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Studies on tobacco leaf ribonuclease III. Its role in the synthesis of tobacco mosaic virus nucleic acid
164
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 33 (1959)
S T U D I E S ON T O B A C C O L E A F R I B O N U C L E A S E I I I . ITS R O L E IN T H E S Y N T H E S I S OF TOBACCO MOSAIC VIRUS NUCLEIC ACID K. K. R E D D I
Virus Laboratory, University o[ Cali/ornia, Berkeley, Cali]. (U.S..4.) (Received September I5th, 1958)
SUMMARY
Ribonuclease activity in tobacco leaves was found to vary with the age of leaf. The old bottom leaves were found to contain more RNase than the top young leaves. The number of lesions appearing on the leaves and also the amount of tobacco mosaic virus synthesized following infection with TMV corresponded with the RNase content of the leaf. There was a significant increase in the RNase content of Turkish tobacco leaves following infection with TMV. The significance of these findings and the possible role of RNase in the synthetic processes were discussed.
INTRODUCTION
Ribonucleases are widely distributed in both the plant and the animal kingdom. They have been isolated from pancreas, tissue homogenates, bacteria and leaves. All the studies were confined to the isolation and study of their degradative properties. The ribonucleases obtained from different sources were shown to differ in the extent of degrading ribonucleic acid. The pancreatic ribonuclease was shown to be highly specific, cleaving only pyrimidine ribonucleaside 3'-phosphate linkages 1-3, while the ribonuclease occurring in bean and tobacco leaves was shown to bring about complete degradation of ribonucleic acid4-L In addition to these studies nucleotide exchange reactions catalyzed b y pancreatic ribonuclease were reported s. The information concerning the in vivo function of ribonuclease is meager. The present studies were undertaken to obtain some information pertaining to the role of ribonuclease occurring in tobacco leaves. Its occurrence in tobacco leaves and its partial purification were first reported b y HOLDEN AND PIRIE 4. It has been recently purified in this laboratory and its mechanism of action has been studied v. The results recorded here suggest that the enzyme in vivo m a y take part in the synthetic processes relating to the formation of tobacco mosaic virus nucleic acid. EXPERIMENTAL
Materials
Tobacco plants used in this investigation were grown in a greenhouse. Commercially obtained yeast ribonucleic acid (YRNA) was purified ~ and this was used as a Re/erences p. ~68/r69.
voL. 33 (1959)
TOBACCO LEAF RIBONUCLEASE. III
165
substrate for ribonuclease (RNase). Tobacco mosaic virus (TMV) was a purified preparation made b y differential centrifugation procedure.
Methods Determination o/ribonuclease activity in tobacco leaves. Immediately after picking the leaves, their midribs were removed, the leaves were cut into small pieces and 3 g portions were placed in a conical centrifuge tube of 12 ml capacity. These were quickly frozen b y placing in a mixture of solid CO 2 and Cellosolve. They were stored in solid CO2 until the time of extraction. Grinding and extraction. Unless otherwise stated all the operations were done at 0°-4 °. The samples after partially thawing were ground using a conical glass pestle shaped to fit the conical centrifuge tube. o.i M sodium acetate at p H 7.0 was used for washing the grinder. The ground material was centrifuged for 20 rain at 3,ooo rev./min. The residue was washed two times with o.1 M sodium acetate at p H 7.0 (approximately 2 ml for each washing). The combined supernatants were centrifuged at IO,OOOrev./min for 15 rain. The clear supernatant was made up to IO ml with o.I M sodium acetate at p H 7.0. The supernatant was centrifuged at 40,000 rev./min in a Spinco 40.3 rotor for one hour. (In the case of infected leaves the pellets were saved for isolation and estimation of TMV and details of this procedure are given below in section "Estimation o/ T M V in in/ected leaves".) To 3 ml of the supernatant were added 6 ml of o.I M sodium acetate buffer at p H 5.1. The mixture was held at o ° for 15 min and centrifuged at io,ooo rev./min for 15 rain. The supernatant was used for the estimation of ribonuclease and protein. Protein estimation. To 6 ml of above supernatant were added 1.5 ml of 5o % trichloroacetic acid at 0% The mixture, after standing at o ° for I h, was centrifuged at io,ooo rev./min for 15 rain. The supernatarlt was discarded. The amount of protein in the precipitate was determined using the procedure of HILLER, MCINTOSH AND VAN SLYKE9. The intensity of the color was measured at 55o m/z in a Beckman spectrophotometer. Estimation o~ ribonudease. Ribonuclease was estimated by measuring in a spectrophotometer, the absorption of the breakdown products which were not precipitated b y acid. The reaction mixture used for the determination of RNase activity unless otherwise stated, was composed of 0.5 ml of 1 % Y R N A in o.I M acetate buffer at p H 5.1, 0.3 ml of above leaf extract and 0.2 ml of o.I M acetate buffer at p H 5.1. Suitable blanks were run side b y side. The rest of the procedure was the same as described previously and the RNase activity was expressed as units per mg of protein% Estimation o / T M V in in/ected leaves. Pellets, obtained when the ground preparations were centrifuged at 40,000 rev./miu (see under section on "Grinding and extraction") were placed overnight at - - lO% Frozen pellets were extracted with o.I M phosphate buffer at p H 7.0 and centrifuged at IO,OOOrev./min. The supernatants were made up to 5 ml with phosphate buffer at p H 7.0. An aliquot of the supernatant was centrifuged at 40,00o rev./min for I h. The pellets were taken up in I ml of distilled water and examined under the electron microscope. The number of TMV rods present in the samples were kindly determined by Mr. JOSEPH TOBY using the procedure of BACKUS AND WILLIAMS1°. Re/erences p. ,68']I69.
