- Email: [email protected]
Isolation and partial characterization of high and low molecular weight acid phosphatases from chicken liver
ISOLATION AND PARTIAL CHARACTERIZATION OF HIGH AND LOW MOLECULAR WEIGHT ACID PHOSPHATASES FROM CHICKEN LIVER FAUSTO PANAKA lstituto di Biologla Ccllularc.
Facolt
Abstract-l. Two acid phosphatase forms were isolated from chicken liver by gel liltration on Scphadcx G- IOO. 2. These enzymes, termed I and II. have similar KJ,,- and I ‘,,,,,,wlucs. but dilycr in molecular v.clght. optimum pH, sensitivity to various inhibitors and substrate specificity. 3. The results were compared with the n~,,,,er~~us htcraturc report\ of rnarn~~~~li~~nacid phosphatasc\.
IihTRODlKTION
The presence of multiple molecular forms of acid phosphatase (EC 3. I .3.2) in various organs of vertebrates has been widely documented [for references see Hollander (1971)]. These enzymes differ in sensitivity to inhibitors (Vanha-Perttula, 1970; Anderson and Toverud, 1979; Di Pietro and Zengerle. 1967: Shibko and Tappel. 1963). substrate requirement (Shibko and Tappel, 1963: Di Pietro and Zengerle, 1967) and molecular size (Saini and Van Etten. 1978;~ b; Heinrikson, 1969). They can, on the basis of molecular weight. be classified as high or low molecular weight acid phosphatases. The enzymes have been characterized in the human placenta (Di Pietro and Zengerle, 1967) and liver (Taga and Van Etten, 1982; Saini and Van Ettcn, 1978af. Similar acid phosphatase forms are present in rat liver (De Araujo et (11.. 1976: Saini and Van Etten 1978b), pig liver (Campbell ct cd., 1973) and bovine liver (Heinrikson. 1969) and brain (Chaimovich and Nome, 1970). The existence of multiple acid phosphatase forms in chicken liver has been reported (Moore and Angeletti. 1961: Panara et a/.. 1980); however a further characterization of these enzymes has not been carried out. In the present paper we describe the isolation and molecular characterization of chicken liver acid phosphatases. Comparisons are also made with earlier literature reports on mammalian enzymes. SIATERIALS AND METHODS
Chickens were killed by decapitation and livers excised immediately and rinsed in 0.25 M cold sucrose. After two changes of sucrose solution the livers were transferred lo cold 0.3 M acetate buffer (pH 6) and homogenized in a Potter-Elvejem homogenizer at 3000 revimin. The final homogenate, containing 2ml of acetate buffer per g of original tissue. was gently stirred at 4 C for 4-6 hr and then centrifuged at 45,000~ for 60 min. The supernatant contuining 75%90”,, of the total activity was then dialysed overnight
1’13
in 50 mM acetate bulTer (pH 6). containing SO mM NaCI: 5 ml of enzyme solution were applied to a column ( 1.8‘* 13 cm) packed with Sephadev G-100 and cquillbratcd with the dialysis buffer. The elutron WDScarried out at 4 C’ ;II unit gravity. Enzyme activity WIS assayed on each 3 ml fraction. Proteins were scanned at 2X0 nm. Fractions containing sullicient enlyme activity wcrc pooled. stored ai 4 C‘ and used for biochemical analycis withln 4X hr.
