Distribution and classification of acylphosphatase isozymes

AK(‘H[VESOF

HIOCHEMISTRV

AND

BIOPHYSICS

Vol. 278, No. 2, May 1, pp. 4377443, 1990

Distribution and Classification Acylphosphatase lsozymes Yusuke Takiko Section

Mizuno,’ Tamura,

Yoichi Ohba,” Hisakazu and Hiroyuki Shiokawa

of Biochemistry,

Institute

of Immunological

of Fujita,”

Yoshikazu

Science, Hokkaido

Kanesaka,

University,

Sapporo 060, Japan

Received August 28,1989, and in revised form December 21,1989

Distributions of acylphosphatase isozymes among organs of several animal species were investigated. Organ extracts of pig and chicken were treated with isozyme-specific antibodies, subjected to electrophoresis on a polyacrylamide gel, then the gel was stained for acylphosphatase activity. Both animals showed three activity bands; one band was named common type isozyme because of its wide distribution in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte, and the other two bands were named muscle type isozymes because of their localization in skeletal muscle. This classification was supported by selective and quantitative reactions of the isozymes to the isozymespecific antibodies. Because the two bands of the muscle type have the same amino acid sequence and differ only in modifications on an -SH group, it is suggested that pig and chicken have only the two major types of acylphosphatase. This conclusion was supported by similar
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Inc.

Acylphosphatase [EC 3.6.1.71 is a small (M, about 11,000) enzyme which specifically catalyzes the hydrolysis of the carboxyl-phosphate bond of acylphosphates. Although the enzyme is present in various organs of animals, the enzymes from mammals and birds have been extensively studied because of the high content of the enzyme and availability of large quantities of skeletal muscle. The amino acid sequence is known for the en’ To whom correspondenceshould be addressedat Section of Biochemistry, Institute of Immunological Science, Hokkaido University, KitaNishi-7, Kita-ku, Sapporo 060, ,Japan. ” Present address: Bioscience Research Laboratories, Nippon Mining Company, Ltd., Toda, Saitama 335, Japan. ” Present address: Laboratory of Molecular Genetics, Cancer Institute, School of Medicine, Hokkaido University, Sapporo 060, Japan. 000:1-9861/90 $3.00 Copyright CC1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

zymes from skeletal muscles of horse (l), pig (2), rabbit (3,4), ox (5), human (6), turkey (7), duck (8), and guinea pig (9). These sequences are similar to each other. On the other hand, Liguri et al. elucidated the primary structure of the acylphosphatase from human erythrocytes (10). This structure differs in 44% of the total positions of amino acid residues from that of human skeletal muscle acylphosphatase. From chicken skeletal muscle, we purified two isozymes of acylphosphatase, named Chl and Ch2 (11). The amino acid sequence of Ch2 differs in 44% of the total positions from that of Chl (12, 13). The sequence of Chl was similar to those of the muscle enzymes from other species. In addition, we purified an acylphosphatase from porcine testis, which is considerably different in amino acid sequence from the porcine skeletal muscle enzyme (14). Then, the following questions arose: How many isozymes of acylphosphatase do animals have? How are they distributed among the organs of animals? To seek answers to these questions, we have developed an activity-staining procedure for acylphosphatase after electrophoresis on a polyacrylamide gel (15). This procedure was employed to analyze acylphosphatase isozymes in porcine organ extracts. There appeared three activity bands in all. The slowest band coincided with that of the purified testis acylphosphatase and the other two bands coincided with those of the monomer and the dimer of the purified muscle acylphosphatase (15). In the present paper we report further assignment and quantification of t,he porcine and the chicken acylphosphatase isozymes by the use of isozyme-specific antibodies, and also report electrophoretie separation and assignment of the isozymes in several other animal species. The results suggest that mammals and birds have two major types of acylphosphatase isozyme; one type is common to many organs and the other is relatively localized in skeletal muscle. MATERIALS Materials. terhouse.

