acetyl-CoA synthetase, is considered to have a low rate of acetate metabolism 15. Both NAD-malate dehydrogenase and NADP-isocitrate dehydrogenase activities are lower in the placenta than in adult liver, whereas 3-hydroxybutyrate dehydrogenase activity is higher than that found in either foetal or adult liver. ATP citrate lyase activity is lower in cotyledon than in either foetal or adult liver, whereas citrate synthase activity is higher in the placenta than in the other two tissues, indicating that the sheep placenta may have a high citric acid cycle activity in addition to a high rate of glycolysis. This investigation was supported by a grant from The Agricultural Research Council. Two of us (E. M. S. and G. C.E.V.) hold postgraduate studentships from the Ministry of Agriculture, Fisheries and Food.
Department of Physiology and Biochemistry, The University,
Reading, RGz 5AQ (GreatBritain)
USHA K. DHAND MARJORIE K. JEACOCK D. A. L. SHEPHERD ELIZABETH M. SMITH G. CAROLE E. VARNAM
I R. SCARISBRICK AND P. D. S. PUGH, Br. Vet. J., r13 (1957) 328. 2 D. P. ALEXANDER, H. G. BRITTON AND D. A. NIXON, J. Physiol., 186 (1966) lOOP. 3 W. E. HUCKABEE, J. METCALFE, H. PRYSTO~,VSKY AND D. H. 13ARRON, Am. J. Physiol., 202 (1962) 193. 4 T. B0CHER AND G. PFLEIDERER, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 1, Academic Press, New York, 1955, p- 4355 I-I. O. BERGMEYER, E. BERNT AND B. HESS, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York and London, I963, p. 736. 6 F. ROSEN, N. R. ROBERTS AND C. A. NICHOL, J. Biol. Chem., 234 (1956) 476 7 Ix[. E. TONHAZY, N. G. WHITE AND W. W. UMBREIT, Arch. Biochem., 28 (195o) 36. 8 Biochemica Information, C. F. Boehringer and Soehne GmbH., Mannheim, 1964, p. 4. 9 F. J. BALLARD AND R. W. HANSON, Biochem. J., 1o 4 (1967) 866. lO M. S. KORNACKER AND J. iv[. LOWENSTEIN, Biochem. J., 94 (1965) 209. I I A. L. LEHNINGER, H. C. SUDDUTH AND J. ]3. WISE, dr. Biol. Chem., 235 (196o) 2450. 12 D. SHEPHERD AND P. 13. GARLAND, Biochem. J., 114 (1969) 597. 13 j . GINSBURG AND M. K. JEACOCK, Biochem. Pharmacol., 16 (1967) 497. 14 M. YOUNG, in A. KLOPPER AND E. DICZFALUSY, Foetus and Placenta, 131ackwell Scientific Publications, Oxford and Edinburgh, 1969, p. I31. 15 E. 1). MAYFIELD, A. 13ENSADOUN AND B. C. JOHNSON, J. Nutr., 89 (1966) 189.
