1-Methyladenosine ribohydrolase in the starfish ovary and its relation to oocyte maturation

Copyright All rights

0 1972 by Academic Press, Inc. of reproduction in any form rererocd

Experimental

l-METHYLADENOSINE AND

Cell Research 75 (1972) 79-88

RIBOHYDROLASE

ITS RELATION H. SHIRAI’

IOcean Research and 230dega Marine

Institute, Laboratory,

TO OOCYTE

IN THE

STARFISH

OVARY

MATURATION

and H. KANATANI’~2

University of Tokyo, Nakdno-ku, and Department of Zoology, Berkeley, Calif. 94720, USA

Tokyo University

164, Japan, of California,

SUMMARY Maturation of isolated oocytes was induced in the supernatant of an incubation mixture consisting of ovarian fragments, or their extract, and sea water containing 1-methyladenosine in the starfishes, Patiria miniata, Asterias amurensis, Asterinapectinifera. Such maturation-inducing activity was ascribed to the production of an active substance derived from 1-methyladenosine by the action of an enzyme present in ovarian tissue. With column chromatography and thin layer chromatography, the active substance was identified as I-methyladenine. The enzyme does not seem to be an ordinary adenosine ribohydrolase, but a new specific enzyme to be called l-methyladenosine ribohydrolase, which splits I-methyladenosine into I-methyladenine and ribose. This enzyme was successfully precipitated from the supernatant of ovarian wall homogenate by adding ammonium sulfate at 0.45 saturation. The optimal pH of the enzyme was found to be about 7.5. Its molecular weight was estimated as 96 000 and its isoelectric point was determined as pH 5.1. Ripe ovaries containing fullgrown oocytes showed higher 1-methyladenosine ribohydrolase activity than that of young ovaries, suggesting that the enzyme activity is dependent on the ripeness of the gonad and that the enzyme is related to both oocyte maturation and spawning in starfishes through its ability to produce 1-methyladenine. A gonad-stimulating hormonal peptide, which induces the production of 1-methyladenine in starfish ovaries, had no effect on the activity of 1-methyladenosine ribohydrolase. This suggests that the gonad-stimulating substance acts in some earlier stage of 1-methyladenine biosynthesis.

1-Methyladenine (I-MA), produced in the maturation [3], this substancefails to induce ovary under the influence of gonad-stimulat- maturation when applied to isolated oocytes ing substance(GSS) releasedfrom the nervous in sea water [3, 71. Oocytes within ligated tissue, has been known as an inducer of ovarian fragments undergo maturation when oocyte maturation and spawning in starfishes treated with l-MAR in vitro 131.These find[l-6]. This substancebrings about maturation ings have led us to assume the presence in of oocytes isolated in sea water as well as the ovarian tissue of an enzyme which splits those in ligated ovarian fragments. When biologically inactive l-MAR into active injected into the coelomic cavity it induces l-MA and ribose. spawning of oocytes which are undergoing This report deals with this enzyme, to be maturation [2,3]. On the other hand, although called I-methyladenosine ribohydrolase (linjected 1-methyladenosine (l-MAR) into MARase), and its possible significance in the coelomic cavity induces spawning, and oocyte maturation in starfishes. oocytes discharged into sea water complete 6-

721811

Exptl

Cell Res 75 (1972)

80

H. Shirai & H. Kanatani

MATERIALS

AND METHODS

Patiria miniata was used in early stages of this work when one of the authors (H. K.) was at the Bodega Marine Laboratory, University of California, in 1969. Asterias amurensis was used in Japan. On the other hand, the main material of the present investigation was Asterina uectinifera. which is closely rehued to Patiria miniita, collected from Hashirimizu (Kanagawa) and Inubozaki (Chiba) and kept in laboratory aquaria supplied with circulating- cold sea water at the Ocean Research Institute, University of Tokyo.

Reagents I-Methyladenosine (l-MAR), 1-methyladenine (lMA) and 5’-adenosine were purchased from Sigma Chemical Co. The water (DDW) used in most of the experiments was deionized on a column of ionexchangers and then glass-distilled. The sea water used was modified Van? Hoff’s artificial sea water (ASW) of the following composition: 2.7% NaCl, 0.07 % KC], 0.10 % CaCl,, 0.34 % MgCl, and 0.21 % MgSOI. Its pH was adjusted with borate buffer (final concentration, 0.02 M or 0.05 M) to 8.2-8.3.