AND

METHODS

Fresh porcine organs were obtained from a local slaughChickens were obtained from the Faculty of Agriculture, 437

438

MIZUNO

Hokkaido University. Organs of feral pigeons (Columba liuia) and crows (Coruus corone) were obtained within a few hours after they were shot down near Sapporo. Organs of dogs, rats, rabbits, and guinea pigs were obtained from laboratory animals immediately after killing. Human tissues were obtained after autopsy 8-15 h after death. Only normal tissues free from apparently morbid parts were used. These organs were extracted immediately or after storage at -20°C. Acylphosphatases from porcine skeletal muscle (16) and testis (14), and two isozymes of acylphosphatase from chicken skeletal muscle (11) were purified as previously described. Other materials were as described previously (17). Extraction of organs. Animal or gans were extracted with 3 vol of deionized water at pH 5.3, and hemoglobin in erythrocyte extracts was removed by the column chromatography on SP-Toyopearl as described previously (15). The yields of the acylphosphatase activity after the chromatography were 30% for the porcine and 60% for the human enzyme. Portions of the extracts were spectrophotometrically assayed for acylphosphatase activity with benzoyl phosphate as substrate (16), and other portions were subjected to immunological and electrophoretie analyses. Antibodies. Rabbit antisera for the porcine skeletal muscle and testis acylphosphatases (14) and those for the two isozymes, Chl and Ch2, from chicken skeletal muscle (11,17) were prepared as previously described. y-Globulin fractions were prepared from the antisera and normal (non-immune) rabbit serum by ammonium sulfate precipitation, at 40% saturation of ammonium sulfate for anti-porcine muscle enzyme serum or at 45% saturation for the other sera. From the y-globulin fractions, specific anti-porcine testis acylphosphatase, anti-porcine muscle acylphosphatase, and anti-Chl antibodies were isolated by affinity chromatographies on the respective antigen-Sepharose columns as previously described for anti-Chl antibody (17). Antigen-antibody reaction. To prepare samples for the electrophoretie analysis, extracts of organs were incubated with the y-globulin fractions at 5°C for 3 h or overnight, then centrifuged for 5 min at 6,000g. The supernatant solutions were subjected to polyacrylamide gel electrophoresis. Quantitative determinations of acylphosphatase isozymes in extracts of the porcine and the chicken organs were carried out essentially by the method described previously (17). The extracts (20 ~1) were incubated with various amounts (2-16 fig) of the affinity-purified antibodies for 2 h at 5°C with shaking. Then, 2-10 pl of the 10% suspension of Staphylosorb (dry bacterial weight corresponded to 50-100 times the weight of the antibodies used) was added and the incubation was continued for another 15 min. After centrifugation, acylphosphatase activity remaining in the supernatant was assayed with benzoyl phosphate as substrate by the spectrophotometric method. The difference of activity between the extract not treated with the antibody and the supernatant after the treatment was assumed to be the activity of the corresponding isozyme bound to the respective antibody used. Because antiXh2 antibody decreased its specific activity during the affinity-purification, the crude y-globulin fraction (up to 180 pg) was used. The specificity of the antibodies for the respective isozymes had been ascertained by the enzyme-linked immunosorbent assay (14,17). The correctness of this quantification method had been checked by the determination of the isozymes after separation by CM-cellulose chromatography (17). Polyacrylamide gel electrophoElectrophoresis and activity stain. resis and activity stain for acylphosphatase were done as previously described (15). Briefly, the extracts were electrophoresed on an 11.25% gel (l-mm thick, g-cm long, pH 4.3) with a constant current of 20 mA for 4 h at 5°C. The gel was then incubated in 50 mM sodium acetate buffer (pH 5.3) containing 10 mM acetyl phosphate and 10 mM lead nitrate, at room temperature with gentle rocking until white bands of lead phosphate appeared. These bands were then stained dark brown in 1% ammonium sulfide solution.

ET AL.

