Regulation of intracellular ribonuclease of Bacillus subtilis by ATP and ADP

202

PRELIMINARY NOTES

BBA 61134

Regulation of intracellular ribonuclease of Bacillus subtilis by ATP and ADP During the course of an investigation on protein biosynthesis in Bacillus subtilis it was found that the activity of intracellular (acid) ribonuclease (ribonueleate nucleotido-2'-transferase (cyclizing), EC 2.7.7.17) was completely inhibited by ATP. Further studies revealed that ADP was another effective inhibitor. The inhibition of pancreatic ribonuclease by ATP, ADP and GDP has been reported ~. The inhibition was, however, only partial even if the concentration of ATP or ADP was very high, and the mode of inhibition was uncompetitive. In contrast to the case of pancreatic ribonuclease, the intracellular ribonuclease activity of B. subtilis was completely inhibited by low concentrations of ATP or ADP, and the mode of inhibition was competitive. The activity of ribonuclease was determined by measuring the absorbance of the acid-soluble fraction at 260 mff using a purified yeast RNA (Sigma Chemical Co., St. Louis, Mo., U.S.A., final 0.3%) and phosphate buffer (final 0.04 M, p H 5.7). Deoxyribonuclease (deoxyribonucleate oligonucleotido-hydrolase, EC 3.1.4.5) activity was measured similarly using heat-denatured calf-thymus DNA (Sigma Chemical Co., final 0.08%) as substrate. Phosphodiesterase (orthophosphoric diester phosphohydrolase, EC 3.1.4.1 ) activity was measured by determining the absorbance of liberated p-nitrophenol at 420 m# using bis-(p-nitrophenyl)-phosphate (Daiichi Pure Chemical Co., Tokyo, Japan), final concn. I mM in 0.04 M phosphate buffer (pH 5,7), as substrate. Ascending paper chromatography of nucleotides was carried out using Solvent I (SoO/0 satd. (NH4)2SO 4 terL-butanol-I M NH4OH (2oo:3:1, v/v/v)) 2 or Solvent U (95% ethanol-i M ammonium acetate (7:3, v/v)) a. B. subdlis strain K was cultured aerobically in I 1 nutrient broth at 3 °o for 20 h (stationary phase). Organisms were collected centrifugally, washed 3 times with o.oi

c 60!

0.12 0.08

E 5O

0

/

°

0

40

© 40

--~30

i20

o 0.04 e4

20

40

Time (rain)

60

1/Es]

(S=mglmt)

I/[S] (S=mg/ml)

F i g . t. T i m e c o u r s e o f R N A d e g r a d a t i o n b y i n t r a c e l l u l a r r i b o n u c l e a s e i n t h e p r e s e n c e o r a b s e n c e o f A T P . T h e t o t a l v o l u m e o f t h e r e a c t i o n m i x t u r e ( p H 5.7), w h i c h c o n t a i n e d o.o 5 m l S - I o 5 f r a c t i o n a n d o . 3 % y e a s t R N A , w a s i . o ml. T h e r e a c t i o n w a s c a r r i e d o u t a t 3 o°. O - ~ O , n o A T P ; k - - A , • lO .4 M A T P . F i g . 2. L i n e w e a v e r - B u r k plot of the inhibition of intracellular ribonuelease by ATP. The assay c o n d i t i o n s w e r e t h e s a m e a s d e s c r i b e d in F i g . i e x c e p t t h a t t h e c o n c e n t r a t i o n o f y e a s t R N A w a s v a r i e d a n d t h e r e a c t i o n t i m e w a s 6 o m i n . O - - O , n o A T P ; & - - A , 2 - IO -7 M A T P . F i g . 3. L i n e w e a v e r - B u r k plot of inhibition of intracellular ribonuclease by ADP. The c o n d i t i o n s w e r e t h e s a m e a s d e s c r i b e d in F i g . 2. Q - - - O , n o A D P ; A - - A , 2 • lO -6 M A D P .

t3iochim. I3iophys. ,4cta, I 3 9 (1967) 2 0 2 - 2 0 4

assay

PRELIMINARY NOTES

203

M Tris-HC1 buffer (pH 7.5, containing o.oi M/~-mercaptoethanol and o.oo2 M MgCl~), suspended in the same buffer and lyzed by lysozyme treatment and osmotic shock. The lysate was centrifuged at lO 5 ooo × g for 9 ° min and the supernatant (S-Io5) was dialyzed against the Tris-HC1 buffer. Tile dialyzed S-Io5 (50 ml) was used as the enzyme preparation. As illustrated in Fig. I, o.I mM ATP strongly inhibited the activity of intracellular ribonuclease. To determine the effect of other nucleotides, various nucleoside triphosphates, nucleoside diphosphates, and nucleoside monophosphates were examined. The results are summarized in Table I. ATP and A D P were highly inhibitory, e.g. I mM of either nucleotide was sufficient for complete inhibition at pH 5.7. The pH value inside B. subtilis cells was concluded to be around 7.0 from the pH value of a cell extract which was prepared by lyzing the organisms in a minimum amount of distilled water. At pH 7.0, the inhibition by ATP and A D P was a little stronger than at pH 5.7, e.g.o.I mM of ATP inhibited the activity of intracellular ribonuclease by 90% and 95% at pH 5.7 and 7.0, respectively. GDP and U T P were also inhibitory. TABLE [ THE

