/ . Biochem., 81, 389-394 (1977)

Kazuei IGARASHI, Hiroshi KUMAGAI, Hirofumi OGUCHI, and Seiyu HIROSE Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho, Chiba, Chiba 280 Received for publication, June 18, 1976

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(Q, and poly(U) ] by polynucleotide phosphorylase [polyribonucleotide : orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(Q>poly(A)>poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of po!y(U) was stimulated only slightly by the addition of spermidine. Although binding of ["Qspermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.

In the preceding paper (7), the effect of polyamines on the activity of E. coli RNase II has been studied. In E. coli the enzymes so far reported which degrade mRNA are RNase II (2-7) and polynucleotide phosphorylase (5, 8, 9), Therefore, as part of a series of studies on the effects of polyamines on RNA degradation, we have investigated the effects of polyamines on the degradation of synthetic polynucleotides by the commercially available polynucleotide phosphorylase of Micrococcus luteus.

were purchased from Boehringer Mannheim GmbH. Yeast RNA (Nutritional Biochemical Co.) was used after purification by acid precipitation and dialysis against distilled water. Polyamines (HC1 salts) were obtained from Sigma Chemical Co. [»H]poly(A), PH]poly(C), and [»H]poly(U) were purchased from Schwarz/Mann. ["C]Spermine was obtained from New England Nuclear Co. Heparin (130units/mg) was a product of Daiichi Pure Chemicals Co. Assay for Polynucleotide Phosphorylase—Assay A: The reaction mixture (0.5 ml) contained 0.1 M Tris-HCl (pH 7.5), 10 mM K.HPO,, 0.5 mM EDTA, MATERIALS AND METHODS 250 fig of yeast RNA or synthetic polynucleotide, Materials—Polynucleotide phosphorylase from 50 /ig of bovine serum albumin, and enzyme, Micrococcus luteus, poly(A), poly(Q, and poly(U) magnesium acetate and polyamines at the specified Vol. 81, No. 2, 1977

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Effects of Polyamines on the Degradation of Ribonucleic Acids by Polynucleotide Phosphorylase of Micrococcus luteus

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K. IGARASHI, H. KUMAGAI, H. OGUCHI, and S. HIROSE

concentrations. After incubation of the mixture at 37° for 2 h, the reaction was terminated by the addition of 0.5 ml of 5 % perchloric acid containing 0.25% uranyl acetate and 0.01 ml of 5% bovine serum albumin. The mixture was centrifuged, after cooling in an ice bath for 30 rrun, and the resulting supernatant was diluted with 4 volumes of water. The acid-soluble nucleotides therein were measured at 260 nm. When poly(C) was used as a substrate, the absorbance at 280 nm was also measured to confirm the value of absorbance at 260 nm. This method was used as a standard assay unless otherwise specified. Assay B: The reaction mixture (0.15 ml), containing 0.1 M Tris-HCl (pH 7.5), 10 HIM K,HPO4, 0.5 mM EDTA, 5 fig of synthetic SHpolynucleotides (specific activity, 1.85 fiC\/ftmo\ of polynucleotide phosphorus), 25 pig of bovine serum albumin, and enzyme, magnesium acetate and polyamines at the specified concentrations, was incubated at 37° for 30 min. The reaction was terminated by the addition of 0.3 ml of 3.75% perchloric acid containing 0.1 % uranyl acetate and 0.01 ml of 5% bovine serum albumin. After cooling in an ice bath for 30 min, the mixture was centrifuged and 0.4 ml of the resulting supernatant was neutralized with 0.8 ml of 0.17 N KOH. After the precipitate had been removed by centrifugation, 1 ml of the supernatant was placed in 10 ml of Bray's solution for counting {10). The counting efficiency was 6-8 x 105 cpm//jG.

tein, respectively. RESULTS Downloaded from https://academic.oup.com/jb/article-abstract/81/2/389/875728 by University of Winnipeg user on 22 January 2019

