3‘; . “ F 3;»: C” I D K. . , . ,’.' *3/31» 1'. I. , ,M. a vro n -. . a u. givi'éé‘f‘i‘lfl’i’ . a?” a? " 3 II1€£I 'Nv I y ,4: ‘ n (a. x; no ’fv‘h’ah'h". ng.r “o ’rv .P b~ p ‘ I .' h n v) i a“? m garmw. . flrymfb #3133" ‘ w ,. r" in :2: 3.539? , up I .r .577- »' ' “,M ' 0 £796! (15:4,: i‘ rvr . 3.‘ I w vi. wafi‘ fikéflfi ”4-73" F}: 'i9r'irfii‘ig 3‘: ‘Mo - v ~J2‘a -, 3 n I .,_~ I'r . r m, . ,v’n» . ”a: 7‘? - 0’)" .‘n ~ ‘ . . . , _ .7 q‘p"~V-"~‘" 31.3.? :,’,-:,"W,_,, r'" I rt: a- "4’ . g . . 7-2-- v>v 4. 4.304‘. 1;: . a . .5” ‘ 7‘ Fr n pig _- 2 "_’>’a’.-'~,§€z,.,fl 3... .,,r"-,-,,.., "gm,”m... , . '33:." try-9 1‘.- I , M 1 a , ”Quiz.- 15: ” ‘.- "at . v.35. x... , 4.04.: , Wflmxuzxm V ,7“ 4mg - u: a ’ “L gfirzzmr #5.: LIBRA 1' R Y Mid’igan State mVCrgfipy' This is to certify that the thesis entitled HORMONAL REGULATION OF PROTEIN SYNTHESIS IN BARLEY ALEURONE LAYERS presented by Warren H . Evins has been accepted towards fulfillment of the requirements for Ph. D. degreein Biochemistry ./éfl-Mw Major professor Date /»3 j/fi/Zflo/fl /'5/ '7 o ABSTRACT HORMONAL REGULATION OF PROTEIN SYNTHESIS IN BARLEY ALEURONE LAYERS BY Warren H. Evins Exogenous addition of gibberellic acid (GA) to barley aleurone cells induces synthesis of d—amylase and other proteins following an 8 to 10 hour lag period. Dur- ing the lag period endoplasmic reticulum (ER) synthesis and polyribosome formation occur, starting at 2 to 4 hours after hormone treatment. Indirect evidence suggests that the GA-induced protein synthesis occurs on ER membrane- bound polysomes and that the polysomes isolated are membrane-bound. Polysome formation reaches a maximal level at 12 to 15 hours after hormone addition. A linear increase in the proportion of ribosomes present as polysomes is seen, reaching a maximum of 76% polysomes at 10 to 11 hours. Total polysomes increase over 2.5 fold, while the percent polysomes, total ribosomes, number of active ribosomes, and rate of protein synthesis double. Although the number of active ribosomes (ribosomes capable of synthesizing Warren H. Evins nascent polypeptides measured by the formation of acid in— soluble 3H-peptidyl puromycin) doubles, the proportion of the total ribosomes that are active is not affected by the hormone. The rate of protein synthesis in 3129 was mea- sured with l4C-amino acids. The high tryptophan/tyrosine ratios of the bulk of the GA-induced proteins was used as a chemical tag to identify the polysomes isolated as the polysomes responsi- ble for the synthesis of the induced enzymes. Polysomes isolated from hormone—treated cells and nascent poly- peptides released by puromycin from these polysomes have higher tryptophan/tyrosine ratios than polysomes and nascent peptides isolated from control tissues. Various treatments which inhibit synthesis of the hormone—induced enzymes inhibit polysome formation. The plant hormone abscisic acid (ABA) prevents GA-induced polysome formation. The removal of GA by washing or the mid-course addition of ABA (2.5 x 10“7 fl) cause a 10 to 15% decrease in the percent polysomes within 2 hours. When ABA is added at the start of the incubation period with 1 pg GA, a 10% decrease in the percent polysomes and no polysome formation occur. Anaerobiosis and actino- mycin D added at the start of the incubation period inhibit both d-amylase synthesis and polysome formation. Fluoro-uracil inhibits both GA responses to a lesser extent. Warren H. Evins GA increases ER synthesis 4 to 8 fold, as measured by determining the amount of 14 C-choline incorporated into a lipid extractable, acid insoluble, semi—purified ER fraction. 6—Methylpurine does not inhibit the recovery of d-amylase synthesis when GA is added back to aleurone layers following GA removal. However, the application of 6-methylpurine to aleurone layers 6 hours prior to the readdition of GA (following GA removal) completely inhibits any recoyery of a-amylase synthesis. The inhi- bition is reversed if 6-methylpurine is removed when GA is added back. HORMONAL REGULATION OF PROTEIN SYNTHESIS IN BARLEY ALEURONE LAYERS BY Warren H. Evins A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1970 ACKNOWLEDGMENT I would like to express my sincere gratitude to Dr. J. E. Varner, my major professor and chairman of my guidance committee. Special thanks are given to Mrs. Rhonda Papaio— annou, Mr. Robert Kever, Mr. Robert Geyer, and Drs. Michael Jost and Frank Dennis. The help of the author's guidance committee: Drs. Anton Lang, Clarence Suelter, Phil Filner, Allan Morris, and Fritz Rottman is also greatly appreciated. The work reported in this thesis was performed in the MSU/AEC Plant Research Laboratory and the Depart- ment of Biochemistry. The author was supported by assistantships from the Department of Biochemistry (State of Michigan) and the AEC Plant Research Laboratory (U.S. Atomic Energy Commission and National Science Foundation). The work performed was supported by the U.S. Atomic Energy Commission (contract AT (ll—l)-l338) and a National Science Foundation grant (GB—8774) to Professor J. E. Varner. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . V LIST OF FIGURES. . . . . . . . . . . . Vii LIST OF ABBREVIATIONS. . . . . . . . . . ix Section I. THE INDUCTION OF POLYSOME FORMATION AND OF TRYPTOPHAN-RICH PROTEINS BY GIBBERELLIC ACID . . . . . . . . . 1 Introduction. . . . . . . . . . 1 Methods . . . . . . . . . . . 4 Incubation. . . . . . . . . . 4 Polyribosome Isolation. . . . . . 5 Polysome Sedimentation. . . . . . 7 Polysome Quantitation . . . . . . 7 RNA Determination . . . . . . . 8 Amino Acid Incorporation . . . . . 8 Trp/Tyr Ratio Determination . . . . lO Polysomal RNA Preparation. . . . . 12 Formation of 3H—Peptidyl Puromycin. . 13 Results . . . . . . . . . . . 14 Protein Synthesis . . . . . . . l4 Polysome Formation . . . . . . . l6 Polysomal Distribution. . . . . . 19 Total Ribosomes . . . . . . . . 22 Effects of RNAase . . . . . . . 23 Sedimentation Coefficients . . . . 32 Active Ribosome Determination . . . 32 Identification of Some of the GA- Induced Enzymes by Use of the Trp/Tyr Ratio . . . . . . . . . . . 43 Discussion . . . . . . . . . . 50 Summary . . . . . . . . . . . 54 Section Page II. GIBBERELLIC ACID CONTROLLED POLYSOME FORMATION: PREVENTION BY ABSCISIC ACID AND ANTI-METABOLITES AND FUNCTIONAL STUDIES 0 O O O O O O O O 0 O I O 56 Introduction. . . . . . . . . . . 56 Methods . . . . . . . . . . . . 58 Results . . . . . . . . . . . . 59 Functional Studies on Polysomes. . . . 62 Removal of GA. . . . . . . . . . 67 Discussion . . . . . . . . . . . 72 Summary . . . . . . . . . . . . 74 III. HORMONE CONTROLLED ENDOPLASMIC RETICULUM SYNTHESIS IN BARLEY ALEURONE CELLS . . . . 76 Introduction. . . . . . . . . . . 76 Methods . . . . . . . . . . . . 77 Results . . . . . . . . . . . . 79 Discussion . . . . . . . . . . . 84 Summary . . . . . . . . . . . . 84 BIBLIOGRAPHY. . . . . . . . . . . . . . 86 iv Table LIST OF TABLES SECTION I The Effect of GA on 14C—Amino Acid Incor- poration by Barley Aleurone Layers . . . Recovery of Polysomes from Mixtures of Rat Liver and Aleurone Layer Ribosomes . . . Sedimentation Coefficients of Polysomes . . Formation of Peptidyl-Puromycin by Ribosomes from Barley Aleurone: Effect of GA and Time of Incubation . . . . . . . . Tryptophan and Tyrosine Composition of Some Proteins. O O O O O O O O O O 0 Location of Acid Precipitable 3H-Tryptophan in Barley Aleurone . . . . . . . . Location and Puromycin Release of Tryptophan-Rich Nascent Polypeptides . SECTION II Effect of Mid—Course Addition of ABA on GA— Induced Polysome Formation. . . . Effect of Fluorouracil and Actinomycin D on d—Amylase Production. . . . . . The Distribution of a-Amylase in the Medium and Homogenate of Barley Aleurone Layers . The Effect of Removal and Readdition of GA on the Distribution of Ribosomes in Polysomes . . . . . . . . . . . Page 15 31 33 42 44 46 49 63 64 68 69 Table Page SECTION III 1. l4C-Choline Incorporation (Acid Insoluble) in Various Cell Fractions . . . . . . 