Preparation of GSS solution To obtain a solution of gonad-stimulating substance (GSS), lyophilized radial nerves [S] were homogenized in a -small amount of cold DDW (40 mg/ml) and centrifuged at 30 000 g for 50 min at 3°C. The supernatant was heated for 15 min in a boiling water bath and then centrifuged at 30 000 g for 30 min. The supernatant was gel-filtrated on a Sephadex G-25 column in 0.1 M sodium chloride. Fractions which showed spawning-inducing activity, when checked with isolated ovarian fragments, were pooled and frozen. This solution was diluted with DDW to an appropriate concentration before use. The amount of GSS was expressed as pg of original dry nerve per ml.

Preparation of I-MARase A routine method for obtaining 1-MARase, which was developed during the course of the investigation described in the vresent vaver. was as follows. Ovaries of Asterina pectkfera were treated with ASW containing 10-O M I-MA (200 to 250 mg/ml ovarian fragments) for 2 h to induce spawning and the ovarian wall material containing a small amount of oocytes was thorouahlv washed with ASW. This was homogenized in cold 0.2 M acetate buffer, pH 6.5 (1 g of original ovary/4 ml) and centrifuged at 20 000 g for 1 h at 3°C. To the supernatant, 0.2 N acetic acid was added to give a pH of 4.6, and then solid ammonium sulfate was added to 0.30 saturation with continuous stirring for 15 min. After standing for at least 1 h the sample was centrifuged at 15 000 g for 30 min. The precipitate, which has little enzymic activity, was discarded. Ammonium sulfate was added to the supernatant to 0.45 saturation and the sample was centrifuged as before. The precipitate Exptl Cell Res 75 (1972)

was washed with 0.2 M acetate buffer (pH 4.6) containing ammonium sulfate at 0.45 saturation and centrifuged again. The precipitate was dissolved in a small amount of DDW and gel-filtrated on a Sephadex G-25 column (5 x 35 cm) in 0.02 M sodium chloride, which was also used as eluant. The flow rate was 35 ml/h and the fraction size was 5 ml. A sample (0.1 ml) taken from each fraction was incubated for 1 h at 25°C with 0.1 ml of 10v3 M l-MAR, 0.1 ml of 0.2 M borate buffer (pH 7.5) and 0.1 ml of a solution containing 100 mM NaCI, 100 mM KC1 and 50 mM MgC12. This was diluted with ASW and assayed with isolated oocytes to determine its maturation-inducing activity. Fractions showing high activity were pooled and designated as 0.45 AS fraction.

Assay of enzyme activity Since l-MAR is split by 1-MARase into l-MA and ribose, assay of the enzyme activity can be performed by determining the amount of either ribose or l-MA produced during incubation. The composition of the reaction mixture and the conditions of incubation will be stated in each exaeriment. For determining the amount of ribose, 0.2 ml of 0.3 N barium hydroxide and 0.2 ml of 5 % zinc sulfate were added to 1 ml of the reaction mixture. After 10 min this was centrifuged at 3 000 rpm for 10 min and the supernatant was filtered through filter paper. The ribose content of 1 ml of the filtrate was determined according to the method of Somogyi [9, lo]. l-MAR was added to the incubation mixture which had not contained it, after adding deproteinizing reagents, and the filtrate obtained from this was used as blank. The amount of l-MA was estimated bv bioassay. After incubation the reaction was stopped either by heating the reaction mixture at 100°C for 15 min or by adding 5 % trichloroacetic acid. A part of the mixture was diluted serially with ASW to various concentrations and usually about 150 to 200 isolated oocytes of Asterina pectinifera were placed in 0.3 to 0.5 ml of test solution. The rate of oocyte maturation as revealed by breakdown of the germinal vesicle was observed after 1 h. The amount of l-MA produced was estimated using authentic l-MA dissolved in ASW at various concentrations as reference standard in every case. Bioassay was found to be more sensitive than ribose determination. The enzyme activity is expressed as ,ng of either ribose or l-MA liberated per mg of protein. Protein determination was carried out according to the method of Lowry et al. [ll], using bovine serum albumin (Nutritional Biochemicals) as reference standard.

Thin layer chromatography The method for thin layer chromatography, to determine whether the active substance produced from l-MAR by enzymic hydrolysis is l-MA, was the same as that described in our previous paper [23].

