ABSTRACT CHARACTERISTICS OF A CELL-FREE PROTEIN SYNTHESIZING SYSTEM ISOLATED FROM LACTATING BOVINE MAMMARY GLANDS by Donald C. Beitz The molecular mechanisms of milk protein synthesis were studied in a cell-free system using defined components. Freshly obtained mammary tissue was immediately frozen,pu1ver- ized, thawed, and extracted with buffer. The microsome and enzyme fractions were then separated by differential centri- fugation. The complete incubation mixture for amino acid incorporation was composed of microsomes, soluble RNA, amino- acyl-sRNA synthetases, ATP and an ATP generating system, amino acids, reducing agent, buffer and salts. Protein synthesis was measured as the incorporation of 014 leucine into TCA precipitable protein. This system incorporated 500 cpm per mg of microsomes per minute or 50 pumoles of leucine over a 40 minute period. Incorporation in the controls was negli- gible. This incorporation of 014 leucine was completely dependent upon microsomes and ATP and only partially depen- dent upon the aminoacyl-sBNA synthetase and sRNA. Incorpora- tion was not affected by deoxyribonuclease, chloramphenicol, poly A, poly U, growth hormone, cortisone, prolactin, estro- gen, or insulin but was inhibited by ribonuclease, puromycin, cycloheximide, sodium fluoride, and sodium deoxycholate. Chromatography of the incubated complete system on Donald C. Beitz Sephadex showed that radioactivity was associated with the microsomes and with a protein fraction of about 32,000 molecular weight. Isotope dilution tests indicated a net synthesis of specific milk proteins--cS-casein, B-lacto- globulin, B-casein, a-lactalbumin, and)(-casein. Radio- autography of precipitin bands of an immunodiffusion test of a complete system incubated for 0 and 40 minutes showed that the latter sample contained labeled dS-casein. This system is then potentially useful in studying control of milk protein synthesis by the lactating dairy cow. CHARACTERISTICS OF A CELL-FREE PROTEIN SYNTHESIZING SYSTEM ISOLATED FROM LACTATING BOVINE MAMMARY GLANDS By ,. C/ ,- \‘d {,k 0/ Donald C} Beitz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Departments of Biochemistry and Dairy Science 1967 go'fnaoi Svit {,2 ACKNOWLEDGMENTS The author wishes to eXpress his sincere appreciation to Professors W. A. Wood and J.VJ. Thomas for their guidance, encouragement, and understanding throughout the course of this work. He is also grateful to Dr. R. S. Emery for his enthusiasm and counsel. His thanks go to Professor A. J. Morris, and H. A. Tucker for serving on his guidance committee and to Mrs. Douglas Randall's help in the preparation of this manuscript. The many discussions and assistance of his fellow graduate students, especially Harvey Mohrenweiser, shall always be remembered. The author is eSpecially grate- ful to his wife, Judy, for typing the rough drafts of this manuscript and to both his wife and son, David, for their encouragement and love throughout the course of this work. The support of the Balston Purina Company and the National Institutes of Health is greatly appreciated. 11 VITA Donald Clarence Beitz was born March 30, 1940 in Stewardson, Illinois. He graduated from Stewardson-Strasburg High School in Stewardson, Illinois, in May of 1958. He received the degree of Bachelor of Science in Agricultural Science from the University of Illinois in June, 1962, and a Master of Science degree from the Dairy Science department at the same institution in October, 1963. He enrolled as a graduate student in the Dairy Science and Biochemistry departments at Michigan State University in September, 1963. For the first two years, he was supported by a Balston Purina fellowship and the latter two years by a National Institutes of Health pre-doctoral fellowship. The require- ments for the Ph.D. degree will be completed in the fall of 1967. Mr. Beitz is a member of the American Association for the Advancement of Science, the American Dairy Science Asso- ciation, Sigma Xi, Alpha Zeta, and Gamma Sigma Delta. He is married and has one child and once"shot an 88 at difficult Forest Akers golf course at East Lansing, Michigan. 111 TABLE OF CONTENTS INTRODUCTION . . . . . . REVIEW OF LITERATURE . . Model of Protein Synthesis . . . . . . . . . Inhibitors of Protein Chloramphenicol . Puromycin . . . . Cycloheximide . . Sodium Fluoride . Bibonuclease . . . Other Inhibitors . Effect of Hormones on Milk Protein Synthesis Synthesis Protein Synthesis . . Arterio-Venous Difference Studies . . . . Studies on Injection of Labeled Precursors In.Vitro Studies . MATERIALS 0 O O O O O O Mammary Tissue Source Chemicals 0 o o o o 0 Equipment . . . . . . MET HOD S O O O O O I O 0 Preparation of Single-Celled Suspensions . . Preparation of Subcellular Fractions . . . Preparation of the Preparation of the Preparation of the AS 0 Enzyme . . . . . Isolation of sRNA from Mammary Tissue . Analysis of Bibosomes Microsomal Fraction PHSEnzymeooco by Sucrose Density Gradient Centrifugatlon o o o o o o o o o o 0 Electron Microphotographs . . . . . . . . Aminoacyl-sRNA Synthetase Assays . . . . . . Properties of Mammary Gland sRNA . . . . . . iv 36 39 39 41 TABLE OF CONTENTS (Continued) Page Bibonuclease Activity Determination . . . . . . . . 42 Isolation of DNA from Mammary Tissue . . . . . . . 43 Protein and Bibosome Determinations . . . . . . . . 44 Assay of Amino Acid Incorporation . . . . . . . . . 44 Identification of the Possible Synthesized PrOteln(S) o o o o o o o o o o o o o o o o o o o o 46 Gel Filtration Studies of Radioactive Product . . . . . . . . . . . . . . 46 Isotope Dilution Studies . . . . . . . . . . . . 47 Immunodiffusion Study . . . . . . . . . . . . . 47 RESULTS . . . . . . . . . . . . . . . . . . . . . . . 51 PART I - CHARACTERIZATION OF COMPONENTS . . . . . . . 51 Characteristics of Ribosomal Fraction . . . . . . . 52 Preparation of the Ribosomal Fraction . . . . . 52 Effect of Additives on Polyribosomal Yield . . . 60 Effect of Incubating Microsomes with Ribonuolease o o o o o o o o o o o o 72 Dependency of Polyribosomal Character Upon Magn681um Ions . g o o o o o 74 Sedimentation Coefficient Determination . . . . 77 Electron Microscopy of Polyribosomal Preparation o 0‘. o o o o o o o o o o o o o o o 78 Properties of Mammary Gland sRNA . . . . . . . . . 81 Determination of Homogeneity and Sedimentation Coefficient . . . . . . . . 81 Esterification of Amino Acids to sRNA . . . . . 83 Enzymatic Activities of Various Fractions . . . . . 86 Aminoacyl-SRNA Synthetase Activity . . . . . . . 86 RibonUCIGaSe ACthity o o o o o o o o o o o o o 90 PART II - IN_VITRO PROTEIN SYNTHESIS . . . . . . . . . 94 Cl“ Leucine Incorporation by a Crude Homogenate . . 94 CI” Leucine Incorporation by Single-Cell Suspensions . . . . . . . . . . 95 Amino Acid Incorporation by the Cell-Free SyStem o o o o o o o o o o o o o o o o o o o o o o 97 TABLE OF CONTENTS (Continued) C14 Incubation . Incorporation of C Microsomes . Leucine Incorporation vs Time of 14 into Mammary Gland The E fect of the Amount of Microsomes on C Leucine Incorporation . . . . . Dependence of Incorporation on the Source of Aminoacyl-SRN Dependence of C1 on SHNA . . SynthetaSeS o o o o o Leucine Incorporation Stability of Microsomes and pH 5 Enzymes to Storage . Dependence of Amino Acid Incorporation on ATP . . . Dependence of Incorporation on Magnesium Ion Concentration Dependence of Incorporation on Amino Acids Comparison of the Incorporation of Different Label Inhibition of C Effect of Polynucleotides on C Incorporation f9» Amino ACidS o o o c Leucine IncorRoration Leucine Effect of Initiation Factor on Clh Leucin Incorporation Effect of Lactogenic Hormones on Clh Leucine Incorporation e Characterization of the Synthesized Protein . Gel Filtration St'lldies o o o o o o o o o Isotope Dilution Tests of the Incubated Standard Assay System Identification Studies by Immunodiffusion DISCUSSION . . . . SUMMARY . . . . . BIBLIOGRAPHY . . . APPENDIX . . . . . vi Page 100 105 111 113 120 123 126 127 135 138 140 154 157 161 166 166 170 175 179 188 190 198 Figure 1. 7. 8. 9. 10. 11. 12. 13. LIST OF FIGURES Preparation of microsomes and pH 5 enzyme from mammary tissue . . . . . . . . . . . . . Isolation of SHNA from mammary tissue . . . . The apparatus for obtaining ribosomal patterns from sucrose density gradients . . . Isolation procedures for as-casein, B-casein, and)(-casein from skimmed milk . . . . . . . Isolation procedures for d-lactalbumin and B-lactoglobulin from the whey fraction of skimmed milk 0 o p o o o o o o o o o o o o o The sedimentation behavior of ribosomes and polyribosomes prepared by homogenizing frozen tissue 0 O O O O O O O O O O O O O O O O O O The sedimentation behavior of ribosomes and polyribosomes prepared with the Virtis homogenizer . ._. . . . . . . . . . . . . . . The sedimentation behavior of ribosomes and polyribosomes prepared by homogenizing tissue with the French press '. . . . . . . . The sedimentation behavior of microsomes before and after treatment with sodium deoxyChOlate o o o o o o o o o c o o o o o o The sedimentation behavior of normal and bentonite-treated ribosomes and poly- ribosomes o o o o o o o o o o o o o 0'. o o c The sedimentation behavior of normal and polyvinyl-treated ribdsomes and poly- rlbcsomes o o o o o o 0.6 o o o o o o c o o 0 Comparison of the sedimentation behavior of normal and Dupanol-treated ribosomes and p01yr1bosomes o o o o o o o o o o o o o o The sedimentation behavior of dissociated and reassociated ribosomes and polyribosomes vii 32 35 38 48 49 54 56 58 62 65 68 7O 75 Figure ng3 14. Sedimentation velocity of mammary gland ribOSOmeSoooooocoooooooooooo 79 15. Electron micrograph of mammary gland polyribosome preparation . . . . . . . . . . . 82 16. Sucrose density gradient pattern of purified mammary gland SRNA . . . . . . . . . . 84 17. C1” leucine incorporation vs time using pH 5 enzyme 0 O O O O O C O O O O O O O O O 0 O 101 C1“ leucine incorporation vs time using As7ofraCt10noooocoocon...oooo 103 18. 19. Effect of amino acid incorporation on the sedimentation behavior of microsomes . . . . . 106 20. Labeli g of ribosomes and polyribosomes with C leucine . . . . . . . . . . . . . . . 109 21. C14 leucine incorporation vs microsome level . 114 22. Cl)+ leucine incorporation vs pH 5 enzyme . . . 118 23. 014 leucine incorporation vs A870 enzyme . . . 121 24. C14 leucine incorporation vs sRNA level . . . . 124 25. 014 leucine incorporation vs magnesium level . 130 26. C3“ leucine incorporation vs magnesium level using "shocked" microsomes . . . . . . . . . . 133 27. Th3 effect of amino acid concentration on C 131101119 incorporation o o o o o o o o o o o 136 14 28. C leucine incorporation vs ribonuclease level-00000000000000.oo'oooo147 29. Inhibition of 014 leucine incorporation by cycloheximide and sodium fluoride . . . . . . . 151 C14 leucine incorporation in the presence of initiation factor . . . . . . . . . . . . . . . 159 30. 31. The effect 2 initiation factor on the time course of C leucine incorporation . . . . . . 162 32. Behavior of the incorporated leucine . . . . . 168 33. Radioautographs of immunodiffusion studies of incubated complete systems . . . . . . . . . . 177 V111 Table 1. 9. 10. 11. 12. 13. 14. 15. 16. LIST OF TABLES Components of the complete amino acid incorporating system . . . . . . . . . . . . . . Incubation of microsomes with ribonuclease . . . Sedimentation coefficients of mammary gland ribosomes O O O O O O 0 O O O O O O O O O O 0 O Esterification of C1“ L-glutamate to mammary gland SRNA o o o o o o c o o o o o o o o o o o 0 Purification of aminoacyl-SHNA synthetase based upon hydroxamate formation . . . . . . . . Aminoacyl-SRNA synthetase activity as measured by radioactive pyrophosphate exchange into ATP . Ribonuclease activities of various fractions . . C14 leucine incorporation by a crude homogenate of mammary tissue . . . . . . . . . . . . . . . Cl“ leucine incorporation by isolated mammary gland cells 0 o o o o o o o o o o o o o o o o 0 Amino acid incorporation by gradient-frac- tionated microsomes o o o o o o o o o o o o o 0 Dependence of ATP for amino acid incorporation . Amino acid incorporation vs type of labeled amino 301d 0 o o o o o o o o o o o o o o o o o 0 Inhibition of Cl“ leucine incorporation by puromy01n o o o o o o o o o o o o o o o o o o 0 Effect of L-chloramphenicol addition on c14 leu01ne incorporation o o o o o o o o o o o o 0 Inhibition of c1“ leucine incorporation by ribonuCleaSe o o o o o o o o o o o o o o o c o 0 Effect of deoxyribonuclease addition to the complete system . . . . . . . . . . . . . . . . ix 45 73 80 87 89 91 92 96 98 112 128 139 142 143 146 149 Table 1. 10. 11. 12. 13. 14. 15. 16. LIST OF TABLES Components of the complete amino acid incorporating system . . . . . . . . . . . . . . Incubation of microsomes with ribonuclease . . . Sedimentation coefficients of mammary gland ribosomes . . . . . . . . . . . . . . . . . . . Esterification of C1“ L-glutamate to mammary gland SRNA O I I O O O O O O O O O O O O O O O 0 Purification of aminoacyl-SRNA synthetase based upon hydroxamate formation . . . . . . . . Aminoacyl-SBNA synthetase activity as measured by radioactive pyrophosphate exchange into ATP . Ribonuclease activities of various fractions . . C1“ leucine incorporation by a crude homogenate of mammary tissue . . . . . . . . . . . . . . . 014 leucine incorporation by isolated mammary gland Cells 0 o o o o o o o o o o o o o o o o 0 Amino acid incorporation by gradient-frac- tionated microsomes . . . . . . . . . . . . . . Dependence of ATP for amino acid incorporation . Amino acid incorporation vs type of labeled amino 801d 0 o o o o o o o o o o o o o o o o o 0 Inhibition of C14 leucine incorporation by puromy01n o o o o o o o o o o o o o o o o o o 0 Effect of L-chloramphenicol addition on C14 leu01ne incorporation o o o o o o o o o o o o 0 Inhibition of cl“ leucine incorporation by ribOHUCIGaSe o o o o o o o o o o o o o o o o o 0 Effect of deoxyribonuclease addition to the complete SyStem o o o o o o o o o o o o o o o 0 ix 45 73 8O 87 89 91 92 96 98 112 128 139 142 143 146 149 Table ° Page 17. Effect of addition of single-stranded DNA, poly A, and poly U to the complete system . . . . 156 18. Effect of hormone additions on C1“ leucine incorporation . . . . . . . . . . . . . . . . . . 165 19. Identification of synthesized protein by isOtOPG d11Ution o o o o o o o o o "o o o o o o o 172 20. Identification of synthesized protein by isotope dilution as conducted by Dr. B. L. Larson.o...................174 Table 1. 2. 3. 4. 5. LIST OF APPENDICES Composition of the thixotropic counting fluid Composition of amino acid mixture . . . . . . Hanks' basic salt solution . . . . . . . . . Composition of Kinard's counting fluid . . . CompOSItion Of Medium A o o o o o o o o o o 0 xi INTRODUCTION Much of our present information concerning protein synthesis has come from the use of a crude cell-free system, developed by Zamecnik, Hoagland, and their associates, which permitted the study of incorporation of labeled amino acids into protein. Such cell-free preparations were obtained initially from rat liver, but have since been derived from other mammalian tissues, microorganisms, and plants. The general features of preparations from all these sources have proven to be essentially similar. I have chosen to adapt this method of study to the protein synthesizing system of the bovine mammary gland. The gland produces a large amount of milk proteins and, hence, should be a very suitable system for the study of many aSpects of protein synthesis. Not only are large quantities of pro- tein continuously formed, but they are also excreted from the cells and thus complications introduced by subsequent catabo- lism largely are absent. I Many workers have studied the overall aspects of milk protein synthesis by arterio-venous differences, injection of labeled amino acids, and perfusion of isolated mammary glands with labeled amino acids. These techniques have proven that the mammary gland utilizes absorbed blood amino acids which account for most of the milk protein synthesis. 