The enzyme activities wcrc assayed v.ith 2.5 mM is-nitrophcnylpho~ph~it~ [IS substrate. 125 mM acctatc bu&r (pF-1 5) and an appropriate amount of enqmc solution in ii linal volume of 0.5 ml. After incubation at 37 C for 5 IO min. the reactlon was btopped bq addition of I.5 ml of I N NaOH and the extinction ofp-nitrophcnol was read at 300 nm. The molar extinction for p-nitrophenol under thehc conditions was 1.78 x IO” M ’ cm ‘. The cnzyrnc activities were expressed as Itmol of ~-nitropheli~~l;~lin per mg of protctn determined by the Lowry r/ nl. ( I95I 1method ucing crystalline serum albumin as standard. The erect of modifiers. alcohols and acctonc on enlyme activity was assayed as abocc in the prcscncc of thcsc compounds at the desired concentrations. The optimum pH was obtained in 125 mM acetate buffer (mngc 3.5--&S), 125 mM Tris--HCl (pH 7-9) or in I40 mM Verona1 acctatc (pH 4-9). Kinetic studies were carried out at 37 C on p-nitrophenylphosphatc as substrate and I75 mM acctatc buffer. pH 5. The K,,,- and I’,,,,,,- values were determined from repeated cxpcrimcnts usin g ;I Lincawer Burk plot; X-IO substrate concentrations ranging from 0.05 IO 0.5 mM were used. Orthophosphatc inhibition wu assayed from 0 to 10 mM potassium phosph~~tc and Iixcd substrate concentrations (S, = 0.5 mM: S2 = 0.1 mM) in acctatc buffer (pH 5). Inhibition constants (K,) wcrc calculated graphically from a Dixon-Webb plot. The hydrolysis rates of various phosphoric chtcr:, were estimated at 37 C in I25 mM acctiltc hulTer (pH 5). IOmM substrate and enzyme solution in ;I final volume of I ml. After 30min the liberated ~~rthophosphatc was assayed according to Saini and Van Etten (I978a). Blanks and controls were run simultaneously on each experiment. Analytical electrophoresis was performed on 7.5”,, acrylamide gel in IOmM histidinc-NaOH buffer (pH X.3) at 3 mA per gel for 2.5 hr 31 4 C (Panara (J/
1214
15 a L IO j z 15 8
Fraction
0
0.
number
protcm.
enzyme activity was stained with I- UI- /Lnaphthol phosphate (Fast Garnet GBC) salt. The estimation of apparent molecular weight was obtained on two independent calibrated columns of Sephadex G-100 (1.8 x 125 cm and I.8 x 85 cm) with the following protein markers: bovine serum albumin dimer (134.000) bovine serum albumin (67.000). ovalbumin (45,000). trypsin (23.500) and lysozime (14.000). Blue Dextran was used to
estimate the void volume. were the same as described
Buffer and above.
elution
procedures
RESULTS
A typical elution pattern of chicken liver extract from Sephadex G-100 is shown in Fig. I. This procedure allows us to separate two acid p-nitrophenylphosphatases. a high molecular weight form termed enzyme I and a low molecular weight form termed enzyme II. The same results were obtained from chicken livers of various strains. When the extract was carried out on material which had been stored at -20 C a third enzymic form appeared between I and II: this accounted for I o-20” (, of the total activity recovered from the column. Preliminary investigations indicate that the intermediate enzyme was a degradation product of acid phosphatase I. On the basis of these findings only fresh livers were used in our experiments. The chromatographic fractions so obtained were pooled separately and stored at 4 C. The studies on enzyme properties were carried out within 4X hr. Under these experimental conditions the acid phosphatase activity of I and II remained unchanged and no interconversion between the two enzymic forms was observed. Acrylamide disc-gel electrophoresis stained with r-naphtol phosphate (Fast Garnet GBC) reveals a single band for enzyme I. On the other hand no bands could be detected on the gel containing enzyme II and stained with c(- or [I-naphthol phosphate. The high and low molecular weight acid /I-nitrophenylphosphatases showed a different behaviour when their enzyme activities were assayed in the presence of various selected compounds. In Table I we report the effect of some modifiers on the activity of enzymes I and II. Both molecular forms were deactivated by Hg’ +1 Cu’* and Mb ions. F and
