THE JOURNAL OF EXPERIMENTAL ZOOLOGY 254:l-5 (1990)

Enzymatic and Immunological Properties of Alkaline Phosphatase of Bullfrog M. GOSEKI, S. OIDA, AND S. SASAKI Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan ABSTRACT

Enzymatic and immunological properties of alkaline phosphatase (ALPase) in several tissues of bullfrog (Rana catesbiana) were investigated. Inhibition and thermal inactivation studies showed that bullfrog ALPases in kidney, liver, and intestine had similar enzymatic properties. In addition, mouse antiserum against bullfrog liver ALPase cross-reacted with kidney and intestine enzymes as well as with liver enzyme. These results suggest that a single phenotype of ALPase exists in all tissues of bullfrog in contrast to two or three isoenzymes in mammals.

Human alkaline phosphatase [ALPase: orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1.1 are genetically classified into three isoenzyme types: 1)kidney/liver/ bone or universal-type, 2) intestinal-type, and 3) placental-type. They were distinguished by differences in primary structure (Badger and Sussman, '76; McKenna et al., '79; Seargent and Stinson, ,791, in antigenicity (McKenna et al., '79) or enzymatic properties such as susceptibility to inhibitors or activators and heat stability. Human placental-type ALPase exists in placentae of hominidae (chimpanzee and orangutan) but not of other primates, including the gibbon (Goldstein et al., '82). In other mammals (mouse, rat, hamster, guinea pig, cat, dog, sheep, pig, cow, and rhesus monkey) ALPase in placenta is classified into universal-type ALPase. Thus the presence of two types (universal and intestinal types) of ALPase isoenzymes was elucidated by biochemical studies (Goldstein et al., '80; Moak and Harris, '79). In our previous study (Sone et al., '84),we confirmed that ALPases in kidney, liver, bone, and placenta have the same properties in the mammals except hominidae. There are not many papers about the properties of ALPases in lower vertebrates. Sorimachi et al. ('83) could not find significant differences in relation to phylogenetic development among kidney ALPases of electric ray, rainbow trout, carp, frog, snake, and pigeon. Maekawa and Yamana ('65) reported that the properties of intestinal ALPases of embryo and adult of Xenopus laevis were almost identical. However, they did not compare the properties of other tissue ALPases or those of other lower vertebrates. In this study, we exam0 1990 WILEY-LISS, INC.

ined ALPases in kidney, liver, and intestine of bullfrog and compared their properties with the enzyme in other vertebrates.

MATERIALS AND METHODS Enzyme preparation Kidney, liver, and intestine of male bullfrogs and tadpoles (just before metamorphosis) were collected. Human placenta was from normal labor, and the other human samples were autopsy materials. Swine tissues were obtained from a local slaughterhouse. ICR random-bred mice (30 g, male), Japanese quail (Corturnzx corturnix; adult, male), four-striate snakes (Elaphe quadrivirgata; 100 cm long, 200 g , male), and carp (Cyprznus carpis; male) were from commercial sources. These tissues were homogenized with distilled water (2 ml/g of tissue) and mixed with l-butanol (1ml/g tissue). Aqueous phase was separated and used immediately. Enzyme assay ALPase activity was determined at 37" C with 10 mM p-nitrophenylphosphate as substrate. Protein concentration was determined according to the method of Lowry e t al. ('51). Specific activities (U/protein mg) of kidney, liver, and intestine ALPases of bullfrog were 0.56, 0.61, and 0.55, respectively (U = pmol p-nitrophenol formedlmin). Inhibition experiments L-Phenylalanine (L-PA) and urea were purchased from Wako Pure Chemical Industries, Received February 17, 1989; revision accepted August 9, 1989