2 Yet, the mechanism of this synthesis and its control are unclear because of lack of biochemical studies. Therefore, an attempt was made to develop a cell-free system which would synthesize milk proteins from amino acids. The attainment of net protein synthesis and identification of the synthesized product were ultimate goals. A cell-free system synthesizing a known protein would permit study of the mechanism of milk protein synthesis and permit elucida- tion of the factors which might limit protein synthesis $2 11232, Application of this knowledge to the lactating cow could give a clearer understanding of the mechanism of con- 1 trol for the quantity and type of milk protein synthesized. A preliminary account of this research was presented at the annual meeting of the American Dairy Science Associa- tion in June, 1967, and an abstract appears in the Journal of Dairy Science 50, 999, 1967. REVIEW OF LITERATURE Studies on protein-synthesizing systems from various organisms have now provided overwhelming evidence that pro- teins are synthesized by a similar mechanism in all organ- isms. The mechanism whereby the different organisms orderly direct the synthesis of peptide bonds from free amino acids is presented in the following discussion of the model system for protein synthesis. Model for Protein Synthesis The individual amino acids are activated in the presence of adenosine triphosphate (ATP), magnesium ions, and specific enzymes (aminoacyl-SRNA synthetases) to form amino- acyl adenylates and inorganic pyrophOSphate (P-P). This reaction constitutes the initial reaction involved in protein synthesis. The aminoacyl-sRNA synthetases which have been par- tially purified from many organisms Show Specificity for individual amino acids (1, 2, 3). For example, aSpartyl- and aSparaginyl-Specific aminoacyl-SRNA synthetases isolated from Lactobacillus arabinosus have been separated by fractionation (4). Amino acid Specificity of the synthetase enzymes was also demonstrated by Herve’and Chapeville (5) who chemically converted sBNA-bound cysteine to sRNA-bound alanine and 3 4 showed that both cysteinyl- and alanyl-SRNA synthetases were unable to "recognize" the complex in which alanine was attached to soluble ribonucleic acid (SRNA). These synthetases catalyze the linkage of the carboxyl group of the amino acid to the phosphate group of the adenyl- ate moiety as an anhydride bond. This activated amino acid is bound to the enzyme. Aminoacyl-SRNA Amino Acid + Adenine-ribose-P-P-P \ Synthetase Enzyme (adenine-ribose-P-amino acid) + P-P For example, aminoacyl-SRNA synthetase-bound threonyl, and valyl adenylate complexes have been isolated (6, 7). The aminoacyl group of the enzyme-bound complex is transferred to amino acid-Specific SRNA'S where the amino acid is attached as an ester linkage to the ribose portion of the terminal adenosine residue. This reaction may be illustrated as follows: AMP-amino acid-enzyme + SHNA ___;.amino acid-SHNA + AMP The end of the SHNA molecule where the amino acid is attached consists of a cytidylate-cytidylate-adenylate sequence (1) while guanylic acid is the predominant 5' terminal nucleotide. The complete nucleotide sequences for tyrosine (8), and alanine sHNA's (9) have been determined. Evidence that several different sBNA's can accept a particular amino acid has been presented by Showing that 1) five leucine-specific sRNA's can be separated from extracts of E. coli (10), and 2) two valyl-SRNA'S have been isolated 5 from yeast (11). In general, however, the purified SRNA'S are Specific to only one amino acid (2). Studies with combinations of SBNA and aminoacyl-sRNA synthetases from different organisms indicate that species differences exist between the enzymes and sBNA's. The SHNA from different Species which accept the same amino acid have different structures (1). The specificity of binding amino acids to SRNA is maintained when synthetases from different Species are used with a particular sRNA, but all of the multiple sRNA's which are Specific for single amino acids may not become esterified with that amino acid by heterolo- gous enzymes. For example, two serine SBNA fractions from E, 22;; are esterified by E, 22;; enzymes, but only one is charged to the extent of 60 per cent by yeast enzymes (1). More recent studies with homologous and heterologous combin- ations of preparations to synthesize aminoacyl-sRNA's sug- gest that the amino acid Specificity between sRNA and amino- acyl-SHNA synthetases is maintained among different organisms such as E, 9211. Neurospora crassa, rat liver, and yeast (12, 13, 14). The aminoacyl-SENA'S react with messenger RNA (mBNA) bound to ribosomes (polyribosomes) at specific positions determined by the nucleotide sequence of the mRNA. This binding process was first demonstrated by the finding of a polyuridylic-dependent binding of phenylalanyl-SRNA to reticulocyte ribosomes (15). The noncovalent binding reac- tion is non-enzymatic and requires magnesium ions and a 6 monovalent cation-~either potassium or ammonium (16). If guanosine triphOSphate (GTP), transfer enzymes, and gluta- thione are added, the bound phenylalanine is incorporated into protein. In contrast to the binding process described above with.§. ggli,.Arlinghaus and coworkers (17) have reported that the binding of aminoacyl-SRNA to reticulocyte ribosomes, prior to peptide bond formation, requires a transfer enzyme as well as GTP. Two enzyme fractions were required for polypeptide synthesis (18, 19). No function has been assigned to these fractions, although one appears to be involved in the hydrol- ysis of GTP as the peptide bond is synthesized in the E, 22;; system (20) and the reticulocyte system (17). These amino- acyl transferring enzyme preparations show considerable species specificity with respect to ribosomes but not with reSpect to aminoacyl-SRNA (14, 21). The aminoacyl-SENA'S are Specifically aligned in rela- tion to the growing peptide chain, that is, the carboxyl terminal amino acid is linked to SBNA. Nucleophilic attack by the d-amino group of the incoming aminoacyl-SHNA on the carboxyl carbon of the peptidyl-SRNA results in the formation of a new peptide bond and the release of the previously attached sRNA. The polypeptide chain containing an addi- tional amino acid residue is linked through the new amino acid to its correSponding SRNA. Polyribosome-bound amino- acyl-SBNA and peptidyl-SRNA are, therefore, intermediates in protein synthesis. 7 Chapeville gt, 9;. (22) have presented evidence that template recognition shows no Specificity with reSpect to the aminoacyl moiety of SRNA. These workers prepared amino- acyl-SBNA compounds in which the amino acid group was modi- fied after incorporation into SRNA and demonstrated that the incorporation of the modified amino acid was dependent on the nature of the SBNA, not on the nature of the amino acid in the hybrid. Hence, the sRNA moiety gives rise to speci- ficity of binding of the aminoacyl-SENA. Wettstein and Noll (23) have found that each rat liver ribosome bound two, and at most, three sRNA molecules. Aminoacyl-SHNA and peptidyl-SHNA were tightly bound, and uncharged SRNA could be removed by washing. Thus, the authors concluded that three distinct types of binding sites exist on ribosomes—-namely decoding, condensing, and exit sites. Thus, during peptide bond synthesis a particular SRNA passes through each of the binding sites. When an amino acid is incorporated, the messenger and the ribosome move one coding unit in relation to each other. A new nucleotide sequence in messenger RNA is, therefore, placed in position to base-pair properly with the next amino- acyl-sRNA. As this process is repeated, the polypeptide chain grows from its N-terminal to its C-terminal residue by the ordered sequential addition of the amino acids of aminoacyl- sRNA's. This ordered sequential addition of amino acids is determined by the nucleotide sequence of the mRNA whose sequence of ribonucleotides was determined by complementation 8 of the deoxyribonucleotide sequence of DNA (1). As peptide bond formation is proceeding, the ribosome moves far enough along the messenger molecule to allow a new ribosome to attach. A second identical peptide chain can now be initiated and synthesized in the path of the second ribo- some in the same way. Consequently, several identical pep- tide chains can be synthesized simultaneously on a polyribo- some complex. Eventually, at the end of the mRNA, the ribo- some, the nascent completed polypeptide chain, and the terminal, esterified sRNA are released from the polyribosome (1). Inhibitors of Protein Synthesis Many antibiotics, enzymes, and inorganic compounds inhibit protein synthesis both in 1112 and in 32339. Inhib- itors which have Specific sites of action have aided greatly in the elucidation of the preceding model of protein synthe- sis. The following discussion will only include those inhibitors which were used in the study reported herein. Chloramphenicol--First shown to inhibit protein syn- thesis in Staphlococcus aureus by Gale and Folkes (24), chloramphenicol has been shown to inhibit protein synthesis both in intact cells and in cell-free systems by a number of different methods and in a wide variety of organisms (25). Chloramphenicol inhibition in microbial systems occurs at a stage subsequent to the attachment of amino acids to sHNA 9 and at a site related to the ribosomal assembly of amino acids on mRNA (26). It neither affects the activation of amino acids (27) nor the transfer of amino acids to SRNA (28), but it inhibits the transfer of activated amino acids from sRNA to ribosomes (29) and interferes with polymeriza- tion of amino acids when synthetic templates are added to E, ggli ribosomes (30). Rendi and Ochoa (31) have shown that this inhibitor does not directly affect RNA synthesis, but interferes with the attachment of mRNA to ribosomes. Although chloramphenicol readily inhibits protein synthesis in microbial systems, protein synthesis in mam- malian systems is markedly resistant. For example, complete inhibition of protein synthesis in E, 22;; cell-free systems can be obtained with 1.5 x 10'“ M.chloramphenicol (30), but in mammalian cell-free systems comparatively little inhibi- tion was obtained with 5-10 x 10"3 M chloramphenicol (32). Also, comparative in.zizg experiments of bacterial and mammalian cells showed that much greater concentrations of the inhibitor are requiraifbr the latter. Experiments utilizing reticulocyte template RNA as a stimulant of protein synthesis in the reticulocyte cell-free system resulted in a marked increase in the incorporation of C14 L-leucine into trichloroacetic acid (TCA) precipitable material. Addition of chloramphenicol inhibited this protein synthesis induced by added template RNA but had comparative little inhibitory effect on the unstimulated protein synthesis SIstem (33)- 10 The differences of response of bacterial and mammalian protein synthesizing systems to chloramphenicol must be related to differences in template RNA turnover and stability since mRNA of mammalian cells is more stable, remaining attached to ribosomes for longer times (26). Indirect evi- dence suggests that chloramphenicol interferes with the func- tion of mRNA; that is, inhibits the attachment of mRNA to ribosomes or possibly directly inactivating mRNA (31, 33, 34). Puromycin--EXperiments by Yarmolinsky and de la Habe (35) firmly established that puromycin inhibited the in_zi£32 synthesis of protein while the suppression of protein synthe- sis by puromycin £2.2l12 was first demonstrated by Gorski gt 2;. (36). Yarmolinsky and de la Habe (35) further showed that the action of puromycin in a cell-free system occurred in the transfer of the amino acids and that esterification of the acids to sRNA was unaffected. Specifically, the anti- biotic was demonstrated to exert its effect at a level involving the sRNA-ribosomal complex, where it could substi- tute for aminoacyl-sRNA and become attached by its amino group to the incomplete polypeptide chain (37). The carboxyl group of puromycin is not available for additional peptide bond formation: therefore, the polypeptide chain could not grow in length. Relatively short chains, each carrying a puromycin molecule, are then released (38). This direct effect of puromycin on the ribosome result- ing in the release of protein was first reported by Morris 11 and Schweet (39, 40) in their studies with leucine incorpora- tion by rabbit reticulocytes. The discharge was non-enzymatic and occurred at 10"3 E,puromycin and was considered to result from the displacement of incomplete globin chains and earlier intermediates from the ribosome. Thus, soluble protein could be freed from ribosomes in the absence of incorporation and without ribosomal breakdown. Cell-free protein synthesizing systems derived from many organisms are inhibited by the same mechanism as pre- viously described. Tissieres and Watson (41) reported that 8 x 10"5 E,puromycin inhibited protein synthesis with the E, 22;; cell-free system. Florini (42) reported that cell- free preparations from rat skeletal muscle did not synthesize protein in the presence of puromycin. Other workers reported that rat liver (43), thymus cell nuclei (44), and rat liver mitochondria (45) cell-free protein synthesizing systems are inhibited by this antibiotic. gyglgheximide--Protein synthesis in many organisms, excluding bacteria, is also inhibited by cycloheximide (46- 50). Two groups of investigators have reported that it does not inhibit the formation of aminoacyl-sRNA but involves the transfer of amino acids from aminoacyl-sRNA to nascent poly- peptide chains (47, 51, 52). Wettstein 23, El- (53) have shown that cycloheximide inhibits the breakdown of polyribo- somes and have suggested that the readout mechanism from mRNA is involved. Recent studies with a reticulocyte cell- 12 free system.by Lin 22, 5;. (54) indicated that cycloheximide inhibits the initiation of new chains on ribosomes. Further, studies on the rate of amino acid incorporation indicate that cycloheximide also decreased the rate of incorporation into nascent polypeptide chains that were initiated in intact cell and remain attached to ribosomes during their isolation. Sodium Fluoride--Marks 533;. 11. (55) and Ravel 21:, 9;. (56) have shown that sodium fluoride inhibits protein syn- thesis in a manner quite similar to that of cycloheximide. They found a decrease in polysomes and an increase in mono- meric ribosomes in rabbit reticulocytes incubated in the presence of sodium fluoride. This dissociation was accom- panied by a loss of nascent polypeptide chains from the ribo- somes. On the other hand, this inhibitor caused little or no detectable dissociation of polyribosomes in the complete cell-free system. Washing out the sodium fluoride from intact reticulocytes permitted protein synthesis to become reestablished (48. 