Molecular weight determination Estimation of the molecular weight of l-MARase was based on the method of Andrews [12], using a Sephadex G-150 column (1.4 x 80 cm) equilibrated

I-Methyladenosine Table 1. Effect miniata

of

Concentration of incubation mixture Oocyte maturation,

%

incubation mixture

of ovary and I-MAR

ribohydrolase

in starfish

on oocyte maturation

in Patiria

l/8

l/16

l/32

l/64

1/12s

control0

lOOfOb

99+1

76klO

25ill

9+2

9+2

a Oocvte maturation in supernatant of incubation mixture without b Mean &SE. of six experiments. with 0.2 M acetate buffer (pH 6.5), which was also used as eluant. An enzyme sample precipitated with ammonium sulfate at 0.45 saturation was dissolved in 1.5 ml of the same buffer and applied to the column at 3°C. The fraction size was 2 ml and the flow rate was: 15 ml/h. The site of elution of the enzyme was determined as follows. After adjusting the pH of a sample taken from each fraction to 7.5 with 0.2 M borate buffer (pH 9.0), 0.9 ml of each sample was incubated with 0.1 ml of 10e2 M l-MAR (pH 7.0) for 1 h at 25°C and then assayed for its maturation-inducing activity with isolated oocytes after dilution with ASW. Ovalbumin (2 x crystallized, Mann Research Lab.), bovine serum albumin (Mann) and Rhus-lactase (gift from Mr Makino) [13] were used as reference standards for molecular weight determination. The sites of elution of ovalbumin and serum albumin were determined by the method of Lowry et al. [ll], and that of lactase by ultraviolet absorption at 280 nm.

fsoelectric point The isoelectric point of 1-MARase was determined by the isoelectric focusing method [14], using 1 % Ampholine carrier ampholytes (pH 3 to 6) (LKBProducter AB) in a 110 ml column at 3°C. Isoelectric focusing was performed at 700 V for 72 h. After isoelectric focusing, fractionation (2 g each) and pH recording were carried out simultaneously with a fraction collector connected with a pH meter and a polyrecorder. The site of elution of the enzyme was determined by bioassay after incubation of the samples taken from each fraction with l-MAR.

RESULTS Effect of incubation mixture of ovary and I-MAR on oocyte maturation Isolated ovarian fragments of Patiria miniata were incubated in ASW containing 1O-4 M l-MAR (200 mg/ml ovary) for 3 h at 25°C and then centrifuged at 4 000 rpm for 10 min. The supernatants were assayed with isolated oocytes by serially diluting them twice with ASW.

81

l-MAR.

The result showed that the maturationinducing substance (MIS) was produced in the incubation mixture of ovaries and l-MAR (table 1); l-MAR seemed to be converted to MIS in the presence of ovarian tissue. In the absence of l-MAR, the ovaries did not produce the active substance (table 1, control). A similar result was also obtained with Asterina pectinifera; in this case S4O/b maturation occurred even at 200-fold dilution under similar experimental conditions. In the next experiment, the effect of ovarian extract was investigated. Ovaries (10 g) of Patiria were incubated in 100 ml of ASW containing 1O-5 M I-MA for 1.5 h at 23°C to induce shedding of most of the oocytes. The spent ovaries were thoroughly washed with ASW and then pressed on filter paper in order to remove the remaining yolky material. The ovarian wall material thus obtained was homogenized in 5 ml of cold deionized water and centrifuged at 14 000 g for 50 min at 2°C. The supernatant was used as a crude enzyme solution. Mixtures consisting of the following components were incubated for 3 h at 25°C: (A) Supernatant 0.5 ml, 2 x 1O-2 M l-MAR 0.1 ml, GSS solution (250 pugof dry nerve/O.5 ml) 0.2 ml, ASW (0.02 M borate-buffered, pH 8.0) 0.4 ml, DDW 0.3 ml; (B) supernatant 0.5 ml, l-MAR 0.1 ml, ASW 0.4 ml, DDW 0.5 ml; (C) supernatant 0.5 ml, GSS 0.2 ml, ASW 0.4 ml, DDW 0.4 ml; (D) GSS 0.2 ml, l-MAR 0.1 ml, ASW 0.4 ml, DDW 0.8 ml; (E) l-MAR 0.1 ml, ASW 0.4 ml, DDW 1 ml; Exptt

Cell Res 75 (1972)

82

H. Shirai & H, Kanatani

Table 2. Effect of incubation mixture of ovarian extract and I-MAR on oocyte maturation in Patiria miniata Components of incubation mixture

Amount of MIS’

(A) (B) (C) (D) (E) (F)

7.98 7.21 0.15 0.02 0.01 0.19

Enzyme + 1-MAR + GSS Enzyme + l-MAR Enzyme + GSS GSS + l-MAR l-MAR Enzyme

a pg l-MA

equivalent per ml incubation

mixture.