INHIBITORY

CLEASE

OF

EFFECT

B. s~.btilis

OF VARIOUS

AS E X P R E S S E D

NUCLEOTIDES IN INHIBITION

ON THE PER

ACTIVITY

OF INTRACELLULAR

RIBONU-

CENT

T h e a s s a y c o n d i t i o n s w e r e t h e s a m e as d e s c r i b e d in Fig. I. T h e r e a c t i o n t i m e w a s 6 o rain. % Inhibition x . xo-3 M

5 "Io-4 M

r ._to-4 M

I "zo -5 M

AT1 ) GTP CTP UTP

ioo 43

97 43 7 54

90 6

74

ADP GDP CDP UDP

11o 83 Io 4

90 15

Ado-5"-P Guo-5'-p Cyd-5'-P Urd-5"-p

3°

IO

Ado-3"-P Guo-3"-p Cyd-3'-P Urd-3'-p

13

3° 53

6 2

7 o

8

6 II

The modes of inhibition of ATP and A D P were competitive as shown in Figs. 2 and 3, respectively. Ks values for ATP and A D P were calculated as 0.24 #M and 0.53 #M, respectively, from Figs. 2 and 3. The Km value was o.o24~'o. The intracellular ribonuclease activity was not derived from polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyltransferase, EC 2.7.7.8 ) because the acid-soluble fraction contained mainly 3'-nucleotides as tested by paper chromatography using Solvent I and II. The possibility of participation of Biochim.

B i o p h y s . A c t a , 139 (1967) 2 o 2 - 2 o 4

204

PRELIMINARY NOTES

phosphodiesterase in the RNA degradation was also excluded by the fact that the activity of phosphodiesterase was completely inhibited by phosphate ion under the assay conditions employed. Recently, the presence of nucleases which preferentially degrade single-stranded nucleic acids in B. subtilis has been reported ~ L The S-Io5 fraction, however, contained little or no deoxyribonuclease activity. These observations strongly suggest that the intracellular ribonuclease activity observed was really that of ribonuclease s. Extracellular (alkaline) ribonuclease of B. subtilis K was not influenced bv ATP and ADP at either pH 5.7 or 7.5. An intracellular inhibitor against extracellular ribonuclease has been reported ", but no inhibitor of intracellular ribonuclease is known. In conclusion, ATP and ADP probably act as natural regulators or inhibitors of intracellular ribonuclease in B. subtilis. The purification of the intracellular ribonuclease is now in progress. The authors are grateful to Drs. (;. TAMVRA and T. BEpPU for their valuable discussion.

Department of Agricultural Chemistry, U~ziversity of Tokyo, Tokyo (Japan)

M. YAMASAKI

K. AI~IMA

1 \V. (;AMBLE AND I.. 1). \Vl~tC;H'r, Biochem..J., 97 (I9()5) 34 o, 2 N. NAKAJIMA, K. ICHIIC_.\WA, M. Ix~:XlXlADAAND E. I:IIJITA, ft. ~qSSOC..'1ft. C h e m . . S ' , c . . ] a / ) a ~ , 35 ( 1 9 6 t ) 797. 3 A . C . ['ALADINI AND L. t:. I.ELO1R, Biochem. J., 5 t ( t 9 5 e) 420. 4 M. NAKAL Z. MINAML T. YAMAZAI¢~ AND A. TSUGITA, ft. Biockem. 7"o/0'o, 57 (1q¢}5) 9~). 5 1. M. KERR, E. A. l}Ra'r'r AND I. R. I.EHMAN, Biockem. Bio/)h)'s. l?es. Comm~tm, 2o ( t 9 0 5 ) t 5 4 . () 1{. ()KAZAKI, T. ()KAZAt,[I AND ]x~. SAKABE, ldiockem. I~iopkys. Res. ()ommun., 22 (1()~)()) ()1 I. 7 1t. C. 13IRN~O~M, .J. Bac/eriol., 9I (190()) i o o 4 . S S. NlSHIMUI~A aXi) 13. ~IARUO, Biochim. Biophvs..Iota, 4 ° ( , 9 6 0 ) 355. 9 J- l~. SMEATON, \V. tl. ILLLIOTT ANI) (;. COLEMAN, Hiockem. Biopk.vs. h'cs. ('ommmz., 15 (I91)5) 3 6.

Received February 27th, 1967 l¢i.ckim. I~iopkys..t cla, i 3 (} ( I 0¢~7) 2o2 - o 4

PN 61135

Localization of lysozyme activity in a Paneth cell granule fraction The identity of the content of the Paneth cell granules is still largely unknown 1. Histochemical studies indicate that these granules are rich in protein 2,3, tryptophan 4 and arginine 5, that disulfide bridges are present 6 and that the overall isoelectric point exceeds lO.3 pH units s. Administration of pilocarpine is known to produce a discharge of granules into the intestinal lumen:. Following the administration of pilocarpine we observed a rapid increase of lysozyme (mucopeptide N-acetylmuramylhydrolase, EC 3.2.1.17) in the small intestinal fluid. As there is some analogy between Biochim. l~iophys..4eta, 139 (~967) 2 o 4 2o 7