.,.__._..,... MODE .0; ACfiGN o: N5 .55N2YLADEMNE IN ma mmamou a“; mpmnou m HIGHER mms WITH SPECIAL REFERENCE 1o. aaoccou (gwgg Wfl VAR mum cv‘ SPARTAN EARLY) Thesis Ear flu Bear» of Ph. D. MECMGAN STATE UNEVERSiTY Vimwdra Tali "i964 311535 This is to certify that the thesis entitled ‘ . . b . . Wade =f Action at N -benzylnden1ne in the InhibiLiun of Rospirnti.n in Higher Plants with Snecinl heferonce to BTUCCHLL (Br8581cn flerncon var itelica cv Spartan garlyl. presented by Virendra Tnli has been accepted towards fulfillment of the requirements for ——I3.llfl__degree in Horticulture 15/. zafiw Major professor S.ll. Wittwer Date / /?6,9( 0-169 LIBRARY Michigan State University ABSTRACT MODE OF ACTION OF N6-BENZYLADENINE IN THE INHIBITION OF RESPIRATION IN HIGHER PLANTS WITH SPECIAL REFERENCE TO BROCCOLI (BRASSICA OLERACEA VAR ITALICA CV. SPARTAN EARLY; by Virendra Tuli The mode whereby N6-benzyladenine (BA), an active kinin, inhibits respiration of higher plants was studied with broccoli and cauliflower explants. Post harvest ap- plications of BA (leO-S‘fl) delayed chlorophyll and caro- tene breakdown of broccoli heads and leaves. This was as- sociated with a delay in the onset of senescence and with respiration inhibition. Effects of BA on respiration were studied using broccoli and cauliflower mitochondria, iso- lated by fractional centrifugation. Adenosine triphosphate (ATP) was found essential for mitochondrial activity of both species when succinate was provided as the substrate. 5 The presence of 3.3x10' y’aa markedly inhibited the oxi- dation of succinate by the isolated mitochondria. The greater retention of chlorophyll and carotene resulted in 14 higher photosynthetic C0 fixation by treated broccoli 2 leaves. Distribution of 14C among the various photosyn- thetic products was determined by autoradio-chromatography. Results suggested that the activity of certain phosphory- lating enzymes (kinases) was impeded by BA. 1 2+ 9 An ianitro system involving hexokinase, ATP, Mg cysteine, glucose, glucose-6-phosphate dehydrogenase and Virendra Tuli--2 nicotinamide-adenine dinucleotide phosphate (NADP+) indi- cated that the reduction of NADP+ was greatly slowed by 3.3xlO-5‘§_BA. This resulted from an inhibition of hexo- kinase activity. It is suggested that structural similar- ities between ATP and BA resulted in a competitive inhibi- tion for an active site on hexokinase. The nature of in- hibition was established by the use of classic enzyme ki- netics. ATP also participates in the enzymic synthesis of glutamine, catalyzed by glutamine synthetase. The in- effectiveness of BA on a 10-fold purification of the enzyme from cauliflower suggested, however, that the inhibition was specific for the kinases. Specificity of BA for the kinases was further established by studying its effects on another in;33££g_system involving pyruvic kinase, +, Mgz+, phospho>>}}}>"’ ’}>>”}’ “l‘l‘l‘“ >}>}}}}> l““l““ >>’}>}}}} }?}}}}}}’ ‘l“““l ’}>}>>}’> }>}’>}>}} ““““‘ }}’ ’},}}} l““““ }>}}}’}>> ""‘h‘>"‘ ‘}‘}‘b >}>}>}>} ““““‘ ”}}}’}}} ll“““‘ }}}>”>>} ““““‘ ,,’>}’}}} ’}”>>} ““““‘ ” }}}’>}} A““‘A‘“‘ ’}}}” "} ““““‘ >”I’}}>’> ””"”} ‘}}}}’}}> }}}’}*}’ “““l“ >‘l”’>}} ‘)’}}’>>’ }>}}}}}>} }}’}}>}> ““““‘ }’}’}”>> >>”>’}, >>}>”>>’ ‘l‘l‘l‘l‘ }>}}’}>’> Figure 5 14 Photosynthetic fixation of CO by BA treated (leO-5 M) 2 and control broccoli leaves. Radioactivity expressed as counts per minute per gram (cpm/gm.) fresh weight. cpm lgm fresh wt. (IO-3) Treated l6 _ (IOppm 8A) l4 _. l2 - Control A IO - of 1 l 1 1 24 48 