I.-( +)tartrate were potent inhibitors of enqmc 1 while the activity of enzyme II was unal‘fected hq these reagents. Iodoacetamide and formaldehyde showed an inhibitor) action on enzyme II but not on enryme I. The presence of Zn” ions lead5 Lo X0” inhibition of enzyme II uhilr ;I slight activation could be observed for en/ylne I. Moderate dilfercnccj wcrc observed with Mp ions and EDT:\ Orthophosphate inhibits both en/~mes <;lyccI-ol. merhanol and ethanol increased the hydrolb\i\ Ir;ltc of ,I nitrophenylphosph~rte t-11en/kmc II. whllc ;I lo\\ I)( activity ws observed IOr en/\ mc I. A more detailed \tud! 0; the acti\it! 01‘ :IVI~! p-nitrophenylphospti~lt~i~c~ I mndII In the prchencc 01 various concentrations of alcohols and acelonc I\ reported in Fig. 2. The result\ indicate that cn,)mc II possesses a phosphotransfer~~~e acti\ it! and LL;~\ more resistant to denaturation induced h? cth;Lnol (,I acetone. Enzyme I was unatfectcd by \ar\inp Itlc* concentrations ol glycerol. The same result\ \~LJI-C obtained at low concentration\ of methanol or ethanol. At higher concentration\ however (about IO” ) a reduction in enzyme acti\it) was obserbrd. Acetone inhibits enzyme I at all the concentration5 assa\cd. Some properties of chicken liver acid ,I-n;tr-c)phenylphosphatases were reported in Table 2. The molecular weight determination gave the apparenr values of X9.000 and 21 .OOO for enzymes I and II. respectively. The variation on 7 repeated experiment\ was about 3”,,. The pH activity curves in acetate buffer show there to be maximum actikit! at pH 4.5--4.X for enzyme I and 5.2 5.5 for en/ymr II. The enzyme activities assayed in Tris HCI or in Verona1 buffer. with and without 3 mM MgCI?. indicate that both enzymes are devoid of any appreciable alkaline phosphatase activity. The Michaelis conbtant of ,Initrophenylphosphate ga1.e the values of 0.16 r IO and 0.10 x 10 ’ M for rnrymcz I and II. rrspcctivel! Inorganic phosphate M~S :I competitive inhihltor 01 both enzymes with a K, of 0.0 x IO ’ M for cn/\mc I and 5.X x IO ‘M for enz\,mc Il. The hydrolysis rate5 oi. high and lo\\ molccula~. weight acid phosphatazcs toward\ a scric\ of phos-
Acid phosphatases
in chicken
1215
liver
Glycerol
1
5
10
15
20
25
Concentrotlon Fig. 2. The effect of glycerol, p-nitrophenylphosphatases from
methanol.
ethanol
and
chicken liver. The values expressed as percentages of control. Bars indicate
phoric esters are given in Table 3. Enzyme I seems to be an aspecific phosphohydrolase and catalyses the dephosphorylation of all the substrates assayed. /j-Glycerophosphate and AMP were preferentially hydrolysed with respect to a-glycerophosphate and ATP. Only p-nitrophenylphosphate and phenylphosphate were hydrolysed by enzyme II. DISCUSSION
The high and low molecular weight acid phosphatases have been isolated and characterized in human (Saini and Van Etten, 1978a; Taga and Van Etten, 1982). bovine (Heinrikson, 1969; Lawrence and Van
I
I
I
I
5
10
15
20
25
(%I
acetone at various concentrations on acid obtained from 5 independent experiments are f SD. 0, Enzyme I: 0. enzyme II.
Etten, l981), pig (Campbell or u/.. 1973) and rat liver (De Araujo et ul., 1976: Saini and Van Etten. 1978b). Similar activities were studied in human placenta (Di Pietro and Zengerle. 1967). human prostate gland (Luchter-Wasyl and Ostrowsky. 1974). rat and bovine brain (Chaimovich and Nome, 1970: De Araujo er ul.. 1976) and rat kidney (De Araujo (‘I rrl.. 1976). This paper now also reports the presence of high and low molecular weight acid phosphatases in the liver of chicken. On the basis of the elution volume from a Sephadex G-100 column they have
Table
3. Relative
rates of hvdrolvsis
of wriow
phosphoric
es(en
Substrates p-Nitrophenylphosphste Table
2. Some physico-chemical
characteristics
I and II of chxken Enzyme
K,,,
K,,
I
0.16
II
0.10
and K, are mM p-nitrophen&min
V m.ll
of acid phosphatases
hver
AMP ATP
mol.
wt
/GGlycerol
phosphate
z-Glycerol
phosphate
K,
PH
0.13
0.9
4.5-4.x
89,000
Phenyl
0.18
5.8
5.2-5.5
21,000
z-Naohthol
concentrations:
VmJr is expressed
per mg of protein.