RESULTS AND DISCUSSION Assignment of acylphosphatase activity bands by pretreatment with antibodies. In the previous paper (15), the acylphosphatase positive bands after gel electrophoresis of the extracts of several porcine organs were assigned to either the muscle isozyme or the testis isozyme by the coincidence of their electrophoretic mobilities with those of the respective purified isozymes. To confirm these assignments, the extracts were first treated with either the anti-porcine muscle acylphosphatase antibody or the anti-porcine testis acylphosphatase antibody, then subjected to the electrophoresis and the activity stain. The results shown in Fig. 1 support the above assignments. Of the three bands of muscle extracts, the lower two bands disappeared after treatment with anti-muscle acylphosphatase antibody, while the upper band, which is common to all of the organs and has the same mobility as the purified testis enzyme, disappeared after the treatment with anti-testis acylphosphatase antibody. The broad bands at the upper edge of the gel, which were newly formed after the antibody treatment, seem to be the soluble antigen-antibody complex. The formation of the enzymatically active soluble complex had been suggested by the observation that the activity of purified acylphosphatases could not be fully precipitated even by excess antibodies. For the quantitative determinations of the isozymes, therefore, the soluble complexes were precipitated with Staphylosorb (heat-killed Staphylococcus aureus) as described below. On the basis of the wide distribution of the upper isozyme reactive to the anti-testis acylphosphatase antibody, we propose to call it “the isozyme common to organs” or “the common type” for short. Thus, the three activity bands of the crude organ extracts were assigned either to the muscle type isozyme or to the common type isozyme, not only by the coincidence in the electrophoretie mobilities but also by the selective bindings to the isozyme-specific antibodies. Quantification of acylphosphatase isozymes in porcine organ extracts. For quantification of the isozymes, the densitometry of the activity bands appeared unreliable because the absorbance of the activity band was proportional to only a narrow range of the enzyme activity. Alternatively, the decrease of the enzymatic activity by the treatment with the isozyme-specific antibodies and Staphylosorb was measured. Staphylosorb was used to adsorb the soluble antigen-antibody complex. As shown in Fig. 2A, the isozyme-specific antibodies decreased the enzymatic activity of the extracts to a certain level, which remained constant while the amount of the antibody was further increased. The differences between the activities with excess antibody and without the addition of the antibodies represent activity of the corresponding isozymes bound to the respective antibodies used. The

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FIG. 1. Zymograms of acylphosphatase in porcine organ extracts before (A) and after (B and C) treatment with antibodies. (A) Extracts of porcine organs were subjected to polyacrylamide gel electrophoresis and the gel was stained for acylphosphatase activity. Only three representative patterns of the zymograms of porcine organs are shown. The full data have been presented in the previous paper (15). The volumes and the amounts of acylphosphatase activity of the extracts used for the electrophoresis were as follows: T, testis (13 ~1, 6.5 nkat); M, skeletal muscle (25 ~1,6.7 nkat); H, heart (25 ~1, 2.3 nkat). (B) and (C) Portions of the porcine organ extracts (6-64 ~1) containing 2-4 nkat of acylphosphatase activity were incubated with either 60 wcgof the anti-porcine muscle acylphosphatase antibody (subscript 1) or 90 Kg of the anti-porcine testis acylphosphatase antibody (subscript 2) for 3 h at 5”C, then centrifuged. The supernatants were subjected to the gel electrophoresis and the gel was stained for acylphosphatase activity. The organs are indicated by their init,ials as follows: T, testis; M, skeletal muscle; B, brain; H, heart; S, spleen; L, liver; K, kidney; E, erythrocyte. The specimens of heart, spleen, liver, and kidney were obtained from one pig and the other organs were from different pigs.

activity of the isozymes precipitated with excess antibodies are shown in Fig. 2B. The muscle type isozyme is predominant in the extract of skeletal muscle and represents about half of the activity in the extracts of heart muscle and liver. The common type isozyme is predominant in the extracts of testis, brain, spleen, kidney, and erythrocytes. The sum of the activity of the two isozymes almost coincides with the activity of each extract before the antibody treatment. The small differences between the activity of the extracts and the sum of those of the two isozymes in spleen, liver, and kidney are probably due to the acid phosphatase artifacts seen in the zymograms (15). Thus, the acylphosphatase activity in the porcine organ extracts is accounted for by the two acylphosphatase isozymes and by minor contributions of the acid phosphatase. Therefore, in the extracts of porcine organs examined, there are only two types of acylphosphatase isozyme, namely the muscle type and the common type.

The procedure Isozymes in dog, human, and rabbit. described above was employed to examine the classification of porcine acylphosphatase isozymes for applicability to other mammalian species. The zymograms of canine organs were similar to those of pig. Three representative patterns are shown in Fig. 3. Of the three bands, the upper band is common to all the organs and it disappeared after the treatment with the anti-porcine testis acylphosphatase antibody; the lower two bands mainly localizing in skeletal and heart muscles disappeared after the treatment with the antiporcine muscle acylphosphatase antibody. However, canine kidney gave a unique band, which migrated to the lower position like the muscle type isozyme but disappeared after the treatment with the anti-porcine testis acylphosphatase antibody, not with the anti-muscle acylphosphatase antibody, The acylphosphatase zymograms of human organs are shown in Fig. 4. In this case, although the total number of bands is three, the relative position of the two iso-