2

M. GOSEKI ET AL.

Ltd., levamisole (LEV) from Aldrich Chemical out according t o the method of Weber and Osborn Company Inc., and L-homoarginine (L-HA)from ('69). Polyacrylamide concentration was a gradient from 5 t o 10%. Standard proteins (highC albiochem. The activity was measured by the addition of molecular-weight and low-molecular-weight calienzyme preparation to 5 mM p-nitrophenylphos- bration kits, Pharmacia Fine Chemicals) were stained with Coomassie brilliant blue and enzyme phate in 500 mM 2-amino-2-methyl-l,3-propanediol-HC1 buffer (pH 10.0) containing 5 mM activity of ALPase was stained by p-naphthylMgClz and various concentrations of L-HA (0-10 phosphate method (Kurahashi and Yoshiki, '72). mM) or LEV (0-1 mM) or L-PA (0-20 mM) in toRESULTS tal volume of 3 ml. The reaction lasted for 10-20 Inhibitory effects of L-homoarginine (L-HA), minutes at 37" C. After the addition of 1 ml of NaOH, absorption at 420 nm was measured. In levamisole (LEV), L-phenylalanine (L-PA), and order t o determine the effect of urea, the enzyme urea are shown in Figure 1. The effects of those was preincubated in 500 mM propanediol buffer inhibitors were quite similar in ALPases of differcontaining various concentrations of urea for 20 minutes, and then substrate solution (final 5 mM) lo was added. "" In the thermostability study, the sample was heated in a waterbath at 56" C or 60" C for various periods, and then substrate solution was added. Enzyme activities obtained at different inhibition concentrations or preheating periods were plotted, and the value required to give 50% inhibition was determined from the curve (Goldstein and Harris, '79). 1' 0 Preparation of antiserum against ALPase Bullfrog liver was homogenized with distilled 20 water and mixed with 1-butanol. The aqueous phase was separated and adjusted to pH 4.9 by the addition of acetic acid, and the precipitate was . 0 O L removed by centrifugation. The supernatant was 2'5 7'5 lo 0 025 05 075 1 rnM quickly adjusted t o pH 6.5 with 1M NaOH. rnM "i. Acetone was added t o the supernatant to a final O°K concentration of 30-50%, and the precipitate that appeared was collected by centrifugation and dried t o acetone powder (Oida et al., '85). Mice were each injected intraperitoneally with a total of 0.1 U of bullfrog liver ALPase emulsified with Freund's complete adjuvant (Difco). The second and third injections with the same amount of enzyme in saline solution were applied intraperitoneally at 10 day intervals. Ten days after the third injection, the animals were bled to collect antiserum. Ouchterlony immunodiffusion Double-gel immunodiffusion was carried out in 1%agarose gel containing 50 mM veronal buffer, pH 8.6, and 0.02% sodium azide (Ouchterlony, '64). Fig. 1. Inhibitory effects of L-homoarginine (L-HA), SDS-polyacrylamide gel electrophoresis levamisole (LEV), L-phenylalanine (L-PA),and urea at variPolyacrylamide gel electrophoresis in the pres- ous concentrations on ALPases of bullfrog tissues. 0 ;kidney. liver. ence of sodium dodecyl sulfate (SDS) was carried A; intestine. 0; "I,

ALKALINE PHOSPHATASE OF BULLFROG

ent bullfrog tissues. As shown in Figure 1, L-HA and L-PA were effective inhibitors t o ALPases in liver, kidney, and intestine of bullfrog, but LEV was not so effective. ALPases in all tissues of bullfrog were not heat-stable, and the activity decreased t o 80% of the initial after 30 minutes of preincubation at 56" C (Fig. 2). Properties of ALPases from several tissues of adult bullfrog and tadpole are summarized in Table 1. Almost the same results were obtained from tadpole tissue ALPases, and only one type can be recognized. Moreover, in our preliminary experiment on the properties of ALPases of various vertebrates (Table 2), ALPases of carp, snake, and quail can be classified clearly into two types. Mouse antiserum against bullfrog liver ALPase cross-reacted with ALPases from bullfrog kidney and intestine in double-diffusion precipitation assay (Fig. 3). It was also suggested immunologically that there is only one type of adult bullfrog ALPase. The molecular weights of bullfrog ALPases were estimated t o range from 135,000 t o 155,000 when calibrated by the migration positions of standard proteins (Fig. 4).These values of molecular weights are almost the same as other reported animal ALPases. Although the main band of kidney ALPase showed the same migration as liver or intestine ALPase, there existed one more high-molecular-weight band that might be due t o aggregation with other membrane components. The relative mobilities of ALPases of adult bullfrog and tadpole were almost identical (data not shown).

DISCUSSION The biochemical properties of bullfrog tissue ALPases (Table 1,Figs. 1 , 2 )were almost similar. The cross-reaction of the mouse antiserum

3

"1.

I0

0

5

10

15

20

min

Tadpole Kidney Liver Intestine Bullfrog Kidney Liver Intestine

30

0

5

10

rnin

Fig. 2. Heat inactivation curves of ALPases of bullfrog tissues. The enzyme solution was preincubated a t 56" C or 60" C, and then enzyme activity was determined 37" C. Symbols as in Figure 1.

against bullfrog liver ALPase with ALPases of kidney and intestine suggested the presence of common antigenic determinants in the enzyme of the three bullfrog tissues. The molecular weights of ALPases from kidney, liver, and intestine of bullfrog were almost identical, ranging from 135,000 to 155,000. Although Dauca et al. ('81) reported that the molecular weights of intestinal brush border ALPases of bullfrog and tadpole were 167,000 2 3,000 and 152,000 2 3,000, respectively, there was no difference in molecular weight of intestinal enzyme in adults and tadpoles in this study. As it is well known that human universal-type ALPases in serum are different in relative mobility on electrophoresis, a slight heterogeneity in the apparent molecular weight of bullfrog ALPases by