(F) supernatant 0.5 ml, ASW 0.4 ml, DDW 0.6 ml. After incubation these mixtures were serially diluted with ASW and assayed for maturation-inducing activity with isolated oocytes (table 2). The results showed mixtures (A) and (B) to be very effective, as compared with the other mixtures. These results suggest that ovary extract contains some enzyme which converts l-MAR to MIS, and that GSS has little effect on this reaction. In order to determine the optimal pH for the activity of this crude enzyme solution, 0.5 ml of the supernatant of ovarian wall homogenate of Patiria obtained in the same way was incubated with 0.1 ml of 1O-2 M l-MAR and 0.4 ml of 0.1 M phosphate buffer, 0.2 M borate buffer or 0.1 M TrisHCl buffer at various pHs for 3 h at 30°C. The reaction mixtures were ice-cooled, serially diluted twice with ASW and assayed with isolated oocytes. From the activity revealed by bioassay, the optimal pH of Patiria enzyme was found to be between 7.0 and 7.5, and none of the buffers appeared to exert any serious inhibition. A similar experiment, conducted three times using ovary extracts (supernatant of the homogenate centrifuged at 75 000 g for 50 min) obtained from different females of Asterina pectinifera, revealed that the optimal pH for Asterina enzyme is about Exptl

Cell Res 75 (1972)

pH 7.5. In these cases0.5 ml of enzyme with 0.9 ml of 0.1 M phosphate buffer and 0.1 ml of 1O-2 M l-MAR was incubated for 2 h at 25°C and the reaction was stopped by heating at 100°C for 10 min. In order to determine whether the active substance (MIS), produced in the incubation mixture of I-MAR and the supernatant of the ovarian wall homogenate, is l-MA, the following experiment was performed. The supernatant (0.5 ml) of ovarian wall homogenate of Asterina pectinifera (250 mg/ml of ovary) was incubated with 0.9 ml of 0.1 M phosphate buffer (pH 7.5) and 0.1 ml of 3 x 1O-3 M l-MAR for 2 h at 25°C. Then, 0.5 ml of the reaction mixture was applied to a Sephadex G-15 column equilibrated with ASW, which was also used as eluant. The maturation-inducing activity of each fraction was assayedwith isolated oocytes. 0.5 ml of 1.34 x 1O-4M l-MA was also applied to the same Sephadex column under the sameconditions, and the activity of each fraction was assayed.

loo-

Es-

50 0

25,

I-

I

I

10

20

30

40

50

60

70

1. Abscissa: fraction number; ordinate: oocyte maturation ( %). Fractionation of incubation mixture of l-MAR and I-MARase of Asterina ovary (A) and of authentic l-MA (B) on a Sephadex G-15 column (0.95 x 97 cm) in ASW (0.05 M borate buffer, pH 8.5). The fraction size was 2 ml. The assay was done with fractions diluted 50 times in both cases.

Fig.

I-Methyladenosine

ribohydrolase

in starfish

83

Fig. 1 shows that the active substance produced in the incubation mixture was eluted in the same fraction as that of l-MA, suggesting that the active substance produced in the reaction mixture is probably l-MA, since this substance has a tendency to be absorbed by Sephadex to some extent, and its elution site is rather specific as compared with common, small-molecular salts. That the active substance produced by the enzymic hydrolysis is l-MA was confirmed by thin layer chromatography. The active fractions obtained from fractionation on a Sephadex G-10 column equilibrated with 0.2 M pyridine acetate buffer, pH 8.4, which was also used as eluant, were pooled and concentrated to dryness. The sample was dissolved in a small amount of DDW and applied to microcrystalline cellulose plates (Aviccl SF) with or without the addition of either authentic l-MA or l-MAR. The samples developed with three different solvent systems [23] gave one distinct spot corresponding to that of I-MA (fig. 2). Effect of testis extract on production of MIS in the presence of l-MAR Testes of Asterina pectinifera were homogenized in cold DDW (250 mg/ml) and centrifuged at 57 000 g for 50 min at 3°C. The supernatant (0.5 ml) was incubated with 0.3 ml of 0.1 M phosphate buffer (pH 7.5), 0.1 ml of 3.3 x 1O-3 M l-MAR and 0.1 ml of DDW (total 1.0 ml) for 2 h at 25°C. The incubation mixture showed maturationinducing activity when assayed with isolated oocytes. Parallel assays with authentic l-MA at various concentrations revealed that the MIS produced in the incubation mixture (1 ml) was equivalent to about 9 pug of l-MA. On the other hand, the same experiment using ovary extract showed that the MIS produced in the incubation mixture was equivalent to about 16 pg of l-MA. When