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FIG. 2. Quantification of acylphosphatase isozymes in porcine organ extracts. The porcine organ extracts were incubated with various amounts of the isozyme-specific antibodies and then with Staphylosorb. After centrifugation, acylphosphatase activity remaining in the supernatant was assayed as described under Materials and Methods. (A) Titration of acylphosphatase isozymes with the affinity-purified antibodies: l , anti-porcine muscle acylphosphatase antibody; 0, anti-porcine testis acylphosphatase antibody. (B) Contents of acylphosphatase isozymes in various organs, determined as shown in (A). The open columns indicate activity of the common type isozyme; the hatched columns the activity of the muscle type isozyme; the horizontal bars, the activity of the extract before the antibody treatment. The organs are indicated by their initials as described in the legend to Fig. 1.

zymes on the gel are contrary to those of pig and dog. The lowest band appeared to be the common type and the upper two bands the muscle type on the basis of the reactivity to the antibodies as most clearly shown for the heart muscle extract (Fig. 4B). The band of erythrocyte and the lowest band of brain seem to be the common type, although the band is broad for erythrocyte and the reaction to the antibody was not complete for brain. This assumption was supported by the similarity in the amino acid composition of acylphosphatase of human

B BI B2K KI K2 M MlM2 FIG. 3. Zymograms of acylphosphatase in canine organ extracts before and after treatment with antibodies. Portions (25 ~1) of organ extracts of a male dog were incubated for 3 h at 5°C with 135 pg of the anti-porcine muscle acylphosphatase antibody, subscript 1, or 140 fig of the anti-porcine testis acylphosphatase antibody, subscript 2, then centrifuged. The supernatants were subjected to gel electrophoresis and the gel was stained for acylphosphatase activity. The extracts (25 ~1) contained the following amounts of acylphosphatase activity: B, brain (3.7 nkat); K, kidney (1.8 nkat); M, skeletal muscle, (8.2 nkat).

erythrocyte (lo), equine brain (ES), and porcine testis (14). Characteristic features of the human zymogram are the dense staining of the muscle type band in brain and the fastest moving band in liver which seems to be the muscle type. The acylphosphatase zymograms of rabbit organs are shown in Fig. 5. Rabbits gave two main bands. The upper band is common to all the organs and the lower band is dense for muscle extract. The lower broad band of the muscle extract disappeared after the treatment with the anti-porcine muscle acylphosphatase antibody. Therefore the band appeared to be the muscle type isozyme. On the other hand, the upper common band of testis was not diminished by either the anti-muscle or the anti-testis acylphosphatase antibodies. Also for the other organ extracts, the lower band disappeared after the treatment with the anti-porcine muscle acylphosphatase antibody, but the upper band was not diminished by either of the two antibodies (data not shown). These results suggest that the anti-porcine testis acylphosphatase antibody does not bind to the rabbit common type isozyme. This low interspecies cross-reactivity of the anti-porcine testis acylphosphatase antibody compared with the antiantibody suggests porcine muscle acylphosphatase larger differences in amino acid sequences of the common type isozymes than those of the muscle type isozymes of pig and rabbit. The rabbit common type acylphosphatase has not yet been purified. For rats and guinea pigs, the zymograms showed only one main band common to organs (data not shown). However, the anti-porcine muscle acylphosphatase anti-

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in human organ extracts before and after treatment with antibodies. Portions (25 ~1) of human organ FIG. 4. Zymograms ofacylphosphatase extracts were treated with the antibodies, then subjected to electrophoresis and staining in the same way as described in the legend for Fig. 3. (A) Zymograms of the human organ extracts before the antibody treatment plus the muscle and erythrocyte extracts after the antibody treatment. The antigen-antibody complex was removed by Staphylosorh from the mixture of erythrocyte extract and antibody before electrophoresis. The lowercase letters indicate different individuals, i.e., a, a 73.year-old male who died of colon cancer; h, a 82-year-old male who died of cancer in oral cavity; c, a 52-year-old female who died of Huntington’s chorea; d, a healthy 43.year-old male. M of a, musculi iliopsoas (6.8 nkat); M ofh, musculi rectus ahdominis (9.2 nkat); B, brain (3.2 nkat); K, kidney (0.6 nkat); L, liver (1.3 nkat); H, heart (1.4 nkat); E, erythrocyte (1.8 nkat). (B) The other organ extracts before and after the antibody treatment. The subscripts 1 and 2 are as described in the legend to Fig. 3.