80 2. Removal of Acid-Insoluble Radioactivity from the Choline-Labeled Microsomal Fraction by Lipid and Nucleic Acid Extraction Procedures . . . . . . . . . . . 81 vi Figure LIST OF FIGURES SECTION I The Effect of GA on Polysome Formation . . . The Distribution of Ribosomes in Poly- ribosomes . . . . . . . . . . . . The Effect of GA on the Total Number of Ribosomes Present in the Polysomal Pellet . Typical Polysome Profiles (Older Isolation Method) and the Effect of RNAase . . . . Typical Polysome Profiles (Newer Isolation Method) and the Effect of RNAase . . . . Calibration of Isokinetic Gradients with Rat Liver Polysomes . . . . . . . . . . The Time Course of 3H-Peptidyl Puromycin Formation by Barley Aleurone Layer Ribosomes . . . . . . . . . . . . Time Course of Ribosome Activity . . . . . Diagram of Expected Trp/Tyr Ratio Results Following Hormone Treatment . . . . . . SECTION II The Effect of ABA on Polyribosome Formation . Incorporation of 32P-Ortho-Phosphoric Acid and l4C-Amino Acids Into Polysomes. . . . The Effect of 6-Methylpurine on d-Amylase Production Upon Removal and Readdition of GA vii Page 18 21 25 27 29 35 37 41 48 61 66 71 Figure Page SECTION III 1. The Effect of GA on the Rate of Endoplasmic Reticulum Synthesis in Barley Aleurone Layers O O O O O O O O O O O O 83 viii LIST OF ABBREVIATIONS The standard abbreviations used in "Abbreviations and Symbols for Chemical Names of Special Interest in Biological Chemistry" of the IUPAC—IUB Combined Commission on Biochemical Nomenclature, published in (1966) Big- chemistry 5: 2485 are used in this thesis. Some of the more frequently used abbreviations are as follows: ABA abscisic acid Act D actinomycin D ATP adenosine triphosphate c curie DDT l,1,l-trichloro-2,2-bis(pfchlorOphenyl)ethane or (dichlorodipheny1trichloroethane) DNA deoxyribonucleic acid EDTA ethylene diamine tetraacetate ER endoplasmic reticulum FU-5 fluoro-uracil g_ gravity GA, —GA, + GA Gibberellic acid, without GA, with GA GB grinding buffer GTP guanosine triphosphate HEPES NfZ—hydroxyethyl-piperazine—N'2-ethanesu1fonic acid lys lysine PEP phospho-enol-pyruvate POPOP 1,4-bis-[2-(4—methy1-5—phenyloxazolyl)]-benzene PPO 2,5-diphenyloxazole RB ribosomal buffer RNA, m, r, t ribonucleic acid, messenger, ribosomal, transfer RNAase ribonuclease E. Svedberg unit (sedimentation coefficient) TCA trichloroacetic acid Trp tryptophan Tyr tyrosine UV ultraviolet ix THE INDUCTION OF POLYSOME FORMATION AND OF TRYPTOPHAN-RICH PROTEINS BY GIBBERELLIC ACID Introduction The barley aleurone is a highly specialized tissue of non-dividing cells of a single type in which the pro- duction of certain enzymes is dependent on the plant hor- mone gibberellic acid (GA) (Varner gt 31., 1965; Cris- peels and Varner, 1967a) and with the exception of abscisic acid (ABA) not influenced by other plant hor- mones (J. E. Varner, unpublished observations; Cleland and McCombs, 1965). The aleurone tissue produces and releases enzymes that degrade the starchy food reserves of the endosperm to supply the embryo with energy and metabolites necessary for the development of the young seedling. These enzyme activities appear or increase in response to GA, and include: d- and B-amylases, catalase, B-hydroxymethyl cellulase, endo-B-glucanase, endopento- sanase, peptidases, protease, RNAase, transaminase, etc. (Brian, 1966; Chrispeels and Varner, 1967a; Paleg, 1960; Yomo 1960a,b,c,d). The increases in d-amylase and protease have been shown to be due to SE.EQXQ synthesis. They occur, however, only after an 8 to 10 hour lag period following GA addition (Chrispeels and Varner, 1967a; Filner and Varner, 1967; Jacobsen and Varner, 1967). Two possible control points for GA action are transcription and translation. The evidence that tran- scription of new RNAs is required for the hormone response is inconclusive. Washing out GA arrests d—amylase syn- thesis (Chrispeels and Varner, 1967b) so that GA does not act only as a trigger, possibly indicating a requirement for continuous RNA synthesis. Inhibition of d—amylase production by 6—methylpurine, an inhibitor of RNA syn- thesis (Chrispeels and Varner, 1967b) may be due to an effect on the level or action of some nucleotide cofactor. Act D inhibits enzyme synthesis when it is added during the lag period, although addition after the lag period inhibits secretion (Chrispeels and Varner, 1967b). How- ever, Act D has effects other than the inhibition of RNA synthesis, such as the inhibition of phOSpholipid syn- thesis (Pastan and Friedman, 1968). Two observations suggest that GA might act during translation. First, the rate of d-amylase synthesis depends on the GA concentration in the range of 10-7 to 10—11 M. Another lag period occurs before the rate of d-amylase synthesis increases following an increase in GA concentration (Chrispeels and Varner, 1967a,b). Second, although GA induces enzyme synthesis, it was reported that there is no increase in the rate of total protein synthesis at 12 hours following hormone treatment (Varner §£_al., 1965). These results suggested further investigation of the possible involvement of GA in one of the steps of translation. One approach is to find out what is happen- ing during the 8 to 10 hour lag period before d-amylase production starts. Perhaps some fundamental biochemical parameter responds to GA earlier than a-amylase synthesis. In addition, it now appears that the previous measurements of protein synthesis were low because of isotope dilution and proteolysis. Two ways to check the increase in protein synthesis not dependent on isotope dilution are to measure the polysomal levels after various treatments and to determine the number of active ribosomes. I now report that polysome formation starts 3 to 4 hours after hormone treatment. The rate of protein synthesis (l4C-amino acid incorporation) doubles toward the end of the lag period and the number of ribosomes increases. The hormone has no effect on the proportion of ribosomes active in protein synthesis. The polysomes are probably bound to the endoplasmic reticulum. The trp-tyr ratio of nascent peptides [d-amylase and other GA-induced proteins are trp—rich (Fischer and Stein, 1960; J. E. Varner, unpublished observations)] was used as a characteristic identification tag to show that the poly- somes were responsible for synthesis of the GA—induced proteins. Methods Incubation Barley (Hordeum vulgare var. Himalaya, supplied by Drs. R. A. Nilan, B. V. Conger, and the Agronomy Club at Washington State University, Pullman) half-seeds were prepared by making 2 transverse incisions, one incision removing the embryo and the other removing the distal tip. The half-seeds were sterilized in about 0.5 ml/half—seed of a sodium hypochlorite solution (reagent grade, diluted 1:5, 4—6+ % NaOCl) in groups of 70-120 half-seeds. After 25 minutes, the solution was stirred, and rinsed 6 times with copious amounts of sterile distilled H20. The half- seeds were preincubated 3 days on moist sterile sand in Petri dishes wrapped in aluminum foil. Two sterile stainless steel spatulas, one with flexible rounded ends, and the other with a rigid square blade, were used to dissect the aleurone layers from the starchy endosperm. All operations were performed in a sterile hood equipped with a UV light. Between 30 and 40 aleurone layers were shaken on a Dubnoff metabolic shaker with 5 ml of incubation medium containing 1 mM Na acetate, pH 4.8, 20 mM CaCl and 2! where required, 1 uM K+ gibberellic acid (GA3), in a 50 ml flask (approximately 80 to 100 oscillations/minute, 25°). At the end