Received June I9th, 197 ° Biochim. Biophys. Acta, 222 (I97 o) 216-218
BBA 23 619 Acid
phosphatasesfrom
different cell types in rat liver
Multiple forms of acid phosphatase in liver have been demonstrated by chromatography 1-~ and by electrophoresis *-~. Most of these studies were performed on the whole liver tissue. In liver, it is known that 60 % of liver cells are parenchymal and 30 % reticuloendothelial ~, and that both cell types possess acid phosphatase acBiochim. Biophys. Acta, 222 (197 o) 218-221
SHORT COMMUNICATIONS
219
tivity histochemically. The present study was undertaken to examine whether each type of liver cells has a different form of acid phosphatase. Male Wistar albino rats weighing approx. 200 g were used. Newborn rats (5th day after birth) and rats with liver tumors induced by feeding a diet containing 0.o6 % 3'-methyl-4-dimethylaminoazobenzene were also used. Animals were killed by decapitation, and organs were removed and homogenized with a glass Teflon homogenizer to make 20 % (w/v) of homogenate in 0.25 M sucrose. The organs used in this study were liver, lymph nodes, spleen and intestinal mucosa, which was obtained by scraping a portion of the intestine. The hepatocyte homogenate was also prepared with the hepatocytes isolated from the liver tissue s. The soluble fractions were prepared b y centrifugation of the tissue homogenates treated with or without addition of o.I % Triton X-Ioo (at IOOOOO × g for 60 rain), or by centrifugation of the homogenate frozen and thawed 6 times (at 30 ooo × g for 30 rain). A soluble fraction (with Triton) from liver was applied to a DEAE-cellulose column equilibrated with o.oi M Tris-maleate buffer (pH 6.3) 3. The enzyme was eluted by application of a linear gradient of NaC1. Two major peaks of enzyme activity were obtained. One was not adsorbed by the column (F I), and the other was eluted at concentration below 0. 5 M NaC1 (F n). These two peaks of enzyme activity were pooled separately. Acid phosphatase activity was determined by the method of ~¢VATTIAUXAND TABLE I ACID PHOSPHATASE ACTIVITIES IN VARIOUS TISSUES AND SOLUBLE FRACTIONS OF LIVER The reaction m i x t u r e consisted of o,oi M s u b s t r a t e and 0.05 M acetate buffer, p H 5.0, and the i n c u b a t i o n was m a d e at 37 ° for IO rain. To obtain the total enzyme activity in the homogenate, T r i t o n X - l o o (o.i °/o) was added to the medium. Specific activity is expressed as nmoles PI formed per min per mg protein. The second s u p e r n a t a n t in E x p t . 3 was p r e p a r e d from the precipitate r e s u s p e n d e d in o.I % Triton, after r e m o v i n g the first s u p e r n a t a n t w i t h o u t Triton.
Preparation and tissue
Expt. I Liver h o m o g e n a t e H e p a t o c y t e suspension Expt. 2 Soluble fraction (with Triton X-ioo) DEAE-cellulose Fraction I Fraction II Expt. 3 Liver h o m o g e n a t e First s u p e r n a t a n t Second s u p e r n a t a n t Expt. 4 N e w b o r n liver Intestinal mucosa L y m p h node Spleen Liver t u m o r (23 weeks) Liver t u m o r (25 weeks)
Specific activity
Phenyl phosphate] Inhibition of fl-glycerophosphate PhP activity (%) NaF
A lloxan
Phenyl phosphate
fl-Glycerophosphate
41.o 35.1
17. I 2o.2
2.39 1.74
57 84
27 o
73.2
31.4
2.33
64
38
89.7 51.2
52.0 16.2
1.72 3.16
87 56
o 62
34.4 34-4 75.4
16,6 2,5 38`8
2.07 13.75 1.94
64 3 79
29 61 28
94.2 53.1 80.5 lO 3.2 83.5 lO4.O
21.6 13. 9 13.1 13.7 16.3 26.8
4.36 3.82 6.14 7.55 5.12 3.88
66 48 34 44 96 57
24 io 33 4i 69 35
(0.05 M) (o.oo5M)
Biochim. Biophys. Acta, 222 (197 o) 218-221
220
SHORT COMMUNICATIONS
DE DUVE9 with phenyl phosphate and fl-glycerophosphate as substrates. Electrophoresis on cellulose-acetate membrane (Sepraphore I I I , Gelman) was carried out using Veronal buffer (pH 8.6, I = 0.o7) at 250 V for 2 h. Other experimental methods are described in the legends to Fig. I and Table I. Table I shows that the phenyl phosphate/fl-glycerophosphate value and the effect of inhibitors were different in various tissues and in the individual cells or fractions from the liver. It seems generally that an enzyme having a high activity ratio of phenyl phosphate/fl-glycerophosphate, such as F I I (Expt. 2) and the first supernatant (Expt. 3), was somewhat inhibited by alloxan, while an enzyme having a low activity ratio of phenyl phosphate//~-glycerophosphate, such as the hepatocyte (Expt. I) and F I (Expt. 2), was strongly inhibited by NaF. Fig. I shows the electrophoretic patterns of acid phosphatase activity in various tissue extracts. At least three bands of enzyme activity were obtained in the adult rat liver. Two bands migrated toward the anode (A, B) and the other one (C) migrated toward the cathode. On the anodic side, a distinct band (B) migrated more slowly than the other less distinct one (A). Sometimes Band C was separated into two bands and an additional faint band was found near the origin at the anodic side (Fig. 2). These patterns varied with tissues and liver fractions.