72 96 Hours after Treatment Figure 6 14 Paper chromatographic distribution of C in treated (leO"5 M) and control broccoli leaves. Autoradiograms prepared of chromatographed extracts 48 hours after treat- ment. A - Uridine diphosphate glucose (UDPG) B - phosphate esters - sucrose — glucose — glycine - serine - aspartate + glutamine citrate - fructose - alanine - malate l" X C-l H :E C) "J I") U 0 l — glycolate Cuba aOIhZOU Table I 32 Effects of BA on the percentage distribution of 14C among the various organic constituents of broccoli leaves at 0, 24, 48 and 72 hours after photosynthetic 14C02 fixation 9...... as; sale 22.2.5 Compound 2: .T.“ s. 2: .9. 2 2 .T. Sucrose 32 26 28.6 26 21.4 27.6 20.9 37 UDPG 12.4 10 8.6 8.7 7.8 9.1 9 9 Glucose — - 0.8 0.3 1.4 0.6 3 1.3 Fructose - - 0.8 0.3 1.4 0.6 3 1.3 Serine 16.5 17.6 19.2 25.2 23.1 30.5 26.5 18.5 Glycine 2.9 3.0 3.6 3.5 3.6 5.3 3.0 10 Glycolate 1.1 1.4 0.8 1.3 0.4 1.2 - 0.6 Citrate 0.7 0.7 0.9 0.1 1.3 0.3 8.0 - Malate 17.4 19.1 15.6 18.5 11.5 12.5 5.4 7.9 Alanine 4.9 5.3 6.5 4.5 8.7 4.2 3.4 4.9 Glutamic 0.2 1.0 0.5 0.3 0.8 - 1.2 - Aspartate 8.0 11.1 9.4 7.7 12.8 3.2 3.0 1.4 P-esters 3.7 5.0 5 4.2 7.2 3.1 11.3 4.2 ‘ - Control " - Treated 33 N6-benzyladenine (BA) and hexokinase activity The reduction in the formation of phosphate esters in treated broccoli leaves (Table I) suggested that the activity of certain phosphorylating enzymes was impeded by BA. An in_yit£g system was thus devised to study the effects of BA on hexokinase activity. Hexokinase catalyzes the phosphorylation of glucose by ATP as follows: (1) Glucose + ATP Hexokinase,Mg%:_ Glucose-6- 7 phosphate + Adenosine diphosphate (ADP). Glucose-6—phosphate is oxidized in the presence of nico- tinamide-adenine dinucleotide phosphate (NADPI), by glucose- 6-phosphate dehydrogenase (GDH): (2) Glucose-6-phosphate + NADP+ GDH 6-phospho- -———-) glucono-S-lactone + NADPH + H*. The two above enzymic reactions were coupled to proceed stoichiometrically and quantitatively. The reduc- tion of NADP* in the second equation was measured spectro- photometrically at 340 mu and served as a measure of hexo- kinase activity. Solutions were prepared as follows, after which they were stored at 4° C, and held in ice during use. Tris buffer (0.05 M; pH 7.6)-—Solutions were made up each day as described under general methods. Glucose (0.1 M)--Three hundred sixty mg. of glucose were dissolved in distilled water and the volume made up to 20 m1. 34 yggnesium chloride (O.l‘§)-—One-ha1f gm. MgC12.6H20 was dissolved in distilled water and the volume made up to 25 ml. Adenosine triphosphate (0.1[EVATP)--Approximately .3H 600 mg. ATP-NaZH 0 were dissolved in distilled water 2 2 and the volume made up to 10 ml. Serum albumin-~Twenty mg. crystalline serum albumin were dissolved in 5 ml. distilled water. The solution served as a protective protein for hexokinase. Nicotinamide-adenine dinucleotide phosphate (1.51:10-3 §;NADP*)-~Approximately 13 mg. NADP-NaH2 were dissolved in distilled water and brought up to 10 ml. gysteine (ca. 0.28 gym-Thirty mg. of cysteine.HCl were dissolved in 0.8 ml. distilled water and neutralized with 0.2 ml.‘§_Na0H. Hexokinase (140 units)-—0ne—half gm. of crystalline yeast hexokinase (Nutritional Biochemicals Co.) was diluted with 0.5 ml. serum albumin solution. One enzyme unit was defined as that amount of protein which caused the phos— phorylation of one micromole of glucose per minute, at 25° C and pH 7.6. Glucose-G-phosphate dehydrogenase (140 units)--An ammonium sulfate suspension of the enzyme (Sigma Chemical Co.) was reconstituted by the addition of 0.1 ml. cold dis- tilled water for each 50 units. One enzyme unit caused the reduction of 1.0 micromole of NADP+ per minute at 25° C and pH 7.6. 