57). Lin 2;, 3;, (54) demonstrated that sodium flouride blocks the initiation of new peptide chains on ribosomes in a reticulocyte cell-free system, suggesting that this inhibitor directly or indirectly interfered with the reattachment of monomeric ribosomes on messenger RNA. Sodium fluoride had no apparent effect on the rate of amino acid incorporation into previously initiated nascent chains. Ribonuclease--Many investigators have found that ribonuclease is a potent inhibitor of the cell-free protein 13 synthesizing systems isolated from many different organisms (58, 59, 60, 61). Incorporation of amino acids into TCA- precipitable protein was inhibited to the extent of 90% by as little as 0.1 ug of pancreatic ribonuclease in the cell- free system derived from reticulocytes (58). Possibly sRNA, ribosomal, and mRNA may have been affected; but preincubation of the purified ribosomes with ribonuclease demonstrated that the effect could have been at the ribosomal level. Later eXperiments with E, 92;; and rat liver ribosomes indicated that this nuclease caused a quantitative degradation of polyribosomes to the monomeric form of ribosomes (62, 63). Because polyribosomes are the major synthetic form of ribo- somes (2), degradation of the mRNA of the polyribosome caused inhibition of protein synthesis. Other Inhibitors-~0ther-compounds that inhibit protein synthesis AE72$££2 are nucleocidin (64), gougerotin (65), blasticidin S (66), amicetin (67), tetracyclines (68), and neomycin B (68). Since these substances were not used in this research, the mechanism of their inhibition of protein synthesis will not be discussed. Effect of Hormones on Protein Synthesis Most tissues in higher animals are to some degree dependent on hormones for their normal growth and development. Knox 22, 3;, (69), who demonstrated that synthesis of certain enzymes was increased in animal cells by the administration 14 of corresponding substrates, observed that the steroid hormone, cortisone, induced the synthesis of adaptive enzymes. These findings provided investigators an eXplanation for the selec- tive regulation of enzyme synthesis by individual hormones. Prior to these experiments, the most prevalent view of hormone action involved a type of hormone-enzyme interaction resulting in enzyme regulation (70). Tata 22, g;, (71), in attempting to describe the mech- anism of thyroxine action, found that it increased the rate of AE.YA22.RNA synthesis in the nucleus and protein synthesis in the cytoplasm. The stimulation of nuclear RNA synthesis occurred prior to any noted change in the amino acid incor- poration activity of ribosomes in the cytoplasm. Similar sequential stimulation of RNA synthesis followed by that of protein synthesis have been recorded for other hormones as well: i.e.. growth hormone acting on liver (72) and muscle (73) and testosterone on prostate gland (74). The observations that hormones affect a change in the rate of protein synthesis ;g_zéngprompted Korner (75), Mueller (76), and Williamqushman 23, 3;. (74) to study the regulation of protein synthesis in liver and accessory sexual tissues by growth hormone, estrogens, and testosterone, reSpectively. These workers showed that the addition of hormones involved in growth and development to cell-free protein synthesizing systems prepared from their respective target organs had no effect on their protein synthesizing capacity. 0n the other hand, the respective hormones, when 15 administered $2,3312, markedly stimulated protein synthesis in the target organ. This stimulatory effect was largely localized in the ribosomes of the target cells. Karlson (77) proposed that hormones act as inducers, which, by combining with appropriate repressors, control mRNA synthesis and thereby regulate enzyme synthesis. Tata (71) proposed two other modes of action: 1) hormones might regulate the rate of transfer of mRNA from the site of syn- thesis into the cytoplasm, and 2) hormones could regulate the type and rate of protein synthesis at the level of trans- lation of mRNA by ribosomes. Hormones such as prolactin, cortisone, estrogen, pro- gesterone, insulin, and growth hormone regulate the synthe- sis of milk by mammary tissue. Insulin, hydrocortisone, and prolactin stimulate synergistically the synthesis of casein- like phosphoproteins and whey proteins in organ cultures of mouse mammary glands. Stimulation was not observed or was minimal when one or more of these hormones were omitted from the culture medium (78. 79). No study has been reported con- cerning the effect of adding hormones to a cell-free protein synthesizing system obtained from this tissue. Milk Protein Synthesig Although the lactating mammary gland is particularly suitable for the study of protein biosynthesis because of its high rate of synthesis and ease with which its product can be 16 characterized, only a few studies have been conducted on the different reaction steps leading to milk proteins. Most of the reported studies have involved arterio-venous (A-V) dif- ferences measurement of amino acid concentration across the mammary gland or perfusion studies designed to delineate precursor-product possibilities. Only a few reports on protein syntehsis in cell-free preparations have appeared. In the absence of specific information it could be postulated that the proteins of milk are formed from either amino acids, peptides, or complete proteins of blood. For many years a debate existed among the first investigators of milk protein synthesis concerning the site of synthesis. To settle this question, investigators utilized the techniques of studying the changes in the level of the possible milk protein precursors in the arterial and venous blood supply systems of the mammary gland. Arterio-Venous Difference Studies--The first attempt to study the precursors of milk proteins by this technique was conducted by Cary (80). Because the concentration of free amino nitrogen was 16-34% lower in the plasma collected from the mammary vein than the concentration in Jugular plasma and only 3-5% lower for dry cows, km; concluded that the A~V differences were in the order of magnitude to be eXpected if all the milk proteins originate completely from the free amino acids of blood. Later Blackwood (81) repeated Cary's work using 17 improved methods of measuring amino nitrogen and arrived at the same conclusion. Calculations of the total weight of amino nitrogen absorbed by the mammary gland versus the amount secreted in the milk led Graham (82) to conclude that blood amino acids could not be the sole source of milk protein. Similar eXperiments conducted by Shaw and Peterson (83) indicated that the blood amino acids could not account for more than 35% of the milk proteins while Nikitin (84) arrived at a value of 45%. Since A-V difference studies pose certain limitations on quantitative conclusions, the findings from these early studies are limited, but indicate that free amino acids are absorbed from the blood by the mammary gland in significant amounts (85). More recently there has been a clear demonstration of uptake of specific amono acids by lactating glands. Borchaert (86) showed that the concentrations of the ten essential amino acids in the blood decreased markedly during a perfusion study. In further studies, absorption of glutamine, glutamic acid, threonine, and serine as measured by A-V differences of lactating cows were shown to provide a significant portion of those same amino acids in casein. Work by Verbeke and Peters (87) showed that lactating bovine glands were capable of absorbing enough arginine, glutamine, isoleucine, leucine, lysine, valine, threonine, and histidine to provide all the respective amino acid residues in milk protein. Taurine, urea, and d-amino-butyric acid were not absorbed. According to this report, absorption could not account for all of the 18 aspartic acid, asparagine, glutamic acid, serine, and proline in the milk proteins. Other workers (88) have also shown that the uptakes of the ten essential amino acids and gluta- mic acid by lactating goat glands were approximately equal to the corresponding output figures. They found that the uptake of serine was consistently less than the output and that the uptake of the other non-essential amino acids were inconsistent. Studies on Injection of Lab§;ed Precursors-~Tracer experiments have been utilized to more accurately study the compounds of the blood which are used by the mammary gland as precursors of milk proteins. Campbell and Work (89) reported that injected radioactive valine and lysine were incorporated into the whey proteins and casein, but the portion of valine and lysine of milk protein that may have originated from plasma proteins could not be estimated. In a later eXperiment these same workers concluded that 90% of the lysine and serine of milk protein came directly from free amino acids in the blood. Furthermore, Askonas(90) also concluded that at least 90% of the valine and lysine residues of crystalline B-lactoglobulin and casein came from a single pool of free amino acids in the mammary gland. Barry (91, 92) concluded that in the lactating goat at least 70% of the lysine, tyrosine, and glutamic acid and glutamine residues of casein were absorbed from the corresponding free amino acids of the plasma. For aSparagine and proline, these percentages amounted to 50%. 19 The possibility that a major fraction of the plasma proteins could be hydrolyzed to a limited extent within the gland and contribute to the pool was not excluded in the above eXperiments. When plasma proteins labeled with Clu glycine were injected into lactating rabbits by Campbell and Work (89), not more than 10% of the glycine or serine of the milk proteins came from these plasma proteins. In a later eXperiment Askonas and coworkers (93) compared the maximum specific radioactivities of milk and plasma pro- teins and found that casein and the whey proteins were over fifty times more radioactive than the plasma proteins. Clearly a significant part of the amino acids of milk proteins did not come from any major fraction of the plasma proteins. The origin of the milk protein amino acids which are not absorbed from the bloodstream in sufficient amounts has been the concern of many research investigators. Volatile fatty acids (e.g. acetate and propionate) were incorporated into aSpartic and glutamic acids of casein by the isolated, perfused cow's udder (94). Black and Kleiber (95) concluded from infusion eXperiments that the carbon of volatile fatty acids, glucose, and of fructose was utilized in the synthe- sis of glutamic acid, aSpartic acid, serine, and alanine. Moreover, acetate and fructose were incorporated, respectively, into proline and glycine. Wood 22, film (96) calculated from their experiments in which glucose cl” was supplied to the mammary gland by injection into the pudic artery, that 20 approximately 25% of the serine in casein was synthesized in the udder. In conclusion, reported eXperiments indicate that the udder obtains the amino acids for milk protein synthesis by two mechanisms: 1) absorption of blood amino acids and 2) by synthesis of amino acids from absorbed precursors. Campbell and Work (89) and Askonas 23. 2;, (93), work- ing with rabbits and goats, found that the total casein iso- lated from milk taken within a few hours after intravenous administration of radioactive amino acids contained higher levels of radioactivity than the total whey proteins. Part of the difference was accounted for by a component of whey proteins--immune globulins--which apparently entered the gland as intact protein. Furthermore, Larson and Gillespie (97) demonstrated that d-casein, B-casein, a-lactalbumin, and B-lactoglobulin were synthesized in the mammary gland from a common free amino acid pool and that the immune globulins, milk serum albumin, and y-casein entered the milk preformed from the blood stream. McCarthy (98) verified portions of the above findings by showing that d-lactalbumin and whole casein were derived from a common pool of free amino acids in the udder. Available chemical and immunolog- ical.evidence indicated that milk serum albumin was identical to albumin of the blood (97. 99). In Vitro Studies--Initial lg Vitro studies of amino acifii incorporation into proteins by Hoagland and his coworkers C100) clearly showed that certain small (15 mp), dense cyto_ 21 plasmic particles were essential for synthesis and, indeed, that the labeled amino acids became attached to the particles, later termed ribosomes. In preparations from mammalian tissues, amino acid-incorporating activity was associated with microsomes which consist of free ribosomes and ribosomes bound to the endoplasmic reticulum. Apparently, the mammary gland contains similar structures since electron micrographs of thin sections of mouse mammary glands have shown that the endoplasmic reticulum of the secretory cell is rather similar to that of the secretory cells in the pancreas (101). The "rough membranes," consisting of lipoprotein with associated electron dense nucleoprotein particles, were particularly prominant. However, areas of smooth membranes, Golgi zones, and secretory granules were also present. All these portions of the cytoplasm probably contribute to the microsome frac- tion isolated from the mammary gland. Bailie and Morton (102) were the first investigators to attempt to isolate microsomes from the mammary gland of dairy cows. In this case mammary gland microsome fractions were isolated, not to study protein synthesis, but to compare various enzymatic activities such as alkaline phOSphatase 1with.the corresponding activity of microsomes found in milk. Brew and Campbell (103) determined the RNA to protein ratio of microsome fraction from guinea pig mammary glands to be 0.35-0.38. Since the ratio obtained from liver microsomal preparations is usually 0.2 (104), this high ratio suggested that the microsome fraction was rich in ribosomes which were 22 not attached to membranes. Sucrose density gradient centri- fugation of such preparations indicated that a substantial peak of free ribosomes was present-—the peak area being greater than the correSponding fraction from liver (103). Fraser and Gutfreund (105) first detected aminoacyl- SRNA synthetase activity in mammary gland tissue isolated from guinea pigs, cows, and rats. Homogenates from all three sources catalyzed the formation of hydroxamates from a com- plete mixture of amino acids; however, the rate of hydrox- amate formation was only 20% above that with tryptophan alone. The same results were obtained using a particle-free super- natant of guinea pig mammary gland as a source of the synthe- tases. Barely significant activity was found for most of the amino acids tested, but tyrosine gave more stimulation over the endogenous level of activity than did most amino acids. Compared to the crude homogenate, the pH 5 enzymes2 retained full activity for coupling leucine and glycine to soluble RNA but would not catalyze an exchange of ATP and radioactively labeled inorganic pyrophosphate. This lack of exchange was uneXpected since amino acids are activated by the following reaction: Enzymes Amino acid + ATP \ \ aminoacyl-AMP + pyrophOSphate Conversely, Bucovaz and Davis (106) found that five of the nineteen common amino acids tested (L-tyrosine, L-trypto- phan, L-isoleucine, L-cysteine, and L-leucine) markedly stimu- lated the ATP and labeled pyrophosphate exchange reaction in 2The pH 5 enzymes are equivalent to the precipitate obtained after adjusting the 105,000 x g supernatant to pH 5. 