cm +
~- ~ } c 0 rigin 1-
"~1
A 8
Fig. I. Electrophoretic p a t t e r n s of acid p h o s p h a t a s e of v a r i o u s r a t tissue e x t r a c t s on t h e cellulosea c e t a t e m e m b r a n e s . Electrophoresis was r u n in Veronal buffer (pH 8.6, I = o.o7) at 25o V for 2 h. Prior to electrophoresis, e:~zymes were c o n c e n t r a t e d b y dialysis a g a i n s t C a r b o w a x 4ooo at 4 °. T h e c o n c e n t r a t e d e n z y m e s were placed at t h e center of t h e strip. A f t e r eleetrophoresis, t h e strips were s t a i n e d on t h e Noble agar (Difco) solution (i °/o), c o n t a i n i n g o.i M a c e t a t e buffer (pH 5.0), I m g / m l s o d i u m a - n a p h t h y l acid p h o s p h a t e (Dajac) a n d 1.5 m g / m l F a s t Blue R R (Sigma). I n c u b a t i o n t i m e was a b o u t 60 nlin at 37 °. (a) a d u l t liver; (b) h e p a t o c y t e s u s p e n s i o n ; (c) n e w b o r n liver; (d) intestinal m u c o s a ; (e) l y m p h node; (f) spleen; (g) liver t u m o r , fed 3 ' - m e t h y l - 4 - d i m e t h y l a m i n o a z o b e n z e n e for 23 weeks; (h) liver t u m o r , 25 weeks; (i) F I fraction b y D E A E - c e l l u l o s e c o l u m n ; (j) F II fraction ; (k) t h e first s u p e r n a t a n t of E x p t . 3 in Table I ; (1) t h e second s u p e r n a t a n t of E x p t . 3. T h e soluble fractions w i t h Triton X - t o o were used in (a), (b), (g), a n d (h), a n d t h e s u p e r n a t a n t fluid of t h e h o m o g e n a t e , frozen a n d t h a w e d 6 times, was used in (c), (d), (e), a n d (f). Fig. 2. T r a c i n g of electrophoretic p a t t e r n in a d u l t r a t liver.
Biochim. Biophys. Acta, 222 (1970) 218-221
SHORT COMMUNICATIONS
221
Band C seems to originate from reticuloendothelial cells, because lymph nodes and spleen exhibited a strong Band C. The first supernatant in Expt. 3 also exhibited a strong Band C. Since the lysosomes in reticuloendothelial cells are too fragile to be separated by mechanical homogenization 1°, it appears that the enzyme in reticuloendothelial cells was released predominantly into the first supernatant. It may be considered that this enzyme has a high activity ratio of phenyl phosphate/fl-glycerophosphate (>3) and is inhibited by alloxan, but not by NaF. Moreover, Band C seems to correspond to the enzyme in F II from the DEAE-cellulose column, to the different form of soluble enzyme reported by SHIBKO AND TAPPEL 3, and also to the enzyme inhibited by the group II compound reported by NEIL AND HORNER11. Band B may originate from parenchymal cells, since it was exhibited markedly in the hepatocyte suspension. This enzyme had a low activity ratio of phenyl phosphate/fl-glycerophosphate ( ~ 2) and its activity was strongly inhibited by NaF. Band B was almost unrecognizable in liver tumor. Band A seemed to be very similar in nature to Band B since both enzymes were not adsorbed on DEAE-cellulose and both were not inhibited by alloxan but were very sensitive to NaF. However, there was a difference between these types in the activity ratio of phenyl phosphate/fl-glycerophosphate, and its value was higher in Band A. This property was deduced from the experiment on the spleen extract, in which the first fraction by DEAE-cellulose column consisted mainly of Band A and the value of phenyl phosphate/fl-glycerophosphate was 3.9- Band A seems to be related to the undifferentiated type of liver cells, because newborn liver, a type of tumor cells (Fig. I, g) exhibited strong Band A. In this connection it is worthy to note that the intestinal mucosa, from which ontogenetically the liver tissue originates, exhibited strong Band A. This study showed (a) that the individual cells in the liver do not necessarily possess all forms of acid phosphatase detected in the liver and (b) the possible existence of different types of enzyme in the different types of cell. The authors are indebted to Drs. M. Mori and K. Dempo for the supply of the materials used in this experiment and thank Mr. T. Horaguchi for his technical assistance.