35 N6—benzyladenine (10"3 §)--Solutions were prepared as described under general methods. Preliminary experiments indicated that the magni- tude of inhibition caused by BA was dependent upon the ATP concentration; hence, kinetic studies with the ATP concen- tration as a variable were conducted. Optical density measurements, relating to the re- duction of NADP+, were made with a Beckman DU spectrophoto- meter at 340 mu, a light path of 1 cm., and a final volume of 3.0 ml. made up with distilled water. The following solutions were pipetted into silica cuvettes in the order listed. 1 — Tris 1.0 ml. 2 - glucose 0.4 ml. 3 - ATP varied 4 - MgCl2 0.1 m1. 5 — cysteine 0.1 m1. 6 - water to make up 3.0 m1. 7 - Hexokinase 0.1 ml. (28 units) 8 - GDH 0.3 ml. (42 units) 9 - BA - 1o — NADP‘ - One treatment consisted of a blank with no addition of BA or NADP*. The control received only NADP+ (0.2 m1.) and the treated BA (0.1 ml.) plus NADP+ (0.2 ml.). After the addition of BA in the treatment, the contents were stirred and equilibrated at 25° C, after 36 which initial optical densities (0.0.) were read against the blank; 0.2 ml. of NADP+ were then added and change in 0.0. measured each 15 seconds. Reaction 2 (page 33) was used as a basis for deter- mining the effect of BA on GDH activity. Components of the $3.325£2_system were the same as those previously de- scribed for hexokinase. Reaction velocities in the presence of BA and vary- ing concentrations of ATP are shown in Fig. 7. A double reciprocal plot of the results of kinetic studies indicat- ing competitive inhibition of hexokinase by BA is shown in Fig. 9. GDH activity was not affected by BA at two different concentrations (Fig. 8). Figure 7 Effect of BA on hexokinase activity. Changes in reaction velocity (£50.D./min.) correspond with the reduction of NADP+ in in_ygt£2 systems containing various concentrations 5 of ATP and 3.3x10’ .11 BA. ON Enos 5222880 a: m. C. _ _ 4m 290. x m.m. <1" 10!x(64nU!w/'0'o V) = moons/x Figure 8 Effect of BA (3.3 and 6.6x10-S g) on glucose-6-phosphate dehydrogenase activity. Changes in optical density corres- pond with the reduction of NADP+ in i2_vitro systems. A 0.0. x :03 60 50 4o 30 20 IO // x7 :7 // °-——O Control // 0----£ 3.3 x 16% 8A A-———A 6.6 x I0'5M BA 1 l I l 30 45 60 75 Time in seconds Figure 9 Inhibition of hexokinase by 3.3xlO-5‘flgBA. The velocities and substrate concentrations (ATP) are plotted as the recip— rocal of their values. The common intercept of the treated 0 and control curves on the %' axis confirms competitive in- hibition. Ks and K are the apparent substrate and inhibitor I constants respectively. l x 10-2. ’1 . V-ax1s should read-V $.23 .2 NO_ x Ho. 8.. 8.. one _ J a _ s. -o: 98.; £4- Zeno: Sc. "my. 3:35: oz lad is me am ambienm o 1n... 3.3% 40 N6—benzyladenine (BA) and glutamine_§ynthetase activity The competitive inhibition of hexokinase by BA sug- gested that BA might inhibit other enzymic reactions where molecular ATP was involved; hence, the activity of glutamine synthetase, in the presence of BA, was tested. The enzymatic synthesis of glutamine occurs as fol- lows: glutamic acid + ammonia + ATP Glutamine synthetaseA ’ glutamine + ADP + phosphate. Ammonia in the above equation can be replaced by hydrazine or hydroxylamine. All three bases react at