of the incubation period, the layers were rinsed 6 times with copious volumes of sterile H20 and blotted on sterile paper towels. All further oper- ations were carried out in the cold room. Care was taken not to warm the homogenate with the hands. All pipets and glassware were handled with plastic gloves and not touched where they would be in contact with the layers or cell fractions, in order to prevent contamination with ribonoclease which might cause degradation of polysomes. Polyribosome Isolation The procedures of Wettstein, Staehelin, and N011 (1963, 1964) as modified using ideas of Leaver and Key (1967) were used for polysome isolation. A prechilled (-20°) porcelain mortar was half—filled with liquid N2 and the layers were added. After the liquid N2 evaporated, the layers were powdered by rapid grinding. The layers were transferred to a chilled homogenizer in an ice bucket (Duall tissue grinder, size E, Kontes Glass Co., Vineland, N.J., empty capacity without pestle approxi- mately 125 ml), which had previously been hand ground with medium, fine, then very fine abrasive (Zip grinding compound, Zip Abrasive Co., Cleveland). As polysome distributions differed with the amount of grinding used to prepare the homogenizer, the same homogenizer was used for all samples. Four ml of "GB" [grinding buffer, containing: 450 mM sucrose, 100 mM HEPES, pH 7.55, with 50 mM K+, 2 mM Mg acetate, 7 mM 2-mercaptoethanol (0.5 ul/ml total volume)] was then added to the homogenizer. The powder was allowed to thaw for 5 minutes and then ground with 2 to 3 strokes and 3 to 5 quarter turns/stroke to the same “feel" and visible consistency. The homogenate was decanted into a cold centrifuge tube. The homogenizer and pestle were rinsed twice with 3 ml of GB. The homogenate was centrifuged at 0° in the Sorvall SS-34 rotor (2 kg for 10 minutes). The super- natant was decanted and Spun at 10 kg for 15 minutes (Method 2). (Earlier studies used centrifugal spins of 5 and then 27 kg (Method 1), but greater polysome recovery and a greater detergent effect are seen with the slower centrifugations.) The supernatant was centrifuged 2 hours through a discontinuous sucrose gradient in the Beckman 65 rotor at full speed. The discontinuous gradient was composed of a bottom layer of 3.5 ml of 1.6 M sucrose buffer [RNAase-free sucrose, Mann Research Laboratories, New York, containing: 50 mM HEPES, pH 7.55, with 25 mM K+, 2 thMg acetate, and 7 mM 2-mercaptoethanol (0.5 ul/ml total volume)], a middle layer of 3.0 ml of 0.6 M sucrose buffer, and the top layer being the 10 kg supernatant in 0.45 M sucrose. The pellet produced will be referred to as the polysomal pellet. The polysomal pellet was resuspended in 0.3 ml "RB" (ribosomal buffer, same as above sucrose buffers, but without any sucrose) with a ground glass pestle (Pyrex no. 7725, supplied by Sargent-Welch Scientific Supply Co., Chicago). Polysome Sedimentation The resuspended polysomal pellets were layered on 0.3-1.0 M isokinetic sucrose gradients as described by Noll (1967). Samples were centrifuged at 0° in Beckman SW65 or SW56 rotors at full speed. The 4.8 ml (with sample added) gradients were centrifuged for 33 minutes in the SW65 rotor, while 40 minutes were required to centrifuge 3.8 ml gradients spun in the SW56 rotor. After centrifugation the bottom of the polyallomer tube was punctured by means of a puncturing device (J&I Technical Specialties, 1535 Cornelia Ave., Waukegan, Illinois 60085). The gradient was pumped through a Helma straight—through 4 mm flow cell in a Gilford spectro— photometer as described by Noll (1969). Polysome profiles were obtained by recording the absorbancy of material in the gradient at 260 nm on the Gilford—Honeywell recorder. (The scan speed was 1 in/min and the pump flow rate was 0.8 ml/min.) Polysome Quantitation The areas of the polysome and monosome regions of the absorbancy scans were determined with a Keuffel l and Esser planimeter graduated in area units of 0.1 square cm. The areas were measured 3 times and averaged. In most samples, 40 aleurone layers were used and the absorbancy scans were recorded at 0.750 A260 units as full scale absorbancy. However, the number of aleurone layers and the full scale absorbancy used for recording the various scans are not the same in all experiments. Therefore, all areas are expressed as the number of area units (0.1 cm2) per 100 aleurone layers, recorded when using 1.0 absorbancy units as the full scale chart deflection (approximately 0.0025 A units/area unit). 260 The polysomal distribution or the "percent poly— somes" was calculated by dividing the area of the polysome (P) region of the sucrose gradient scans by the sum of the areas of the polysomal and monosomal (M) regions (% P/P+M) . RNA Determination RNA was determined by the spectrophotometric method of Warburg and Christian (1942) in the Cary 15 double- beam spectrophotometer using an aliquot of the resus- pended polysomal pellets. The results are expressed as pg RNA per 100 aleurone layers. Amino Acid Incorporation Incorporation of l4C—amino acid was performed with 10 aleurone layers and 0.5 uc of a mixture of 15 14C— uniformly labeled amino acids (reconstituted algal protein Q‘s. .. hydrolysate, Volk Radiochemical Corp. or International Chemical and Nuclear Corp., Irvine, Cal.). Carrier—free amino acid was added to the aleurone layers at the start of the incubation period. The aleurone layers were ground and the homogenate of the aleurone layers and the medium were prepared as described by Chrispeels and Varner (1967a). In one experiment, an ethanol extract was prepared by washing the pellets 3 times with 1 m1 of 70% ethanol and combining the washings. Carrier bovine serum albumen (crystalline, A grade, Calbiochem, Los Angeles, Cal.) was added to each fraction (25 ug). The homogenate, medium, and extract were brought to "10%" TCA by the addition of 50% (wzv) TCA and allowed to sit over- night in the cold room. The precipitate was collected on Millipore filters (0.45 p, 25 mm diameter, Millipore Filter Corp., Bedford, Mass.), washed with at least 30 m1 of 5% TCA containing carrier amino acids (neutralized casein acid hydrolysate, Calbiochem), dried for several hours at 70°, and counted with 10 ml of scintillation fluid “A" (49 PPO + 100 mg POPOP per 1 of toluene) in a Beckman scintillation counter. In one experiment, amino acid incorporation was determined in the presence of carrier amino acids. Tryptophan, phenylalanine, and cysteine were added to a mixture of the 17 other protein amino acids to make a freshly prepared solution. Each amino acid (A grade, Calbiochem) was present at a concentration of 5 x 10—4 M. 10 Trp/Tyr Ratio Determination One uc of L-l4C-tyr (uniform label, New England Nuclear Corp., Boston, sp. act. 362 mc/mmole) and 5 uc of L-3H-trp (general label, New England Nuclear Corp., sp. act. 5.4 c/mmole, supplied in 50% ethanol solution) were added to the aleurone layers for 2 hours, beginning 8 hours after the start of incubation. In some experi— ments, the labels were present for all 10 hours. Cell fractions were prepared as described above (see amino acid incorporation). In another series of experiments, a 16 minute pulse of labeled trp and tyr was given to the aleurone layers so that the aleurone layers were incubated a total of 10 hours. 