23 an aparticulate fraction of rat mammary glands. Purification of this soluble fraction by Norit treatment not only lowered the endogenous exchange rate but also resulted in the observa- tion that fifteen of the nineteen tested amino acids stimu- lated the exchange reaction. Using the aparticulate fraction, L-leucine and L-glutamic acid significantly increased the rate of exchange. Passage of the aparticulate fraction through Sephadex G-25 resulted in a 4-fold increase in the exchange rate due to tyrosine addition over the rate obtained by Norit treatment. Based on these studies, it is not known whether the mammary gland contains aminoacyl-sRNA synthetases Specific for each amino acid as has been found for other organisms. Bucovaz and Davis (106) also showed that in rat mam- mary tissue the specific activity of the leucine and glutamic acid-activating enzymes increased in the early stages of lac- tation and decreased toward the latter stages of lactation. These data suggested that as the demand for protein biosyn- thesis by the mammary gland increased, there was a concomitant increase in the ability of the intact gland to activate amino acids. Fraser £3, 2;. (107) studied the esterification of activated amino acids to sRNA in mammary tissue preparations Clu-labeled amino acids that were by measuring the amount of incorporated into the pH 5 enzyme fraction. Under their experimental conditions, significant quantities of amino acids became esterified to the sRNA contained in the pH 5 enzymes. When rat liver sRNA was added to the guinea pig mammary gland 24 pH 5 fraction, the amount of glycine coupled to sRNA was pro- portional to the total sRNA concentration. Inspection of the graphs of amino acid concentration versus a measure of bound amino acid residues indicated a biphasic process. First, there was a rapid increase of amino acid-sRNA forma- tion with increasing concentration of amino acids up to about 20 mE.followed by a slower continuous increase in amino acid-sRNA formation which was proportional to amino acid concentrations. At high concentrations, amino acids combined to the extent of one residue per sRNA molecule. Further incubation of the total pH 5 fraction with ATP and glycine or leucine did not significantly diminish the reac- tion of sRNA with valine which suggests sRNA Specificity. These investigators found that this reaction may be reversed by enzymatic removal of the amino-acyl-AMP (the precursor of amino acid-sRNA). Only the L isomer of the amino acid was esterified to the sRNA. Gutfreund (108) concluded that the acylation of amino acids to the sRNA and not the formation of the aminoacyl-AMP was the rate limiting step in amino acid-sRNA formation. Fraser and Gutfreund (105) reported the first demon- stration that subcellular fractions of mammary tissue pos- sessed the ability to incorporate amino acids into TCA- precipitable material. They incubated a homogenate of lac- tating guinea pig mammary gland with radioactive amino acids and fractionated the resultant incubation mixture. Most of the radioactivity was associated with the mitochondrial 25 rather than the microsomal fraction. Because addition of ribonuclease inhibited synthesis in the mitochondrial frac- tion, incorporation by this fraction was considered to be due to contamination by microsomes. Later Turba and Hilpert (109) demonstrated that the microsomal fraction isolated from lactating guinea pig mammary glands incorporated amino acids into protein by an ATP-dependent mechanism. More recently Brew and Campbell (103) incubated slices of lactating guinea pig mammary gland with radioactive amino acids and they, too, found that the mitochondrial fraction was the most active. However, the mitochondrial and micro- somal fractions were equally active when incubated with labeled amino acids, pH 5 enzyme, and an energy generating system. The combined mitochondrial and microsomal fractions incorporated labeled amino acids into d-lactalbumin. This incorporation depended on the presence of energy and resulted in synthesis of complete molecules of d-lactalbumin from radioactive amino acids based on usual peptide analyses of the hydrolyzed product. MATERIALS Mammary Tissue Source The mammary tissue utilized in this study came from lactating cows and was obtained from Van Alstine's slaughter house in Okemos, Michigan. All cows were completely milked prior to slaughtering. Since the cows usually available at this source were generally below average in production, special arrangements were made to obtain normal mammary tissue of normal cows from the dairy herd of Michigan State University. Higher rates of amino acid incorporation were obtained with this source presumably because better control of milk production levels, udder health, and freedom of milk in the tissue preparations was possible. Chemicals The chemicals used in this study and their sources are listed in the following paragraphs. Amino acids-A grade, adenosine triphosphate (ATP)-disodium salt, guanosine triphos- phate (GTP)-trisodium salt, protamine sulfate, bovine growth hormone (1.0 USP units/mg), recrystallized bovine pancreatic insulin (23.4 IU/mg), bovine lactogenic hormone or prolactin (grade B, 20 IU/mg), ammonium salt of polyuridylic acid, and potassium penicillin C were obtained from Calbiochem, Los 26 27 Angeles. Trisodium salt of phosphoenol pyruvic acid, crys- talline pyruvate kinase, tris (hydroxymethyl) aminomethane (TriseHCl buffer), cycloheximide, crystalline bovine serum albumin, and sodium deoxycholate were preparations of the Sigma Chemical Company. Cortisone acetate, 2 X crystallized deoxyribonuclease, and puromycin dihydrochloride were obtained from Nutritional Biochemicals and reduced gluta- thione and polyvinyl sulfate, potassium salt, were purchased from General Biochemicals. Crude collagenase (125-200 units/ mg), and electrophoretically purified horseradish peroxidase were obtained from Worthington Biochemical Corporation. Polyadenylic acid, potassium salt, was purchased from Miles Chemical Company and crystalline L-chloramphenicol was obtained from Parke, Davis and Company. Bentonite (325 mesh) was purchased from E. H. Sargent Company. L-Leucine-U-Clu (200-250 uc/umole), sodium pyrophos- 32 phate-P were obtained from New England Nuclear Corporation while L-lysine-U-Clu (1.17 uc/umole) and L-phenylalanine-U- C1“ (1.3 uc/umole) were purchased from Volk Radiochemical Company. 2,5-Diphenyloxazole (PPO) and 1.4-di[2-(5-phenyl- oxazolyli]-benzene (POPOP) were preparations of the Packard Instrument Co. Inc. Sephadex G-75 was obtained from Pharmacia, Limited. Fresh milk was supplied by the Michigan State University dairy herd. Dr. J. R. Brunner of the Department of Food Science at Michigan State University donated antibodies against whole casein, proteose-peptones, dS-casein and)<-casein. The pro- teins were isolated and purified by methods devised by the 28 following investigators: whole casein, McKenzie and Wake (110). dS-casein, Thompson and Kiddy (111),)(-casein, Swaisgood 23, El: (112), and proteose-peptones by Brunner and Thompson (113). Antibodies against these purified proteins were prepared according to the method of Kabat and Mayer (114). Ten mg of each of these proteins were sus- pended in complete Freund's adjuvant which was purchased from Difco Laboratories and then injected intramuscularly into rabbits. Ten mg of each of the milk proteins sus- pended in incomplete Freund's adjuvant (minus the antibody production stimulant) were injected at two week intervals twice more into the same respective rabbits. At the end of the six week eXposure period, the rabbits were each bled by heart puncture and the blood sera containing the anti- body were collected. Equipment Some of the homogenizations of mammary tissue were performed in a Virtis "45" high Speed homogenizer and in a French high pressure tissue press. All radioactivities were determined in a series 3000 Packard TriCarb scintillation counter. The Spinco Model L and the Model B-60 International Preparative ultracentrifuge were used for sucrose density gradient centrifugation and microsome isolation. Sedimenta- tion velocity was determined with a Model E Spinco ultra- centrifuge equipped with sclieren optics. A Gilford record- 29 ing Spectrometer was used for measuring enzyme activities by Spectrophotometric methods. METHODS Preparation of Single-Cell Suspensions Single-cell suSpensions of mammary tissue were pre- pared according to the method of Eoner 23, film (115). One gram of the washed mammary tissue was suspended in 3 ml of Hanks' basic salt solution (see Appendix) which contained 3 mg of collagenase per ml. This suspension was incubated for 1 hour at 37°C with constant stirring. The tissue was 1) recovered by centrifuging the suspension for 10 minutes at 5,000 x g, 2) resuspended in the same volume of col- lagenase solution as used in the previous mixture, 3) incu- bated for one hour at 37°C 4) again recovered by centrifuga- tion, and 5) washed twice with the salt solution by centri- fugation. This process resulted in a suspension of intact mammary cells, secretory and otherwise, which was devoid of the normal connective tissue. Preparation of Subceglular Fractions For the preparation of the subcellular fractions, the mammary tissue was homogenized by the following procedure. The relatively milk-free tissue was cut with scissors into small pieces and washed with a Medium A salt solution (see Appendix Table 5) and frozen with Dry Ice as soon as possible. 30 31 After tranSporting the tissue to the laboratory, it was fur- ther frozen with liquid nitrogen and then powdered with a mortar and pestle in a cold room. The procedure for preparation of the microsomes and the aminoacyl-sRNA synthetases (pH 5 enzyme) is presented in Figure 1. The suspension of thawed, homogenized tissue was suspended in 2 to 3 volumes of Medium A and centrifuged at 10,000 x g for 10 minutes at 4°C to remove mitochondria, nuclei, and cell debris. The supernatant solution was then centrifuged for 90 minutes at 105,000 x g at 4°C in a sw 3o rotor for the Spinco Model L centrifuge. The aparticulate supernatant solution was utilized for the preparation of the aminoacyl-sRNA synthetases and the pellet was used as the microsomal fraction. Preparation of the Migrosoma; Fraction-~The procedures for microsome preparation were adapted from those utilized by Korner (60) who isolated a similar fraction from rat liver. The pellet was suSpended with the aid of a Broeck homogenizer in oneethird the original volume of Medium A. This cpalescent solution was again centrifuged at 105,000 x g. The washed microsomes were resuspended in Medium A to a final concentra- tion of 8 to 12 mg of ribosomes per ml based on the extinc- tion coefficient of 11.3 absorbancy units at 260 mu per mg ribosomes per ml (116). This solution was subsequently used for studying amino acid incorporation by the cellefree system. The ribosomes, i.e., particles not bound to the endoplasmic 32 PREPARATION OF MICROSOMES AND pH 5 ENZYME Tissue washed, frozen, homogenized, and suspended in Medium A solution centrifuge at 10,000 x g--10 min. r W Supernatant Cell debris and Mitochondria (discard) centrifuge at 105,000 x g--90 min. VT v Supernatant Microsomes Adjust to ResusPended in pH 5 Medium A Centrifuge Recentrifuge Precipitate Washed microsomes Redissolve in Redissolve in 0.05 E,phos. Medium A buffer, pH 7.5 Adjust to pH 5 \LCentrifuge \V Washed Precipitate Microsomes Redissolve in 0.05 M- phOSo buffer, pH 7.5 pH 5 enzyme Figure 1. Preparation of microsomes and pH 5 enzyme from mammary tissue. 33 reticulum, were prepared from this microsome solution by the addition of a 10% solution of sodium deoxycholate to a final concentration of 0.25%. The ribosomes were reisolated by centrifuging the solution at 105,000 x g for 90 minutes at 4°C. This procedure usually resulted in the isolation of about 250 mg of microsomes per 100 g of tissue. Preparation of pH~5 Enzyme--Aminoacyl-SRNA synthetases were isolated by acid precipitation of pH 5 from the apar- ticulate fraction (supernatant obtained after the 105,000 x g centrifugation) according to the method of Hoagland pp, 3;, (117, see Figure 2). This fraction was held at 0°C and was adjusted to pH 5.0 by the addition of 1 E acetic acid. After gently stirring the solution for 10 minutes, the mixture was centrifuged for 10 minutes at 30,000 x g. The pellet was dissolved in a volume of 0.05 E,phosphate buffer (pH 7.5) equal to that of the original solution. The solution was again adjusted to pH 5.0 with 1 E,acetic acid and centrifuged as before. The resulting pellet was dissolved in phOSphate buffer to give a final concentration of 4 to 8 mg of protein per ml. This solution--the pH 5 enzyme fraction—-was subse- quently utilized in the amino acid incorporation eXperiments. When it became necessary to remove the sRNA which contamin- ated this preparation, neutralized protamine sulfate was added to a final concentration of 1.0 mg per ml of the aparticulate fraction. The precipitate was then removed by centrifugation at 30,000 x g for 10 minutes at 4°C. The pH 34 5 enzyme was prepared from this solution as previously described. Usually 500 mg of pH 5 enzyme was isolated from each 100 g of tissue. Preparation of the A820 Enzyme--Aminoacyl-sRNA syn- thetases were also isolated from the aparticulate solution by ammonium sulfate fractionation according to the procedure of Allen and Schweet (58). Powdered ammonium sulfate was added slowly to the aparticulate supernatant solution to 40% saturation. After gently stirring for 1 hour, the mixture was centrifuged, the precipitate discarded, and the superna- tant solution was adjusted to 70% of saturation with solid ammonium sulfate. After standing for 2 hours with occasional stirring, the precipitate was collected by centrifugation at 20,000 x g for 15 minutes and the supernatant was discarded. The precipitate, termed the A870 enzyme, was suSpended in 0.05 E_phOSphate buffer at pH 7.5 and utilized for amino acid incorporation studies. Isolation of sRNA from Mammary Tissue--The procedure for precipitation of the sRNA (see Figure 2) was based on the work of Rosenbaum and Brown (118). Mammary tissue frozen at -20°C was sliced into small pieces and homogenized in the Virtis "45" homogenizer for 1 minute at full Speed. The tissue was suspended in one volume of redistilled, water- saturated phenol, one volume of 0.14 E NaCl-0.01 E_phOSphate buffer at pH 7.0, and 0.02 volume of 1% EDTA at 0°C. The homogenate was centrifuged at 5,000 x g and separated into 35 PREPARATION OF sRNA Tissue + Phenol + EDTA + 0.01 E_phos. buffer pH 7.0 Homogenize and centrifuge W ‘W Water Phase Phenol Phase (discard) Wash 3x with diethyl ether Water Phase Make to 1 111 NaCl \I, \l/ Supernatant Precipitate (discard) Add 2 vol EtOH i/ Precipitate Supernatant (discard) Dissolve, reprecipitate Dissolve, and dialyze sRNA Solution Figure 2. Isolation of sRNA from mammary tissue. 36 two phases. After the phenol was removed from the water phase by ether extraction, sodium chloride was added to a concentration of 1 E, The solution was kept at 4°C over- night. The white flocculent precipitate was removed by centrifuging the solution for 10 minutes at 20,000 x g. Two volumes of absolute ethanol were added to the superna- tant solution to precipitate the sRNA. The precipitate was collected by centrifugation, redissolved, reprecipitated with ethanol, and dialyzed against several changes of 0.01 E phosphate buffer, pH 7.4. This solution containing approximately 1 mg of sRNA per ml was subsequently utilized in amino acid incorporation eXperiments. The concentration was determined by the relationship: 1 mg per ml equals 24 absorbance units [260 mm and 1 cm light path (119)]. Usually 25 mg of purified sRNA was prepared from each 100 g of mammary tissue. Analysis of Ribpsomespy Sucrose Density Gradient Centrifugation Analysis of the degree of aggregation of ribosomes was performed by sucrose density gradient centrifugation. One-tenth ml of a ribosome and polyribosome solution con- taining ten absorbancy units (260 mu) Per ml was layered on a linear 10 to 34% sucrose gradients (4.8 ml total) prepared in an apparatus similar to that described by Martin and Ames (120). The gradient solution contained, in addi- tion to the sucrose, 0.005 E MgClZ, 0.025 E KCl, and 0.01 E 37 Tris-HCl buffer, pH 7.4. The gradient was centrifuged for 90 minutes at 39,000 rpm in a Spinco SW swinging bucket rotor and allowed to stop without braking. After centrifugation, the ribosomal sedimentation pattern was obtained by adapting the Gilford recording spectrometer as shown in Figure 3. When the gradient had been placed in the holder, toluene containing B-carotene as an indicator was pumped by means of a Sage pump into the void Space above the gradient and into the outlet tube. When the air was completely expelled, this part of the system was then closed. The gradient tube was punctured at the bottom by the adjustable needle which was connected via teflon tubing to the flow cell. This tubing, the flow cell, and the outlet tubing were previously filled with 34% sucrose. After baseline adjustment on the recorder and opening the outlet valve, the toluene was pumped into the gradient tube at a rate of 5.0 ml per hour. The recorder was adjusted to 1.0 absorbancy unit full scale and the chart speed was 0.1 inch per minute. The Special Gilford flow cell with a pin hole cross section had a 1 cm light path. Teflon tubing having an internal diameter of 0.053 cm was used and the Spectrometer wavelength was set at the 260 mu. Two eXperiments involving sucrose density gradient centrifugation were performed with the type SB-283 rotor for the International Preparative Ultracentrifuge, Model B-60. The rotor was operated at 40,000 rpm for 1 hour. All procedures were identical to that previously described