Fig. 2. Thin layer chromatography of the active fraction on Avicel SF plate. Solvent system: isopropanol/ hydrochloric acid/water (65 : 16.7: 18.3 by volume). Detection of the spots was performed with ultraviolet light (254 nm). A, authentic l-MAR alone; B, active fraction with authentic l-MAR; C, active fraction alone; D, active fraction with authentic I-MA; E, authentic l-MA alone.

0.1 M Tris-HCl buffer was used instead of phosphate buffer, production of MIS from l-MAR in the presence of testis extract was also observed. These experiments show that the enzyme is also present in the testis. Enzyme activity in relation to ripenessof ovary In order to determine whether the ripeness of the ovary has some relation to the enzyme activity, two groups of ovaries, small and Exptl

Cell Res 75 (1972)

84 H. Shirai & H. Kanatani well-developed, were used as enzyme source. The well-developed ovaries have large alveoli and contain mainly fullgrown oocytes with large germinal vesicles. The follicles around the oocytes are thin and consist of a single cell layer. Young ovaries have slender alveoli and contain a large number of small oocytes surrounded by thick and compact follicles. Enzyme solution, consisting of the supernatant of an ovarian homogenate (250 mg wet ovary/ml DDW), was prepared from four groups each of young and well-developed ovaries of Asterina. The enzyme solution (0.1 ml) was incubated with 0.6 ml of 0.1 M phosphate buffer (pH 7.5), 0.1 ml of 1O-2 M l-MAR and 0.2 ml of DDW at 20°C for 10 min; the reaction was arrested with 0.2 ml of 10% trichloroacetic acid, The enzyme solutions were diluted with ASW to various concentrations and assayed with isolated oocytes. It was found that the incubation mixture of the enzyme from well-developed ovary contained 0.030* 0.005 ug/ml of l-MA (Mean* S.E.), whereas that of the enzyme from young ovaries contained 0.015 k 0.003 pg/ml, indicating that the enzyme activity (amount of enzyme) increases with the growth of the ovary.

Enzyme activity of the precipitate at 0.45 saturation of ammonium sulfate In order to remove nucleosides, nucleotides and sugars which might be contained in the supernatant of ovarian wall homogenate, the enzyme was precipitated with ammonium sulfate. Ovaries (32.6 g) of Patiria miniata were treated for 2 h with ASW containing 2 x 1O-6 M l-MA and then washed with sea water. The ovarian wall material was separated from the discharged oocytes by filtration with gauze. Deionized water (16.3 ml) was added and the ovarian wall material was homogenized. The homogenate was centriExpil

Cell Res 75 (1972)

fuged at 56 000 g for 50 min at 2°C. Solid ammonium sulfate was added to the supernatant (18 ml) to give 0.45 saturation at 2°C. After 1.5 h this preparation was centrifuged at 14 000 g for 30 min. The precipitate was dissolved in 5 ml of 0.05 M phosphate buffer (pH 7.0) and gel-filtrated on a Sephadex G-25 column (1.5 x 25 cm) equilibrated with the same buffer, at 3°C. Fractions of the sample containing the first large peak of protein were pooled and used as enzyme solution. A mixture of 1 ml of the enzyme solution, 0.8 ml of 0.1 M phosphate buffer (pH 7.0) and 0.2 ml of 1O-2 M I-MAR was incubated for 2 h at 25°C. After incubation, the maturation-inducing activity was assayed with isolated oocytes by two serial dilutions with ASW. This incubation mixture was found to be very effective; 88% maturation was observed even after 32 770-fold dilution, whereas both the incubation mixture without enzyme solution (phosphate buffer and lMAR alone) and that without I-MAR (phosphate buffer and enzyme solution alone) had little effect in inducing oocyte maturation. Further precipitation of the same supernatant sample with ammonium sulfate up to 0.95 saturation and similar treatment and bioassay of the precipitate showed the protein fraction to have very weak activity in inducing oocyte maturation as compared with that of the fraction precipitated by 0.45 saturated ammonium sulfate; after incubation maturation-inducing activity was observed only up to 64-fold dilution. In another experiment, supernatani of an ovarian homogenate of Asterias amurensis was fraciionated by successively adding solid ammonium sulfate at 0.30, 0.45, 0.55 and 0.75 saturation. It was found that most of the enzyme was precipitated by adding ammonium sulfate at 0.45 saturation to the supernatant of 0.30saturated sample.