Effects of Polyamines on the Breakdown of Various Substrates by Polynucleotide Phosphorylase —As shown in Fig. 1, spermidine could stimulate the breakdown of all the substrates tested. In the presence of 1 mM Mg2+ and 100 mM Tris-HCl, pH 7.5 (A-D), the breakdown of yeast RNA, poly(C), poly(U), and poly(A) was stimulated by 4 mM spermidine (O) by 2.27-, 2.67-, 1.34-, and 1.91fold, respectively. In the presence of 1 mM Mg1+ and 100mM Tris-HCl, pH 8 5 (E-H), the breakdown of these substrates was stimulated by 4 mM spermidine (O) by 1.89-, 2.12-, 1.70-, and 1.64fold, respectively. From these results, it was found that the breakdown of poly(C), among the substrates tested, was most markedly affected by spermidine. The difference in degree of stimulation by spermidine of all the substrates tested except for poly(U) decreased as the pH was increased. The stimulation of hydrolysis by sperrrudine gradually decreased with increase of Mg2+ concentration. In the absence of Mgs+, regardless of the amount of polyamines, no hydrolysis of the synthetic polynucleotides could be observed (data not shown). When EDTA was removed from the reaction mixture, essentially the same stimulatory effect by spermidine was obtained. As shown in Fig. 2, the stimulation of breakdown of PHJpoIytQ, [SH> poIy(U), and [3H]poly(A) by 0.8 mM spermidine Binding of [uC]Spermne to Yeast RNA or was 1.69-, 1.39-, and 1.41-fold, respectively, at 1 Polynucleotide Phosphorylase as Determined by mM MgJ+. The data in Figs. 1 and 2 indicate that Sephadex G-50 Gel Filtration—The reaction mixture the concentration of spermidine effective for the (0.5 ml) used to study the interaction of spermine stimulation of polynucleotide hydrolysis was dewith yeast RNA, bovine pancreatic RNase A creased when the concentration of substrate was (Sigma Chemical Co., Type I-A), or polynucleotide decreased from 500 /Jg/ml to 33 ftg/m\; with larger phosphorylase contained 10 mM Tris-HCl (pH 7.5), amounts of substrate very little or no stimulation 4 0.2 mM P C]spermine (specific activity, 1.24/JCI/ was observed with 0.2 mM spsrmidine. ^mol) and either 500 fig of yeast RNA or 1 mg of enzyme. After incubation at 37° for 15 min, the Table I shows the effects of other polyamines reaction mixture was subjected to gel filtration on and K+ on the breakdown of synthetic polynucleoa Sephadex G-50 column (1x23 cm) previously tides by polynucleotide phosphorylase. Putresequilibrated with 10 mM Tris-HCl (pH 7.5). The cine had the same effects as spermidine, although a column was eluted with the same buffer. A 0.1 ml higher concentration was required to produce the aliquot of each 1 ml fraction was placed on a paper same degree of stimulation. Spermine had an disc (25 mm diameter) and the radioactivity was effect similar to that of spermidine on the breakcounted. The absorbance was determined at down of poly(Q and poly(U), but the breakdown either 260 or 280 nm using samples diluted 5-fold of poly(A) was not stimulated, as precipitation or 3-fold to measure the amount of RNA or pro- occurred during incubation. The presence of 0.1 M /. Bwchem.

391

EFFECT OF POLYAMINES ON POLYNUCLEOTIDE PHOSPHORYLASE 03

02

T5

°

UJ 03

02

0.1

0

1

2

3 4

2

3 4 5 1 2 Mg 2 + ( m M )

3

A 5

1 2

3 4 5

Fig. 1. Activity of polynucleotide phosphorylase toward yeast RNA and synthetic polynucleotides in the presence of various concentrations of spermidine. The assays were carried out under standard conditions except that spermidine and Mgs+ were added to the reaction mixture as specified in the figure. For the yeast RNA (A, E), poly(Q (B, F), poly(U) (C, G), and poly(A) (D, H) experiments, 75, 15, 10, and 10 /ig of polynucleotide phosphorylase were used, respectively. A-D, pH 7.5; E-F, pH 8.5. • , no spermidine added; X, 2 mM spermidine; O, 4 mM spermidine; A, 8 mM spermidine. * Precipitation occurred during incubation.