except 38 ad sobdm was mdmhamso can wcdaaomnoa now no mmopofim Scam msamppma Hmaomobda wSAEHSpno sow .Qoapoom mpOSpoz oz» mo pump on» Hancocka baa .proHomaw hpamsoo mapmswaam 639 .m madman 39 that approximately 10 absorbance units (260 mu) of ribo- somes and polyribosomes were layered onto the 12 ml sucrose gradient and the flow rate through the flow cell was 10 ml per hour. Electron Microphotographs--The ribosome sample used for electron microscopy consisted of a solution from the polyribosome fraction of a sucrose gradient. Droplets of these ribosome samples were placed on Formvar-coated copper grids. The excess fluid was removed by touching filter paper to the edge of the grid and extensively rinsing each grid with solutions of decreasing concentrations of sucrose, but each solution contained 0.025 E HCl, 0.005 EMgClZ, and 0.01 E’Tris-HCl buffer, pH 7.5. The polyribosomes on the copper grids were shadowed in a Kenny shadow caster with oxidized tungsten, and then photographed in a RCA, type EMU 2A, electron microscope. Aminoacyl-SRNA Synthetase Assayg Two general methods were used to assay aminoacyl-sRNA synthetase activity in various fractions isolated from mam- mary gland tissue. They were 1) the radioactive pyrophos- 'phate exchange with ATP by the method of Bucovaz and Davis (106) and 2) aminoacyl hydroxamate formation according to the procedure of Berg (121). The reaction mixture used for the measurement of aminoacyl-sRNA synthetase activity by ATP-PP32 exchange 40 contained 100 umoles of Tris-H01 buffer, pH 7.5, 5 umoles of ATP, 5 umoles of sodium pyrophOSphate containing approximately 100,000 cpm, 5 umoles of MgClZ, 0.5 ml of the 0.02 E_amino acid mixture whose composition was similar to that of whole casein, 50 umoles of KF, and 1 mg of enzyme, all in a total volume of 1.0 ml. After the incubation, two ml of trichloroacetic acid (10% w/v) was added to stop the reaction. Two hundred mg of acid-washed Norit A were added per ml of the supernatant. The Norit A was removed by centrifugation at 2,000 x g and washed with 4 ml of 0.1 E‘sodium acetate (pH 4.5). The Norit A was next washed 1) with 0.05 E’sodium acetate-0.1 E sodium pyrophoSphate solution (pH 4.5), 2) three times with a 0.1 E sodium acetate, and 3) finally with distilled water. The washed Norit A was then suspended in 5 ml of 1 E HCl heated at 100°C for 15 minutes and then centrifuged. An aliquot of the supernatant solution was suSpended in Kinard's solution for scintillation counting (see Appendix). The inorganic phosphate content was measured by the method of Fiske and SubbaRow (122). Aminoacyl-SRNA synthetase activity in various prepara- tions was also measured by aminoacyl hydroxamate formation. The assay mixture contained 10 umoles of ATP, 0.7 ml of the 0.02 E.amino acid mixture whose composition was comparable to that of whole casein, 10 umoles of MgClz, 20 umoles of Tris-HCl buffer, pH 7.4, the enzyme fraction and 1000 umoles of neutralized hydroxylamine in a total volume of 3 ml. The 41 control contained no amino acids. The mixture was incubated for 30 minutes and then the reactions were stopped by adding 1.4 ml of 100% trichloracetic acid (w/v). After 0.6 ml of 2 E_FeCl was added, the mixture was centrifuged at 2,000 x 3 g and the absorbancy of the supernatant at 520 mu was deter- mined. These results were converted to the amount of hydrox- amate formed by comparison to a standard curve prepared with succinic anhydride. Properties of Mammary Glgnd sRNA The homogeneity and sedimentation coefficient of sRNA was determined by sucrose density gradient centrifugation according to the method described by Martin and Ames (120). Linear gradients of 5 to 20% sucrose solutions containing 0.005 M.M3C12v 0.025 E|KCl, and 0.01 E Tris-HCl buffer at pH 7.4 were centrifuged 15 hours in an SW 39 Spinco swinging bucket rotor at 4°C and then fractionated by collecting 6 drop fractions. Peroxidase and cytochrome c were utilized as markers of known sedimentation coefficients and they were assayed according to the method deve10ped by Worthington Biochemical Corporation and by measuring absorbance at 415 mu, respectively. Esterification of amino acids to the purified sRNA was studied by the method of Fraser 23, 2;. (107). The incuba- tion mixture contained 0.15 mg sRNA, 15 umoles of MgClZ, 300 umoles of Tris-RC1 buffer, pH 7.4, 0.1 ml of 0.02 E amino acid mixture whose composition was comparable to that of whole 42 casein minus glutamate, 0.02 no of L-glutamate-U-C14 (1.5 uc/umole), and 11 to 15 mg of the enzyme fraction in a volume of 3.0 ml. This mixture minus sRNA served as the control. The systems were incubated for 30 minutes at 37°C and the reaction was stopped by acid precipitation at pH 5.0. The precipitate was 1) collected by centrifu- gation at 30.000 x g for 10 minutes, 2) dissolved in 10 ml of 0.01 E phosphate buffer, pH 7.0, and 3) extracted twice with an equal volume of water-saturated phenol. Two volumes of absolute ethanol were combined with the aqueous phase. The precipitate was collected by centrifugation and the radioactivity was determined by dissolving an aliquot in Kinard's counting fluid (see Appendix Table 4). / Ribonuclease Activity Determination Ribonuclease activity of the various fractions of the mammary tissue was measured by the method of Kalnitsky 23, El- (123). The incubation mixture contained 10 mg of yeast RNA, the enzyme fraction to be tested, 10 umoles of MgClZ, in a volume of 2.0 ml of 0.01 E_phosphate buffer, pH 7.5. The solution was incubated for 30 minutes at 37°C. The reaction was stopped by the addition of 1.0 m1 of a solution of 0.75 g of uranyl acetate dissolved in 25% per- chloric acid. After standing for 15 minutes, the precipi- tate was collected by centrifugation at 2,000 x g and the absorbance at 260 mm was determined on the supernatant. 43 Unincubated samples served as controls for each enzyme preparation. Isolation of DNA from Mammary Tissue Mammary gland DNA was prepared according to the method of Chargaff as follows (124): Two hundred grams of fresh mammary tissue was suSpended in 400 ml of 0.1 E,NaCl- 0.05 E_sodium citrate, pH 7, and homogenized at 4°C for 30 seconds in a Waring Blender. All subsequent operations were also performed at O to 4°C. The sediment obtained from centrifuging the homogenate at 2,000 x g was washed twice with the above buffer. The washed sediment was com- bined with 1,250 ml of a sodium chloride solution (10% w/v) and gently stirred overnight. After a second Similar extrac- tion, the combined supernatants were injected into two volumes of 95% ethanol. The nucleohistone precipitate was spooled out, washed successively with 70% and 80% ethanol, and then dissolved in 2,500 ml of a 10% sodium chloride solution. The proteins were removed by extracting this solution eight times with chloroform-amyl alchohol (3:1). After injecting the deproteinized solution into 2 volumes of 95% ethanol, the precipitate was spooled out and washed with 70%, 80%, and 100% ethanol in succession. The final yield was 450 mg of air-dried DNA. 44 Protein and Riggsome Determinations Protein concentrations in the various preparations were determined by the method of Warburg and Christian (125). Ribosome and polyribosome content of various solutions was determined by the method of Tso’and'Vinograd(116). They found that 1 mg of ribosomes per ml have an absorbance of 11.3 at 260 mu and 1 cm light path. Assay of Amino Acid Incorporation 1° leucine incor- The complete system for measuring C poration was composed of the components (each adjusted to pH 7.4) listed in Table 1. Unless specified, the complete system was incubated for 40 minutes at 37°C and contained the pH 5 enzyme as a source of aminoacyl-sRNA synthetases. The incubation period was considered to-have been initiated when the final component, i.e., the labeled amino acid, was added to the incubation mixture. Six mg of bovine serum albumin was added to the mixture immediately before stopping the reactions by adding 10 ml of 5% trichloracetic acid (TCA). The proteins in the zero time sample was precipitated immediately after the addition of the labeled amino acid. The TCA-precipitated proteins of each incubation mixture were washed and counted according to the method cf Casjens and Morris (65). Briefly, this procedure involved 1) washing the precipitated proteins twice with 5% TCA, 2) dissolving the precipitates in 0.5 ml of 1 E sodium hydroxide, 3) 45 TABLE 1 Components of the compiete amino acid incorporating system The amounts of the components in this table were uti- lized in each ml of the complete incubation mixture. The specific amounts of microsomes, enzyme, and labeled amino acid are quoted with each eXperiment. the amino acid mixture was comparable to the amino acid com- position of whole casein as analyzed by Jenness and Patton (126). The composition of Component Amount per ml Microsomes o o o o o o a pH 5 enzyme or A37O enzyme sRNA . . . . . . . . . . ATP . . . . . . . . . . GTP . . . . . . . . . . PhOSphoenolpyruvate . . Pyruvate kinase . . . . KCl . . . . . . . . . . MgClZ . . . . . . . . . Glutathione (reduced) . Amino acids minus leucine _-(see Appendix for composition) C1“, leuoine o . o ,. (200 to 250 uc/umole) Tris-H01 buffer, pH 7.4 0.3-0.6 ms 1 to 2 mg 50 us 1.0 umole 0.25 umole 5.0 umole 40 us 50 umoles 5.0 umoles 20 umoles 1.0 umole 1.0 to 1.5 no 50 umoles 46 reprecipitating the proteins with TCA, 4) washing the preci- pitates again with 5% TCA, and 5) dehydrating the precipi- tates with washes of acetone containing 0.1 E ROI, of acetone-RC1 and ether (4:1), and finally ether alone. The dried samples were transferred to a scintillation vial and dissolved in 0.5 ml of 1 E sodium hydroxide. Thixotropic counting solution (see Appendix Table 1) was mixed with the dissolved sample for the determination of radioactivity. Identification of the Possible Synthesized P;oteins(s) Gel Filtration Studies of Radioacpive Product--Column chromatography on Sephadex G-75 was utilized.to assess the approximate molecular weight and homogeneity of the proteins that may have been synthesized in the cell-free system. The Sephadex gel was packed in a column of 1 x 70 cm dimensions and equilibrated with 0.05 E phosphate buffer, pH 7.5. Two and one-half ml of the 40 minute incubated complete system containing peroxidase and cytochrome c as standard proteins of known molecular weights were applied to the column. One ml of the 0 time sample containing the same markers was also analyzed by the same procedures. The 0.05 E phosphate buffer, pH 7.5, was allowed to flow through the column at a rate of approximately 3 ml per hour. One ml fractions were collected. Peroxidase activity and cytochrome c in the collected frac- tions were assayed by the methods previously described. Radioactivity was determined by combining 0.25 ml of each fraction with 0.5 m1 of 1 E sodium hydroxide and suspending 47 the resulting solution in the thixotropic counting solution (see Appendix Table 1). Determination of the molecular weights of the unknown proteins was based on the method described by Andrews (12?). Isotope DiEption Studies--Partial identification of the synthesized protein in the incubated complete system was also performed by isotope dilution studies. Incubation mixtures which were incubated for 0 and 40 minutes were combined with fresh skimmed milk. Precipitation of the caseins and subsequent isolation of individual casein frac- tions-~as, B, and)(,--were performed according to the method of McKenzie and Wake (110) as shown in schematic form in Figure 4. Isolation of d-lactalbumin and B-lactoglobulin was conducted according to the method of Aschaffenburg and Drewry (128) as shown in Figure 5. Aliquots of each protein was then dissolved in sodium hydroxide and the resulting solution was suspended in the thixotropic counting solution for radioactivity determinations. Immunodiffusion Study--Antigen-antibody reactions utilizing the technique of immunodiffusion according to the method of Ouchterlony (114) were performed to more adequately define the protein that was synthesized in the cell-free system. One percent ion-free agar dissolved in 0.15 E,NaCl and 0.15 E_phosphate buffer (pH 7.2) was poured into a 2 mm thick layer in a petri dish and allowed to solidify. The antigen (0.05 ml) was placed in the center well while the 48 Sample + Skimmed Milk 67 Precipitate lWaSh 3x with H 0 2 Precipitate precipitate \LDissolve and 1 twice at 4.6 Precipitate Dissolve in 3.3 E_urea Lower to pH 4.9 Centrifuge ' Precipitate Dissolve Dialyze Add CaCl to 0.2. 11 Centrifu e Adjust pH to 4.6 f Centrifuge Supernatant (Whey Proteins) 0’ Supernatant Dialyze B-Casein I Supernatant Adjust to pH 7.0 Add ethanol to 25% Centrifuge \ \ W’ Precipitate Dissolve Dialyze (is-Caseinl Supernatant Adjust to pH 3.5 Add ethanol of 50% Adjust to pH 4.6 Centrifuge PreoIpitate (discard) \ Precipitate Dissolve Dialyze [ghCaseinj .Figure 4. Isolation procedures for d .thase n from skimmed milk. W Supernatant (discard) S-casein, B-casein, and 49 Whey Proteins Adjust to 8H 7.6 Warm to 40 C Add anhydrous Na SO“ (20 g/1OO ml3 Centrifuge _ Supernatant Precipitate (discard) lAdjust to pH 2.0 Precipitate Supernatant Dissolve l/Dialyze Adjust to pH 3.5 -lacto lobulin Precipitate Supernatant (discard) Dissolve Adjust to pH 4.0 Let stand overnight . v t Precipitate Supernatant Add (NH ) SO Dissolve (11.5g/ 00ml) Adjust to pH 6.6 w t Add equal vol. sat'd Precipitate Supernatant (NHu)2804 sol'n -l (discard) v \l/ Supernatant Precipitate (discard) until cloudy Let stand overnight V t Precipitate Supernatant (discard) Dissolve Dialyze [d-lactalbumirfl Figure 5. Isolation procedures for d-lactalbumin and B-lactoglobulin from the whey fraction of skimmed milk. 5O antibodies against the four proteins (cs-casein, whole casein, X-casein, and proteose-peptone fraction) were placed in wells equidistant (10 mm) from the center well. The petri dish was placed in a room temperature, water vapor-saturated chamber. Immunodiffusion was permitted to proceed for three days. The unprecipitated protein was then leached from the agar by repeatedly placing 0.15 E NaCl-0.15 fl'phOSphate buffer at pH 7.2 over the agar for a period of 12 hours. After air-drying the agar containing the precipitin bands, the position of these bands was recorded. Next the agar sheet was placed in contact with Kodak No Screen Medical X—Ray film for 30 days. The eXposed X-ray film was developed by normal photographic methods. RESULTS PART I CHARACTERIZATION OF COMPONENTS The aim of this work was to develop a cell-free sys- tem for synthesis of specific milk proteins utilizing com- ponents of bovine origin isolated from lactating mammary glands. Synthesis of specific proteins would be an essen- tial property of any system that might later be used to study the control of synthesis of milk proteins. Many characteristics of this system have been studied and their presentation is organized in the following manner. First, each of the purified cellular components which was utilized in the complete protein synthesizing system was partially characterized. These include microsomes, sRNA, and the aminoacylusRNA synthetase preparations. Subsequent studies utilizing these components for amino acid incorpora- tion will then be discussed. This latter section includes a description of eXperiments designed to determine 1) the dependence of the complete process on each of the components of the complete system, 2) inhibition of amino acid incor- poration by known protein synthesis inhibitors, 3) possible stimulation by polynucleotides and hormones, and 4) type of 51 52 protein synthesized by the complete system.3 Characteristics of the Ribosome Fraction Preparation of the Ribosomal Fraction--The presence of polyribosomes, i.e., aggregates of ribosomes held together by strands of RNA, has been demonstrated in many different cell types in various species (1, 2, 3). Polyribosomes have been shown to be required in cell-free systems that are cap- able of synthesizing amino acids (129). These fragile struc- tures are extremely sensitive to ribonuclease and mechanical forces. Since polyribosomes are required for cell-free pro- tein synthesis, procedures for their preparation from lactat- ing bovine mammary tissue were investigated. In order to determine which method of preparation yielded a higher per- centage of polyribosomes, mammary tissue was disrupted by 3Note: Throughout this thesis, except in some Figure titles, the following terminology will be used: Ribosomes--Particles consisting of the single or monomeric ribosome. Polyribosomes--Particles consisting of more than one ribosome. Ribosomal fraction-~A solution which contains both ribosomes and polyribosomes and contains sodium deoxycholate (0.25% w/v) to solubulize the endoplas- mic reticulum. Microsomes--The particles in the microsomal fraction and consisting of free ribosomes, free polyribosomes and membrane-bound ribosomes and polyribosomes. As a matter of convenience, in certain titles of Figures, the word "ribosomes" refers to both ribosomes and polyribosomes as defined above. 