A procedure has been described to determine picomole levels

of adenosine3',5'-cyclic monophosphate (CAMP) in plant materials. The purification of CAMP from other

interfering nucleotide and phenolic compounds is made

using paper and column chromatography. The resulting CAMP

is enzymatically transformed to ATP and estimated by its luminescent reaction with iuciferase. The advantages of this method have been discussed in the light of purification and assay of CAMP. The content of CAMP in lettuce, barley seeds, cabbage leaves, and cultivated mushrooms has been estimated by this procedur

Narayanan, A

Pradet, A

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SHORT COMMUNICATIONSKt .L tf i — C o m p o u n d s  S c r e e n e d  f o r  T h e ir  E f f e c t  o n  P y r u v ic  P  A c id  O x id a t io n  i n  R a t  B r a in  H o m o g e n a t e s'ycorh experiment was done in duplicate. Results represent mean ;X e s  of per cent inhibition calculated from 2  separate expen­d s  inhibition was determined by the decrease in Oa uptake/ H i  f f l 2  wet weight o f tissue/hr. All compounds were used a t a 1  final concentration of 2 mM )2-A l k y l -3 -a r y l -6 - /7 -f l u o r o -4 (3 H )-q u in a z o l o n e s  a n dCom­pound R i R,T h io q u in a z o l o n e s  Rs R 4  R 5  R* XInhibi­tion0/No.1 CHj H H H F H O/o17.742 C H 3 H F H F H O 11.643 C H , H F H ' F H s 34.51” 4 CH , H H F F H 0 56.595 C H , H H CCH 3 F H 0 17.846 C H j H H OC 2 Hs F H 0 '41.997 CH 8 H H O Q H s F H s 36.788 c 2h 5 H H H F H 0 66.259 "CsHs H F H F HQ._ l-5r611 0 C s H s H H F F H 0 5.851 1 c 2 h 6 H H Cl F H 0 17.681 2 C 2H s H CFa H F H 0 29.9313 c 2 h s H CH 3 H F H 0 67.2214 CsH 5 H CHs H F H s 50.4415 CHS H H H H F 0 80.3016 c h 3 H H H H F s 29.7617 c h 3 H H Br H F 0 16.7418 c h 8 H CFS H H F 0 55.7419 C H S H C F, % H F s 5.182 0 CFa H H F H 0 4;66(Nos 15, 16 and 18), showed promising inhibitory effects.Compounds having a fluorine substituent either in 6 - or 7-position with a 2-methyl substituent (Nos 3, 4, 6, 7, 15, 16, and 18) and a 2-ethyl substi­tuent (Nos 8, 12, 13 and 14) showed significant activity, while a compound having a 2-trifluoromethyl substituent (No. 20) showed very poor inhibitory effect. This activity is very low as compared to its 2-methyl analog (No. 1) and 2-ethyl analog (No. 8). Thus, the replacement of 2-CH s or 2-CaH5 group by a 2-CFa group decreases the activity markedly.In  the quinazolone series, a marked decrease in CNS activity, has been observed when a C =  O group was replaced by a C = S  group in 4-position of the quinazolone ring system4. In  this series, the replacement o f a C = 0  group by a C —S group in 4-position o f the quinazolone ring _ system also resulted in marked decrease in inhibition (compare the pair of com pounds:'6, 7; 13, 14; 15, 16; 18, 19— Table 1) except compound No. 3 as compared to No. 2.Quinazolones, having an unsubstituted phenylring in 3-position, displayed significant activity (compounds No. 8 and No. 15) except compound No. 1. Similarly, a few of the 3-substituted phenyl derivatives (compounds No. 4, No. 13 and No. 18) were found to  be highly active. From  these data, it is difficult to make a definite generalization about the role of substituents in the 3-phenyl ring.The general observations are that fluorine substi­tution, either in 6 - or 7— position, results in enhancing the inhibitory effect up to  some extent.The authors thank Prof. R. C. M ehrotra, Head of the Department, for providing facilities and Prof. S. S. Parmar, Department of Pharmacology, K. G. Medical College, Lucknow, for help in evaluating the biological activity of the compounds.References1. P a rm a r ,  S. S. & S e th , P . K . ,  Can. J. Biochem., 43 (1965),1179.2. Shuk la , J. S ., Sin g h , H . H . & P armar, S. S ., Can. J. Chem.,3. Joshi^K .