3H-trp and l4C--tyr were used to determine the trp/tyr ratio. The reverse experiment was also l4C (side chain label, New performed using L—trp-3- England Nuclear Corp., sp. act. 22.8 mc/mmole) and L-tyr- 3,5-3H (New England Nuclear Corp., sp. act. 16.5 c/mmole, supplied in sterile 50% ethanol solution). The pulse was given in order to label nascent peptides that are attached to the polysomes. At 9 hours 45 minutes after the start of incubation, the aleurone layers were transferred to a clean, sterile 25 m1 erlenmeyer flask after rinsing 3 times with sterile H20 and blotting on sterile paper towels. One hundred ul of a mixture containing the carrier—free trp and tyr labels and incubation medium :GA was spread evenly over the 40 aleurone layers. All ll transfer operations were performed in the sterile transfer hood. The flask was replaced on the Dubnoff metabolic shaker at 25°. It was removed from the shaker after the labeling period, rinsed 6 times with sterile H20, and the layers were blotted. The polysomal pellet was prepared as described. The polysomal pellet was resuspended in 0.2 m1 of RB and 0.15 ul of a 10—2 M stock solution of puromycin (nutritional Biochemical Corp., Cleveland) was added (7.5 x 10'4 M). The suspension was allowed to react with the puromycin for 30 minutes at 0° in an ice bucket as described by Redman and Sabatini (1966) and Redman (1967). The suspension was layered onto a discontinuous sucrose buffer gradient and spun in the Beckman SW65 or SW56 rotors at full speed for 2 hours. The discontinuous gradient consisted of a bottom layer of 1.6 M sucrose buffer, a middle layer of 0.3 M sucrose buffer, and the sample in buffer alone. The middle layer was 2.0 ml in the SW56 rotor tube, and 2.5 ml in the SW65 rotor tube; the total volumes were 3.8 and 4.8 ml, respectively. After centrifugation, the top 2 layers were re- moved with a disposable Pasteur pipet, the bottom layer was decanted, and the pellet was taken up in 5% TCA. The tOp and bottom supernatant fractions were counted with 18 ml of Bray's scintillation fluid (Bray, 1960) in the Beckman liquid scintillation counter. The pellet was 12 collected on a Millipore filter, washed with 25 ml of 5% TCA, dried for several hours at 70°, and counted with 10 ml of scintillation fluid "A". 4 4 In some experiments, 5 x 10— M L—trp and 5 x 10‘ M L-tyr were added to the first discontinuous sucrose ultracentrifugal gradients before preparation to the polysomal pellet. These carrier amino acids were added in order to remove some of the background counts present in the supernatant fractions. Polysomal RNA Preparation The polysomal pellet was resuspended with a ground glass homogenizer in 0.5 volumes of 0.1 M EDTA - Na2 and 0.5 volumes of RB + 2—mercaptoethanol (total volume 0.3 ml) and was incubated for 30 minutes with EDTA at 0°. No further release of polysomal RNA occurs after this time. The suspension was layered onto continuous 15—30% isokinetic 10 mM EDTA - NaZ—sucrose buffer gradients and spun in the SW65 or SW56 rotors of the Beckman ultra- centrifuge at full speed. A spin of 3.75 hours was used to separate the heavy and light ribosomal subunits from 5 S and tRNA. In order to separate the mRNA region, the heavy ribosomal subunit was pelleted during 9—13 hour runs. The sucrose gradients were prepared by the N011 method using 15% and 37% sucrose buffer-EDTA solutions. 13 The tube was punctured as described and scanned in the Gilford spectrophotometer. Fractions were col- lected with an LKB fraction collector or by hand. Twenty- five pg of carrier DNA was added to each fraction, followed by 50% (w:v) TCA to a final concentration of 10%. The sample was allowed to sit in the cold room overnight. The precipitate was collected on Millipore filters washed with at least 30 ml of 5% TCA with 0.1 M carrier phosphate, dried at 70°, and counted in the Beck- man scintillation counter. Formation of 3H—peptidyl Puromycin The first step in the assay of the number of active ribosomes is the reaction between 3H—puromycin and the polysome—bound nascent peptidyl tRNA. The methods of Wool and Kurihara (1967) were used. Ribosomes isolated from aleurone cells were incubated at 37° in a reaction mixture (0.500 ml total volume) that contained: 37.7 pM HEPES, pH 7.55, with 18.8 NA K+, 50 PM Mg acetate, 7.3 mM 2— mercaptoethanol (0.5 pl/ml total volume), 5 mM ATP, 50 pM GTP, 1 mM PEP, and 10 pg pyruvate kinase. In most experi— ments 5 pc of puromycin (puromycin methoxy-3H (N)- dihydrochloride, specific activity 1.11 c/mmole) was used. The reaction was terminated by the addition of 5 ml of 10% TCA, filtered on a Millipore filter, and washed with 5 50 ml of 5% TCA containing 5 x 10— M carrier puromycin HCl. The filter was dried at 70°, and counted with 10 m1 14 of scintillation fluid in the Beckman scintillation counter. Most of the control experiments performed by Wool and Kurihara with rat muscle ribosomes were repeated with aleurone layer ribosomes. The amount of RNA present in each of the resus- pended polysomal pellets assayed for active ribosomes was measured spectrophotometrically by A260/A280 ratios as previously described. This allowed the determination of the specific activity of active ribosomes or the cpm 3H—peptidyl puromycin formed per pg RNA. The following parameters were determined or calcu- lated: (a) total number of ribosomes, (b) total molecules of peptidyl puromycin formed, and (c) the ratio (b)/(a) to obtain the molecules 3H-peptidyl puromycin/ribosome from which the number or percent active ribosomes is easily obtained. Results Protein Synthesis The incorporation of a carrier—free mixture of l4C—amino acids was measured in order to estimate the rate of protein synthesis (Table 1). At all times, there is more labeled protein in the homogenates of the control samples; whereas, the hormone treated samples have more labeled protein in the medium. After 8 hours of v... 15 .He m masHo> Hmuou m.HH N.OOH <0I O.vo H.Hm O.MH n.mOH ¢w+ mcHom ocHEm HoHuumo QHHB .musoc w . . N.NmH m.mv «.mmH mH «OI . . m.mm m.mm O.H¢H HuHmcom ommoHosconHH mo moHumH omm<\ 0N4 map OcHHDmmoE ma ANOOHV GBHHmHHQU paw OHSQHMB Ho pocuofi esp mchs mHHBOHHpoEOHocmonuommm UOCHEHOHOO mmz muome OQOHSOHB OOH\ Ectom <0I l um 092 v no... m2340> Eotom omo Eotom (0| 0 muosz v I 000.0 Eotom no... MZDJO> \\ O \\ fl \ \ \ oauHH Hon xm HO HO .Amqm + 40 +O mmEommHom Ho>HH you + muohmH mcousmHm moHHmn ON mo .Adoflv mHmMMH mconsmHm mmHHmn ow mo mmHmEmm oumoHHQSQ« 31 . . N.em Nam 46 I am . . o.me was <6 + em O.HN mam m.m+ O.He 0mm mam + <0 I am N.H . mm 0.4: O.Hm ewe mam + «o + rm o.eoa eemH . . m.em eemH mosomsHom Ho>HH umm moEOmmHom Hm>HH coum>oomm wEOmmHom moEomocoz «mend Ham on can moEOmmHom Ho>HH umm soapsnaupmao w mmEOmmHom o oEOmmHom Ho>HH ppm mo w mo mmud dEomeom. moEommHom tmmEOmonHm HommH mcousoH¢ paw Hm>HH umm mo mmusprS Scum moEOmmHom mo >Hm>ooom N MHmfle 32 cells shows an increase compared to the distribution in the control aleurone tissue. However, when rat liver polysomes are mixed with hormone-treated aleurone layer polysomes, the resulting polysomal distribution is lower than that in the unmixed aleurone tissue. This reduction is due to the high percentage breakdown of polysomes and the consequent increase in monosomes. It is clear that there is greater polysome breakdown in polysomes from the hormone—treated tissue. Therefore, the lack of polysomes in the control samples is not due to greater polysome degradation. Sedimentation Coefficients In order to further characterize the polysomes and determine the size and number of ribosomes present in the polysomal peaks the sedimentation coefficients of the barley polysomes, monosomes, and large ribosomal sub- unit (Table 3) were determined by comparison with the known sedimentation values of the rat liver polysomes, monosome, and large ribosomal subunit (Noll, 1967) by the method of Stutz and Rawson (1968). Calibration of the isokinetic gradients used with rat liver polysomes (Figure 6) and mixing experiments were performed. Active Ribosome Determination Barley aleurone ribosomes incubated with 3H- puromycin formed radioactive 3H-peptidyl puromycin rapidly (Figure 7). The formation of 3H-peptidyl 33 TABLE 3 Sedimentation Coefficient of Polysomes* n-mer Rat Liver Barley Aleurone Large ribosomal subunit 60 60 l 80 80.5 2 119 117 3 152 150 4 180 179 5 207 207 6 230 233 *The sedimentation coefficients of barley aleurone polysomes were determined by comparison with the known sedimentation values of rat liver polysomes using iso- kinetic gradient centrifugation and mixing experiments. Similar results were obtained in 3 experiments. 