53 the Virtis "45" tissue homogenizer, the French press, and by breaking frozen tissue with a mortar and pestle. After the initial homogenization of the tissue by each of the techniques, the microsomes were isolated as described in the Methods section. Sodium deoxycholate was added to the microsome suspensions to a final concentration of 0.25% (w/v) to solubulize the endoplasmic reticulum. An aliquot of the solution from each of the three tissue prepa- rations was subjected to sucrose density gradient centrifuga- tion and the extent of aggregation of the ribosomes was determined by scanning the gradient at 260 mu (see Methods). Patterns of the individual ribosomal preparations (Figures 6, 7, and 8) show a sharply defined peak of single ribosomes. Ribosomes in this fraction were determined to be monomer units by comparison of this mammary ribosome pattern with similar density gradient patterns of reticulo- cyte ribosomes where the state of aggregation has been thoroughly investigated (129). The absorbing material to the left of this large ribosome peak represents a series of polyribosome components. Figures 6, 7, and 8 demonstrate that the homogenization of the tissue in the frozen state produced a microsome fraction which contained a higher pro- portion of polyribosomes than was found in the microsome fractions isolated after the tissue had been homogenized with the Virtis "45" or the French press. Since polyribo- somes are necessary for protein synthesis, it is important to obtain a preparation with the highest proportion of 54 Figure 6. The sedimentation behavior of ribosomes and polyribosomes prepared by homogenizing frozen tissue. The ribosomal fraction containing 0.25% DOC (w/v) was isolated from tissue which had been frozen in the liquid nitrogen and disrupted with the aid of a mortar and pestle (see Methods). Seven-tenths of an absorbance unit (260 mu) of ribosomes and polyribosomes dissolved in 0.1 ml of Medium A (see Appendix Table 5) was layered onto a linear sucrose gradient of 10 to 34% sucrose containing 10 mEDTris-HCl buffer (pH 7.4), 25 mE_ KCl, and 5 mE|MgCl (final volume 4.8 ml). The gradient was centrifuged 1.5 hoar at 39.000 rpm in a SW 39 rotor at 4°C. The contents of the centrifuge tube was then analyzed optically for ribosomal and polyribosomal content (see Methods). 55 RIBOSOMES PREPARED BY HOMOGENIZING FROZEN TISSUE -O.5O I ABSORBANCE “-0.25 )2 ‘ 1 1 1 1 l 2 3 4 EFFLUENT VOLUME (ml) 56 Figure 7. The sedimentation behavior of ribosomes and polyribosomes prepared with a Virtis "45" homogenizer. The ribosomal fractions containing 0.25% DOC (w/v) were prepared from tissue which had been disrupted with the Virtis "45" homogenizer by methods described in the Methods section. Eight-tenths of an absorbance unit of ribosomes and polyribo- somes were analyzed for ribosomal and polyribosomal content as described in Figure 6. 57 RIBOSOMES PREPARED WITH VIRTIS HOMOGENIZER -O.50 ABSORBANCE -O.25 I( i i 1 1 I 2 3 4 EFFLUENT VOLUME (m0 58 Figure 8. The sedimentation behavior of ribosomes and polyribosomes prepared by homogenizing tissue with the French press. The ribosomal fraction containing 0.25% DOC (w/v) was isolated from tissue which had been disrupted by utilizing the Aminco-French pressure cell at 12,000 to 16,000 psi (see Methods). Eight-tenths of an absorbance unit of ribosomes and polyribosomes were analyzed for ribosomal and polyribo- somal content as described in Figure 6. 59 RIBOSOMES PREPARED BY HOMOGENIZING TISSUE WITH FRENCH PRESS ~o.50 Lu 0 2 <1 — m a: o a) m <1 -o.25 L L l J 1 2 3 4 EFFLUENT VOLUME (ml) 6o polyribosomes. Therefore, the procedure of homogenizing frozen tissue became the method of choice for all the future studies. Effect of AddEEives on Po;yribosome Yield--Electron micrographs have shown that in the liver and pancreas, ribo- somes are primarily attached to an intracellular lipoprotein membrane, the endoplasmic reticulum, and a small proportion apparently is free in the cytoplasm (130). Palade and Siekevitz (131) demonstrated that pancreatic ribosomes are released from the endoplasmic reticulum while maintaining their structural integrity by treating the microsomal frac— tion with sodium deoxycholate (DOC). Burka (132) has recently demonstrated that the free ribosomes of the reticulocyte ribosomal fraction are broken down by deoxy- cholate. Insignificant breakdown of ribosomes and polyribo- somes was observed when DOC was added to rat liver microsomes at levels varying from 0.3% to 1.2% (w/v); however, concen- trations higher than 1.66% destroyed the ribosomal particles (63). Because sodium deoxycholate releases the bound ribo- somes from the endoplasmic reticulum, this anionic detergent was used to ascertain whether mammary gland ribosomes existed as bound and/or free, and whether this treatment altered the sedimentation behavior of the various aggregates of ribosomes. Sodium deoxycholate (DOC) was combined with an aliquot of a microsome solution to a final concentration of 0.25% (w/v). Equal absorbancy units at 260 mu of the DOC-treated and the non-treated solutions were each subjected to sucrose 61 density gradient centrifugation and the pattern of ribosome sedimentation was determined as previously described (see Methods). The results of this experiment are illustrated in Figure 9. The ribosomal pattern of the microsome prepara- tion showed that unbound ribosomes and polyribosomes were present since not all the 260 mu absorbing material sedi- mented to the bottom of the centrifuge tube. Treatment of the microsome preparation with deoxycholate resulted in a substantial increase in the amount of each of the ribosomal peaks, thereby illustrating a release of ribosomes from the endoplasmic reticulum. Also, the DOC treatment did not interfere with the ribosomal structures or the binding of messenger RNA to the ribosomes since the polyribosomal peak heights were also substantially increased. The proportion of polyribosomes in a given preparation of microsomes is dependent on the activity of ribonuclease in the solution. Because polyribosomes are necessary for protein synthesis, it is desirable to reduce the action of ribonuclease to a minimum. Ribonuclease has been shown to bind to bentonite and thereby become inactivated (133). With this fact in mind, it was decided to determine if treatment of the crude homogenate of mammary tissue with bentonite would increase the yield of polyribosomes. Bentonite, previously washed and sized by the method of Fraenkel-Conrat and Singer (133), was added at a concen- tration of 10 mg per ml to the crude homogenate. The 62 Figure 9. The sedimentation behavior of microsomes before and after treatment with sodium deoxycholate. These preparations were isolated from a crude homogenate which had been prepared by powdering the frozen mammary tissue (see Methods). The microsome fraction was clarified by adding DOC to a final concentration of 0.25% (w/v) to prepare the ribosomal fraction (endoplasmic reticulum solubulized). One absorbance unit (260 mu) of both prepa- rations were layered onto a linear sucrose gradient which was subsequently centrifuged and analyzed for ribosomal and polyribosomal content as described in Figure 6. 63 MICROSOMES TREATED WITH SODIUM ” DEOXYCHOLATE L-o.eo 1 ABSORBANCE TREATED \ J l I l v" I 2 3 4 EFFLUENT VOLUME (ml) 64 microsomes were isolated as usual and the endoplasmic reticu- lum was solubulized with 0.25% (w/v) of DOC. Analysis by sucrose density gradient centrifugation was conducted as described in the Methods section. Sucrose density gradient patterns of normal and bentonite-treated ribosomes are shown in Figure 10. Ribo- somal patterns of the two preparations were equivalent with respect to the proportion of polyribosomes, illustrating that bentonite treatment was not advantageous. Bentonite-treated ribosomes and polyribosomes equivalent to 1.10 absorbancy units at 260 mu were centrifuged while 0.7 absorbancy units of normal ribosomes and polyribosomes were centrifuged to obtain ribosomal patterns. Yet, the pattern of the normal ribosomes and polyribosomes showed the most 260 mu absorbing material. This suggests that binding of a portion of the ribosomes to bentonite had occurred and consequently these were not Scanned by the usual gradient analysis. Tester and Dure (134) have explained a similar loss of ribosomes as the formation of a ribosome-bentonite complex formed by salt binding of negatively charged ribosomes to the negatively charged surface of bentonite through magnesium ions. In summary, bentonite did not increase the proportion of polyribosomes in a given microsome preparation, and there- fore, this procedure was not utilized in subsequent studies. In addition to bentonite, polyvinyl sulfate also has been shown to inactivate ribonuclease (135). Therefore, it was logical to examine whether this inhibitor, if added to 65 Figure 10. The sedimentation behavior of normal and bentonite-treated ribosomes and polyribosomes. The normal ribosomal fraction was isolated from a crude homogenate which had been prepared by‘powdering the frozen mammary tissue (see Methods). The bentonite-treated ribosomes and polyribosomes were isolated from a similar crude homogenate containing washed bentonite at a concentration of 6 pg per 10 ml of protein. Five-tenths absorbance unit (260 mu) of the normal ribosomes and polyribosomes (0.1 ml) and 0.42 absorbance unit of the same preparation treated with bentonite (0.1 ml) were layered onto linear sucrose gradi- ents. These gradients were subsequently centrifuged and analyzed for ribosomal and polyribosomal content as described in Figure 6. 66 NORMAL vs BENTONITE TREATED RIBOSOMES —O.75 ABSORBANCE / J I l 67 the crude homogenate, would increaSe the proportion of poly- ribosomes in the ultimate microsome fraction. Polyvinyl sulfate was added to the crude homogenate at a 0.01 E concentration. The ribosomes and polyribosomes were isolated and analyzed by the usual sucrose density gradient centrifugation (see Methods). The results of the experiment are presented in Figure 11. Comparison of poly- vinyl sulfate-treated ribosomes and polyribosomes with the control illustrates that a partial destruction of the ribo- nucleoprotein structure of the ribosomes had occurred. Each ribosomal peak in the treated sample was drastically reduced in area and a ribosomal subunit smaller in size than the monomer ribosome was produced. Consequently, this ribonuclease inhibitor was not utilized in further future experiments. Since Dupanol might decrease ribonuclease activity and thus produce a higher proportion of polyribosomes in the microsome fraction, this possibility was next investi- gated. Microsomes were prepared as usual (see Methods). Dupanol was added at a concentration of 1% (w/v) to an aliquot of the microsome solution. Sucrose density gradient centrifugation of the normal DOC-treated microsomes and the Dupanol-treated microsomes was conducted as usual. Results of this experiment are presented in Figure 12. Since no ribosomal peaks were observed in the treated sample, it was concluded that Dupanol completely destroyed the physi- cal structure of the ribosomes. Hall and Doty (136) have 68 Figure 11. The sedimentation behavior of normal and polyvinyl sulfate-treated ribosomes and polyribosomes. The normal ribosomal fraction was isolated from a crude homo— genate as described in Figure 10. The polyvinyl sulfate- treated ribosomes and polyribosomes were isolated from a similar crude homogenate which contained polyvinyl sulfate at a concentration of 0.01 E, Five-tenths absorbance unit (260 mm) of the normal ribosomal fraction in 0.1 ml of Medium A (see Appendix Table 5) and 1.1 absorbance unit of the polyvinyl sulfate-treated ribosomes and polyribosomes dissolved in 0.1 ml of Medium A were layered onto linear sucrose gradients which were subsequently centrifuged and analyzed for ribosomal and polyribosomal content as in Figure 6. 69 NORMAL vs POLYVINYL SULFATE TREATED INBOSOMES "-0.75 3 -050 NORMAL———e ABSORBANCE 70 Figure 12. Comparison of the sedimentation behavior of normal and Dupanol-treated ribosomes and polyribosomes. The normal ribosomal fraction was isolated from a crude homogenate (same as Figure 10) which had been prepared by powdering the frozen mammary tissue (see Methods). The Dupanol-treated ribosomes and polyribosomes were isolated from a similar crude homogenate by treating the microsome fraction with Dupanol (1% w/v). Five-tenths absorbance unit (260 mu) of the Dupanol-treated microsomes and 0.5 absorbance unit of the normal ribosomes dissolved in 0.1 ml of Medium A were layered onto linear sucrose gradients and subsequently centrifuged and analyzed for ribosomal and polyribosomal content as described in Figure 6. 71 NORMAL vs DUPANOL TREATED RIBOSOMES L—O.75 ~4150 ABSORBANCE -<125 TREATED a9” ’ ’ %”’ EFFLUENT VOLUME (ml) 72 shown that this anionic detergent readily attacked liver ribosomes by dissociating them into an RNA fraction and a protein-detergent complex fraction. Quite obviously, this procedure could not be of use in future eXperiments. Effect of Incubating Microsomes with Ribonuclease-- Polyribosomes have been shown to be degraded to monomeric forms by the action of ribonuclease (58, 63). With this in mind, a study of the effect of ribonuclease on mammary gland ribosomes and polyribosomes was undertaken. One ml solutions of Medium A (see Appendix Table 5) containing 0.82 mg of microsomes and 0.01 ug of crystalline pancreatic ribonuclease were incubated at 37°C for various times. In another study, 30 times more ribonuclease was added to the incubation medium. In both experiments the samples were cooled rapidly and subjected to the usual sucrose density gradient analysis. Data of this experiment is presented in Table 2. There was a time-dependent degradation of the polyribosome peaks with a Concomitant increase in the peak representing the monomeric form of ribosomes. Incubation of the micro- somes with the higher level of ribonuclease (1 ug RNase per 2.7 mg of microsomes) resulted in extensive ribosomal degradation. After 5 minutes of incubation, all the ribo- somes were destroyed. These results demonstrate that polyribosomes and even ribosomes are unstable in the presence of ribonuclease at 73 TABLE 2 Incubation of microsomes with ribonuclease The reaction mixture contained 0.82 mg of microsomes and 0.01 ug of ribonuclease per one ml of Medium A (see Appendix Table 5). The column on the extreme right Show results which refer to a reaction mixture containing 0.3 ug of ribonuclease and 0.82 mg of microsomes per ml. After incubation at 37°C, aliquots were subjected to sucrose density gradient centrifugation (see section on Methods) and the areas under each peak was calculated and compared to the total area. Ribosome size was calculated from the sedimentation rate in the sucrose gradient according to Martin and Ames (120) and using the reticulocyte monomeric ribosome (78 S, 129) as a standard. Ribosome Size Time of Incubation (min.) O-1-/ 1y ' 5-1/ 1 03/ 5-2/ ”v 80 S 74.4%2/ 78.8 92.4 91.2 Trace A4120 S 22.2 19.0 7.6 7.8 None PU190 S 3.3 2m3 .Trace None None 2'-/1 ug of RNase added per 82 mg of ribosomes and polyribosomes 2 . -/1 ug of RNase added per 2.7 mg of ribosomes and polyribosomes 2/Value represents perbent of total area under ribosomal peaks 74 37°C. Also the dissociation of polyribosomes to ribosomes illustrates that the former are held together by RNA. Depgndencygpf Polypibosom§;_Character Upon Magnesium Egggr-Previous workers (63) have demonstrated that magnesium ion concentration of at least 1 mmolar was necessary for stability of rat liver polyribosomes. Magnesium concentra- tion of 0.1 mE'or less resulted in a considerable decrease in the amount of polyribosomes as analyzed by sucrose gradi- ent density centrifugation. The purpose of the following experiment was to determine whether mammary gland polyribo- somes possess a similar dependence on magnesium ion concen- trations. An aliquot of a microsome pellet was suspended in a volume of 0.5 E,KCl equal to the volume of the original microsome solution and recentrifuged at 105,000 x g for 90 minutes. The resultant microsomes were suSpended at a final concentration of about 1 mg per ml in Medium A which con- tained no magnesium chloride. In order to determine the effect of adding magnesium back to these "shocked" microsomes, magnesium chloride was added to a final concentration of 8 mE, These three preparations of microsomes containing the three levels of magnesium chloride-~0, 4, and 8 mEr-were subjected to sucrose density gradient centrifugation. The results of this study are presented in Figure 13. In the absence of magnesium ion, ribOsomes in all states of aggregation were changed to a slower moving component which 75 Figure 13. The sedimentation behavior of dissociated and reassociated microsomes. The normal microsomes were isolated by the procedure described by the Methods section. The dissociated microsomes were prepared by suSpending the microsomes in O. 5 E KCl, reisolating the dissociated micro- somes by centrifugation at 105, 000 x g for 90 minutes and suSpending them in Medium A (see Appendix Table 5) minus MgClZ. The microsomes were reassociated by adjusting an aliquot of this latter preparation to 8 mE MgCl . Twelve and six-tenths absorbance units (260 mu) of the normal micro- somes (0.1 ml), 9. 