as LIMOI
of
All
phosphate phosuhate
substrates values
were
5mM
are the means
m the incubation of three expenments
medium
Percentage
Acid phosphatases Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. h/0/. Clrcvn. 193, 265-275. Luchter-Waayl E. and Ostrowsky W. (1974) Subunit structure of human prostatic acid phosphatace. Bioc,/tr/~~. hiophp .4ctrr 365, 349-359. Moore B. W. and Angcletti P. II. (1961) Chromatographic heterogenetty of some enzymes in normal tissues and tumors. ,4,1/r. ,Y. 1’. Act/t/. Sr,i. 94, 65%667. Panara F.. Cirotto C.. Arangi I. and Barbcrini L. (lY7X) Acid and alkaltne phosphatase heterogeneity m liver. heart and tntcstine of the adult chtck. Ev/~r,vicvrritr 34, 983-984. Panara F.. Cirotto C.. Arangi I. and Barberint L. (1980) Localization of actd phosphatases in the ccl1 fractions 01 chick liver. E.vprricv~/itr 36, I IY- 120. Saini M. S. and Van Etten R. L. (lY7Xa) A homogeneous isoenzymc of human liver acid phosphatasc. .4&s Bioc~/Ie/,t. Bi0/I/lj~.\. 191. 613 624.
in chicken
liver
1117
Saini M. S. and Van Etten R. L. (lY78b) Dimeric nature and aminoacid composition of homogeneous canine prostrattc. human hver and rat liver acid phosphatase isoenzymes. Specificity and pH dependence of the canine enzyme. Bbxhirrr. hio&rr.\. Ac(tr 526. 46X-47X. Shibko S. and Tanncl A. L. (lY63) Acid nhosnhatase of the lysosomal and’ soluble fraction of rat li;,er. &r~hi,~r. hiqJ/rrc. /lctc/ 73. 7686. Taga E. M. and Van Etten R. L. (19X2) Human likcr acid phosphatases: purification and properties of II lovmolecular-weight isocnrymc. .4rc+\ B/oc,hcv~r. H/~J/II,\. 214, 505-515. Tanizaki M. M.. Bittencourt H. M. S. and Chatmo\tch H. (1977) Acttvation of low, molecular weight actd phosphatase from bobinc brain by purtncs and glycerol. B;oc~/I;~,I.hiop/r\,.\. ,4c /
Facolt
Abstract-l. Two acid phosphatase forms were isolated from chicken liver by gel liltration on Scphadcx G- IOO. 2. These enzymes, termed I and II. have similar KJ,,- and I ‘,,,,,,wlucs. but dilycr in molecular v.clght. optimum pH, sensitivity to various inhibitors and substrate specificity. 3. The results were compared with the n~,,,,er~~us htcraturc report\ of rnarn~~~~li~~nacid phosphatasc\.
IihTRODlKTION
The presence of multiple molecular forms of acid phosphatase (EC 3. I .3.2) in various organs of vertebrates has been widely documented [for references see Hollander (1971)]. These enzymes differ in sensitivity to inhibitors (Vanha-Perttula, 1970; Anderson and Toverud, 1979; Di Pietro and Zengerle. 1967: Shibko and Tappel. 1963). substrate requirement (Shibko and Tappel, 1963: Di Pietro and Zengerle, 1967) and molecular size (Saini and Van Etten. 1978;~ b; Heinrikson, 1969). They can, on the basis of molecular weight. be classified as high or low molecular weight acid phosphatases. The enzymes have been characterized in the human placenta (Di Pietro and Zengerle, 1967) and liver (Taga and Van Etten, 1982; Saini and Van Ettcn, 1978af. Similar acid phosphatase forms are present in rat liver (De Araujo et (11.. 1976: Saini and Van Etten 1978b), pig liver (Campbell ct cd., 1973) and bovine liver (Heinrikson. 1969) and brain (Chaimovich and Nome, 1970). The existence of multiple acid phosphatase forms in chicken liver has been reported (Moore and Angeletti. 1961: Panara et a/.. 1980); however a further characterization of these enzymes has not been carried out. In the present paper we describe the isolation and molecular characterization of chicken liver acid phosphatases. Comparisons are also made with earlier literature reports on mammalian enzymes. SIATERIALS AND METHODS
Chickens were killed by decapitation and livers excised immediately and rinsed in 0.25 M cold sucrose. After two changes of sucrose solution the livers were transferred lo cold 0.3 M acetate buffer (pH 6) and homogenized in a Potter-Elvejem homogenizer at 3000 revimin. The final homogenate, containing 2ml of acetate buffer per g of original tissue. was gently stirred at 4 C for 4-6 hr and then centrifuged at 45,000~ for 60 min. The supernatant contuining 75%90”,, of the total activity was then dialysed overnight
1’13
in 50 mM acetate bulTer (pH 6). containing SO mM NaCI: 5 ml of enzyme solution were applied to a column ( 1.8‘* 13 cm) packed with Sephadev G-100 and cquillbratcd with the dialysis buffer. The elutron WDScarried out at 4 C’ ;II unit gravity. Enzyme activity WIS assayed on each 3 ml fraction. Proteins were scanned at 2X0 nm. Fractions containing sullicient enlyme activity wcrc pooled. stored ai 4 C‘ and used for biochemical analycis withln 4X hr.