^C . & Singh, V. K ., Indian J. Cheml, 11 (1973),430.4. Bia n ch i, C. & D avid , A ., / .  Pharm. Pharmac., 12 (1960),501.Isolation & Determination of Adenosine 3', 5'-Cydic Monophosphate from Plant TissuesA. N a r a y a n a nPlant Physiology Division, International Crops Research Institute for the Semi-arid Tropics Hyderabad 500016 &A .  J^RADET ’Station de Physiologie et de Biochemie Vegetales, I.N .R .A ., Pont-de-La-Maye, FranceManuscript received 28 July  1973; revised manuscript received 12 September 1973A procedure has been described to determine picomole levels of adenosine3',5'-cyclic monophosphate (CAM P) in plant materials. The purification of CAM P from other interfering nucleotide and phenolic compounds is made using paper and column chromatography. The resulting CAMP is enzymatically transformed to ATP and estimated by its lumi­nescent reaction with iuciferase. The advantages of this method have been discussed in the light of purification and assay of CAM P. The content of CAMP in lettuce, barley seeds, cabbage leaves, and cultivated mushrooms has been estimated by this procedure.T’£[E discovery of adenosine 3',5'-cyclic mono­phosphate (CAMP) in liver tissues1 has initiated a number o f investigations on its distribution in mammalian tissues2-4, bacteria*-7, slime molds8 and plant tissues9-13. Although the biological role o f CAMP is well documented in animals, bacteria and slime molds, no intensive studies have been reported in the case of higher plants, probably for the following analytical difficulties : (a) concentration o f CAMP is extremely low as compared to  that of the other nucleotides present in higher plants; (b) separa­tion of such low amounts of CAMP from other po­tentially interfering nucleotides; and_ (c) interference of phenolic compounds present in the extract with the enzymatic assay of CAMP.Taking all these analytical difficulties into account, a practical and sensitive method for the287INDIAN J. EXP. BIOL., VOL. 12, MAY 1974estimation o f CAMP has been developed, especially for plant tissues.The method, as described in this paper, involves fixation, extraction and separation o f CAMP from other interfering nucleotides by paper chromatogra­phy, elimination o f phenolic compounds by the use o f column chromatography (Dowex-50-H+or anhy­drous aluminium oxide) and finally enzymatic estima­tion. The validity of the method is checked on lettuce barley seeds, cabbage leaves, cultivated mushrooms' and ra t lung.Plant tissues ( 0.2 to 1.0 g ) were frozen in liquidnitrogen .and ground with 'an ultra-turrax a t __20°Cwith 10 ml o f ethyl ether containing 0.98 e of trichloroacetic acid (TCA) d irectly ,in  a stainless el ^ entri  uge tube held in an alcohol bath at —20 C. The temperature of the fixed sample was slowly raised to 0°C and the homogenate was centrifuged. The supernatant was collected in a separating funnel kept at 0CC. The residue was extracted with 10 ml o f 0.1 N TCA and centrifuged- the supernatant was added to the first ether extract.’eIl^ ated by shakin3 with ether. H-CAM P (0.02 fiCi, sp. activity 18 Ci/mmole;i ^ ° / f IV D r From aSeot> C.E.A., Paris) was added to TCA free extract which was then concentra­ted to approximately 1 ml by evaporation under reduced pressure at 35°C and was used directly for paper chromatography.^ 5 r eSCending paper chromatography on W hatman oMM paper was used. The 1 ml concentrate was applied as a narrow band along the startingI m p  T h t>  ( n l i i a n *  _________________I____________0  ■ -  ________ ®r  -  * --— u i v u g  m e  s u n t i u gline. The solvent isopopamoi=ci>nc. NH«UH-HaO ( /  . 