TABLE 1 . Properties of alkaline phosphatases from several tissues of tadpole and bullfrog' Tissues

25

Concentration or min required to 50% original activity LEV L-HA L-PA Urea 56°C 60" C (mM) (mM) (mM) (MI (mid (min) 0.91 0.94 1.13

2.88 2.31 4.75

13.9 12.4 8.7

0.9 1.7 1.6

10.7 6.5 6.3

6.5 5.7 3.9

>1 >1 >1

3.10 2.60 3.09

12.6 13.6 6.8

2.7 2.8 2.3

3.9 3.3 2.1

1.4 1.8 0.9

LEV = levamisole; L-HA= L-homoarginine; L-PA= L-phenylalanine.

M. GOSEKI ET AL.

4

TABLE 2. Properties of alkaline phosphatases from several tissues of various vertebrates Tissues (mM) Kidney /liver' LEV2 L-HA L-PA Intestine LEV L-HA L-PA Placenta LEV L-HA L-PA

Human

Swine

Mouse

Quail

Snake

Bullfrog

0.03 1.53 > 20

0.03 1.95 > 20

0.05 2.64 > 20

0.30 2.01 11.8

0.63 6.79 > 20

>1 2.85 13.1

> 1 > 10 1.9

>1

>1

> 1

> 10

> 10

> 10

2.1

3.7

4.1

> 1 > 10 > 20

> 1 3.09 6.8

0.93

0.04 2.38 > 20

0.06 0.89 > 20

> 10 2.3

Carp 0.37 3.92 15.5

>1

> 10 > 20

'Mean values of kidney and liver. 'Concentration required to 5 0 8 original activity.

Fig. 3. Double-diffusion Ouchterlony precipitation bands stained by the P-naphthylphosphate method for the enzyme activity. Center Well: mouse antiserum against bullfrog liver ALPase. Outer wells: K, bullfrog kidney ALPase. L, bullfrog liver ALPase. I, bullfrog intestinal ALPase. s, physiological saline solution.

Fig. 4. SDS-polyacrylamide gel electrophoresis of bullfrog tissue ALPase. The enzymatic activities are stained by the pnaphthylphosphate method, A: High-molecular-weight Caliibration standard proteins (ferritin: 220,000; albumin: 67,000, catalase: 60,000; lactate dehydrogenase: 36,000). B: Bullfrog liver ALPase. C: Bullfrog intestinal ALPase. D: Bullfrog kidney ALPase, E: Low-molecular-weight calibration standard proteins (phosphorylase b: 94,000; albumin: 67,000; ovalbumin: 43,000; carbonic anhydrase: 30,000).

Dauca et al. ('81) may be explained by differences in their sugar contents. There are several reports on the difference in intestinal ALPase in human adult and fetus (Vockley and Harris, '84); but the present data suggest that there is only one type of tissue ALPase in bullfrog even after metamorphosis (Table 1). In a preliminary experiment, we investigated ALPases of other amphibians. Liver, kidney, and intestinal ALPases of Xenopus Zaeuis and a newt

(Triturus pyrrhogaster) were found to have similar properties in inhibition by LEV, L-HA,and LPA and their ALPases did not separate into two types such as those of carp or snake. L-HAand LEV were effective inhibitors for universal type ALPase, whereas L-PA was more effective than L-HA on intestinal and human placental ALPases. Although ALPases in tissues of carp, bullfrog, snake, and quail differed slightly from each other, they can be classified clearly into two types in carp, snake, and quail; but bullfrog

ALKALINE PHOSPHATASE OF BULLFROG

ALPases in kidney, liver, and intestine had no difference, indicating the presence of a single type. cDNA analysis showed high homology among human ALPase isoenzymes. Weiss et al. ('86) reported that homology between human universal and placental ALPase or Escherichia coli enzyme was 52% and 25%, respectively. Henthorn et al. ('87) reported that human intestinal ALPase had 86.5% identity t o placental ALPase and 56.6% t o universal-type ALPase in amino acid composition. According to those pieces of evidence, we suppose that ALPase genes evolved from a single ancestral gene; the original type of ALPase has been developed and differentiated into several types such as universal, intestinal, and placental types. All of the types of ALPases may have the same basic physiological function because Noguchi and Yamashita ('87) reported that both universal- and intestinal-type ALPases were found in rabbit kidney and liver. The discrepancy of a single type of ALPase present in bullfrog and other amphibian tissues and two types of ALPase existing in carp, which is classified lower than the bullfrog, is highly interesting. It may be explained either by retrogression or by phenotypic suppression of the gene for intestinal-type ALPase in the bullfrog. According to Komoda and others ('861, another type of ALPase similar t o universal-type was present in fetal rat intestine in addition to intestinal ALPase. Therefore, it might be reasonable t o presume that universal-type ALPase is acting in bullfrog intestine as replacement for the lacking intestinal-type enzyme. More study is necessary in order to conclude that only one gene for ALPase would be present in bullfrog. The phylogeny of ALPase isoenzymes will be revealed by genetic study in the future.