I

5 A

B

c

Q-

H4 t/>

ui ^3 o UJ

d2 < p o i UJ x n 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

Fig. 2. Activity of polynucleotide phosphorylase toward synthetic 'H-polynucleotides in the presence of various concentrations of spermidine. The assays (Assay B) were carried out as described in "MATERIALS AND METHODS" except that spermidine and Mg1+ were added to the reaction mixture as specified in the figure. For the poly(C) (A), poly(U) (B), and poly(A) ( Q experiments, 10, 7, and 7 fig of polynucleotide phosphorylase were used, respectively. • , no spermidine added; X, 0.2 mM spermidine; O, 0.4 mM spermidine; A , 0.8 mM spermidine.

Vol. 81, No. 2, 1977

KC1 had a slight stimulatory effect on poly(Q hydrolysis at 1 mM Mg2+ and poly(A) hydrolysis at 2 and 5 mM Mg1+. Similar effects were observed with 100 mM NHjQ. The optimal concentration of monovalent cations for stimulatory effect was 100-200 mM. As shown in Table II, spermidine was able to stimulate the hydrolysis of polynucleotides when Mgl+ was replaced by Mn1+; however, the degree of stimulation was less. The effect of spermidine on the breakdown of synthetic polynucleotides was then studied in the presence of 0.1 M KC1 and Mga+ (Fig. 3). The stimulation of the breakdown of poly(Q, poly(U), and poly(A) by 4 mM spermidine was 1.58-, 1.18-, and 1.48-fold, respectively, at 1 mM Mg1+. These results suggest that the stimulation of the breakdown of synthetic polynucleotides by spermidine was decreased by monovalent cations. Effect of Spermidine on the Activity of Polynucleotide Phosphorylase in the Presence of Heparin —It was of interest to determine whether the stimulation of hydrolysis of polynucleotides by spermidine was affected by various RNase in-

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01

K. IGARASHI, H. KUMAGAI, H. OGUCHI, and S. HIROSE

392

TABLE I. Activity of polynucleotide phosphorylase toward synthetic polynucleotides in the presence of K+ or polyamines. The assays were carried out under standard conditions except that K + , Mg1+, and polyamines were added to the reaction mixture as specified in the table.

Mg1+

K+

Spermine

Putrescine

Poly O

Poly U«

Poly A»

0 0 0 0 20 40

.030 .040 .064 .074 .064 .076

.056 .059 .071 .063 .067 .067

.082 .099 .099 .042" .151 .163

0 0 0 0 20 40

.058 .068 .103 .112 .118 .142

.111 .120 .104 .102 .121 .123

.173 .222 .184 .085" .263 .281

.175 .173 .162

0

40

.079 .074 .133 .156

.209 .275

1

0 0 0

.169

.328

1 1 1 1 1 1

0 100 0 0 0 0

0 0 0.5

2 2 2 2 2 2

0 100 0 0 0 0

0

5 5 5 5

0 100 0 0

Substrate used.

Absorbance at 260 nm

1 0 0

0 0.5 1 0 0 0 0

.102b

" Precipitation occurred during incubation.