34 Figure 6.--Calibration of isokinetic gradients with rat liver polysomes. [Rat liver polysomes of known sedi- mentation coefficients were centrifuged through 0.3 to 1.0 M sucrose gradients in the Beckman SW56 rotor. The position of each peak was plotted XE; its sedimentation coefficient. A straight line was obtained when gradients in the SW65 rotor were used. Each point represents the average of 6 replicate samples. Similar curves were ob- tained in 5 experiments (although some curves showed less deviation from linearity near the top of the gradient).] 35 R SW56 3 l IOO , S/far 50 I 300 36 Figure 7.--The time course of 3H-peptidyl puromycin formation by barley aleurone layer ribosomes. [The ribo- somes (from 10 layers) were incubated with 5 pc of 3H- puromycin (see Methods) and the 3H-puromycin peptides were precipitated and washed with TCA and collected on Millipore filters. Each point represents the average of 4 replicate samples. Similar results were obtained in 2 experiments.] 37 3000 000 *- 2 52685.. I323: I000 . Eco 45 30 Time (min) I5 38 puromycin is essentially completed after a reaction period of 30 minutes, which was used as the standard 37° incu- bation time for all of the assays. Does GA cause an activation of the ribosomes? An active ribosome is a ribosome that is synthesizing nascent protein and consequently has nascent peptidyl tRNA as a structural part of the ribosome. Puromycin added £3 vitro inhibits protein synthesis, but allows an active ribosome to make 1 peptide bond releasing the nascent puromycin peptide. However, a few active ribosomes could react with puromycin again if reinitiation occurs. Previous results indicated that $2 vitro amino acid incorporation was dependent on the addition of a pH 5.0 enzyme fraction to isolated microsomes. Puromycin inhibited 80% of the cell-free amino acid incorporation (Mrs. Rhoda Papaioannou and Evins, unpublished obser- vations). It is likely, therefore, that only one peptide bond is formed per active ribosome. Ribosomes were isolated from aleurone cells that were incubated for various times in the presence or absence of GA. The amount of RNA present in duplicate aliquots was determined. The sample was then divided into 4 equal parts: 2 assays and 2 blanks. Two duplicates were assayed for the amount of 3H-peptidyl puromycin formed during a 30 minute incubation period. The peptidyl puromycin formation reaction in the other 2 duplicates 39 was immediately terminated with the addition of 5 m1 of 10% TCA as soon as the 3H-puromycin was added (zero time blank). Control samples with 3H-puromycin and assay medium were incubated without ribosomes. The no ribo- some and zero time blank incorporations were subtracted from the counts incorporated during the 30 minute incu- bation period. The effect of GA on the change in the number of active ribosomes in the barley aleurone cells with time was measured. The specific activity of TCA precipitable 3H-peptidyl puromycin presented in Figure 8 is a relative measure of the number of active ribosomes. A peak of ribosome activity occurs around 4 hours after the start of incubation. This increase in ribosome activity is, however, not due to hormone action as both the control and GA-treated tissues show the same time course of ribo— some activation; it may be a hydration or injury effect. Subsequent to 4 hours, the ribosome activity decreases in both the control and hormone-treated aleurone cells. The results expressed in Figure 8 are on a per ribosome basis. There is more total ribosome activity in the hormone- treated tissues. There are also more ribosomes present, but the activity of each ribosome does not depend on the presence of GA. Table 4 shows the effect of GA and time of incu— bation on the percent of active ribosomes. Although there H.mucwEHHmmxo N GH pochqu mums mqumoH HMHHEHm .monEmm onoHHmoH O HO ommuo>m map mucwmmHQoH ucHom comm .mHHmoHHHoEouonmonwoomm coHSmmmE mp3 GHQEmm homo mo HOSUHHM cm cH «zm mo pcsoam one .GHOSEOHSQ HoHHHmo OchHmucoo <05 wm QHHB Ombmms cam mHouHHm oHOQHHHHZ co pmpomHHoo was GHOMEOHDQ HMUHHmomImm mHnSHOmcH HUB .cHo>EOH:m Imm mo 01 m cHHB mGOHHmnsocH opscHE om Ho AMGBHQV oHDGHE O OGHBOHHOH ohm um ©o>Mmmm mHmB mmHmEMm oHMOHHQSQ .Ampogpoz paw pxou oomv pmcHEHouop was .ucmmon mmEOmonHH m>Huom mo Hogans mcH mo whammofi m .AfizmH m:\Emov cHomEousm HmpHumwmIm mo muH>Hpoc OHMHoomm ocBH .%HH>HHom wEOmOQHH mo onusoo wEHBII.m oHSOHm 40 m 41 20 I0 Time (hrs) - ID 1 l n v O O O O O O O o 0 0 0 O o o m In <1- !0 N _ VNHJ brI/ quKqumd Mpudad—H deo sawosoqu engiov .mucmEHHmmxo N cH Umchqu wHoB mHHSmmH HmHHEHm .mmHBOHHmsp 030 mo omnum>m map mum muHSmmH HH¢ .Amponumz can mep mmmv pump msoH>mHm Eonm coumHDOHmo mHoB mmEomonHH m>Huom Hcmoumm mnH 0cm cmEHow aHo%Eousm HmcHummmIm mo mmHsomHoE mo Hmnfisc m£B« 42 m m.mm m.vmm OO.H OH w.om O.me om.m NH m.nm m.omm mm.m m H.mm O.mOH On.m v w.m 0.0mH mHOH x ow. O 40+ O.mm «.mmH mm.H OH b.Hv m.OhH mm.H NH m.vm O.mmH Hm.m m m.mm m.mOH mm.m v h.m 0.0mH mHOH x ow. O «0| mmEOmOQHm coauom o>auo¢ dzm m: caomEousm HmcHumomlm Amunv oEHB Hcmfipmmne . . m coHucbsocH w moHsooHoz scoHHmnsocH mo mEHB 0cm HO HO uommmm "mCOHSmH¢ hoHHmm Eoum moEomonHm ma zHohfionsmIHmpHpmom mo coHumEHom v mqmfiB 43 is slightly more ribosomal activity present in the GA- treated cells early in the incubation period, the activity per ribosome decreases faster in the hormone-treated cells. At 16 hours the control tissue has 55% more activity per ribosome than the hormone-treated tissue. It is clear that the hormone causes an increase in total ribosomes and also in total polysomes. The expected hormone-mediated increase in the number of active ribosomes is seen in the above experiments. However, these results indicate that the activity of each ribosome is not affected by the hormone; the hormone does not cause ribosome activation. Identification of Some of the GA—Induced Enzymes by Use of the Trp/Tyr Ratio Fischer and Stein (1960) noted that although d-amylases display no striking similarities in their rather average amino acid composition, they are rich in trp and tyr which accounts for their high extinction coefficients at 280 nm. The tyr-to-trp ratios of a-amylases vary markedly, however, providing them with distinctive ab- sorption spectra. Many hydrolytic enzymes are rich in trp (Table 5). Barley d-amylase has a high trp/tyr ratio (J. E. Varner, personal communication). The distribution between medium and homogenate .4 of 3H-trp incorporated into TCA-precipitable protein of 44 TABLE 5 Tryptophan and Tyrosine Composition of Some Proteins Moles Amino Acid in Protein Reference Source ( MW 3 Total 100' 00° 9 Of Trp/Tyr x 10 ) N r Trp Ty): Catalase 1 Equine liver 250 nd nd 34 . 4 . Trp Synthetase l Escherichia coli 29 . 5 17. 5 0 23. 9 0 Carbonic Anhydrsse-I l Bovme erythrocytes 31 14. 9 mi 21.0 . . Carbonic Anhydrase-II 1 Bovine erythrocytes 31 16 . 1 nd 19. 4 . Aas 1 Bovine pancreas l2 . 7 17 . 8 nd 42 . 3 . Ass 1 Bovine pancreas 12 . 7 nd nd 40. 7 . . Enolase 1 Yeast 67.2 17 3 7.8 22.4 .35 Enclose 1 Yeast 67.2 nd 10.7 21.6 .50 a-Amylsse 8 Bacillus subtilis 48.7 16.23 30.5 50.0 .61 Pspain l P ya 20 34 16.10 22 9 81 3 28 Papain 1 Papaya 20.34 16. 10 20 6 66 7 31 Chymot sinogen A 1 Bov1ne 25. l 16. 27 9 16 3 l. 7 Chymotrypsinogen B l Bovxne l6. 2 25 7 12 6 Csrboxypeptidsse A 1 Bovine 34. 3 15. 4 17 7 57. 2 . 31 ’1 Csrboxypeptidase A 1 Bovine 34. 3 nd l6 9 ; Carboxypeptidase B 1 Bovrne 34 nd 1 64 . 