I-Methyladenosine Some properties

Effect of the enzyme on adenosine. The substrate specificity of this enzyme was investigated using adenosine as substrate in order to know whether the enzyme is specific for l-MAR. A similar preparation of 0.45 AS - 3.0 .

20:

/\

./

lo./I\ .

0

0

. -2.0 0

o

in starfish

85

of the enzyme

Optimal PH. The optimal pH of the enzyme was determined with 0.45 AS Fraction (see Materials and Methods). The fraction (0.4 ml) was incubated for 100 min at 25°C with 0.4 ml of 0.2 M borate buffer at various pHs, 0.1 ml of 0.1 M l-MAR (pH 7.0) and 0.1 ml of a solution containing 100 mM NaCl, 100 mM KC1 and 50 mM MgCl,. The reaction was arrested by adding barium hydroxide and zinc sulfate. The optimal pH of the enzyme was found to be about 7.5 with respect to the liberation of ribose (fig. 3). That the optimal pH of the enzyme is 7.5 was also shown when the reaction was arrested by heating the incubation mixtures and assaying for the production of maturation-inducing substance, l-MA (fig. 3). The linearity of the reaction was determined by the amount of ribose liberated at pH 7.5 under the same conditions. Release of ribose from l-MAR occurred at the same rate up to about 160 min under the conditions used (fig. 4).

.

ribohydrolase

0 -1.0 0

Fig. 3. Abscissa: pH; ordinate: amount of ribose liberated (yg ribosejhjmg protein) (fefr) and MIS liberated (pg 1-methyladenine equivalent/h/mg protein) (right). Optimal pH of I-MARase.

40

80

120

160

4. Abscissa: incubation time (min); ordinate: amount of ribose liberated (,ug ribose/mg protein). Linearity of enzymic liberation of ribose from lMAR.

Fig.

Fraction was used as enzyme solution. The composition of the reaction mixture and the conditions of incubation were the same as those employed for determining the optimal pH, except that 5 x lO-2 M adenosine was used as the substrate instead of l-MAR. As a result, it was found that 10.3+ 1.3 pg of ribose was liberated per h per mg protein from l-MAR and 0.320.2 ,ug from adenosine, suggesting that the enzyme is not an ordinary purine nucleosidase or adenosine ribohydrolase [ 151 which splits adenosine into adenine and ribose, but a new specific enzyme to be called 1-methyladenosine ribohydrolase (1-MARase). Effect of heating on I-MARase activity. A brief survey of the effect of heating on the enzymic activity was conducted using an enzyme preparation obtained from gelfiltration of 0.45 AS Fraction on a Sephadex G-150 column. It was found that the activity decreased somewhat after standing for 1 h at 35°C and after heating at 55°C for 1 h the activity decreased to about one-tenth. The enzyme activity did not change after standing for 1 h at 25°C. Molecular weight. The molecular weight of 1-MARase of Asterina pectinifera was determined with gel-filtration on a Sephadex G-l 50 Expti

Cell Res 75 (1972)

86 H. Shirai & H. Kanatani

loo4\

I 3

. ’ . . ‘. . I I’ 4 5 6 78910

20

30

Fig. 5. Abscissa: molecular weight (X 10”); ordinate: elution volume (ml). Molecular weight determination of I-MARase of Aiterina pectinifera by gel-filtration on a Sephadex G-150 column. A, ovalbumin; B, bovine serum albumin; C, Rhus-lactase; ---, elution volume of I-MARase.