ACKNOWLEDGMENTS The authors thank Dr. K. Sugimoto (Dept. of Oral Physiology, Tokyo Medical and Dental University) for providing bullfrogs. LITERATURE CITED Badger, K.S., and H.H. Sussman (1976) Structural evidence that human liver and placental alkaline phosphatase isoenzymes are coded by different genes. Proc. Natl. Acad. Sci. U.S.A., 73:2201-2205. Dauca, M., J. Hourdry, J.S. Hugon, and D. Menard (1981) Amphibian intestinal brush border membranes. Comp. Biochem. Physiol., 69:15-22.

5

Goldstein, D.J., and H. Harris (1979) Human placental alkaline phosphatase differs from that of other species. Nature, 280:602-605. Goldstein, D.J., C.E. Rogers and H. Harris (1980) Expression of alkaline loci in mammalian tissues. Proc. Natl. Acad. Sci. U.S.A., 77:2857-2860. Goldstein, D.J., C.E. Rogers, and H. Harris (1982) Evolution of alkaline phosphatases in primates. Proc. Natl. Acad. Sci. U.S.A., 79:879-883. Henthorn, P.S., M. Raducha, Y.H. Edwards, M.J. Weiss, C. Slaughter, M.A. Lafferty, and H. Harris (1987) Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: Close homology to placental alkaline phosphatase. Proc. Natl. Acad. Sci. U.S.A., 84:1234-1238. Komoda, T., I. Koyama, A. Nagata, Y. Sakagishi, K. Deschryver-Kecskemeti and D.H. Alpers (1986) Ontogenic and phylogenic studies of intestinal, hepatic, and placental alkaline phosphatases. Gastroenterology, 91:277-286. Kurahashi, Y., and S. Yoshiki (1972) Electron microscopic localization of alkaline phosphatase in the enamel organ of the young rat. Arch. Oral. Biol., 17:155-163. Lowry, O.H., N.J. Rosebruogh, A.L. Farr and R.J. Randall (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem., 193:265-275. Maekawa, H., and K. Yamana (1965) Alkaline phosphatase isoenzymes of Xenopus laevis embryos and tissues. J. Exp. Zool., 192:155-164. McKenna, M.J., T.A. Hamilton, and H.H. Sussman (1979) Comparison of human alkaline phosphatase isoenzymes. Biochem. J., 181:67-73. Moak, G., and H. Harris (1979) Lack of homology between dog and human placental alkaline phosphatases. Proc. Natl. Acad. Sci. U.S.A., 76:1984-1951. Noguchi, T., and Y. Yamashita (1987) The rabbit differs from other mammalian in the tissue distribution of alkaline phosphatase isoenzymes. Biochem. Biophys. Res. Commun., 143:15-19. Oida, S., M. Goseki, and S. Sasaki (1985) Purification and subunit structure of alkaline phosphatase from bovine enamel organ. Arch. Oral. Biol., 30:193-196. Ouchterlony, 0. (1964) Gel diffusion techniques. In: Immunological Methods. J.F. Ackroyd, ed. Blackwell, Oxford. Seargent, L.E., and R.A. Stinson (1979) Evidence that three structural genes code for human alkaline phosphatases. Nature, 281:152-154. Sane (Goseki), M., S. Oida, and S. Sasaki (1984) Comparative studies on immunological properties of kidney-type alkaline phosphatases in various mammals. JBMM, 2:114119. Sorimachi, K., H. Mizuno, R. Konno, A. Niwa, Y. Yasumura, and S. Uchiyama (1983) Alkaline phosphatase in various animal species and heat stability of enzyme in catfish (Silurus astotus) kidney. Zoological Magazine, 92:226-230. Vockley, J., and H. Harris (1984) Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography. Biochem. J., 21 7:535-541. Weber, K., and M. Osborn (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel eletrophoresis. J. Biol. Chem., 244:4406-4412. Weiss, M.J., P.S. Henthorn, M.A. Lafferty, C. Slaughter, M. Raducha, and H. Harris 11986) Isolation and characterization of a cDNA encoding a human liveriboneikidney-type alkaline phosphatase. Proc. Natl. Acad. Sci. U.S.A., 83: 7182-7186.

Enzymatic and immunological properties of alkaline phosphatase of bullfrog.

Enzymatic and immunological properties of alkaline phosphatase (ALPase) in several tissues of bullfrog (Rana catesbeiana) were investigated. Inhibitio...
476KB Sizes 0 Downloads 0 Views