0 . 45 * Carboxypeptidsse B 1 Porcme 34 . 3 15 . 5 25. 9 7 Pepsin l Bov1ne 35 14.9 17.2 51.9 33 Insulin 1 Bovine 5. 7 d 0 .0 0 Insulin A-component 1 Bovine . . 15 88 0 69.5 0 Insulin B-component l Bov1ne . . 15 63 0 . 4 0 Glucagon 1 Bovxne 3.6 17 45 28.0 58.6 .48 Hemoglobin a—Chain 1 Human 15.1 nd 5.9 18.7 .32 Hemoglobin B-Chain 1 man 15. nd 11.1 17.8 .62 Apoferritin 1 Horse spleen 480 16. 3 4.4 27.8 .16 ytochrome C 1 Horse heart 12.5 16.8 7.5 2 .2 .28 Cytochrome C 1 Horse heart 12.1 15.98 8.3 29.7 .28 Cytochrome C 1 Horse heart 12.1 ' 15.91 8.3 3 .4 26 Serum bumin 1 nd 0.9 25. 7 04 B-Lsctoglobulin AB 1 Bovine 37 7 nd 12 . 8 21. 5 60 B-Lsctoglobulin A 1 Bovrne 37. 7 nd 12 . 8 21. 4 60 B-Lactoglobulin B 1 Bovine 37 nd 12 . 7 20 . 9 Lactalbumin-u 1 Bovine 15.5 15 86 34. 3 29.7 1.2 as ‘ 1 Bovine . . 9.8 40.3 Histone 1 Calf Thymus 15.5 17.4 o 20.4 0 Histone A 1 Calf Thymus 15.5 15.4 0 4.1 0 Histone 1 Calf Thymus 15.5 16.9 0 24.1 0 Nucleohistone l Calf Thymus 10. 0 16. 9 nd 20. 5 Silkl-‘ibroin 1 Bomb x mori . . 18.3 2.0 66.6 03 Tropomyosi 1 Pinns 50 18.1 0 14 o Collae 1 Python skin . 16.19 0 1.7 0 Collagen-Elastoldin l Shark f . . 18 2 O 9. 5 0 a- la 2 Bacillus masceran 140 . . 22' 56' 39 a- 1s:e 3 Asper gi iilus ni er sacid stable 58 . . ll' 31' . 35 a-Anyllse 3 acid unstable 61 . . 12' 34' .35 u-Anylase 4 Aspergillus oryzae 51.8 . . 10' 31' .32 . u- lsse 5 Bar ey a eurone 45 . . 10' 10' 1. 0 Cytochrome C 6 Various 12.1 . . 1-2' 4—5' .20—.50 Ferredoxin 6 Clostridium pasterianum 6 . . 0' 1* 0 Perredoxin 6 C. Eutyricum 6 . . 0' 0' 0 ucose Oxidase 7 Aspergillus niger 150 . . 22" 5' 4 4 Hemoglobin a 6 Various 15. l . . 1' 3' . 33 Hemoglobin B 6 Various 15.9 . . 2' 2-4' 1.0-0.5 Hemoglobin Y 6 uman . . . . 3' 2" l 5 Hemoglobin 6 6 Human . . . . 2' 3' . 66 Lye zym 6 Chicken 14.3 . . 6' 3' 2.0 Hyoglobine 6 Sperm whale 17. 8 . . 2' 3' . 66 RNAase 6 Bovine 12. 7 . . 0' 6' 0 RNAal 6 Aspergillus oryzae 11.1 . . l' 9' .11 Trypsinogen 6 Bovine 24 . . 4' 10' . 40 ‘Residues of amino acid in the total protein. References: (l) Tristam and Smith (1963); (2) Pinto and Campbell (1968); (3) Minoda e_t al. (1969), (4) Stein et al; .(1960); (S) J. E. Varner; (6) Da ayhotf and lick (1968); (7) Pazur 3 g. (1965):— (87 Fischer and Stein TI96 45 barley aleurone cells is shown in Table 6. Proteins formed in the presence of labeled trp and tyr have 36% higher trp/tyr ratios in the homogenate, 3.5 times higher trp/tyr ratios in the medium, and more trp incorporated in hormone-treated aleurone layers. These results suggest that the bulk of the GA-induced proteins are trp—rich and have trp—tyr ratios. The results demonstrate that the trp/tyr ratio can be used to identify at least some of the GA—induced enzymes. Double label experiments were performed to show that the polysomes isolated from GA-treated cells were the polysomes responsible for synthesis of the GA-induced proteins (Figure 9). Growing polypeptide chains of the GA—induced hydrolases should have a higher trp/tyr ratio than nascent peptides synthesized in the absence of the hormone. Puromycin release and recovery of the GA—induced peptides would demonstrate that they have higher trp/tyr ratios than nascent peptides of the control polysomes. Table 7 shows that ribosomes from GA-treated cells have significantly higher trp/tyr ratios than ribosomes from control cells. Puromycin release of the nascent peptides causes a large decrease in the trp/tyr ratio of the ribosomes isolated from GA-treated cells. The released puromycin peptides are recovered in the supernatant and have a higher trp/tyr ratio. Contaminating labeled amino acids are also found in the supernatant. Release of 46 TABLE 6 Location of Acid Precipitable 3H-Tryptophan in Barley Aleurone* Homogenate Medium Treatment Trp/Tyr Trp/Tyr cmp Ratio cmp Ratio +GA 48,000 .87 9600 4.78 —GA 32,400 .64 5200 1.35 *Triplicate samples of 10 aleurone layers were incubated at 25° in the pregence and absence of 1 pM GA. Between 8—10 hours 5 pc of H-tryptophan (trp) and 1 pc of l4C-tyrosine (tyr) were added. Cell fractions were prepared (see Methods), and the TCA precipitates were collected on Millipore filters and washed with 5% TCA containing carrier amino acids. The samples were counted on a Beckman 3 channel liquid scintillation counter and the tr /tyr ratios were calculated using specially pre- pared H and 14C standards. .H.mcoHnoHconm onn nan oonmo mnHSmon Hosuoo one .csonm ono mnHomon oonoomxo one .Amv nconmcnomom onn cH oochEon nHoefionsmIHmpHnmom ucoomoz .nmv ncoHpmnm omonUSm mooanncoomHo o nmsonnu noHnoOSMHnncoo an chmm conooHHoo onoB moEomeHom one .mooHpmom ncoomoc omooHon on poms mmB nHo%Eon5m .poanOmH onoB mofieuno poosocHI<0 mcHNHmonu Icmm moEOmmHom MH Haul A <0+V ocoEnon onn mo oocomonm onn cH 0Hnon n>n\mnn nonoonw o o>on oHsonm moEOmoan one .UoHoHOmH mo3 HoHHom HoEOmoaneHom one .do mo oonomno no oocomonm onn cH AncoEHnomxo omno>onv nenImm oco mnnIUOH no nnmnv ochonmquvH 0cm Amnuv nonmoummnnlmm nnHB poHnom COHHmndocH noon OH o no monscHE OH umMH onn mcHnoo ponmndocH onoB mnomoH oconsoHH\mnH ponoomXo mo EmnmoHQII.m onsmHm 47 48 E38950. 323322. 23»on EEoanam 185835.31 E Eoceoaam 65623. a :C. cosmetic... not. Scented mAw <0+ozom so Eaten. o2 182855.26“... <0I A <0 + atom mso>on 38:24 \ "|‘ / 5f SE 2.13.1.9... 0232 49 TABLE 7 Location and Puromycin Release of Tryptophan- rich Nascent Polypeptides* Trp/Tyr Ratio Treatment :22:: Pellet Supernatant +GA 3H/14C 1.35 1 0.34 1.55 i 0.58 +GA + Puromycin 0.47 i 0 24 2.19 i 0 59 -GA 3H/l4C 0.317 1 0 315 2.34 i 0.72 -GA + Puromycin 0.17 i 0 l6 1 53 i 0 20 l +GA l4C/3H 1.59 i 0.24 0.44 1‘ 0.02 +GA + Puromycin 1.02 i 0 12 0 70 i 0 08 —GA l4C/3H 0.91 i 0.07 0.56 i 0.18 -GA + Puromycin 0.96 i 0 08 0.34 i 0.09 P < 0.1 for significant differences. *Forty aleurone layers were incubated during the last 16 minutes of a 10 hour incubation period with 25 uc of 3H—tryptophan (trp) and 5 uc of -tyrosine (tyr) or in the reverse experiment with 4 uc 1of C- trp and 20 no of 3H-tyr at 25° in the presence and absence of 1 uM GA. The polysomal pellets were treated with 7.5 x 10 4 M_ puro- mycin and the released nascent peptidyl puromycin was separated from the ribosomes by centrifugation through a discontinuous sucrose gradient. In the reverse experiment, 5 X 10' 4 M L-trp and 5 x 10"4 M L- tyr were added to the first discontinuous gradients to reduce background counts. The samples were counted on a Beckman 3 channel liquid scintillation counter (the pellet was precipitated with 10% TCA, collected on a Millipore filter, and washed with 5% TCA containing carrier amino acids-—see Methods, and the supernatant was counted directly with Bray's scintil— lation fluid) and the trp/tyr ratios were calculated using specially prepared 3H and C standards. 