166
K.K. REDDI
VOL. 33 (I959)
Experiments RNase activity in young and old leaves. Two varieties of healthy tobacco plant s, Turkish and Holmes were used in these experiments. They were approximately twelve weeks old and were grown under similar conditions. Large bottom leaves and the top leaves, immediately after picking, were traced on a paper and their areas were measured with a planimeter. The ribonuclease and protein in these leaves were determined using the procedures described above and the results are given in Table I. TABLE
I
RIBONUCLEASE ACTIVITY IN YOUNG AND OLD TOBACCO LEAVES Expt. No.
Variety o~ tobacco
Number o/ plants
i 2
Holmes Holmes
1 2
Turkish Turkish
Lea[ size, cm "z
RNase units per m~ protein
Top
Bottom
Top
Bottom
5 5
18 32
I 12 lO2
146 176
823 284
5 5
17.5
86
189 446
742 I 176
Lesion/ormation as affected by the age o/ lea/. The plants used in these studies were the Holmes variety of tobacco. They were grown under similar conditions and were approximately of the same size. Carborundum was applied on all the leaves of the plants and tobacco mosaic virus in a concentration of 0.2/~g/ml in o.I M phosphate buffer at pH 7.0 was rubbed on the leaves (in second and third experiments the concentration of TMV was 0.05/xg/ml). When the lesions were visible, the time of which varied from 7 ° to 96 h depending upon the weather conditions, the areas of the leaves and the number of lesions on them were determined. The results are given in Table II. Synthesis o/tobacco mosaic virus as affected by the age o/lea/. Turkish tobacco plants used in these studies were grown under similar conditions and were approximately TABLE
II
TOBACCO MOSAIC VIRUS INFECTION AS AFFliCTED BY AGE OF LEAF Expt. No.
Re/erences p. ±68[~69.
Variety o/tobacco
Position o/lea/on plant ]rom top
Number o/plants
Lea/size, cm~
Total No. o] lesions
Holmes
A t3 (] D
4 4 4 4
58.8 99 .2 lO7.4 84.7
22 268 IO35 516
Holmes
A B C D
6 6 6 6
23.7 63.9 81.9 66.8
3 29 335 357
Holmes
A B C D
6 6 6 6
13- r 31.o 70.2 i o i .3
16 53 265 690
VOL. 33 (1959)
TOBACCO LEAF RIBONUCLEASE. III
167
twelve weeks old. Carborundum was applied on all the leaves of the plants and tobacco mosaic virus in a concentration of 0.2 t~g/ml in o.I M phosphate buffer at pH 7.0 was rubbed on the leaves. After five days one large leaf from each plant and top young leaves were picked. Immediately they were traced on a paper and their areas were measured with a planimeter. The ribonuclease and protein present in them were determined using the procedures described above. The TMV present in the leaves was isolated as described above and the number of rods present in each sample was determined using the technique of electron microscopy1°. The results are given in Table I21. TABLE III R I B O N U C L E A S E A C T I V I T Y IN T U R K I S H TOBACCO L E A F AS A F F E C T E D BY TOP AND I~OTTOM T U R K I S H TOBACCO LEAVES
Expt. No.