the same rate (24). With hydroxylamine, a hydroxamic acid (glutamyl- hydroxamic acid) may be produced, the estimation of which provides a convenient colorimetric test (54). Preparation of solutions and reagents Sodiumgglutamate (O.S‘§)-—Four hundred forty mg. of sodium glutamate (MW 176) were dissolved in distilled water and made Up to 50 m1. ASE (0.05 fl)~-Six hundred mg. of ATP-Na2H2.3H20 were dissolved in distilled water and made up to 20 ml. Tris buffer, BA, and Cysteine-~Prepared as in the hexokinase experiments. flagnesium sulfate (1 gum-Twelve gm. anhydrous M9504 were dissolved in distilled water and made up to 100 ml. Hydroxylamine (4‘fl; pH 6.4)-—Prepared by nearly neutralizing NHZOH.HC1 (28%) with an equal volume of 14% 41 NaOH (3.5 fl), so that the resulting solution remained slightly acidic. This labile solution was prepared daily. Hydroxylamine (1 y; pH 7.6)--Prepared by dissolving 0.33 gm. NH OH.HC1 in Tris (pH 7.6) and brought up to a 2 volume of 10 m1. Acetate buffer (0.1 35 pH S.4)--One-tenth molar solutions of acetic acid and sodium acetate were mixed in 1:4 proportions. Ferric chloride (S%)--A solution of FeC13.6H20 was made in 0.1 §_HC1. I Trichloracetic acid (12% w/v)--This solution was prepared with distilled water. Glutamylghydroxamic acid standard curve--One-half gm. of L-glutamine was dissolved in 4 m1. of 2 fighydroxyla— mine, in a 10 m1. volumetric flask. Ten minutes were al-. lowed for the conversion of glutamine to glutamyl-hydrox- amic acid. The flask was then filled to the 10 ml. mark with distilled water. Aliquots were pipetted into stand- ardized colorimetric tubes containing 1 ml. acetate buffer (pH 5.4) and the volume adjusted to 3 ml. at room tempera- ture. Thereafter, 1 ml. each of HCl, TCA, and ferric chloride solution was added in the indicated order. Absorp- tion of the purple iron complex formed was measured at 540 my on a Bausch and Lomb colorimeter (54). Protein standard curve—-Constructed after the method of Lowry _e_§ _a_]_._. (55). Glutamine synthetase activity was determined according 42 to Elliott (24). Incubations were carried out at 30° C for 20 minutes. The system consisted of the following com- ponents for the control. Each treatment was replicated three times . Tris 0.5 m1. Na-glutamate 0.5 m1. ATP 0.5 m1. MgSO4 0.1 m1. Cysteine 0.1 ml. NHZOH (pH 7.6) 0.1 m1. Enzyme 0.3 m1. Water 0.15 ml. BA _ 2.25 ml. Treated samples were provided with 0.1 ml. BA (final con- centration ca. 4.4x10-5 3) and only 0.05 ml. of water. Immediately following incubation, 1 ml. aliquots were drawn into centrifuge tubes containing 1 ml. of 4 fl hydroxylamine and 1 ml. acetate buffer. After 10 minutes, when the glutamine formed during incubation had been con- verted to glutamyl-hydroxamic acid, 1 ml. each of HCl, TCA, and FeCl2 was added respectively. The precipitated protein was removed by centrifugation and the supernatant decanted into colorimetric tubes. Glutamyl-hydroxamic acid was meas- ured quantitatively using an appropriate standard. One enzyme unit was arbitrarily defined as the production of 43 1.50 micromoles of glutamyl-hydroxamic acid in 20 minutes at 30° C. The procedure used in the purification of glutamine synthetase was a modification of that proposed by Elliott (24). Temperatures were maintained at 0°—4° C. Centrifuga- tion was carried out at 10,000x g, for 15 minutes at 0° C. Stage 1 - Extraction--Washed cauliflower curd (100 gm.) was homogenized in a Servall omni-mixer with 200 m1. of 0.1 flNaHCO3 for 1 minute. Twelve gm. of M9804 