1.5 : 15  vol vol). was allowed to reach the lower end of the paper within 20 h r a t 24°C in a constant temperature room. The chromatograph was air dried and the location o f CAMP was detected by the presence o f 3H-CAMP. The strip corres­ponding to CAMP was eluted with 50 Vf th?5o°A T d„ p o n fn tra ted  under reduced pressure11 ^  ^ t concentrated solution wasyellowish in colour.The concentrated solution containing CAMP and the remaining non-cyclic adenine nucleotide and phenolic compounds was purified by one o f the two following procedures.f  c^ omal° s r a p h y ~  The concentrated ^ lu tio n  (about 0.5 ml) was chromatographed onS w f t f   ^ a 200-400 mesh) column13 (6 x 0 .6  cm) and was eluted with distilled water The first 3 ml fraction contained almost all inhibiting rA M i?0 ™ w kch inhibit *he enzymatic assay of *, n fraction contained the CAMPand the column retained traces o f 5'-AM P if present m  the extract. 's o l * chromatography ~  The concentrated solution (about 0.5 ml) was passed through a column anhydrous aluminium oxide prepared with dis-Ztih m  The column was elu^ dwith 10 m M  tns-H C l buffer (pH 7 4) The first > ml£ f a Cf°n£ain^  thS V c o ip o u n Swerl fubse<*uent enzymatic assaywere retained by the columnfllnm!lf-elUateSM0btatinedeitherfrom  ion-exchange or iS ?  ° ?  chromat°graphy were concentrated to O nf ^ ^ f 5 SUre t0 dryness and taken in 0.2 o f TAM P water‘ Tiie enzymatic estimationof CAMP was done on this final purified colourless288 -  . -extract. An aliquot of this extract was counted -Beckman LS 100 Liquid Scintillation systenfai ? J  coefficient o f loss was calculated. the-*Enzymatic estimation o f  C AM P  — CAMP in the extract was hydrolysed to 5'-AMP incubation with 3'-5'-cyclic nucleotide phosohodiV ^  terase from beef heart*w« The resulfag was enzymatically converted into ATP on i n c u b a t ^  with adenylate kinase, pyruvate kinase and ^ phoenolpyruvate17 (enzymes were bought f W *  Sigma Chemical Co.). The level o f ATP was d S ? *  mined using a crude extract o f firefly tails1’ t I  ;aimOUntS as l0^  as °-5 P M ole 'oP  7  \ u  h  s,ample was assayed m duplicate witlr and■without phosphodiesterase plus an added s ta n ­dard o f CAMP. To a 30 ^1 sample was added 20 S  ot reaction mixture containing tris-buffer 0 35 mole (pH  7 4), M gS04, 1.1 mole; K 3S 0 4 19  mole; phosphoenolpyruvate, 1.7 mole; EDTA 0.02 mole; adenylate kinase; 6.25 ^ g ; pyruvate kinas^'6 ug, and phosphodiesterase, 20 m U when present’ Ih e  reaction mixture was incubated first at 37°c fm 30 min then at 25°C for 1 hr. I t  was found that the conversion o f CAMP to AMP takes place at 37*C and the succeeding conversion was complete if the incubation was carried out at 25°C for 1 hr When the incubation was done at 37°C for 1* hr continuously a slight hydrolysis o f ATP was noticed- A t the end o f the incubation, ATP levels were determined. The final result was corrected for the loss o f CAMP during the extraction process.________ ______________________ of_ adenylate-... nucleotides — ^£he-possibility o f CAMP formation from cyclization of ATP, ADP and AM P during extraction and separa-A?5nWial Vi?n ? ed. by the addit*on o f ATP, ADP and AM P labelled with 32P in the a-position (labelled nucleotides were obtained from C. E. A., France).j  PurPose, 1 g o f lettuce seeds was imbibed in distilled water for 2 h r at 20?C and fixed with liquid nitrogen as described above. After fixation, extrac­tion and separation, A TPa^P (10? cpm), A DP a^P  (10^ cpm) and A M Pa^P (10* cpm) were added: Immediately after the separation o f CAMP onf m n  t i^e sPot corresponding toCAMP was eluted and counted. No radioactivity was detectable in this spot. "The analytical procedure described was utilized to measure