I 2
Lea/size, ¢m~
RNase units per mg protein
No. o] T M V rods per g wet lea]
Top
Bottom
Top
Bottom
Top
Bottom
9.1 12.7
96.9 94.9
3Ol 237
2137 3194
3 . 1 " i o 12 4 .1"1o13
9 . 6 " l O 12 i 1 . 2 "1o13
RNase activity in lea~ as agected by tobacco mosaic virus in/ection. Turkish tobacco plants approximately 12 weeks old were used in these studies. Forty healthy plants were divided into two groups aa~d carborundum was placed on all the leaves. To one group (control) o.I M phosphate buffer at pH 7.0 was applied and to the other group tobacco mosaic virus in a concentration of 0.2 t,g/ml was applied. Five days after infection one leaf from each plant of the same size and at the same position of the plant was selected. RNase activity and protein were estimated as described above and the results are summarized in Table IV. TABLE
IV
R I B O N U C L E A S E ACTIVITY IN T U R K I S H TOBACCO L E A F AS A F F E C T E D TOBACCO MOSAIC V I R U S I N F E C T I O N
Number ot estimations
2 2 2 2
Number o/plants
5 5 5 5
RNase units per mg protein Control
In]eeted
830 1178 1123 883
1272 1283 15oo 1291
RESULTS AND DlSClJSSlON The RNase activity in bottom leaves was significantly higher than that in top young leaves (Table I). When the leaves were infected with TMV, there were more lesions on the bottom leaves than on the top leaves (Table II). Similar results were also noticed when Xanthi variety of tobacco plants were used. In the case of Turkish tobacco plants in which TMV multiplies systemically, more virus was found in the bottom leaves than in the top leaves (Table III). Thus the extent of TMV formation appears to parallel the RNase activity of the leaves. Relerences
p. 168]r69.
168
K.K. REDDI
VOL. 33 (I959)
Following infection with TMV there was a s u b s t a n t i a l increase in R N a s e a c t i v i t y of the leaves (Table IV). Since the R N a s e a c t i v i t y of t h e leaf depends u p o n t h e size, age a n d its location on t h e plant, in an e x p e r i m e n t of this t y p e one has to b e a r this in m i n d in selecting leaves from infected a n d control p l a n t s for e n z y m e assays. The increases in R N a s e a c t i v i t y of the leaf following infection w i t h TMV is either due to r e m o v a l of inhibitors or f o r m a t i o n of new enzyme. Several a u t h o r s referred to changes in e n z y m e levels caused b y virus infection. WOODS 11,12 r e p o r t e d the d i s t u r b a n c e s of oxidase a n d p e r o x i d a s e s y s t e m s in p l a n t s infected with TMV. Increases in diastase were r e p o r t e d in t o b a c c o infected with TMV 13. WYND 14 described increases in i n v e r t a s e a n d changes in oxidizing enzymes. HOLDEN AND TRACEY15 also r e p o r t e d changes in p e c t a s e a n d p r o t e a s e activities resulting from systemic infections. The correlation between TMV synthesis a n d R N a s e a c t i v i t y in t h e leaf o b s e r v e d in the p r e s e n t studies is suggestive t h a t R N a s e t a k e s p a r t in t h e s y n t h e t i c processes leading to the f o r m a t i o n of viral nucleic acid. Since TMV does n o t possess a n y k n o w n i n d e p e n d e n t e n z y m e activities*, it is r a p i d l y f o r m e d in locations of the p l a n t where such activities n e e d e d for its synthesis are s i t u a t e d . Since the f o r m a t i o n of a nucleop r o t e i n possessing t h e p r o p e r t i e s of TMV is not t h e n o r m a l function of tobacco leaf, the m a t e r i a l s n e e d e d for its synthesis m a y n o t be p r e s e n t in the host cell in a d e q u a t e a m o u n t s a n d also in a suitable form. Since the TMV was f o r m e d more in lower leaves of the p l a n t where n o r m a l l y the catabolic processes are a t a higher level t h a n the s y n t h e t i c processes, it can be a s s u m e d t h a t TMV is f o r m e d to a large e x t e n t out of b r e a k d o w n of p r e f o r m e d leaf p r o t e i n a n d nucleic acid. The role of R N a s e in the infected leaves p o s s i b l y will be in the b r e a k d o w n of leaf R N A a n d the s y n t h e s i s of viral R N A b y a process of r e a r r a n g e m e n t of b r e a k d o w n p r o d u c t s thus bringing a b o u t the necessary changes in nucleotide sequences, since the biological p r o p e r t y of a nucleic acid has been shown to be d e t e r m i n e d b y t h e nucleotide sequences in its molecule ~s, 19. A s t u d y of correlation between various e n z y m e activities arid TMV synthesis in the leaf will help in e l u c i d a t i n g the different reactions i n v o l v e d in the synthesis of TMV. ACKNOWLEDGEMENTS