were then stirred in and the precipitate allowed to settle over— night at 0° C. The supernatant fluid was poured off and the remaining suspension centrifuged. The two supernatant fluids were combined. Aliquots were used for estimating protein content and glutamine synthetase activity, in the presence and absence of BA. Stage 2 - Fractionation with ammonium sulfate--The extract was adjusted to pH 6.5 by the addition of 2 g KH2P04 and 0.33 gm. of solid ammonium sulfate per ml. The precipitate was allowed to settle overnight at 0° C and the supernatant discarded. The precipitate was suspended in 220 ml. of cold distilled water and brought to pH 7.2 by the addition of'fl NaOH. The thick suspension was put into cellophane tubes, and dialyzed with stirring against two changes of 7 liters of cold distilled water, for about 40 hours. A small sample of the cloudy dialysate was cen- trifuged. This product was used for protein determination and enzyme assay. 44 Stage 3 - Treatment with Protamine--The dialyzed extract was treated with a 2 per cent solution of protamine sulfate, until a small sample, after centrifugation, gave no further precipitate on the addition of a drop of protamine solution. About 200 ml. were required. The inactive pre- cipitate was centrifuged and the supernatant retained for protein and enzyme assay. Stage 4 - Second ammonium sulfate fractionation—- The above supernatant was adjusted to pH 7.6 by the addi- tion of phosphate buffer (prepared as described under gen- eral methods) and 300 m1. of cold, saturated ammonium sul- fate were added. The bulky inactive precipitate was removed by centrifugation and 240 ml. of the cold, saturated ammonium sulfate solution were added to the supernatant. The result- ing precipitate was centrifuged and redissolved in cold HPO water, with the addition of §_K to a pH of 7.3. A 2 4 small sample of redissolved precipitate was used for pro- tein and enzyme assay. Stage 5 - Dialysis-—The remainder of the above sus- pension was dialyzed with stirring against three changes of 7 liters of distilled water for about 30 hours. The precipitate was discarded after centrifugation and the supernatant retained for protein and enzyme assay. Stage 6 - Third ammonium sulfate fractionation-— The above supernatant was adjusted to pH 7.4 by the addi— tion of phosphate buffer and 50 m1. of cold, saturated am- monium sulfate were then added. The precipitate was discarded_ 45 and a further 50 ml. of the saturated ammonium sulfate were added to the supernatant and left overnight at 0° C. The precipitate was centrifuged and redissolved in cold distilled water. .§.K2HPO4 was added to bring the pH of the redissolved precipitate to 7.3. This was tested for protein and gluta- mine synthetase activity. . After stage 6, a 10-fold purification of the enzyme, glutamine synthetase had been obtained. A purification summary is presented in Table II. The comparative activity of glutamine synthetase at each purification stage, in the presence and absence of BA, is portrayed by Fig. 10. 46 2 m 0.00 00.0 T0 000 0.0 om H . 0H 0.00 00.0 m e.0 Hem 0.0 0N m. 0 0.0m 03.0 Ta 00... 0.0 0m H . 0.0 N.0m m0H.0 m 0.H ham m.0 mm m. m 0.0a 00H.0 0.H 00m 0.0 0m m. . m.~ 0.0a 0mH.0 0.0 0.H 00m 0.0 on .w 0 a.m 000.0 0.0 00m 0.0 N00 m. . N.H 0.0 000.0 00 a.m 00m 0.0 N00 .m m 0.0 mmm.0 0.0 N.aam m.0 mom m. . m.H 0.0a mmm.0 50 0.0a 0.000 0.0 mom m. m m.0 0.H 0.00 00HH «.0 man emummue . a 0.0 0.