5 units of dissociated microsomes (0.1 ml) and 9.5 units of reassociated microsomes (0.1 ml) were layered on linear sucrose gradients of 10 to 34% sucrose con- taining 10 mE Tris-HCl buffer (pH 7. 4), 25 mE KCl, and 5 mE MgClZ (final— volume 12.0 ml). The gradients— were centrifuged 1 hour at 40, 000 rpm in the Model SB-283 rotor at 40 C in the Model B-60 International Preparative Ultracentrifuge. The contents of the centrifuge tubes were then analyzed for ribo- somal and polyribosomal content (see section on Methods). Note--the disassociated ribosome peak may actually be "swelled" ribosomes as described in the text. 76 DISASSOCIATION AND REASSOCIA- TION OF RIBOSOMES I'. DISASSOC.\ 1'1. I I I I ’° ~050 ABSORBANCE NORMAL . .— —-—"-—— —— l I I I 2 4 6 8 EFFLUENT VOLUME (mD 77 was equivalent to a 60 S particle. These particles may be a subunit of an 80 S ribosome or may represent a particle of the same molecular weight as the 80 S particle but having an altered (swollen) conformation. This possibility has pre- viously been suggested for reticulocyte ribosomes subjected to similar treatment (145). This approximate sedimentation coefficient is based on 1) the calculation that the major peak in the normal preparation is an 80 S particle with mono- meric reticulocyte ribosome of 78 S used as standard (129), 2) the method of calculation of sedimentation coefficients from known values as reported by Martin and Ames (120). When magnesium chloride was added to these "shocked" micro- somes at a concentration of 8 mE, the ribosomes and polyribo- somes reappeared in the gradient pattern and the peak repre- senting the slower moving component decreased in height. Thus, magnesium ion is necessary to preserve the integrity of the ribosomes and the polyribosomes. Removing magnesium ions from the solution containing both ribosomes and polyribosomes results in ribosomal dissOciation or "swelling". However, these ribosomal "Subunits" may be reassociated (altered) in such a way as to form ribosomes and even polyribosomes by bringing the magnesium ion con- centration back to 8 mE. Sedimentation Coefficient Determination-~In order to substantiate the calculation of sedimentation coefficients 78 from the sucrose density gradient data, two preparations of ribosomes suSpended in Medium A and containing 0 and 4 mE magnesium chloride were subjected to sedimentation velocity measurements in the analytical ultracentrifuge. The solu- tion of ribosomes and polyribosomes containing magnesium chloride also contained DOC at a concentration of 0.25% (w/v) to solubulize the endoplasmic reticulum. The sedi- mentation patterns are presented in Figure 14 and the data for the calculations are presented in Table 3. The sedimentation coefficients of these two samples of ribosomes were similar to that calculated from the sucrose gradient data when utilizing the monomeric reticulo- cyte ribosomes as a 78 S standard (129). The monomeric ribosomes were found by this experiment to possess a sedi— mentation coefficient, corrected to 20°C and water as the solvent, of 79 while the ribosomes in the solution contain- w 20 for normal mammary gland ribosomes compares well with the ing no magnesium possesses an S of 54. The value of 79 S of 75 reported for pancreatic ribosomes (137) and 83 w 20 reported for rat liver ribosomes (138). Electron Microscopy of Polyribosomalggreparation-- Warner 32, El! (129) have verified the existence of polymers of ribosomes in reticulocyte preparations by electron micro- scopy. A similar test was conducted with the mammary gland ribosomes in order to obtain more information concerning the integrity of the preparations. 79 .ao om.n was monogamoa Amado one Odds: zodpmpoa mo sopsoo one scam so Nm.m we: mosoaomoh Hosea can use mm.am mes Hopomw soapmoamacwma mama decade one .9 z\po\ao u m ”codename Heapscacmmao wsazoflflom can 809% oopmHSOHso was Acodmmspsoosoo ohms op oopooaaoo pony paedoamhooo soapepsOa [doom map was pcpmasoamo was good nose mo psoaoboa do mpaooaob one .Apfiooaoe aaadxsa peacock on: Omahaapsoo on» songs mopssda 0H was .m .w .3 pm Semen cams msaoppsa moaoaasom one mo madmawoposa one .owSMAAp:oomeHs Hmoapmamss m Homo: oosaam SH ads 0:0.m: pm oOwSmHaono ones msoapsaom omega ho Ha mapsmplwam .saoppmd soapmpsoadpom songs can an popsomoaaoa one one He had we m.w mo soapehpsoosoo e as Naowz o: wsHQaSpsoo 4 adapt: ad poeaommdp who: HUM z m.o ad doosommam soon on: Sean: mofiomohoaav mofioonOda sooxoonma one .oaewam can ad samppoa sofipmpaoadpom Hozoa map an oopsommadoa ma noHpmHSQOHQ mane .Ha nod ms m.m mo soapmapswosoo a pm opwHOSOhNood Rmm.o waasaspsoo Am OHDSB Napsoaa< oomv < adapt: EH ombaommao one: moaomonahhaoa one moaomopaa Hmaaoz .moaomondh madam hamaama mo hpdooaob soapmpsoaadom .EH onawam 80 TABLE 3 Sedimentation coeffgcients of mammaryggland ribosomes The normally prepared ribosomes and polyribosomes were dissolved at a concentration of 2.5 mg per ml in Medium A (see Appendix Table 5) containing 0.25% deoxycholate and "shocked" ribosomes (microsomes suspended in 0.5 E KCl solu- tion) were dissolved in Medium A containing no MgClZ at a concentration of 2.5 mg/ml. Six-tenths ml of these solutions were centrifuged at 42,040 rpm in a Spinco Model E analytical ultracentrifuge and the sedimentation velocity was monitored by photographing the schieren optics pattern at time inter- vals. Viscosity of the solutions was determined with the Ostwald viscometer in order to correct the calculated S valve to 20°C and water as a solvent. I __- J — Sample Viscosity of Sedimentation Coefficient Solution at 4°C Uncorrected S Corrected to Water and 20°C W (820 Normal Ribosomes 3.51 22.6 79.2 Shocked Ribosomes 3.47 16.1 56.0 81 For this study, ribosomes were prepared and centri- fuged in a sucrose gradient as previously described (see section on Methods). That fraction of the gradient which contained material sedimenting faster than the ribosomes was isolated, and aliquots of this solution were analyzed by electron microscopy as previously discussed (see section on Methods). As shown in Figure 15, particles were indeed observed in the polyribosome fraction. They are also of the correct dimensions (200-250 A in diameter, 129) to be ribosomes. Some particles appear to exist as ribosomes while many are seen in various states of aggregation. Some polyribosomes appear to be linearly arranged as if attached to a strand of messenger RNA. groperties of Mammarngland sRNA Determination of Homogeneityfand Sedimentation Coef- ficient-~In addition to the ribosomal fraction, sRNA is required in cell-free protein synthesizing systems. Activated amino acids become esterified to the sRNA and are thereby transferred to the ribosomal complex. In order to determine the Specific requirement Of sRNA in a mammary gland cell- free system, the RNA must be purified to remove other RNA's present in mammary tissue. Mammary gland sRNA was purified according to method of Rosenbaum and Brown (118) and assayed for purity by sucrose density gradient centrifugation. Horse radish 82 Figure 15. Electron micrograph of mammary gland polyribosome preparations. One absorbance unit (260 mu) of normally prepared ribosomes and polyribosomes were layered onto a linear sucrose gradient (10 to 34%) containing 10 mE Tris-H01 buffer (pH 7. 4), 25 mE KCl, ands mE MgCl (total— volume, 4. 8 ml). The gradient— was centrifuged at 39, 000 rpm for 1.5 hours and the polyribosome fraction was isolated and used for the electron microscopic analysis. Droplets of this polyribosome sample was placed on Formvarcoated.copper grids and washed several times (see section on Methods). The grids were then shadowed with tungsten oxide and photo- graphed in_an RCA, type EMU 2A, electron microscope. Mag- nification of the photograph is approximately 45,000. 83 peroxidase was used as a marker of known sedimentation coef- ficient. Results of this study are illustrated in Figure 16 and indicate that the sRNA preparation was homogeneous and free of other RNA's such as ribosomal RNA. Calculation of the sedimentation coefficient by the method of Martin and Ames (120) gave an 830 value of 4.1. Esterification of Amino Acids to sRNA—~If the iso- lated mammary gland sRNA is to function as an intermediate in protein synthesis in a manner Similar to that found in other cell-free systems (2), enzymatic esterification of amino acids to the sRNA must occur. This property was tested by the following eXperiment. The incubation mixture of 3.0 ml volume contained 0.15 mg of sRNA, 15 umoles of ATP, 15 umoles of MgClZ, 300 umoles of Tris-RC1 buffer, pH 7.4, a total of 2 umoles of an amino acid mixture minus glutamate whose composition was equal to that of whole casein (see Appendix Table 2), and 0.02 no of L-glutamate-U-C1° possessing a Specific activity of 9.5 no per umole. The esterification was catalyzed either by aminoacyl-sRNA synthetases precipitated at pH 5 (pH 5 enzyme) or by the same enzymes isolated by ammonium Sulfate fractionation (AS7o enzyme). Isolation procedures 0f“both enzyme fractions were described in the Methods sec- tion. This incubation mixture minus the addition of sRNA Served as a control. The system was incubated for 30 minutes at 37°C and then the sRNA was reisolated to determine 84 .mconpcz songs debauched mm posdaaopop was msoapomam meoflamb can ad hpa>apom ommoaNoaom .Amafiv 1E com um oosmpaomnm map 8099 oosaaaopoo was Soapomaa Some mo pampsoo szmm one .sOdpomad Hod macho o wsasampsoo mmoapomam mm Oped booabao swap mama 095p m&dmahpdoo on» do mpsopsoo one .00: pm 90909 mm 3m m ca ads ooo.mm pm mason ma oOwSMHAPSOo mos psoaomaw one ..ps6Hoammooo Soapmpsoafioom sacsx mo Hogans e we cmooaxosod shanty nomaonlwn m cosdmpsoo omao Soapsaom gzmn Mme .AHs mh: oasaob Hmsdgv waves as m can .Ao.a man access dominate as on .Hoa as mm meannessoo oncpodw Rom on m ablazeaomaw omoaosm amosda o opso poached was Ao.m mav seamen mpmnamosd z Ho.o mo Ha H.o ad co>aommap azmm mo Ans ommv means recompense osaz .Amaav ssoam one admnsomom do eczema on» op madcaoooo mammap ascends scum oedmdaSQ one ocpmaoma was 42mm 639 .szmm madam harness oedeHSQ do chopped psoaomnw Andaman mmoaoam .mH madman 85 mmmEDZ 20:043.... ON 0. O. m BONVBHOSBV 42mm 02415 >m<§§<§ 09...:de do 2mm._:_.._._mzwn_ memODm 0.. L O.NL 86 the radioactivity as also described in the Methods section. The results of this eXperiment are presented in Table 4. Both the AS and the pH 5 enzyme catalyzed the 70 esterification of glutamate and the isolated sRNA. Under the conditions used, the AS enzyme catalyzed the ester- 70 ification of 0.1% of the added glutamate while the pH 5 enzyme catalyzed the esterification of 0.17% at the end of the 30 minute incubation. These data indicate that the purified mammary gland sRNA can be charged with amino acids and thus may serve as an intermediate in milk protein synthesis. It has been assumed that, in addition to glutamyl-SRNA, sRNA's Specific for every other amino acid found in proteins are present in the preparation. Enzymatic Activities o£_Various Fractions Aminoacyl-SRNA Synthetase Activity--Before amino acids can be incorporated into proteins, activation by the forma- tion of the aminoacyl-AMP compound must occur. This acti- vated amino acid must then react with sRNA to form an amino- acyl-sRNA. Both reactions have been shown to be catalyzed by the aminoacyl-sRNA synthetaSes. Aminoacyl-SRNA had previously been shown to be synthesized by the mammary gland preparations. The next study was designed to demonstrate the initial reaction in aminoacyl-sRNA synthesis; namely, the activation of amino acids. 87 TABLE 4 ESterification of L-gEutamate-U-C1° to mammary gland sRNA The 3.0 ml incubation mixture contained 0.15% of sRNA, 15 umoles of ATP, 15 umoles of MgClz, 300 umoles of Tris-HCl buffer, pH 7. 4, 2 umoles of mixed amino acids minus glutamate (sefu Appendix Table 2 for composition), 0. 02 no of L-glutamate- (9. 