I wish to t h a n k Dr. W . M. STANLEY for his interest a n d e n c o u r a g e m e n t a n d also Mrs. MARGARET FIKRAT for her technical assistance in these studies. This i n v e s t i g a t i o n was s u p p o r t e d b y g r a n t s from the Rockefeller F o u n d a t i o n a n d t h e U n i t e d S t a t e s Public H e a l t h Service. REFERENCES 1 R. MARKHAMAND J. D. SMITH, Biochem. J., 52 (1952) 552. DEKKER AND A. R. TODD, J. Chem. Soc., (2952) 27I 5. a E. VOLKIN AND W. E. COHN, J. Biol. Chem., 2o5 (1953) 767. 4 M. HOLDEN AND 1'4. W. PIRIE, Biochem. J., 6o (t955) 39. 5 R. MARKHAMAND J. L. STROMINGER,Biochem. J., 64 (1956) 46p. 6 W. FRISCH-1NIGGEMEYERAND K. •. REDDI, Biochim. Biophys. Acta, 26 (1957) 4o. 7 K. K. REDDI, Biochim. Biophys. ~4cta, 28 (1958) 386. 8 L. A. HEPPEL AND ~. R. WHITFIELD, Biochem. J., 60 ( I 9 5 5 ) 1. 9 A. HILLER, J . R. MClNTOSH AND D. D. VAN SLYKE,.]. Clin. Invest., 4 (1927} 235. 2 D . H . BROWN, C. A .
* BINKLEY16 reported that TMV has a transpeptidase activity. Examination of this finding in this laboratory revealed that transpeptidase activity is not an intrinsic property of TMV since only impure preparations of virus possess this activity and not the purified preparationsiv.
VOL. 33 (1959)
TOBACCO LEAF RIBONUCLEASE. III
169
10 R. C. BACKUS AND R. C. WILLIAMS, J. Appl. Phys., 21 (195o) i i . n A. F. WOODS, Centr. Bakteriol. Parasitenk., 2 (1899) 745. 12 A. F. WOODS, Science, i i (19oo) 17. la M. LODTKE, Phytopathol. Z., 2 (193 o) 341. 14 F. L. \¥YND, Botan. Rev., 9 (1943) 395. 15 i~{. HOLDEN AND M. V. TRACEY, Biochem. J., 43 (1948) 151is F. BI.'~KLEY, Proe. Roy. Soc. (London), B, 142 (1954) 17o17 G. M. CHRISTENSEN, personal c o m m u n i c a t i o n . 19 K. K. REDDI, Biochim. Biophys. Acta, 25 (1957) 528. 19 K. K. REDDI, Biochim. Biophys. Acta, in the press.
S T U D I E S ON T H E E L E C T R O N T R A N S P O R T
SYSTEM
X X . CHEMICAL A N D PHYSICAL P R O P E R T I E S OF T H E COENZYME Q FAMILY OF COMPOUNDS R. L. L E S T E R * , Y. H A T E F I * , C. x,VIDMER* AND E. L. C R A N E W i t h t h e technical assistance of E L I Z A B E T H M. W E L C H AND W A N D A F. F E C H N E R
Institute/or Enzyme Research, University o/ Wisconsin, Madison, Wisc. (U.S.A.) (Received J u l y 2 i s t , 1958)
SUMMARY
The chemical and physical properties of four members of a closely related group of compounds are described. These compounds, which function as oxidation-reduction coenzymes in terminal electron transport, have been isolated from beef heart mitochondria, Azotobacter vinelandii and Torula utilis. These compounds are shown to be similar in that each contains the same quinonoid chromophore, probably dialkoxylated. These compounds have different molecular weights, and this difference is at least in part explained b y the composition of the mono-unsaturated isoprenoid side chain(s) ; that is, these compounds differ in the number of such isoprenoid units which occur in the side chain. The formulae of these four compounds which best fit the available data are CssHssO 4, Cs~Hs00 4, C48H~O 4 and C4~H~O~.
INTRODUCTIO N
In the previous communications of this series, evidence was presented for the coenzyme role of a lipid material in the oxidation of succinic acid b y beef heart particles 1, 3. I t was shown that this compound, coenzyme Q, was present in particles of beef heart with electron transport properties and t h a t with removal of the compound by isooctane extraction the capacity to catalyze the oxidation of succinate was lost * Postdoctoral trainee of t h e U n i v e r s i t y of Wisconsin, I n s t i t u t e for E n z y m e Research. P r e s e n t address of CARL WIDMER : H u m b o l d t State College, Arcata, California.