the levels o f CAMP in surface sterilized imbibed lettuce and barley seeds, cabbage leaves, cultivated mushrooms and rat lung. The results are as follows :TissueLettuce seeds Barley seeds Cabbage leaves Cultivated mushrooms R at lung R at lung18CAM P (pmole)3 1 04 2 02000^'  250b 7700b 7200”aper gram dry weight bper gram wet weightSince the levels o f CAMP in plant tissues are low as compared to those o f animal tissues1*-2* and bacteria the am ount o f plant tissue needed for analysis is large. However, we could easily proceed with a sample o f 0.25 g because o f the simplified procedure that we have developed. The loss during the analysis was estimated from isotopic dilutionSHORT COMMUNICATIONSiising 3H-CAMP. The recovery varied from 30 to: . 'r h e  paper chromatography step is labourious, nevertheless, it is highly necessary in the case of lant tissues to remove salts and the major part ot W c v c lic  adenine nucleotides and phenolic com- Sourids. .The solvent system used in the paper chro­matography has two advantages : (1)_ Because of S ?  volatile nature, the CAMP is- obtained free of 'salts and other chemicals., (ii) The CAMP is separated from all other possibly interfering adenine nucleoti- ij.es.and particularly adenosine diphosphate ribose / a d PR) which can liberate 5'-AMP by the action i f  cyclic nucleotide phosphodiesterase19’2". The R f values o f ATP, ADP, AMP, CAMP and ADPR •in this solvent system are 0.05, 0.11, 0.25, 0.55 and 015 respectively. Since enzymatic estimation ot CAMP involves a phophodiesterase, it is preferable -to' use this particular solvent for the separation.The extract eluted from the paper chromatography was always yellowish in colour because of the presence of phenolic compounds. The final purification step on the Dowex-50W column or A120 3 column removes 'the inhibitory substances. When the enzymatic assay was performed in the presence of a known , amount of CAMP, no inhibitory effect was observed. Also, when phosphodiesterase _ was not added to -the incubation mixture practically no nucleotide could be detected. This clearly shows that the puri­fication achieved with this method is perfectly ade­quate fo r-the  enzymatic analysis of CAMP.CAMP values for ra t lung as determined by_this_ procedure in comparison with those obtained by the phosphorylase activation assay18 are given above. The two values are in good agreement. *Because our- solvent for paper chromatography contains concentrated ammonia, the possibility ot CAMP formation by cyclization of ATP, ADP or 5'-AMP had to be considered. I t  has been noted that there was no formation o f CAMP from none of the adenylate nucleotide. .Thus, this analytical procedure can be sately employed for the determination o f CAMP in all tissues.References16 Jo h n s o n ,R . A .,H a rd m a n , J. G B r o a d u s ,  A. E. & S u th e r-  l a n d , E .  W ., 35 (1970), 91.17. P radet, A ., Physiol. Veget., 5 (1967), 209, _18 M u ra d , F ., R all, T. W . & Vaughan , M ., Biochem. biophys. Acta, 192 (1969), 430. ,19. G oldberg, N. D ., Larner, J. & O Toole, A. G .s Anal.Biochem., 28 (1969), 523. •20. G o ld b e rg ,  N . D . &  O ’T o o le , A . G .,m  M ethods o f  biochemi­cal analysis, Vol. 20, edited by D . Ghck (Wiley Inter- science, New York), 1971. .21. Posner, J. B ., H ammermeister, K . E ., Bratw old , G . E.& K rebs, E . G ., Biochemistry, 3 (1964), 1040.1. S u th e r la n d ,  E . W . &  R a l l ,  T. W ., / .  