The enzyme activities wcrc assayed v.ith 2.5 mM is-nitrophcnylpho~ph~it~ [IS substrate. 125 mM acctatc bu&r (pF-1 5) and an appropriate amount of enqmc solution in ii linal volume of 0.5 ml. After incubation at 37 C for 5 IO min. the reactlon was btopped bq addition of I.5 ml of I N NaOH and the extinction ofp-nitrophcnol was read at 300 nm. The molar extinction for p-nitrophenol under thehc conditions was 1.78 x IO” M ’ cm ‘. The cnzyrnc activities were expressed as Itmol of ~-nitropheli~~l;~lin per mg of protctn determined by the Lowry r/ nl. ( I95I 1method ucing crystalline serum albumin as standard. The erect of modifiers. alcohols and acctonc on enlyme activity was assayed as abocc in the prcscncc of thcsc compounds at the desired concentrations. The optimum pH was obtained in 125 mM acetate buffer (mngc 3.5--&S), 125 mM Tris--HCl (pH 7-9) or in I40 mM Verona1 acctatc (pH 4-9). Kinetic studies were carried out at 37 C on p-nitrophenylphosphatc as substrate and I75 mM acctatc buffer. pH 5. The K,,,- and I’,,,,,,- values were determined from repeated cxpcrimcnts usin g ;I Lincawer Burk plot; X-IO substrate concentrations ranging from 0.05 IO 0.5 mM were used. Orthophosphatc inhibition wu assayed from 0 to 10 mM potassium phosph~~tc and Iixcd substrate concentrations (S, = 0.5 mM: S2 = 0.1 mM) in acctatc buffer (pH 5). Inhibition constants (K,) wcrc calculated graphically from a Dixon-Webb plot. The hydrolysis rates of various phosphoric chtcr:, were estimated at 37 C in I25 mM acctiltc hulTer (pH 5). IOmM substrate and enzyme solution in ;I final volume of I ml. After 30min the liberated ~~rthophosphatc was assayed according to Saini and Van Etten (I978a). Blanks and controls were run simultaneously on each experiment. Analytical electrophoresis was performed on 7.5”,, acrylamide gel in IOmM histidinc-NaOH buffer (pH X.3) at 3 mA per gel for 2.5 hr 31 4 C (Panara (J/
1214
15 a L IO j z 15 8
Fraction
0
0.
number
protcm.
enzyme activity was stained with I- UI- /Lnaphthol phosphate (Fast Garnet GBC) salt. The estimation of apparent molecular weight was obtained on two independent calibrated columns of Sephadex G-100 (1.8 x 125 cm and I.8 x 85 cm) with the following protein markers: bovine serum albumin dimer (134.000) bovine serum albumin (67.000). ovalbumin (45,000). trypsin (23.500) and lysozime (14.000). Blue Dextran was used to
estimate the void volume. were the same as described
Buffer and above.