Re/erences p. ±84[±85.
BIOCHIMICA ET BIOPHYSICA ACTA
VOL. 33 (1959)
S T U D I E S ON T O B A C C O L E A F R I B O N U C L E A S E I I I . ITS R O L E IN T H E S Y N T H E S I S OF TOBACCO MOSAIC VIRUS NUCLEIC ACID K. K. R E D D I
Virus Laboratory, University o[ Cali/ornia, Berkeley, Cali]. (U.S..4.) (Received September I5th, 1958)
SUMMARY
Ribonuclease activity in tobacco leaves was found to vary with the age of leaf. The old bottom leaves were found to contain more RNase than the top young leaves. The number of lesions appearing on the leaves and also the amount of tobacco mosaic virus synthesized following infection with TMV corresponded with the RNase content of the leaf. There was a significant increase in the RNase content of Turkish tobacco leaves following infection with TMV. The significance of these findings and the possible role of RNase in the synthetic processes were discussed.
INTRODUCTION
Ribonucleases are widely distributed in both the plant and the animal kingdom. They have been isolated from pancreas, tissue homogenates, bacteria and leaves. All the studies were confined to the isolation and study of their degradative properties. The ribonucleases obtained from different sources were shown to differ in the extent of degrading ribonucleic acid. The pancreatic ribonuclease was shown to be highly specific, cleaving only pyrimidine ribonucleaside 3'-phosphate linkages 1-3, while the ribonuclease occurring in bean and tobacco leaves was shown to bring about complete degradation of ribonucleic acid4-L In addition to these studies nucleotide exchange reactions catalyzed b y pancreatic ribonuclease were reported s. The information concerning the in vivo function of ribonuclease is meager. The present studies were undertaken to obtain some information pertaining to the role of ribonuclease occurring in tobacco leaves. Its occurrence in tobacco leaves and its partial purification were first reported b y HOLDEN AND PIRIE 4. It has been recently purified in this laboratory and its mechanism of action has been studied v. The results recorded here suggest that the enzyme in vivo m a y take part in the synthetic processes relating to the formation of tobacco mosaic virus nucleic acid. EXPERIMENTAL
Materials
Tobacco plants used in this investigation were grown in a greenhouse. Commercially obtained yeast ribonucleic acid (YRNA) was purified ~ and this was used as a Re/erences p. ~68/r69.
voL. 33 (1959)
TOBACCO LEAF RIBONUCLEASE. III
165
substrate for ribonuclease (RNase). Tobacco mosaic virus (TMV) was a purified preparation made b y differential centrifugation procedure.
Methods Determination o/ribonuclease activity in tobacco leaves. Immediately after picking the leaves, their midribs were removed, the leaves were cut into small pieces and 3 g portions were placed in a conical centrifuge tube of 12 ml capacity. These were quickly frozen b y placing in a mixture of solid CO 2 and Cellosolve. They were stored in solid CO2 until the time of extraction. Grinding and extraction. Unless otherwise stated all the operations were done at 0°-4 °. The samples after partially thawing were ground using a conical glass pestle shaped to fit the conical centrifuge tube. o.i M sodium acetate at p H 7.0 was used for washing the grinder. The ground material was centrifuged for 20 rain at 3,ooo rev./min. The residue was washed two times with o.1 M sodium acetate at p H 7.0 (approximately 2 ml for each washing). The combined supernatants were centrifuged at IO,OOOrev./min for 15 rain. The clear supernatant was made up to IO ml with o.I M sodium acetate at p H 7.0. The supernatant was centrifuged at 40,000 rev./min in a Spinco 40.3 rotor for one hour. (In the case of infected leaves the pellets were saved for isolation and estimation of TMV and details of this procedure are given below in section "Estimation o/ T M V in in/ected leaves".) To 3 ml of the supernatant were added 6 ml of o.I M sodium acetate buffer at p H 5.1. The mixture was held at o ° for 15 min and centrifuged at io,ooo rev./min for 15 rain. The supernatant was used for the estimation of ribonuclease and protein. Protein estimation. To 6 ml of above supernatant were added 1.5 ml of 5o % trichloroacetic acid at 0% The mixture, after standing at o ° for I h, was centrifuged at io,ooo rev./min for 15 rain. The supernatarlt was discarded. The amount of protein in the precipitate was determined using the procedure of HILLER, MCINTOSH AND VAN SLYKE9. The intensity of the color was measured at 55o m/z in a Beckman spectrophotometer. Estimation o~ ribonudease. Ribonuclease was estimated by measuring in a spectrophotometer, the absorption of the breakdown products which were not precipitated b y acid. The reaction mixture used for the determination of RNase activity unless otherwise stated, was composed of 0.5 ml of 1 % Y R N A in o.I M acetate buffer at p H 5.1, 0.3 ml of above leaf extract and 0.2 ml of o.I M acetate buffer at p H 5.1. Suitable blanks were run side b y side. The rest of the procedure was the same as described previously and the RNase activity was expressed as units per mg of protein% Estimation o / T M V in in/ected leaves. Pellets, obtained when the ground preparations were centrifuged at 40,000 rev./miu (see under section on "Grinding and extraction") were placed overnight at - - lO% Frozen pellets were extracted with o.I M phosphate buffer at p H 7.0 and centrifuged at IO,OOOrev./min. The supernatants were made up to 5 ml with phosphate buffer at p H 7.0. An aliquot of the supernatant was centrifuged at 40,00o rev./min for I h. The pellets were taken up in I ml of distilled water and examined under the electron microscope. The number of TMV rods present in the samples were kindly determined by Mr. JOSEPH TOBY using the procedure of BACKUS AND WILLIAMS1°. Re/erences p. ,68']I69.