body with Staphylosorb precipitated at most 80 and 60% of acylphosphatase activity in the extracts of rat skeletal muscle and heart muscle, respectively. From guinea pig brain and testis extracts, the anti-porcine testis acylphosphatase antibody with Staphylosorb precipitated at most 70 and 60%, respectively, of acylphosphatase activity. Therefore it seems likely that each of these animals have the two isozymes of the same electrophoretic mobility. An electrophoretic or chromatographic system of higher resolution is needed for direct demonstration of the two isozymes in these animals.

MjM2M B H L K T Tj T2 FIG. 5. Zymograms of acylphosphatase in rabbit organ extracts. Organs of a 2-month-old male rabbit were extracted with 3 vol of water. Twenty-five microliters of the extracts containing the following amounts of acylphosphatase activity were subjected to gel electrophoresis followed by activity staining: M, muscle (6.2 nkat); B, brain (2.8 nkat); H, heart (2.2 nkat); L, liver (1.1 nkat); K, kidney (0.9 nkat); T, testis (1.5 nkat). The electrophoresis was performed for 7 h. The subscripts 1 and 2 are as described in the legend to Fig. 3.

Isozymes in birds. The acylphosphatase zymograms of birds are shown in Fig. 6A. For chicken, only the zymogram of skeletal muscle is shown as a representative. Of the three bands in total, the upper two bands mainly localized in the skeletal and heart muscles disappeared after the treatment with the anti-Chl antibody, while the lowest band common to all the organs disappeared after the treatment with the antiCh2 antibody (( 15) and data not shown). Therefore the upper two bands are Chl and the lowest band is Ch2. These assignments were confirmed by the coincidence of the mobility with purified Chl and Ch2 (15). On the basis of the tissue distribution and the homology of the primary structure between Chl and the mammalian muscle type enzymes and that between Ch2 and the human erythrocyte enzyme (13), we conclude that Chl corresponds to the muscle type and Ch2 corresponds to the common type. Quantification of acylphosphatase isozymes in chicken organ extracts are shown in Fig. 7. The sum of the activity of Chl and Ch2 almost coincides with the activity of each extract before the antibody treatment. Therefore, there are only two types of acylphosphatase isozyme, namely the muscle type (Chl) and the common type (Ch2), in the extracts of chicken organs examined. This is the same conclusion as for the porcine organs. However, the localization of the two types of isozyme in chicken is not so extreme as in the porcine organs, e.g., the common type forms a relatively larger part of the activity in the chicken skeletal muscle than in the porcine skeletal muscle. The zymograms of pigeon organs and crow muscle show three bands in total (Fig. 6A). The upper two bands

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BoBlB2 Moo;4; KoKI K2lykM’42 FIG. 6. Acylphosphatase zymograms of birds. (A) Organ extracts from a pigeon, a crow, and a chicken (40 ~1 for pigeon liver and 25 ~1 for the other organs) were subjected to gel electrophoresis and the gel was stained for acylphosphatase activity. The extracts contained the following amounts of acylphosphatase activity. Pigeon organ extracts: Mb, breast muscle (9.3 nkat); Ml, leg muscle (8.0 nkat); H, heart (5.7 nkat); K, kidney (3.0 nkat); B, brain (2.7 nkat); L, liver (1.9 nkat). Crow organ extracts: Mb, breast muscle (4.8 nkat); Ml, leg muscle (6.2 nkat); Ch, chicken leg muscle (5.2 nkat). (B) Portions of the extracts (25 ~1) were incubated overnight at 5°C with 100 fig of the following y-globulins: subscript 0, normal y-globulin; subscript 1, the anti-Chl antibody; subscript 2, the antiXh2 antibody. Larger amounts of antibody were used for B, (180 pg) and M, (150 fig) of pigeon. After centrifugation the supernatants were subjected to gel electrophoresis and the gel was stained for acylphosphatase activity

mainly localized in the skeletal and heart muscles were assigned as the muscle type isozyme on the basis of their disappearance after the anti-Chl antibody treatment (Fig. 6B). However, the lowest band common to the organs was not diminished by the anti-Ch2 antibody treatment. It seems likely that the common type acylphosphatases of pigeon and crow are too different in amino acid sequence from Ch2 to cross-react with the anti-Ch2 antibody. Thus, higher divergences of amino acid sequence in the common type isozymes than in the muscle type isozymes among birds are suggested, compared to that of mammals seen above. The common types are also variable in their electrophoretic mobilities, from the lowest mobility of the porcine common type to the highest mobility of the pigeon