elution
procedures
RESULTS
A typical elution pattern of chicken liver extract from Sephadex G-100 is shown in Fig. I. This procedure allows us to separate two acid p-nitrophenylphosphatases. a high molecular weight form termed enzyme I and a low molecular weight form termed enzyme II. The same results were obtained from chicken livers of various strains. When the extract was carried out on material which had been stored at -20 C a third enzymic form appeared between I and II: this accounted for I o-20” (, of the total activity recovered from the column. Preliminary investigations indicate that the intermediate enzyme was a degradation product of acid phosphatase I. On the basis of these findings only fresh livers were used in our experiments. The chromatographic fractions so obtained were pooled separately and stored at 4 C. The studies on enzyme properties were carried out within 4X hr. Under these experimental conditions the acid phosphatase activity of I and II remained unchanged and no interconversion between the two enzymic forms was observed. Acrylamide disc-gel electrophoresis stained with r-naphtol phosphate (Fast Garnet GBC) reveals a single band for enzyme I. On the other hand no bands could be detected on the gel containing enzyme II and stained with c(- or [I-naphthol phosphate. The high and low molecular weight acid /I-nitrophenylphosphatases showed a different behaviour when their enzyme activities were assayed in the presence of various selected compounds. In Table I we report the effect of some modifiers on the activity of enzymes I and II. Both molecular forms were deactivated by Hg’ +1 Cu’* and Mb ions. F and
I.-( +)tartrate were potent inhibitors of enqmc 1 while the activity of enzyme II was unal‘fected hq these reagents. Iodoacetamide and formaldehyde showed an inhibitor) action on enzyme II but not on enryme I. The presence of Zn” ions lead5 Lo X0” inhibition of enzyme II uhilr ;I slight activation could be observed for en/ylne I. Moderate dilfercnccj wcrc observed with Mp ions and EDT:\ Orthophosphate inhibits both en/~mes <;lyccI-ol. merhanol and ethanol increased the hydrolb\i\ Ir;ltc of ,I nitrophenylphosph~rte t-11en/kmc II. whllc ;I lo\\ I)( activity ws observed IOr en/\ mc I. A more detailed \tud! 0; the acti\it! 01‘ :IVI~! p-nitrophenylphospti~lt~i~c~ I mndII In the prchencc 01 various concentrations of alcohols and acelonc I\ reported in Fig. 2. The result\ indicate that cn,)mc II possesses a phosphotransfer~~~e acti\ it! and LL;~\ more resistant to denaturation induced h? cth;Lnol (,I acetone. Enzyme I was unatfectcd by \ar\inp Itlc* concentrations ol glycerol. The same result\ \~LJI-C obtained at low concentration\ of methanol or ethanol. At higher concentration\ however (about IO” ) a reduction in enzyme acti\it) was obserbrd. Acetone inhibits enzyme I at all the concentration5 assa\cd. Some properties of chicken liver acid ,I-n;tr-c)phenylphosphatases were reported in Table 2. The molecular weight determination gave the apparenr values of X9.000 and 21 .OOO for enzymes I and II. respectively. The variation on 7 repeated experiment\ was about 3”,,. The pH activity curves in acetate buffer show there to be maximum actikit! at pH 4.5--4.X for enzyme I and 5.2 5.5 for en/ymr II. The enzyme activities assayed in Tris HCI or in Verona1 buffer. with and without 3 mM MgCI?. indicate that both enzymes are devoid of any appreciable alkaline phosphatase activity. The Michaelis conbtant of ,Initrophenylphosphate ga1.e the values of 0.16 r IO and 0.10 x 10 ’ M for rnrymcz I and II. rrspcctivel! Inorganic phosphate M~S :I competitive inhihltor 01 both enzymes with a K, of 0.0 x IO ’ M for cn/\mc I and 5.X x IO ‘M for enz\,mc Il. The hydrolysis rate5 oi. high and lo\\ molccula~. weight acid phosphatazcs toward\ a scric\ of phos-
Acid phosphatases
in chicken
1215
liver
Glycerol
1
5
10
15
20
25
Concentrotlon Fig. 2. The effect of glycerol, p-nitrophenylphosphatases from
methanol.
ethanol
and
chicken liver. The values expressed as percentages of control. Bars indicate
phoric esters are given in Table 3. Enzyme I seems to be an aspecific phosphohydrolase and catalyses the dephosphorylation of all the substrates assayed. /j-Glycerophosphate and AMP were preferentially hydrolysed with respect to a-glycerophosphate and ATP. Only p-nitrophenylphosphate and phenylphosphate were hydrolysed by enzyme II. DISCUSSION
The high and low molecular weight acid phosphatases have been isolated and characterized in human (Saini and Van Etten, 1978a; Taga and Van Etten, 1982). bovine (Heinrikson, 1969; Lawrence and Van
I
I
I
I
5
10
15
20
25
(%I
acetone at various concentrations on acid obtained from 5 independent experiments are f SD. 0, Enzyme I: 0. enzyme II.