column using ovalbumin, bovine serum albumin and Rhus-lactase as reference standards. The sample of 1-MARase used was 0.45 AS Fraction. 1-MARase was eluted between serum albumin and lactase. After gel-filtration, the elution volume was plotted against the log (molecular weight) of the reference standards, and the molecular weight of 1-MARase was estimated by its elution volume (fig. 5). The experimental points for ovalbumin, serum albumin and lactase lie on a straight line; their molecular weights are known to be 45 000 for ovalbumin [12], 67 000 for bovine serum albumin [12], and 104 000 for Rhlrs-lactase [13]. From this result the molecular weight of I-MARase of Asterina ovary was found to be 96 000. Isoelectric point. To determine the isoelectric point of 1-MARase, partial purification of the enzyme was performed in the following way. The 0.45 AS fraction obtained from 15 g of wet ovary of Asterina was dissolved in 0.2 M acetate buffer (pH 6.5) and gelfiltrated on a Sephadex G-150 column (2.5 x 80 cm) equilibrated with the same buffer, which was used as eluant. The active fractions were pooled and the pH was adjusted to 4.6 by adding 0.2 M acetic acid. Exptl

Cell Res 75 (1972)

The enzyme was again precipitated by adding ammonium sulfate at 0.45 saturation. The precipitate was dissolved in about 10 ml of 0.02 M phosphate buffer (pH 6.5) and applied on a DEAE-Sephadex A-25 column (1.4 x 24 cm) equilibrated with the same buffer. The active fractions (25 ml) eluted with the same buffer containing 0.1 M sodium chloride were dialysed against 1 “,‘o glycine and one-third of the dialysed material was used as the sample for each run of isoelectric focusing. After isoelectric focusing, each fraction was dialysed against DDW. A sample (0.4 ml) taken from each dialysed sample was incubated with 0.5 ml of 0.2 M borate buffer (pH 7.5) and 0.1 ml of 1O-2 M l-MAR for 1 h at 25°C and then assayedwith isolated oocytes after dilution with ASW. From these experiments the isoelectric point of 1-MARase was found to be pH 5.1 at 3°C.

Effect of GSS on I-MARase activity. Since starfish gonad tissue produces I-MA under the influence of GSS [I], there is a possibility that GSS may increase the activity of IMARase. This was examined using 0.45 AS Fraction as the enzyme solution. A mixture consisting of 0.4 ml of enzyme solution, 0.4 ml of 0.2 M borate buffer (pH 7.5), 0.1 ml of 0.1 M l-MAR, 0.1 ml of salt solution containing 100 mM NaCl, 100 mM KC1 and 50 mM MgCI,, and 0.1 ml of desalted GSS solution (1.2 mg/ml) was incubated at 25°C for 100min. For the control the samereaction mixture containing 0.1 ml of DDW instead of GSS solution was used. After incubation, the reaction was stopped by adding barium hydroxide and zinc sulfate, and then 0.1 ml of DDW was added to the experimental mixture and 0.1 ml of GSS solution to the control. Determination of the amount of ribose liberated in the reaction mixture re-