“ 00H N.N~ omen 0.0 00H Houeeoo H emeeeuze Amoec A.He\mec tame» a Aeoac .He\muaeo A.Hec A.He0 usegummue 00000 team suneauu< tampons meets poseaae .Ho> unmnumam Hmuoe GOHOMUHMHHDQ mo mmmum comm um memncm ms» c0 9 The activity of PK was measured by the decrease in optical density at 340 mu, from the oxidation of NADH (re- duced nicotinamide-adenine dinucleotide). Quantitative conversion was assured because of the equilibria of the reactions catalyzed by PK and LDH. The following solutions were prepared using deion- ized glass distilled water, and maintained as in the hexo- kinase experiments. 3 Tris buffer (0.05 E; pH 7.6) and y; (10" gym-See general methods. Magnesium chloride (0.1 §)--Prepared by dissolving 0.19 gm. MgCl2 in water and made up to a volume of 20 ml. Potassium chloride (0.5 fl)--The chemical (0.75 gm.) was dissolved in water and brought up to 20 ml. Adenosine diphosphate (0.1 §_ADP)--The solution 49 consisted of 0.511 gm. of ADP-Na dissolved in 10 m1. of 3 water. Phospho(enol)pyruvate (0.1 fl)--Solution prepared by dissolving 0.234 gm. PEP-Na in 10 ml. of water. 3 Reduced nicotinamide-adenine dinucleotide (ca. 0.01 y NADH)--Prepared by dissolving 35 mg. NADH-Na in 5 m1. 2 Tris buffer (pH 7.6). Pyruvic kinase (Sigma Chemical Co., 2,500 units)-- A unit was defined as that amount of enzyme which catalyzed the conversion of 250 micromoles of PEP to pyruvate per minute at a pH of 7.6 and at 25° C. The enzyme suspension was reconstituted by the addition of 0.1 m1. Tris (pH 7.6) for each 50 units. Lactic dehydrogenase (Sigma Chemical Co., 10,000 units)--Each unit was that amount required to catalyze the conversion of 400 micromoles of NADH per minute at a pH of 7.6 and at 25° C. The suspension was reconstituted by the addition of 0.1 m1. Tris (pH 7.6) per 200 units. Optical density (O.D.) values utilized for kinetic measurements were obtained with a Beckman DU spectrophoto- meter at 340 mu, a light path of 1 cm., and a final volume of 3 m1. made up with water. Measurements were made against a blank, using silica cuvettes. Components of the blank were pipetted into the cuvettes, in the indicated order. Tris 1.0 ml. KCl 0.1 m1. MgCl 0.2 ml. 2 50 ADP varied PEP 0.2 ml. NADH — ‘DGD water to make up 3.0 ml. BA _ LDH 0.4 ml. (200 units) PK 0.1 ml. (50 units) The control samples received only NADH (0.2 ml.), and the treated received NADH (0.2 ml.), plus BA (0.1 m1.). The final concentration of BA was 3.3x10-5 3? After the addition of lactic dehydrogenase (LDH), the contents were mixed by inversion and allowed to equili- brate for three minutes at 25° C. When the O.D. was con- stant, pyruvic kinase (PK) was added and O.D. measured every 30 seconds. Reaction velocities in the presence of BA and varying concentrations of ADP are shown in Fig. 11. Explor- atory experiments indicated that LDH activity was not af- fected by BA. Double reciprocal plots of the velocities and substrate concentrations of ADP, portraying the nature of BA inhibition of pyruvic kinase activity, are presented in Fig. 12. ‘Deionized glass distilled water. Figure 11 Effect of BA on pyruvic kinase activity. Changes in velocity (£>O.D./min.) correspond to the oxidation of NADH in in yitgg systems containing various concentrations of ADP and 3.3x10"5 fl BA. ON «0 2.0-0. x 0.0. m. 130: 8:22.200 a0< o. _ ,‘OIX(a;nugw/ 'C] '0 v) : MyooIaA Figure 12 Double reciprocal plots of the velocities‘ and substrate concentrations (ADP) illustrate competitive inhibition of 5 are the apparent pyruvic kinase by 3.3x10" fl BA. KS and K I substrate and inhibitor dissociation constants respectively. should read % x 10-2.