166
K.K. REDDI
VOL. 33 (I959)
Experiments RNase activity in young and old leaves. Two varieties of healthy tobacco plant s, Turkish and Holmes were used in these experiments. They were approximately twelve weeks old and were grown under similar conditions. Large bottom leaves and the top leaves, immediately after picking, were traced on a paper and their areas were measured with a planimeter. The ribonuclease and protein in these leaves were determined using the procedures described above and the results are given in Table I. TABLE
I
RIBONUCLEASE ACTIVITY IN YOUNG AND OLD TOBACCO LEAVES Expt. No.
Variety o~ tobacco
Number o/ plants
i 2
Holmes Holmes
1 2
Turkish Turkish
Lea[ size, cm "z
RNase units per m~ protein
Top
Bottom
Top
Bottom
5 5
18 32
I 12 lO2
146 176
823 284
5 5
17.5
86
189 446
742 I 176
Lesion/ormation as affected by the age o/ lea/. The plants used in these studies were the Holmes variety of tobacco. They were grown under similar conditions and were approximately of the same size. Carborundum was applied on all the leaves of the plants and tobacco mosaic virus in a concentration of 0.2/~g/ml in o.I M phosphate buffer at pH 7.0 was rubbed on the leaves (in second and third experiments the concentration of TMV was 0.05/xg/ml). When the lesions were visible, the time of which varied from 7 ° to 96 h depending upon the weather conditions, the areas of the leaves and the number of lesions on them were determined. The results are given in Table II. Synthesis o/tobacco mosaic virus as affected by the age o/lea/. Turkish tobacco plants used in these studies were grown under similar conditions and were approximately TABLE
II
TOBACCO MOSAIC VIRUS INFECTION AS AFFliCTED BY AGE OF LEAF Expt. No.
Re/erences p. ±68[~69.
Variety o/tobacco
Position o/lea/on plant ]rom top
Number o/plants
Lea/size, cm~
Total No. o] lesions
Holmes
A t3 (] D
4 4 4 4
58.8 99 .2 lO7.4 84.7
22 268 IO35 516
Holmes
A B C D
6 6 6 6
23.7 63.9 81.9 66.8
3 29 335 357
Holmes
A B C D
6 6 6 6
13- r 31.o 70.2 i o i .3
16 53 265 690
VOL. 33 (1959)
TOBACCO LEAF RIBONUCLEASE. III
167
twelve weeks old. Carborundum was applied on all the leaves of the plants and tobacco mosaic virus in a concentration of 0.2 t~g/ml in o.I M phosphate buffer at pH 7.0 was rubbed on the leaves. After five days one large leaf from each plant and top young leaves were picked. Immediately they were traced on a paper and their areas were measured with a planimeter. The ribonuclease and protein present in them were determined using the procedures described above. The TMV present in the leaves was isolated as described above and the number of rods present in each sample was determined using the technique of electron microscopy1°. The results are given in Table I21. TABLE III R I B O N U C L E A S E A C T I V I T Y IN T U R K I S H TOBACCO L E A F AS A F F E C T E D BY TOP AND I~OTTOM T U R K I S H TOBACCO LEAVES
Expt. No.