Etten, l981), pig (Campbell or u/.. 1973) and rat liver (De Araujo et ul., 1976: Saini and Van Etten. 1978b). Similar activities were studied in human placenta (Di Pietro and Zengerle. 1967). human prostate gland (Luchter-Wasyl and Ostrowsky. 1974). rat and bovine brain (Chaimovich and Nome, 1970: De Araujo er ul.. 1976) and rat kidney (De Araujo (‘I rrl.. 1976). This paper now also reports the presence of high and low molecular weight acid phosphatases in the liver of chicken. On the basis of the elution volume from a Sephadex G-100 column they have
Table
3. Relative
rates of hvdrolvsis
of wriow
phosphoric
es(en
Substrates p-Nitrophenylphosphste Table
2. Some physico-chemical
characteristics
I and II of chxken Enzyme
K,,,
K,,
I
0.16
II
0.10
and K, are mM p-nitrophen&min
V m.ll
of acid phosphatases
hver
AMP ATP
mol.
wt
/GGlycerol
phosphate
z-Glycerol
phosphate
K,
PH
0.13
0.9
4.5-4.x
89,000
Phenyl
0.18
5.8
5.2-5.5
21,000
z-Naohthol
concentrations:
VmJr is expressed
per mg of protein.
as LIMOI
of
All
phosphate phosuhate
substrates values
were
5mM
are the means
m the incubation of three expenments
medium
Percentage
Acid phosphatases Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. h/0/. Clrcvn. 193, 265-275. Luchter-Waayl E. and Ostrowsky W. (1974) Subunit structure of human prostatic acid phosphatace. Bioc,/tr/~~. hiophp .4ctrr 365, 349-359. Moore B. W. and Angcletti P. II. (1961) Chromatographic heterogenetty of some enzymes in normal tissues and tumors. ,4,1/r. ,Y. 1’. Act/t/. Sr,i. 94, 65%667. Panara F.. Cirotto C.. Arangi I. and Barbcrini L. (lY7X) Acid and alkaltne phosphatase heterogeneity m liver. heart and tntcstine of the adult chtck. Ev/~r,vicvrritr 34, 983-984. Panara F.. Cirotto C.. Arangi I. and Barberint L. (1980) Localization of actd phosphatases in the ccl1 fractions 01 chick liver. E.vprricv~/itr 36, I IY- 120. Saini M. S. and Van Etten R. L. (lY7Xa) A homogeneous isoenzymc of human liver acid phosphatasc. .4&s Bioc~/Ie/,t. Bi0/I/lj~.\. 191. 613 624.
in chicken
liver
1117
Saini M. S. and Van Etten R. L. (lY78b) Dimeric nature and aminoacid composition of homogeneous canine prostrattc. human hver and rat liver acid phosphatase isoenzymes. Specificity and pH dependence of the canine enzyme. Bbxhirrr. hio&rr.\. Ac(tr 526. 46X-47X. Shibko S. and Tanncl A. L. (lY63) Acid nhosnhatase of the lysosomal and’ soluble fraction of rat li;,er. &r~hi,~r. hiqJ/rrc. /lctc/ 73. 7686. Taga E. M. and Van Etten R. L. (19X2) Human likcr acid phosphatases: purification and properties of II lovmolecular-weight isocnrymc. .4rc+\ B/oc,hcv~r. H/~J/II,\. 214, 505-515. Tanizaki M. M.. Bittencourt H. M. S. and Chatmo\tch H. (1977) Acttvation of low, molecular weight actd phosphatase from bobinc brain by purtncs and glycerol. B;oc~/I;~,I.hiop/r\,.\. ,4c /