Department of Pathology, Sapporo Medical College, Sapporo (Japan) I 2 3 4 5 6 7 8 9 IO 1I
A. KANEKO T. IKEDA T. ONO~
B. W. MOORE AND P. U. ANGELI~TTI,Ann. N . Y . Acad. Sci., 94 (1961) 659. T. BARKA, J. Histochem. Cytochem., 9 (1961) 564. S. SHIBKO AND A. L. TAPPEL, Biochim. Biophys. Acta, 73 (1963) 76. T. BARKA, J. Histochem. Cytochem., 9 (196I) .542. L. BECKMAN AND G. BECKMAN, Biochem. Genet., i (1967) 145. L. ROZENSZAJN, Y. EPSTEIN, D. SHOHAM AND I. ARBER, J. Lab. Clin. Med., 72 (1968) 786. a . DAOUST AND A. CANTERO, Cancer Res., 19 (1959) 757. S. T. JACOB AND V. M. BHARGAVA, Exptl. Cell Res., 27 (1962) 453. R. WATTIAUX AND C. DE DOVE, Biochem. J., 63 (1956) 606. J. CoNcHIE, A. J. HAY AND G. A. LEvvY, Biochem. J., 79 (1961) 324 • M. W. NEIL AND M. W. HoRNER, Biochem. J., 92 (1964) 217 .
Received June 3oth, 197o
Biochim. Biophys. Acta, 222 (197 o) 218-221
SHORT COMMUNICATIONS
the rate of acetate utilisation by the placenta is low, particularly as adult ruminant liver, which has 2.5 times greater activity of acetyl-CoA synthetase, is considered to have a low rate of acetate metabolism 15. Both NAD-malate dehydrogenase and NADP-isocitrate dehydrogenase activities are lower in the placenta than in adult liver, whereas 3-hydroxybutyrate dehydrogenase activity is higher than that found in either foetal or adult liver. ATP citrate lyase activity is lower in cotyledon than in either foetal or adult liver, whereas citrate synthase activity is higher in the placenta than in the other two tissues, indicating that the sheep placenta may have a high citric acid cycle activity in addition to a high rate of glycolysis. This investigation was supported by a grant from The Agricultural Research Council. Two of us (E. M. S. and G. C.E.V.) hold postgraduate studentships from the Ministry of Agriculture, Fisheries and Food.
Department of Physiology and Biochemistry, The University,
Reading, RGz 5AQ (GreatBritain)
USHA K. DHAND MARJORIE K. JEACOCK D. A. L. SHEPHERD ELIZABETH M. SMITH G. CAROLE E. VARNAM
I R. SCARISBRICK AND P. D. S. PUGH, Br. Vet. J., r13 (1957) 328. 2 D. P. ALEXANDER, H. G. BRITTON AND D. A. NIXON, J. Physiol., 186 (1966) lOOP. 3 W. E. HUCKABEE, J. METCALFE, H. PRYSTO~,VSKY AND D. H. 13ARRON, Am. J. Physiol., 202 (1962) 193. 4 T. B0CHER AND G. PFLEIDERER, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 1, Academic Press, New York, 1955, p- 4355 I-I. O. BERGMEYER, E. BERNT AND B. HESS, in H. U. BERGMEYER, Methods of Enzymatic Analysis, Academic Press, New York and London, I963, p. 736. 6 F. ROSEN, N. R. ROBERTS AND C. A. NICHOL, J. Biol. Chem., 234 (1956) 476 7 Ix[. E. TONHAZY, N. G. WHITE AND W. W. UMBREIT, Arch. Biochem., 28 (195o) 36. 8 Biochemica Information, C. F. Boehringer and Soehne GmbH., Mannheim, 1964, p. 4. 9 F. J. BALLARD AND R. W. HANSON, Biochem. J., 1o 4 (1967) 866. lO M. S. KORNACKER AND J. iv[. LOWENSTEIN, Biochem. J., 94 (1965) 209. I I A. L. LEHNINGER, H. C. SUDDUTH AND J. ]3. WISE, dr. Biol. Chem., 235 (196o) 2450. 12 D. SHEPHERD AND P. 13. GARLAND, Biochem. J., 114 (1969) 597. 13 j . GINSBURG AND M. K. JEACOCK, Biochem. Pharmacol., 16 (1967) 497. 14 M. YOUNG, in A. KLOPPER AND E. DICZFALUSY, Foetus and Placenta, 131ackwell Scientific Publications, Oxford and Edinburgh, 1969, p. I31. 15 E. 1). MAYFIELD, A. 13ENSADOUN AND B. C. JOHNSON, J. Nutr., 89 (1966) 189.