I 2
Lea/size, ¢m~
RNase units per mg protein
No. o] T M V rods per g wet lea]
Top
Bottom
Top
Bottom
Top
Bottom
9.1 12.7
96.9 94.9
3Ol 237
2137 3194
3 . 1 " i o 12 4 .1"1o13
9 . 6 " l O 12 i 1 . 2 "1o13
RNase activity in lea~ as agected by tobacco mosaic virus in/ection. Turkish tobacco plants approximately 12 weeks old were used in these studies. Forty healthy plants were divided into two groups aa~d carborundum was placed on all the leaves. To one group (control) o.I M phosphate buffer at pH 7.0 was applied and to the other group tobacco mosaic virus in a concentration of 0.2 t,g/ml was applied. Five days after infection one leaf from each plant of the same size and at the same position of the plant was selected. RNase activity and protein were estimated as described above and the results are summarized in Table IV. TABLE
IV
R I B O N U C L E A S E ACTIVITY IN T U R K I S H TOBACCO L E A F AS A F F E C T E D TOBACCO MOSAIC V I R U S I N F E C T I O N
Number ot estimations
2 2 2 2
Number o/plants
5 5 5 5
RNase units per mg protein Control
In]eeted
830 1178 1123 883
1272 1283 15oo 1291
RESULTS AND DlSClJSSlON The RNase activity in bottom leaves was significantly higher than that in top young leaves (Table I). When the leaves were infected with TMV, there were more lesions on the bottom leaves than on the top leaves (Table II). Similar results were also noticed when Xanthi variety of tobacco plants were used. In the case of Turkish tobacco plants in which TMV multiplies systemically, more virus was found in the bottom leaves than in the top leaves (Table III). Thus the extent of TMV formation appears to parallel the RNase activity of the leaves. Relerences
p. 168]r69.
168
K.K. REDDI
VOL. 33 (I959)
Following infection with TMV there was a s u b s t a n t i a l increase in R N a s e a c t i v i t y of the leaves (Table IV). Since the R N a s e a c t i v i t y of t h e leaf depends u p o n t h e size, age a n d its location on t h e plant, in an e x p e r i m e n t of this t y p e one has to b e a r this in m i n d in selecting leaves from infected a n d control p l a n t s for e n z y m e assays. The increases in R N a s e a c t i v i t y of the leaf following infection w i t h TMV is either due to r e m o v a l of inhibitors or f o r m a t i o n of new enzyme. Several a u t h o r s referred to changes in e n z y m e levels caused b y virus infection. WOODS 11,12 r e p o r t e d the d i s t u r b a n c e s of oxidase a n d p e r o x i d a s e s y s t e m s in p l a n t s infected with TMV. Increases in diastase were r e p o r t e d in t o b a c c o infected with TMV 13. WYND 14 described increases in i n v e r t a s e a n d changes in oxidizing enzymes. HOLDEN AND TRACEY15 also r e p o r t e d changes in p e c t a s e a n d p r o t e a s e activities resulting from systemic infections. The correlation between TMV synthesis a n d R N a s e a c t i v i t y in t h e leaf o b s e r v e d in the p r e s e n t studies is suggestive t h a t R N a s e t a k e s p a r t in t h e s y n t h e t i c processes leading to the f o r m a t i o n of viral nucleic acid. Since TMV does n o t possess a n y k n o w n i n d e p e n d e n t e n z y m e activities*, it is r a p i d l y f o r m e d in locations of the p l a n t where such activities n e e d e d for its synthesis are s i t u a t e d . Since the f o r m a t i o n of a nucleop r o t e i n possessing t h e p r o p e r t i e s of TMV is not t h e n o r m a l function of tobacco leaf, the m a t e r i a l s n e e d e d for its synthesis m a y n o t be p r e s e n t in the host cell in a d e q u a t e a m o u n t s a n d also in a suitable form. Since the TMV was f o r m e d more in lower leaves of the p l a n t where n o r m a l l y the catabolic processes are a t a higher level t h a n the s y n t h e t i c processes, it can be a s s u m e d t h a t TMV is f o r m e d to a large e x t e n t out of b r e a k d o w n of p r e f o r m e d leaf p r o t e i n a n d nucleic acid. The role of R N a s e in the infected leaves p o s s i b l y will be in the b r e a k d o w n of leaf R N A a n d the s y n t h e s i s of viral R N A b y a process of r e a r r a n g e m e n t of b r e a k d o w n p r o d u c t s thus bringing a b o u t the necessary changes in nucleotide sequences, since the biological p r o p e r t y of a nucleic acid has been shown to be d e t e r m i n e d b y t h e nucleotide sequences in its molecule ~s, 19. A s t u d y of correlation between various e n z y m e activities arid TMV synthesis in the leaf will help in e l u c i d a t i n g the different reactions i n v o l v e d in the synthesis of TMV. ACKNOWLEDGEMENTS