50 nascent peptides from non-treated cells reduces the trp/ tyr ratio in the supernatant. The reverse experiments shown in Table 7 give the same results. The ratios in the supernatant are lower primarily because of the reduction of contaminating label by the addition of carrier trp and tyr. All important differences are significant at P < 0.1. Discussion An increase in polysomes is one of the earliest events to be seen after the application of GA and is pre— sumably responsible for the hormone induction of specific enzyme synthesis. A silent period of 3 to 4 hours pre- cedes polysome formation during which the synthesis of the ER to which the polysomes are attached, occurs (section 3). The actual synthesis of a—amylase occurs at 8 to 10 hours, although under certain conditions a—amylase production can be seen at 6 hours (P. C. Lin, personal communication). This short lag period between polysome formation and the appearance of measurable a-amylase activity might be related to the secretion processes. GA causes a 2.5 fold increase in polysome for- mation within 12 to 15 hours after hormone addition. The large amount of polysome formation induced by GA is due to the fact that the aleurone system consists of a 51 single cell type and that every cell most probably responds to the added hormone. Polysome formation has been demonstrated in a number of growing systems, after fertilization (Humphreys, 1969; Monroy, 1970), during germination (Marcus gt 21., 1966), and during H O imbibition (Marcus and Feeley, 2 1965; Barker and Rieber, 1967; Sturami gt 31., 1968; Mascarenhas and Bell, 1969). But hormone—induced poly— some formation generally occurs at long times after addition of the hormone, during which time growth and development of the cells continue. Many hormones induce the synthesis of specific enzymes. The more direct effects of the hormone on polysome formation are minimal, in that the polysomes responsible for the synthesis of the hormone-induced enzymes represent only a small per* cent of the total polysomes. For example, lactogenic hormone causes a doubling of polysomes over a 96 hour period (Gaye and Denamur, 1969). The increase in the number of ribosomes isolated as polysomes is due both to ribosome synthesis and aggregation (increase in the percent polysomes). It is necessary to be reasonably certain that the increase is not due to changes in recovery, in spite of the fact that there is more RNAase in the hormone—treated cells. No increase in recovery was seen with different isolation methods, including the use of various RNAase inhibitors, 52 detergents, and different methods of isolation, centri— fugation, and homogenization (unpublished results). Polysome recovery increases in GA—treated cells following isolation in 0.5% Na deoxycholate when gentle homogenization is used. Not all cells are broken and the total polysome recovery is less than that following normal homogenization. The relationship between the amount and distribution of polysomes isolated from GA— treated and control tissue does not change. The polysomes responsible for the synthesis of GA—induced enzymes are probably ER—bound. Electron micrographs show a considerable number of bound ribo— somes (van der Eb and Nieuwdorp, 1967; Jones, 1969a,b; Vigil and Ruddat, in press). Secretory proteins are thought to be synthesized on membrane—bound polysomes (Palade and Porter, 1966; Siekevitz and Palade, 1966; Redman, 1968; Priestley gt gt., 1969; Takagi gt gl., 1969; Andrews and Tata, 1969; Ganoza and Williams, 1969). The increased recovery of polysomes following membrane solubilization by detergents can be used to estimate the amount of ER—bound ribosomes. However, the cell walls of aleurone cells are very thick, up to 1/2 of the cell by volume. Fairly vigorous homogenization is necessary to break the cells. This homogenization probably breaks membranes and releases membrane—bound polysomes. 53 The rate of protein synthesis doubles following hormone treatment. This increase was measured by the addition of a mixture of 15 amino acids. Varner gt gt. (1965) did not see any increase; however, they measured the incorporation of only one amino acid into half—seeds (endosperms) at later times. The number of active ribosomes also doubles. This determination of the rate of protein synthesis is unaffected by the problems of isotope dilution affecting the above measurements. Many different types of experiments have estab— 1ished that the polysomes isolated were those responsible for GA-induced enzyme synthesis. The high trp/tyr ratio of the nascent peptides released from polysomes isolated from hormone—treated cells indicates that nascent GA- induced proteins are bound to these polysomes. Polysomes isolated from hormone-treated cells have substantially more a—amylase activity associated with them than the control polysomes (Evins, unpublished observations). In addition, anaerobic conditions inhibiting a—amylase synthesis inhibit polysome formation, washing out GA reduces the number of ribosomes in polysomes, ABA, a plant hormone that prevents aeamylase synthesis but not RNA or protein synthesis, prevents polysome formation, and inhibitors of a—amylase synthesis inhibit polysome formation (section 2). 54 The events occurring during the lag period before the appearance of hormone—induced enzyme activity are now « better understood. However, further investigation is needed before the primary site and mechanism of gibberellin action in the aleurone system is completely understood. Summary Gibberellic acid (GA) causes the formation of polysomes and an increase in the proportion of ribosomes present as polysomes during the 8 to 10 hour lag period of d-amylase induction. Polysome formation starts at 3 to 4 hours and reaches a maximal level at 12 to 15 hours after hormone addition. A linear increase in the percent polysomes is seen between 3 to 4 hours and 10 to 11 hours, reaching a maximum of 76% polysomes. The percent poly- somes more than doubles following GA treatment, while polysome formation increases over 2.5 fold. An increase in total ribosomes of almost twofold occurs. The recovery of polysomes from mixtures of rat liver and aleurone layer ribosomes was much less using polysomes isolated from hormone—treated aleurone cells indicating that the stimulation of polysome formation occurs despite an increase in the amount of ribonuclease present in GA—treated cells. Indirect evidence suggests that the GA—induced protein synthesis occurs on endo— plasmic reticular membrane-bound polysomes and that the polysomes isolated are membrane-bound. 55 Although the number of active ribosomes (ribosomes capable of synthesizing nascent polypeptides, measured by . the formation of acid insoluble 3H—peptidyl puromycin) doubles at 12 hours following hormone treatment, the proportion of the total ribosomes that are active is not affected. The incorporation of l4C-amino acids into acid insoluble material was used to show a doubling in the rate of protein synthesis within 8 hours of hormone treatment. The bulk of the GA-induced proteins are tryptophan- rich and have high tryptophan/tyrosine (trp/tyr) ratios. Polysomes isolated from hormone-treated cells and nascent polypeptides released by puromycin from these polysomes have higher trp/tyr ratios than polysomes and nascent peptides isolated from control tissues. II GIBBERELLIC ACID CONTROLLED POLYSOME FORMATION: PREVENTION BY ABSCISIC ACID AND ANTI- METABOLITES AND FUNCTIONAL STUDIES Introduction The response of barley aleurone layers to exo- genous gibberellin has been described in recent articles (Chrispeels and Varner, l967a,b; Filner gt gt., 1969; Varner and Johri, 1968). A dramatic increase in the gg ggyg synthesis of a-amylase and protease follows the addition of the hormone after an 8 to 10 hour lag period (Filner and Varner, 1967; Jacobsen and Varner, 1967). Recently, it has been demonstrated that GA—treatment also increases the formation of polysomes, the percentage of ribosomes present in polysomes, the rate of protein syn- thesis, and the rate of synthesis of the endoplasmic reticulum (ER). Most of these effects occur within 2 to 4 hours after hormone application, that is preceding the induction of hydrolytic enzymes (sections 1 and 3). Using the distinctive trp/tyr ratio of a-amylase and the high trp content of some of the other GA—induced proteins as a 56 57 chemical identification tag, it was moreover shown that the polysomes produced in the presence of GA were, in fact, the polysomes responsible for the synthesis of the GA—induced proteins. Several lines of evidence have sug- gested that the polysomes are membrane-bound. ABA (recent review, Millborrow, 1969), a plant hormone recently characterized and synthesized in 1965 (Cornforth gt gt., 1965; Ohkuma gt gl., 1965), acts antagonistically