Received June I9th, 197 ° Biochim. Biophys. Acta, 222 (I97 o) 216-218
BBA 23 619 Acid
phosphatasesfrom
different cell types in rat liver
Multiple forms of acid phosphatase in liver have been demonstrated by chromatography 1-~ and by electrophoresis *-~. Most of these studies were performed on the whole liver tissue. In liver, it is known that 60 % of liver cells are parenchymal and 30 % reticuloendothelial ~, and that both cell types possess acid phosphatase acBiochim. Biophys. Acta, 222 (197 o) 218-221
SHORT COMMUNICATIONS
219
tivity histochemically. The present study was undertaken to examine whether each type of liver cells has a different form of acid phosphatase. Male Wistar albino rats weighing approx. 200 g were used. Newborn rats (5th day after birth) and rats with liver tumors induced by feeding a diet containing 0.o6 % 3'-methyl-4-dimethylaminoazobenzene were also used. Animals were killed by decapitation, and organs were removed and homogenized with a glass Teflon homogenizer to make 20 % (w/v) of homogenate in 0.25 M sucrose. The organs used in this study were liver, lymph nodes, spleen and intestinal mucosa, which was obtained by scraping a portion of the intestine. The hepatocyte homogenate was also prepared with the hepatocytes isolated from the liver tissue s. The soluble fractions were prepared b y centrifugation of the tissue homogenates treated with or without addition of o.I % Triton X-Ioo (at IOOOOO × g for 60 rain), or by centrifugation of the homogenate frozen and thawed 6 times (at 30 ooo × g for 30 rain). A soluble fraction (with Triton) from liver was applied to a DEAE-cellulose column equilibrated with o.oi M Tris-maleate buffer (pH 6.3) 3. The enzyme was eluted by application of a linear gradient of NaC1. Two major peaks of enzyme activity were obtained. One was not adsorbed by the column (F I), and the other was eluted at concentration below 0. 5 M NaC1 (F n). These two peaks of enzyme activity were pooled separately. Acid phosphatase activity was determined by the method of ~¢VATTIAUXAND TABLE I ACID PHOSPHATASE ACTIVITIES IN VARIOUS TISSUES AND SOLUBLE FRACTIONS OF LIVER The reaction m i x t u r e consisted of o,oi M s u b s t r a t e and 0.05 M acetate buffer, p H 5.0, and the i n c u b a t i o n was m a d e at 37 ° for IO rain. To obtain the total enzyme activity in the homogenate, T r i t o n X - l o o (o.i °/o) was added to the medium. Specific activity is expressed as nmoles PI formed per min per mg protein. The second s u p e r n a t a n t in E x p t . 3 was p r e p a r e d from the precipitate r e s u s p e n d e d in o.I % Triton, after r e m o v i n g the first s u p e r n a t a n t w i t h o u t Triton.