I wish to t h a n k Dr. W . M. STANLEY for his interest a n d e n c o u r a g e m e n t a n d also Mrs. MARGARET FIKRAT for her technical assistance in these studies. This i n v e s t i g a t i o n was s u p p o r t e d b y g r a n t s from the Rockefeller F o u n d a t i o n a n d t h e U n i t e d S t a t e s Public H e a l t h Service. REFERENCES 1 R. MARKHAMAND J. D. SMITH, Biochem. J., 52 (1952) 552. DEKKER AND A. R. TODD, J. Chem. Soc., (2952) 27I 5. a E. VOLKIN AND W. E. COHN, J. Biol. Chem., 2o5 (1953) 767. 4 M. HOLDEN AND 1'4. W. PIRIE, Biochem. J., 6o (t955) 39. 5 R. MARKHAMAND J. L. STROMINGER,Biochem. J., 64 (1956) 46p. 6 W. FRISCH-1NIGGEMEYERAND K. •. REDDI, Biochim. Biophys. Acta, 26 (1957) 4o. 7 K. K. REDDI, Biochim. Biophys. ~4cta, 28 (1958) 386. 8 L. A. HEPPEL AND ~. R. WHITFIELD, Biochem. J., 60 ( I 9 5 5 ) 1. 9 A. HILLER, J . R. MClNTOSH AND D. D. VAN SLYKE,.]. Clin. Invest., 4 (1927} 235. 2 D . H . BROWN, C. A .
* BINKLEY16 reported that TMV has a transpeptidase activity. Examination of this finding in this laboratory revealed that transpeptidase activity is not an intrinsic property of TMV since only impure preparations of virus possess this activity and not the purified preparationsiv.
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10 R. C. BACKUS AND R. C. WILLIAMS, J. Appl. Phys., 21 (195o) i i . n A. F. WOODS, Centr. Bakteriol. Parasitenk., 2 (1899) 745. 12 A. F. WOODS, Science, i i (19oo) 17. la M. LODTKE, Phytopathol. Z., 2 (193 o) 341. 14 F. L. \¥YND, Botan. Rev., 9 (1943) 395. 15 i~{. HOLDEN AND M. V. TRACEY, Biochem. J., 43 (1948) 151is F. BI.'~KLEY, Proe. Roy. Soc. (London), B, 142 (1954) 17o17 G. M. CHRISTENSEN, personal c o m m u n i c a t i o n . 19 K. K. REDDI, Biochim. Biophys. Acta, 25 (1957) 528. 19 K. K. REDDI, Biochim. Biophys. Acta, in the press.
S T U D I E S ON T H E E L E C T R O N T R A N S P O R T
SYSTEM
X X . CHEMICAL A N D PHYSICAL P R O P E R T I E S OF T H E COENZYME Q FAMILY OF COMPOUNDS R. L. L E S T E R * , Y. H A T E F I * , C. x,VIDMER* AND E. L. C R A N E W i t h t h e technical assistance of E L I Z A B E T H M. W E L C H AND W A N D A F. F E C H N E R
Institute/or Enzyme Research, University o/ Wisconsin, Madison, Wisc. (U.S.A.) (Received J u l y 2 i s t , 1958)
SUMMARY
The chemical and physical properties of four members of a closely related group of compounds are described. These compounds, which function as oxidation-reduction coenzymes in terminal electron transport, have been isolated from beef heart mitochondria, Azotobacter vinelandii and Torula utilis. These compounds are shown to be similar in that each contains the same quinonoid chromophore, probably dialkoxylated. These compounds have different molecular weights, and this difference is at least in part explained b y the composition of the mono-unsaturated isoprenoid side chain(s) ; that is, these compounds differ in the number of such isoprenoid units which occur in the side chain. The formulae of these four compounds which best fit the available data are CssHssO 4, Cs~Hs00 4, C48H~O 4 and C4~H~O~.
INTRODUCTIO N
In the previous communications of this series, evidence was presented for the coenzyme role of a lipid material in the oxidation of succinic acid b y beef heart particles 1, 3. I t was shown that this compound, coenzyme Q, was present in particles of beef heart with electron transport properties and t h a t with removal of the compound by isooctane extraction the capacity to catalyze the oxidation of succinate was lost * Postdoctoral trainee of t h e U n i v e r s i t y of Wisconsin, I n s t i t u t e for E n z y m e Research. P r e s e n t address of CARL WIDMER : H u m b o l d t State College, Arcata, California.
Re/erences p. ±84[±85.