TABLE II. Activity of polynucleotide phosphorylase toward synthetic polynucleotides in the presence of Mn'* and spermidine. The assays were carried out under standard conditions except that 1 mM Mn1* instead of Mg1+ and vanous concentrations of spermidine were added to the reaction mixture. Ions (ITIM)

Mn1+

Spermidine

1 1 1 1

0 1 2 4

Absorbance at 260 nm Poly O Poly U» .056 .083 .093 .095

.088 .103 .104 .107

Poly A» .137 .199 .194 .129"

• • b Set: Table I.

hibitors (11-13). A slight inhibition by poly(G) of hydrolysis of polynucleotides was observed, while heparin clearly inhibited the hydrolysis of

polynucleotides (data not shown). As shown in Fig. 4, spermidine stimulated the breakdown of poly(C) and poly(A) markedly in the presence of heparin. Under the same conditions, spermidine stimulated the hydrolysis of poly(U) to a much smaller but still significant extent. When 2 mM Mg1+ instead of 1 mM Mg I+ was added to the reaction mixture, essentially the same results were obtained. Binding of [liC]Spermine to Yeast RNA or Polynucleotide Phosphorylase—In a previous communication (14), we reported that polyamines affect the activities of RNases which hydrolyze RNA at pyrimidine nucleotides through the binding of the polyamines to the nucleic acids. By gel filtration (Table III), a slight binding of P'qspermine to polynucleotide phosphorylase was also observed; the amount of spermine bound to the enzyme was much less than that to RNA. Since the polynucleotide phosphorylase was essentially

J. Biochem.

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Ions (mM)

EFFECT OF POLYAMTNES ON POLYNUCLEOTIDE PHOSPHORYLASE

393

TABLE m . Binding of P4C]spermine to yeast RNA or polynucleotide phosphorylase. The radioactivity bound to yeast RNA, bovine pancreatic RNase A [EC 3.1.4.22], or polynucleotide phosphorylase was measured as described in "MATERIALS AND METHODS."

4 5 0 1

2 3 4 5 0 1

2 3 4 5

Mg 2 * ( m M )

Pig. 3. Activity of polynucleotide phosphorylase to•ward synthetic polynucleotides in the presence of spermidine and K + . The assays were carried out under •standard conditions except that 100 mM K + and various •concentrations of Mg1+ and spermidine were added to the reaction mixture. The concentrations of polynucleotide phosphorylase were as described in the legend to Fig. 2. A, poly(Q; B, poly(U); C, poly(A). • , no •spermidine added; x , 2 mM spermidine; O, 4mM spermidine. 200 150 100

LJ IT

50

0

1 2 3 A SPERMIDINE (mM)

Fig. 4. Effect of spermidine on the activity of polynucleotide phosphorylase in the presence of heparin. The assays were carried out under standard conditions •except that 40 /ig of heparin, 1 mM Mg1+, and various amounts of spermidine were added to the reaction mixture. For the poly(Q (x), poly(U) ( • ) , and poly(A) Biochem. Biophys. Res. Commun. 67, 407-413 16. Cohen, S.S. (1971) Introduction to the Polyamines p. 74, Prentice-Hall, Englewood Cliffs, N J . 17. Igarashi, K., Hara, K., Watanabe, Y., Hirose, S., & Takeda, Y. (1975) Biochem. Biophys. Res. Commun. 64, 897-904 18. Singer, M.F. & Tolbert, G. (1965) Biochemistry 4, 1319-1330 19. Grunberg-Manago, M. (1963) Progr. Nucleic Acid Res. 1, 94-133 20. Levy, C.C, Hieter, P.A , & LeGendre, S.M. (1974> / . Biol. Chem. 249, 6762-6769

/ . Biochem.

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can bind to RNase II. It has been reported that Citrobacter RNase, which hydrolyzes RNA at uridylic acid residues, can bind to polyamines (20). If polyamines influence the degradation of mRNA in vivo, the base compositions of mRNA's during various phases of the growth cycle of bacteria should differ. Studies to investigate this possibility are now in progress in our laboratory

Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus.

/ . Biochem., 81, 389-394 (1977) Kazuei IGARASHI, Hiroshi KUMAGAI, Hirofumi OGUCHI, and Seiyu HIROSE Faculty of Pharmaceutical Sciences, Chiba Univer...
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