Preparation and tissue
Expt. I Liver h o m o g e n a t e H e p a t o c y t e suspension Expt. 2 Soluble fraction (with Triton X-ioo) DEAE-cellulose Fraction I Fraction II Expt. 3 Liver h o m o g e n a t e First s u p e r n a t a n t Second s u p e r n a t a n t Expt. 4 N e w b o r n liver Intestinal mucosa L y m p h node Spleen Liver t u m o r (23 weeks) Liver t u m o r (25 weeks)
Specific activity
Phenyl phosphate] Inhibition of fl-glycerophosphate PhP activity (%) NaF
A lloxan
Phenyl phosphate
fl-Glycerophosphate
41.o 35.1
17. I 2o.2
2.39 1.74
57 84
27 o
73.2
31.4
2.33
64
38
89.7 51.2
52.0 16.2
1.72 3.16
87 56
o 62
34.4 34-4 75.4
16,6 2,5 38`8
2.07 13.75 1.94
64 3 79
29 61 28
94.2 53.1 80.5 lO 3.2 83.5 lO4.O
21.6 13. 9 13.1 13.7 16.3 26.8
4.36 3.82 6.14 7.55 5.12 3.88
66 48 34 44 96 57
24 io 33 4i 69 35
(0.05 M) (o.oo5M)
Biochim. Biophys. Acta, 222 (197 o) 218-221
220
SHORT COMMUNICATIONS
DE DUVE9 with phenyl phosphate and fl-glycerophosphate as substrates. Electrophoresis on cellulose-acetate membrane (Sepraphore I I I , Gelman) was carried out using Veronal buffer (pH 8.6, I = 0.o7) at 250 V for 2 h. Other experimental methods are described in the legends to Fig. I and Table I. Table I shows that the phenyl phosphate/fl-glycerophosphate value and the effect of inhibitors were different in various tissues and in the individual cells or fractions from the liver. It seems generally that an enzyme having a high activity ratio of phenyl phosphate/fl-glycerophosphate, such as F I I (Expt. 2) and the first supernatant (Expt. 3), was somewhat inhibited by alloxan, while an enzyme having a low activity ratio of phenyl phosphate//~-glycerophosphate, such as the hepatocyte (Expt. I) and F I (Expt. 2), was strongly inhibited by NaF. Fig. I shows the electrophoretic patterns of acid phosphatase activity in various tissue extracts. At least three bands of enzyme activity were obtained in the adult rat liver. Two bands migrated toward the anode (A, B) and the other one (C) migrated toward the cathode. On the anodic side, a distinct band (B) migrated more slowly than the other less distinct one (A). Sometimes Band C was separated into two bands and an additional faint band was found near the origin at the anodic side (Fig. 2). These patterns varied with tissues and liver fractions.
cm +
~- ~ } c 0 rigin 1-
"~1
A 8
Fig. I. Electrophoretic p a t t e r n s of acid p h o s p h a t a s e of v a r i o u s r a t tissue e x t r a c t s on t h e cellulosea c e t a t e m e m b r a n e s . Electrophoresis was r u n in Veronal buffer (pH 8.6, I = o.o7) at 25o V for 2 h. Prior to electrophoresis, e:~zymes were c o n c e n t r a t e d b y dialysis a g a i n s t C a r b o w a x 4ooo at 4 °. T h e c o n c e n t r a t e d e n z y m e s were placed at t h e center of t h e strip. A f t e r eleetrophoresis, t h e strips were s t a i n e d on t h e Noble agar (Difco) solution (i °/o), c o n t a i n i n g o.i M a c e t a t e buffer (pH 5.0), I m g / m l s o d i u m a - n a p h t h y l acid p h o s p h a t e (Dajac) a n d 1.5 m g / m l F a s t Blue R R (Sigma). I n c u b a t i o n t i m e was a b o u t 60 nlin at 37 °. (a) a d u l t liver; (b) h e p a t o c y t e s u s p e n s i o n ; (c) n e w b o r n liver; (d) intestinal m u c o s a ; (e) l y m p h node; (f) spleen; (g) liver t u m o r , fed 3 ' - m e t h y l - 4 - d i m e t h y l a m i n o a z o b e n z e n e for 23 weeks; (h) liver t u m o r , 25 weeks; (i) F I fraction b y D E A E - c e l l u l o s e c o l u m n ; (j) F II fraction ; (k) t h e first s u p e r n a t a n t of E x p t . 3 in Table I ; (1) t h e second s u p e r n a t a n t of E x p t . 3. T h e soluble fractions w i t h Triton X - t o o were used in (a), (b), (g), a n d (h), a n d t h e s u p e r n a t a n t fluid of t h e h o m o g e n a t e , frozen a n d t h a w e d 6 times, was used in (c), (d), (e), a n d (f). Fig. 2. T r a c i n g of electrophoretic p a t t e r n in a d u l t r a t liver.