to GA (Thomas, 1965; Aspinall gt gl., 1967). In many systems ABA may inhibit specific RNA synthesis (Khan and Anojulu, 1970; Khan and Heit, 1969; Ihle and Dure, 1970). Inhibition of polysome formation in Fraxinus excelsior by the inhibition of RNA synthesis was indicated by electron microscopical and autoradio- graphic studies (Villiers, 1968). One method to realize the primary goals of the study of hormone action, the determination of both the mechanism and the location of the primary site of action, is to investigate early effects of hormone administration. The early release of soluble carbohydrate and several phosphatases has been noted (Pollard and Singh, 1968). I have tried to trace the cause of an early fundamental biochemical change, the fig ggyg synthesis of a-amylase. The present results show that the formation of polysomes and the control of ER synthesis, which occur near the beginning of the lag period, are required steps necessary for the synthesis of a—amylase in response to GA. I now 58 report that inhibitors of a—amylase synthesis, such as anaerobiosis, specific metabolic inhibitors, and removal of GA inhibit or decrease polysome formation. The addition of ABA to hormone-treated aleurone layers pre- vents polysome formation. Methods Aleurone layers were prepared as described in Section 1 and incubated for various times with 1 mM Na acetate pH 4.8, 20 mM_CaCl and where required 1 uM K+ 2, GA3, in a 50 m1 flask on a Dubnoff metabolic shaker at 25°. The homogenate and medium fractions and d-amylase assays were performed as described by Chrispeels and Varner (1967a). Polysomes were prepared as described previously (Section 1). Wash out experiments were per- formed by removing the flask containing the aleurone layers from the shaker, rinsing the aleurone layers 6 times with sterile distilled H20 under a UV sterilized hood, blotting on sterile paper towels, rinsing and shaking 10 times with sterile distilled H O, and then 2 rapidly shaking the layers for 30 minutes in buffer without GA. The wash out and transfer steps were re— peated 5 times. Incorporation experiments were performed with carrier-free l4C—amino acid mixture (a mixture of 15 amino acids from an algal protein hydrolysate, Inter- national Chemical and Nuclear Corp.) and carrier-free 59 32P-ortho-phosphoric acid (ICN). The layers were removed from the flask in the sterile hood, rinsed with sterile distilled H O, blotted on sterile paper towels, and trans- 2 ferred to a sterile 25 m1 flask. 32P-ortho—phosphoric l4 . . . . . . C-amino ac1d mixture were mixed w1th medium acid or with or without the hormone in a total volume of 100-150 ul/sample and spread evenly over the layers during the last 60 or 30 (32F), or 16 or 7 (14C) minutes of the incu— bation period. Fractions were collected dropwise by hand, 25 ug of carrier DNA or bovine serum albumen were added, 50% TCA (w:v) was added to a final concentration of 10%, and the samples were allowed to sit overnight in the cold room. The precipitates were collected on Millipore filters, washed with 30 ml of 5% TCA containing either 0.1 M phosphate or casein acid hydrolysate, dried at 70°, and counted on a Beckman scintillation counter in scintil- lation fluid "A" (4 g PPO + 100 mg POPOP per 1 of toluene). Results ABA added at the start of the incubation period prevents the GA—enhanced polysome formation and also causes a decrease in the amount of polysomes present in the con- trol tissue (Figure 1). Not only is there a change in the amount of polysomes, but the percent of the ribosomes that can be isolated as polysomes decreases by 10% in the GA-treated samples, whereas no effect is seen in the control tissue. Similar results were seen when ABA is _—..—_ _ .mucmafluomxo m CH Umcflmuno mums muasmmu Hwaflfiflm .muswflpmum meHUSm 0a .umzflxowfl 2 O.H 0p m.o EH Uwcflfiuoump wuoB moaflmoum mEOmhaom Q M £0 0mm um A £28m 528m ‘1 x a ’ m 65w mum mumnfids HH< .mcon £0 QDH3 Ho AD pomv Q aflomfiocfluod Ho Abmv HHOMHDOHOSHE Ucm A2 mloav «o QDHB mason vm Umumnsocfl whoa mummma oconsmam cme+ +.sv m.m+ m.mv H.6m H.mH a 60¢ Hs\m: OOH + «0+ m.m m.em v.ss H.ms m.mm am_ms m.m + 46+ 0 m.Hm m.mm m.s «.ms 46+ cOflDHQHQQH Edflwmz CH HMDOB mbmcmmofiom SSHGGE w mmmHmfiflla w DcmEummHB onwawfifila m1 «sofluospoum mmmHhfidla so Q cfiowfiocflpod 6cm HHOMHSOHOSHm mo uommmm N mqmflfi 65 H.mquE IHHmmxw m CH pmMHmubo mumz muHSmmH HCHHEHm .mCoEHOC 0CD mo QOCmem 0CD CH ADV Ho .Aflw 21 HV mCoEHOC 6CD mo mOCmmmHm 0CD CH Amv H©®DMQ50CH mHmNMH EOHM UmummmHm mmEOwhHom CH «zm OHCH UwpwyomHOOCH mpCCoo pHom OHHOCQmOCm IOCDHOICNM pCm .mmpHummdeom uCoommC ODCH wwpmnomwooCH mquoo wHom OCHEm IUvH .mHHMOHm >0CMQHOQO 6CD mBOCm m mHCmHm .mHmDHHm mHOQHHHHE Co UwuooHHoo mumz mCoHuomym .mHm>HuowmmmH .mouCCHE b no ow How xmmHm HwhmECmHnm HE mm m CH mumme 0CD Hm>o mHCm>o pmonmm was oECHo> Hapop H1 com 0H 00H CH ECHUwE HpomoHpmu owaIHoHHHmo .H mHCmHm CH meHHommp mm UmCHEHmumU onmz mmHHmoHQ pCm mum>MH oCOHCmHm wwHuma ow Eonw UoDmHOmH meB mmfiommHomH .meommHom ouCH mUHom OCHEMIUvH pCm UHom UHHOCmmOCQIOCDHOImmm mo CoHpmHOQHOOCHII.N mHCmHm 66 —3 Ncpm 32p (xlO )_ n — _ _ _ — — -— r- 51 '1' N '1’ fl _ —' - I l | l l I I I ‘ cpm TCA insoluble l4’C- amino acid (xlO'Z) I l l I l l l '0. 9! '- O o O wuoszv cpm 32p(xlo'3) y __ __no__ __N__ ___—___ ____° $l_ 11‘, T 3' '1' N": ‘0 _ :- i f \ l I I u l 1 cpm TCA insoluble I4C—amino ocid(x|0_2) ; 26;";. J."- < o 4. § b. <1 '0. o 4‘ d;-._ Q- <1\§ O...“ ‘— ~V\\O‘"n <\%: b l l l i l l I l V v IQ N —. 0 d 0 0' ‘3 muosazv Top VOLUME Bottom 8 ,— VOLUME Bottom 67 monosomal regions in control tissues. The appearance of l4C-amino acid counts in the monosome region and the region of smaller polysomes is probably due to degradation. More a-amylase activity is found in the polysomal pellets isolated from GA—treated cells. However, it is difficult to show that this increase in activity is due to enzyme bound to the polysomes and not enzyme contamination from another fraction. Removal of GA GA was removed from the aleurone cells by numerous washings in an attempt to correlate the resulting decrease in the amount of a-amylase synthesized (see below) with an effect on polysomes. Removal of GA by washing causes a reduction in the number of ribosomes isolated as polysomes (Table 4). Adding back GA causes the percent polysomes to return to the same level found in polysomes isolated from unwashed GA—treated tissue. The amount of a-amylase synthesized decreases when GA is removed. The amount of a-amylase present in the medium (Figure 3a) parallels the total a—amylase production in all cases (not shown). When GA is added back to the layers following its removal,a-amylase synthesis starts without the original 8-10 hour lag period. 6-Methy1purine (1 mM) does not prevent the recovery of a-amylase synthesis upon readdition of the hormone. 68 TABLE 3 The Distribution of a-Amylase in the Medium and Homogenate of Barley Aleurone Layers* pg a—Amylase Produced Treatment (Eéfiis) Total Medium Homogenate —GA 8 4.0 3.2 .8 16 12.4 10.8 1.6 24 17.5 15.2 2.3 +GA 8 7.4 5.8 1.6 16 101.0 88.5 12.5 24 133.2 125.6 7.6 +GA + 1 mM 6- Methylpurine 24 3.8 2.8 1.0 *Ten aleurone layers were incubated at 25° for the times specified in the medium described in Table l with 1 uM GA3 and 1 mM 6- -methy1purine, if present. All results are —the average of duplicate samples. obtained in 3 experiments. Similar results were .mpCoEHummxo m CH poCHmqu oHoB mpHsmoH HmHHEHm .mmHmEdm m mo mmmuo>m on ohm muHSmmH HH< .oCoEHOC oCD mo CoHquUmmu H0\pCm Hm>oEoH wCu mCHBOHHow UoCHEHoDoC mmz Hflz+mv\m IIwConon AZV oEowOCoE pCm oEOmmHom 0CD mo mmon mCu mo 85m oCD hm popH>Hp huuoEHCMHQ >9 UwCHEHoumU ConoH Amv GEOmeom oCu m0 momma COHDCQHHpme HmEOmOQHuhHom oCB+ >.wv . . . . mH m.mv . . . . 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