I-Methyladenosine

ribohydrolase

in starfish

87

The data presented here clearly demonstrate the presence of an enzyme which splits I-methyladenosine into I-methyladenine and ribose. Since the enzyme, 1-methyladenosine ,ug ribose/h/mg Composition of incubation ribohydrolase (I-MARase), does not act mixture protein on adenosine but acts specifically on l-MAR it seemsto play an important role in oocyte Enzyme + I-MAR-t GSS 12.6k0.5’ maturation in starfish in the sensethat the Enzyme -!-I-MAR 14.1 & 1.8 enzyme produces 1-MA. a Mesn +S.E. of five experiments. That l-MAR induces oocyte maturation when applied to the ovary has been shown vealed that GSS had no effect on I-MARase in at least six starfish species, but it has no activity, as shown in table 3. effect on isolated oocytes [3, 71. This fact supports the idea that l-MAR is the immediate precursor of l-MA and that 1-MARase DISCUSSION is of universal occurrence in starfish ovaries. That oocyte maturation and spawning in Production of MIS has been shown in starfish starfish are under hormonal control is well testis [21], and the presence of I-MARase established [16]. A gonad-stimulating hor- in testis is also shown in the present study. monal peptide, GSS, released from nervous That the activity of this enzyme is high tissue at the time of spawning acts on the in ripe ovaries which contain a larger number ovary to produce the direct trigger of these of fullgrown oocytes as compared with that phenomena, maturation-inducing substance, in small ovaries which have many small MIS [17, 181. MIS has been isolated from oocytes suggests that the enzyme activity Asterias amurensisand identified as l-methylcorrelates with the state of ripeness of the adenine (l-MA) [l]. Among a number of gonad. adenine derivatives so far examined, only As to the distribution of 1-MARase, our I-MA and 1-ethyladenine show MIS activity previous data [22] suggest that the follicles when applied to isolated oocytes [3]. Since around fullgrown oocytes contain this enmethylated adenines other than l-methylzyme; follicles incubated with GSS produce adenine, I-methylhypoxanthine and l-methylMIS, probably I-MA. Fullgrown oocytes guanine have no effect in inducing oocyte without follicles show no such activity. maturation, the structural requirements for Schuetz also suggested the presence of a the induction of oocyte maturation postulated ribonucleosidase which cleaves ribose from are a short alkyl radical such as methyl or l-MAR in the ovarian wall tissues (nonethyl attached to the N1 site and an imino oocyte) in Asterias forbesi [7]. radical at the C, site of the purine nucleus Although 1-MARase is important in pro[3]. Since 1-ethyladenine was not detected ducing l-MA, GSS has no relation to the in the course of purifying MIS (I), and since action of this enzyme, as demonstrated in the ethionine inhibited the production of MIS present paper. The role of this hormonal when ovaries were incubated with GSS [19, peptide in producing l-MA seemsto be to 201, I-ethyladenine does not seemto be pro- activate the transfer of methyl group to the duced, suggesting that 1-methyladenine is N, site of the purine nucleus of a precursor the only MIS produced in the starfish gonad. of l-MAR [20]. Table 3. Effect of GSS on enzyme activity of I-methyladenosine ribohydrolase in Asterina pectinifera

Exptl

Cell

Res 75 (1972)

88 H. Shirai & H. Kanatani We wish to express our gratitude to Dr J. C. Dan for reading the manuscript-and to Dr H. A. Bern for his interest. One of the authors (H. K.) is indebted to the Director and the staff of the Bodega Marine Laboratory of the University of California for placing the research facilities of the laboratory at his disposal. Thanks are also due to Mr Makino for suuplvina Rhus-lactase. This work has been supported in part by grants-inaid from the Ministry of Education and Kaiseikai, by US National Science Foundation Grant GB-6424 to Dr Bern and by a fellowship to H. K. from the US Population Council. -

REFERENCES 1. Kanatani, H, Shirai, H, Nakanishi, K & Kurokawa, T, Nature 221 (1969) 273. 2. Kanatani, H, Exptl cell res 57 (1969) 333. 3. Kanatani, H & Shirai, H, Development, growth and differentiation 13 (1971) 53. 4. Stevens, M, Exptl cell res 59 (1970) 482. 5. Bryan, J & Sato, H, Exptl cell res 59 (1970) 371. 6. Schroeder, P C, Naturwiss 58 (1971) 270. 7. Schuetz, A W, Exptl cell res 66 (1971) 5.

Exptl

Cell

Res 75 (1972)

8. Kanatani, H & Ohguri, M, Biol bull 131 (1966) 104. 9. Somogyi, M, J biol them 160 (1945) 69. 10. - Ibid 195 (1952) 19. 11. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 12. Andrews, P, Biochem j 91 (1964) 222. 13. Makino, N & Ogura, Y, J biochem 69 (1971) 91. 14. Vesterberg, 0 & Svensson, H, Acta them Stand 20 (1966) 820. 15. Wang, T P, Methods in enzymology (ed S P Colowick & N 0 Kaplan) vol. 2, p. 456. Academic Press, New York (1955). 16. Kanatani. H. Commentarii pontificia academia scientiarum 2~No. 27 (1970) 1: 17. Schuetz, A W & Biggers, J D, Exptl cell res 46 (1967) 624. 18. Kanatani, H & Shirai, H, Nature 216 (1967) 284. 19. - Gen camp endocrinol, suppl. 3. In press. 20. Shirai, H, Kanatani, H & Taguchi, S, Science 175 (1972) 1366. 21. Kanatanij H & Shirai, H, Development, growth and differentiation 12 (1970) 119. 22. Hirai, S & Kanatani, H, Exptl cell res 67 (1971) 224. 23. Shirai, H, Exptl cell res 74 (1972) 124. Received April 22, 1972