Biochim. Biophys. Acta, 222 (1970) 218-221
SHORT COMMUNICATIONS
221
Band C seems to originate from reticuloendothelial cells, because lymph nodes and spleen exhibited a strong Band C. The first supernatant in Expt. 3 also exhibited a strong Band C. Since the lysosomes in reticuloendothelial cells are too fragile to be separated by mechanical homogenization 1°, it appears that the enzyme in reticuloendothelial cells was released predominantly into the first supernatant. It may be considered that this enzyme has a high activity ratio of phenyl phosphate/fl-glycerophosphate (>3) and is inhibited by alloxan, but not by NaF. Moreover, Band C seems to correspond to the enzyme in F II from the DEAE-cellulose column, to the different form of soluble enzyme reported by SHIBKO AND TAPPEL 3, and also to the enzyme inhibited by the group II compound reported by NEIL AND HORNER11. Band B may originate from parenchymal cells, since it was exhibited markedly in the hepatocyte suspension. This enzyme had a low activity ratio of phenyl phosphate/fl-glycerophosphate ( ~ 2) and its activity was strongly inhibited by NaF. Band B was almost unrecognizable in liver tumor. Band A seemed to be very similar in nature to Band B since both enzymes were not adsorbed on DEAE-cellulose and both were not inhibited by alloxan but were very sensitive to NaF. However, there was a difference between these types in the activity ratio of phenyl phosphate/fl-glycerophosphate, and its value was higher in Band A. This property was deduced from the experiment on the spleen extract, in which the first fraction by DEAE-cellulose column consisted mainly of Band A and the value of phenyl phosphate/fl-glycerophosphate was 3.9- Band A seems to be related to the undifferentiated type of liver cells, because newborn liver, a type of tumor cells (Fig. I, g) exhibited strong Band A. In this connection it is worthy to note that the intestinal mucosa, from which ontogenetically the liver tissue originates, exhibited strong Band A. This study showed (a) that the individual cells in the liver do not necessarily possess all forms of acid phosphatase detected in the liver and (b) the possible existence of different types of enzyme in the different types of cell. The authors are indebted to Drs. M. Mori and K. Dempo for the supply of the materials used in this experiment and thank Mr. T. Horaguchi for his technical assistance.
Department of Pathology, Sapporo Medical College, Sapporo (Japan) I 2 3 4 5 6 7 8 9 IO 1I
A. KANEKO T. IKEDA T. ONO~
B. W. MOORE AND P. U. ANGELI~TTI,Ann. N . Y . Acad. Sci., 94 (1961) 659. T. BARKA, J. Histochem. Cytochem., 9 (1961) 564. S. SHIBKO AND A. L. TAPPEL, Biochim. Biophys. Acta, 73 (1963) 76. T. BARKA, J. Histochem. Cytochem., 9 (196I) .542. L. BECKMAN AND G. BECKMAN, Biochem. Genet., i (1967) 145. L. ROZENSZAJN, Y. EPSTEIN, D. SHOHAM AND I. ARBER, J. Lab. Clin. Med., 72 (1968) 786. a . DAOUST AND A. CANTERO, Cancer Res., 19 (1959) 757. S. T. JACOB AND V. M. BHARGAVA, Exptl. Cell Res., 27 (1962) 453. R. WATTIAUX AND C. DE DOVE, Biochem. J., 63 (1956) 606. J. CoNcHIE, A. J. HAY AND G. A. LEvvY, Biochem. J., 79 (1961) 324 • M. W. NEIL AND M. W. HoRNER, Biochem. J., 92 (1964) 217 .
Received June 3oth, 197o
Biochim. Biophys. Acta, 222 (197 o) 218-221