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FIG. 7. Concentrations of acylphosphatase isozymes in chicken organs. Activities were determined in a similar manner as shown in Fig. 2A. Open column, activity of the common type isozyme absorbed by antiCh2 antibody; hatched column, activity of the muscle type isozyme absorbed by anti-Chl antibody; horizontal bar, activity of the extract before the antibody treatment. The organs are indicated by their initials as described in the legend to Fig. 6 plus T, testis; S, spleen; G, gizzard.

common type. Because the molecular weight of the enzymes so far purified is fairly constant, the variable mobilities are due to variable basicity of the common type isozymes. The homology of amino acid sequences between the human erythrocyte enzyme and Ch2 is 62%, while that between the human skeletal muscle enzyme and Chl is 82% (13). These data support the above presumption. In conclusion, the mammals and the birds so far examined have only two main types of acylphosphatase, namely, the common type and the muscle type, although each type may consist of two or more structurally similar proteins. There actually were minor variants in the canine kidney and the human liver, which bind to the isozyme-specific antibodies but have different electrophoretie mobilities from those of the isozymes in other organs. Moreover there is always a possibility that a single activity band contains two similar but slightly different proteins, which can bind to the same isozymespecific antibody. In fact, we have isolated two molecular forms of the common type acylphosphatase from both testis and brain of pig, which are slightly different in amino acid sequence (to be published). As for intracellular distribution of acylphosphatase, we have shown that both Chl and Ch2 of chicken muscle exist in the cytosol, not in mitochondria (17). Although membrane-bound acylphosphatase activity has been shown in rabbit skeletal muscle, it is distinct from the soluble acylphosphatases (19). Acylphosphatase is generally high in testis, skeletal muscle, brain, and heart muscle, but low in spleen, liver, kidney, and erythrocytes. Among the high-content acyl-

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phosphatase organs, skeletal muscle was uniquely rich in the muscle type isozyme, testis and brain were rich in the common type isozyme, and heart muscle contained both isozymes in similar amounts. From these results it seems that acylphosphatase is needed for the organs consuming much energy and that the muscle type isozyme is more suitable than the common type isozyme for some characteristic feature of skeletal muscle metabolism. Further work is needed to clearly determine the physiological roles of the two acylphosphatase isozymes.

-7 UF

A., and Ramponi, Camici, G., Manao,

3. Kizaki, T., Takasawa, T., Mizuno, Biochem. (Tokyo) 97, 1155-1161.

Y., and Shiokawa,

4. Manao. G., Camici, G., Cappugi, G., Stefani,

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M., Liguri,

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G. (1984) Mol. Biol. Med. 2,369-

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T., and Shio-

A., Stefani,

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241, 418-

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11.

G. (1980) J.

G., Modesti,

M., Cappugi, G., and Ramponi,

We thank Dr. Lafayette H. Noda, Meriden, NH 03770, for critically reading the manuscript, Dr. Yuko Kikuchi, School of Medicine, Hokkaido University, for providing the human tissue specimens, and D. V. M. Sachiya Kudo, Ebetsu Meat Inspection Center of Hokkaido, for providing the porcine organs. This work was supported in part by the Special Grant-in-Aid for Promotion of Education and Science in Hokkaido University provided by the Ministry of Education, Science and Culture.

G., and Ramponi,

G. (1985) Arch. Biochem. Biophys.

5’ pugi, G., and Ramponi, G. (1986) Ital. J. Biochem. 35,1-15. 6, Manao, G., Camici, G., Modesti, A., Liguri, G., Berti, A., Stefani,

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ISOZYMES

424.

ACKNOWLEDGMENTS

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17.

105,173-177. I~., 18. Stefani, M., Berti, A., Camici, G., Manao, G., Degl’lnnocenti, Prakash, G., Marzocchini, R., and Ramponi, G. (1988) FEBS Lett.

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