bio I. Chem., 2322. R obison, G . A ., Butcher, R . W . &  Sutherland , E . W .,A. Rev. Biochem., 37 (1968), 149.3. H ardman, J. G .,  R obison, G . A . &  Sutherland , E . w .,A. Rev. Physiol., 33 (1971), 311. . n c n n4. J ost, J. P . & R ickenberg , H.V., A. Rev. Biochem., 4  (1971),5. Id e , M ., Y oshim oto , A . &  O k ab ayash i, T ., J- B ad ., 946. M akm an,31R . S. & S u th e r la n d ,  E . W ., J. biol. Chem.,1. P A m N ,9L & P e rlm a n , R ., Science N . Y.,8. K onun , T. M ., Barkley, D . S ., Chang , Y . Y . & Bonner ,J. T ., Am. Naturalist, 102 (1968), 225.9. P o l l a r d ,  C . J . ,  Biochem. biophys. Acta. 201 (1970), 511.1 0 .  N a r a y a n a n ,  A . ,  V e r m e e r s c h , J .  &  P r a d e t  A . ,  c. r .held Stanc Acad. Sci., Paris, 271 (1970), 2402.11. Aza r , S. &  Krishna  M u rti, Biochem. Biophys. Kes.Commun., 43 (1971), 58. i n  ClQ7n12. Salom on, D . & M a sc a re n h a s , P ., Life Set., 10 (19/1),07913 K r ish n a , G .,  W eiss, B. &  B ro d ie , B ., J. Pharm. exp. Ther., 163 (1968), 379.14. R amachandran, J ., Anal. Biochem., 43 (1971), 22 /.15. B re c k e n rid g e , B. M cL ., Proc. natn. Acad. Sci., Vb, to.(1964), 1580.E vidence  o f  E n d o red u p lica tio n  in  C a llu s  C u ltu re s  o fCrepis suffreniana Steud.B. D. S in g h *Crop Science Department, University of Saskatchewan, Saskatoon, Saskatchewan, CanadaManuscript received 27 August 1973; revised manuscript received 1 November 1973Cytology of callus cultures initiated from shoot and leaf tissues of C. suffreniana on agar B5 medium with 2 .2 5 x lO '9M  2, 4-D, was studied. Initially, only diploid cells were present, and later tetraploid, octaploid and aneuploid cells were also present. A mutant culture was isolated and was found to be highly polyploid with chromosome numbers ranging from 22-52. These and other findings suggest that the occurrence of endoreduplication m vivo is not a  prerequisite for it to occur in vitro.PARTANEN1’2 has postulated that the nuclear.....  w ^avinur of plant tissues cultured in vitrois in large part a reflection of their in vivo potectiali- ties Thus a plant tissue culture can be expected to become polyploid provided that endoreduplication occurs in vivo, and that the conditions to elicit such a response are present in vitro. Conversely, it the differentiation o f the tissues in vivo occurs without endoreduplication, the tissue cultures may be expected to remain diploid. A  survey of the literature shows that in most of the tissue cultures studied cytogenetically, such a relationship does exist . However, there are a few notable exceptions. Endoreduplication is known to occur in Medicago sativa L.4, Glycine max L.4, Haplopappus graciUis Nutt. (Gray)3, and Vicia hajastana Grossh. _ tissues in vivo. The tissue cultures of these species have been demonstrated to possess only diploid ’ or a small frequency of polyploid3’6’7 cells. On the other hand, Crepis capillaris (L.) Wallr. tissues do not undergo endoreduplication in vivo. A high incidence o f polyploidy, however, has been reported in the tissue cultures of this species ’ .In the present study, the chromosome numbers, of callus cultures of Crepis suffreniana Steud., a species which' most likely differentiates without endoreduplication. were recorded over a period of 18 months to find out if these cultures would becomeP°*Callus cultures were initiated from shoot and leaf tissues of C. suffreniana on agar B5 medium1® contain­ing 2 25 X 10 '6Af 2,4-D. The calli were subcultured every 5-6 weeks and kept in a cul^ r® r0^  w:ltb a constant temperature (27-28°C) and light (2000 lux).♦Present address : Department of Genetics and PlantBreeding, Faculty of Agriculture, Banaras Hindu University Varanasi 221005289'

Isolation and determination of adenosine 3',5'-cyclic monophosphate from plant tissues

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Isolation and determination of adenosine 3',5'-cyclic monophosphate from plant tissues

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