5 uc/umole) and the enzyme fraction. The preparation of the enzyme fractions were described in the Methods section. The reaction mixture was incubated for 30 minutes at 37°C and the sRNA isolated and assayed for radioactivity (see Methods for isolation procedures). mg of mg l/ 2 % 014 glutamate Preparation enzyme sRNA net dpmr/ esterified to sRNA AS7O enzyme 15.0 0.15 567 0.104% pH 5 enzyme 11.0 0.15 901 0.166% i/Based on 24 absorbance units (260 mu for 1 mg sRNA/ml (119) E/Corrected for the control containing no added sRNA 88 Partial purification of aminoacyl-sRNA synthetases by acid precipitation was studied by measuring the extent of hydroxamate formation of amino acid which was catalyzed by the various fractions. The complete incubation mixture con- tained 10 umoles of ATP, 0.7 ml of the 0.02 E amino acid mixture whose composition was comparable to that of whole casein, 10 umoles of MgClZ, 20 umoles of Tris-H01 buffer, pH 7.4, the enzyme fraction, and 1000 umoles of neutralized hydroxylamine in a total volume of 3 ml. After incubation for 30 minutes, the amount of amino acid hydroxamates formed was measured as described by Berg (121). Results of this study are presented in Table 5. The Specific activity of the pH 5 precipitate was nearly 20 times that of the crude homogenate. Insignificant activity remained in the pH 5 supernatant indicating that enzymes which activate all the amino acids were precipitated at pH 5. Aminoacyl-SRNA synthetase activity was also measured in the pH 5 and AS enzyme fractions by determining the 0 rate of exchange 0: inorganic pyrophOSphate into ATP accord— ing to the method of Bucovaz and Davis (106). The one ml incubation mixture contained 100 umoles of Tris-RC1 buffer, pH 7.4, 5 umoles of ATP, 5 umoles of sodium pyrophosphate containing 100,000 cpm, 5 umoles of MgCl 0.5 ml of the 29 0.02 E amino acid mixture whose composition was similar to that of Whole casein, 50 umoles KF, and 1 mg of the enzyme fraction. The mixture was incubated for 30 minutes. After the reaction was stopped by the addition of trichloroacetic 89 TABLE 5 Purification of aminoacyl-sRNA synthetases based upon hydroxamate formation The fractions which were1assayed for aminoacyl-sRNA synthetase activity were isolated from mammary tissue as described in the section on Methods. The incubation mix- ture contained 10 umoles of ATP, 0.7 ml of the 0.02 E amino acid mixture (see Appendix Table 2 for composition), 10 umoles of MgClZ, 20 umoles of Tris-RC1 buffer, pH 7.4, 1000 umoles of neutralized hydroxylamine, and aliquots of the various mammary tissue fractions in a final volume of 3.0 ml. The measurement of the aminoacyl-hydroxamates formed was described in the Methods section. “— h- —‘ r Fraction Controli/ Complete Protein Specific (mg) Activit Crude Homogenate 3.702/ 4.20 77.0 .0144 15,000 x g Supernatant 2.06 3.50 34.0 .042 105,000 x g Supernatant 2.70 3.50 12.0 .033 pH 5 Supernatant 1.52 1.72 10.5 .0192 10.4 .260 pH 5 Precipitate 1.60 4.32 __fi l/Incubation mixture minus amino acids 2 -/Specific activity units: umoles/mg protein/hour 2/All values are eXpressed as umoles hydroxamates formed per hour 90 acid, Norit A was added to separate the ATP from the reaction mixture. After several washed of the Norit A, the specific radioactivity of the bound ATP was determined by procedures described in the Methods section. The activities of the pH 5 enzyme and the A370 enzyme are presented in Table 6. Although both enzymes catalyzed the exchange reaction, the A870 enzyme possessed 3 times higher specific activity than the pH 5 enzyme. Therefore, these results suggest that the AS enzyme would be the 70 preparation of choice, but results in the next study give reasons for utilizing the pH 5 enzyme in future acid incor- poration studies. Ribonuclease Activity-~Many investigators have shown that ribonuclease is a potent inhibitor of protein synthesis in cell-free systems (58-61). Accordingly, to obtain maximal amino acid incorporation by cell-free systems, components must possess minimal ribonuclease activity. The subsequent study was conducted to ascertain the ribonuclease activity of the different preparations from the mammary gland. Both the pH 5 and the AS enzymes were 70 prepared by the usual procedures (see section on Methods) and assayed for ribonuclease activity according to the method of Kalnitsky (123) by measuring the release of acid- soluble nucleotides from the RNA substrate. The ribonuclease activity of many fractionscfi‘mammary tissue is shown in Table 7. Concerning the pH 5 enZyme 91 TABLE 6 Aminoacyl-sRNA synthetase activityias measured by radioactive pyrophosphate exchange into ATP The isolation procedures for the preparation of these enzyme fractions were described in the Methods section. The incubation mixture contained 100 umoles of Tris-HCl buffer, pH 7.4, 5 umoles of ATP, 5 umoles of sodium pyrophosphate containing 100,000 cpm, 5 umoles of MgClZ, 0.5 ml of the amino acid mixture (see Appendix Table 2), 50 umoles of KF, and 1 mg of the enzyme preparation. Both enzyme preparations were dissolved in 0.05 M_phosphate buffer, pH 7.4. The reaction mixture was incubated 30 minutes and the amount of radioac- tivity exchanged into the ATP was measured (see Methods for procedure). 1 T Enzyme Controli/ Complete Protein Specific2 (mg) Activity_/ pH 5 enzyme 1.212/ 3.642/ 1.0 2.43 A870 enzyme 0.3582/ 8.2u2/ 1.0 7.89 , i/Control contained no added amino acids .2_/ 2/ Specific activity units = “moles/mg protein/hour Values are expressed as umoles pyrophosphate exchanged per hour 92 TABLE 7 Ribonuclease activities of various fractions The procedures for preparing fractions which were assayed for ribonuclease activity and the procedure for assaying ribonuclease activity are described in the Methods section. The rate is expressed as the amount of acid soluble nucleotides released from RNA per unit of time. Preparation Specific Activity (units_//mg proteins) Crude Homogenate . . . . J . . . . . . . . . . 0.033 15,000 x g Supernatant . . . . . . . .e. . . . 0.072 105,000 x g Supernatant . . . . . . . . . . . 0.076 I. pH 5 enzyme Supernatantg/ . . . . . . . 0.40 pH 5 enzyme Precipitateg/ . . . . . . . 0.070 pH 5 enzyme Supernatantz/ . . . . . . . 0.330 pH 5 enZyme Precipitatez/ . . . . . . . 0.045 II. 40% Ammonium Sulphate Precipitate . . . 0.065 70% Ammonium Sulfate Supernatant . . . 0.077 70% Ammonium Sulfate Precipitate (AS7o enzyme) . . . . . . . . . . . . . 0.460 E/Unit = amount of acid-soluble nucleotides that give an increase in absorbancy of 1 unit per minute at 260 mu (1 cm light path) E/Results of first pH 5 precipitation 2/Results of reprecipitation of the first pH 5 precipitate 93 preparation, most of the ribonuclease was in the pH 5 enzyme supernatant. A reprecipatation of pH 5 enzyme further reduced the ribonuclease activity in the pH 5 enzyme precipitate. In contrast, most of the ribonuclease remained with the AS 70 enzyme rather than being removed by fractionation. PART II INIVITRO PROTEIN SYNTHESIS C11+ Leucine Incorporation by,a Crude Homogenate The previous discussion dealt with the cellular com- ponents which comprise a cell-free system that synthesizes protein. The second part of this thesis will relate to experiments concerning amino acid incorporation by a com- plete protein synthesizing system composed of the previously characterized cellular components plus other non-cellular constituents. However, before studying amino acid incorpor- ation by the complete system composed of defined components, incorporation was first verified in crude preparations of mammary tissue; e.g. crude homogenates and single-cell sus- pensions. To study amino acid incorporation in a crude homogenate, fresh mammary tissue was chopped into small pieces and washed three times with Hanks' basic salt solution (see Appendix Table 3). The tissue was again extensively macerated with scissors and suSpended in Hanks' solution at a concentration of 0.5 8 Per ml. Three-tenths of a gram of tissue was incu- 'bated with 0.35 mg of penicillin G, and 2.0 no of L-leucine- Uncll+ (251 uc/umole) in a final volume of 2 ml. Aliquots were taken after incubating the mixture at 37°C for 0, 1, and 94 95 2 hours. The reactions were stopped by adding 5% trichloro- acetic acid. The protein precipitates were washed twice with 5% trichloroacetic acid, dissolved with 1 _N_ sodium hydroxide, reprecipitated with 5% trichloroacetic acid, dehydrated with acetone and diethyl ether and assayed for radioactivity by the method of Casjens and Morris (65). This particular system incorporated 16.9, 144.12, and 162.6 unmoles of the added leucine per gram of tissue for 0, 1, and 2 hours reSpectively (Table 8). Because the effective Specific activity of the LLleucine-U-014 was sig- nificantly reduced by 1) the intracellular pool of free leucine and 2) the quantity of non-synthetic connective tissue present, the observed amount of incorporation repre- sents only a part of the total amount of protein synthesis actually occurring. But these results do show that mammary gland tissue which has been isolated from the animal, homo- genized, and incubated at 37°C can synthesize protein. ‘Clu Leucine Incorporation by Single-Cell SuSpensions Ebner 22, 2;! (115) reported that a single cell sus- pension of mammary tissue was capable of synthesizing pro- tein. Therefore, it was decided to measure the amount of C14 leucine incorporated into trichloroacetic acid-precipi- tated material and, in addition, to compare its level of incorporation to that found above with the crude homogenate. One gram of collagenase—isolated cells (see section on Methods) were suSpended in 4.0 m1 of Hanks' solution 96 TABLE 8 C1“ leucine incorporation by_a crude homogenate of mammary tissue Each incubation mixture contained 0.3 g of homogenized tiasue, 0.35 ms of penicillin G, and 2.0 no of L-leucine-U- C (251 uc/umole) in a final volume of 2 ml. The mixture was incubated for the various times at 37°C with intermittent shaking. Amino acid incorporation was assayed according to the method of Casjens and Morris (65) as described in the Methods section. The cpm incorporated were corrected fior quenching. The efficiency of counting the external C1 benzoic acid standard was 82%, Incubation Time cpm pumoles incorporated (hours) Incorporated per g tissue 0 2,863 16.9 1 19,532 144.2 2 22,057 162.6f 97 (see Appendix Table 3) which also contained 40 uc of L-leucine- U-C14 (0.9 uc/umole) and 0.4 ml of the 0.2 M_amino acid mix— ture minus leucine (see Appendix Table 2). One ml aliquots were taken after 0, 1, 10 and 60 minutes of incubation at 37°C. The reaction was stopped by the addition of trichloroacetic acid (5% w/v) and the radioactivity associated with the pro- tein was determined as described in the previous eXperiment. The results of this study are presented in Table 9. The level of incorporation at 60 minutes was about seven times the 0 time control. The 60 minute level of incorpora- tion of leucine (corrected for 0 time values) in the single cell suspension was 40 times greater than the amount of leucine incorporation by the crude homogenate. The amount of synthesis per gram of tissue for the crude homogenate was reduced an unknown amount by the presence of nonsynthetic connective tissue. Yet, both crude systems did show signifi- cant leucine incorporation and suggested that partial purifi- cation and recombination of the correct cellular components would perform similarly. Amino Acid Incorporation by the Cell-Free System The study of milk protein synthesis was then extended to a cell-free system utilizing the components which were defined in the first sections of the thesis. A discussion CI4 of many characteristics of leucine incorporation by the cell-free system such as time, microsome, energy, and 98 TABLE 9 C14 leucine incorporation by isolated mammary gland cells The incubation mixture contained 1 g of collagenase- isolated cells (see Methods for preparation procedures), 4 ml of Hanks' basic salt solution a described in Appendix Table 3, and 40 uc of L-leucine-U-C1 (0.98 uc/umole). One ml aliquots were removed at time intervals from the incuba- tion mixture and assayed for amino acid incorporation. Efficiency of counting an external standard was 72%. 1 -— Time uumoles leucine (min.) cpm/g cells incorporated . . g cells 0 1565 ' 5 1097 1 1955 1372 10 3250 2280 60 9940 6970 99 magnesium dependence will be presented. This discussion will be completed by the partial identification of the proteins that were synthesized by this cell-free system. Amino acid incorporation was studied in the complete system composed of the following components dissolved in 1 ml volume: Microsomes (0.3 to 0.6 mg), sRNA (50 pg), pH 5 enzyme (1 to 2 mg), ATP (1 umole), GTP (0.25 umole), phos- phoenol pyruvate (5 umoles), pyruvate kinase (40 us). KCl .(50 umoles), MgCl2 (5 umoles), reduced glutathione (20 umoles), amino acid mixture minus leucine whose composition was compar- able to that of whole casein (1.0 umole total), Tris-H01 buffer, pH 7.4, (50 umoles), and L-leucine-U-Clu (1 to 1.5 no, specific activity 200 to 250 uc/umole). This complete system was incubated at 37°C for 40 minutes. The reaction was stopped by the addition of 6 mg of carrier protein (bovine serum albumin) and 10 ml of trichloroacetic acid (5% w/v). The precipitated protein was washed twice more with trichloroacetic acid, dissolved in 1 M sodium hydroxide, reprecipitated and washed once again with trichloroacetic acid, dehydrated with acetone-HCl and ether, and assayed for radioactivity according to the method of Casjens and Morris (65). Unless specified, this will be the composition of the complete system which will be utilized in later experiments. Once such variation was the inclusion of the A870 enzyme instead of the pH 5 enzyme in certain eXperiments. 100 014 Leucine Incorporation vs Tinm of Incubation--The time dependence of C1” leucine incorporation was determined by stopping the reactions at 0, 5, 10, 20, 40, and 60 minutes by the addition of trichloroacetic acid (5% w/v) and measuring the radioactivity that was associated with the washed precipitate (Figure 17). Incorporation into tri- chloroacetic acid precipitable material was linear for the first twenty minutes and was nearly completed after 40 minutes. However, when the complete system was incubated overnight, incorporation reached a very high level probably due to bacterial growth. Microbial growth appeared to con- tribute insignificantly to the observed incorporation in the short term eXperiments since the addition of 35 ug of peni- cillin G did not reduce the incorporation of leucine. The amount of radioactivity at 40 minutes was usually one hundred to five hundred times that at 0 time. When the A870 enzyme was utilized instead of the pH 5 enzyme, incorporation increased linearly with time for 10 minutes and reached a maximum at 40 minutes (Figure 18). The reason for the cessation of incorporation is not known since the addition of more components after incorpora- tion had ceased was not performed. However, the failure may have resulted from the lack of integrity of the ribosomes and polyribosomes. In order to test this hypothesis, the following eXperiment was conducted. The complete system was incubated for 0, 10, and 40 minutes at 37°C. After incuba- tion aliquots of the mixtures were subjected to the usual 101 .Amoonpmz so soapomm oomv moonpoa deans can an dosaaaopmo mm: soapmaonaoozd oaom 02Ha¢ 0:» mo mpcdoam adam: on» one AmHo81\01 o.Hv ofihuso n ma .Awa :o.ov moaomoaoda confidence 0mm .oahnsm m mm wsam: mad» m» soapmaoahooza oCaoSoH .mpsosoaaoo amnpo onoaeaaeseanq .Aws N.mv pads ammmm camoqmpm 039 :HO .ma oaswam 102 00 On 3235.5 ME; 0*» 0m 0m 0_ _ i _ m2: m> zofiqmoamooz. mzaamj so _ _ _ OOmK 000.0 _ 000.NN 00 0.0m amona'l M0 ("400) caivaoemoom 103 Figure 18. C14 leucine incorporation vs time using fraction. The standard assay mixture contained miffio- $0283 (1. 4 mg),A enzyme (1.7 mg), and L-leucine-U-C /unge), and the usual amounts of the other (1. 5 no, 210 no Amino acid incorporation was determined by the components. usual procedures (see section on Methods). INCORPORATED (Cpm) C" LEUCINE IODOO 7,500 '5000 2,500 104 I I I c" LEUCINE INCORPORATION vs nwm 42 IO 20 30 40 TIME (minutes) 105 sucrose density gradient centrifugation for analysis of the ribosomal components (Figure 19). The 0, 10, and 40 minute sample had incorporated 158, 6,770, and 19,183 cpm, reSpec- tively, when assayed by the usual method. The results demon- strate a time-dependent degradation of the ribosomes asso- ciated with each peak of the ribosomal pattern. Although it is impossible to decide whether this degradation was a direct result of protein synthesis, as described by Noll 22.‘§l. (138), or the result of ribonuclease activity, the ribosomes and polyribosomes were unstable under these condi- tions. This observed instability suggests that incorporation ceases after 40 minutes of incubation because the amount of active polyribosomes and ribosomes present in the assay mix- ture were insufficient to sustain incorporation. These results agree with those of Korner (60), who postulated that amino acid incorporation reached a maximum level after one hour because liver microsomes were unstable to incubation of the complete system at 37°C., Allenind Schweet (58), Sing 22, 2;. (139), and Talal (61) also reported that amino acid incorporation reached a maximum after 1 hour of incubation in the reticulocyte, thyroid, and spleen cell-free protein synthesizing systems, respectively. However, no Specific causes for this phenomenon was observed or proposed. Incorporation of Cl” Leucine into Mammary Gland MicrosOmes--Studies in mammalian reticulocyte, liver, and 106 Figure 19. Effect of amino acid incorporation on the sedimentation behavior of microsomes. The standard assay mix- ture containe microsomes (0.4 mg), pH 5 enzyme (1.2 mg), L-leucine-U-C 4 (1.25 no, 210 uc/umole), and the usual amounts Of the other constituents. The mixtures were incubated for 0, 10, and 40 minutes and then equal aliquots of each sample (0.1 ml) were layered onto a linear sucrose gradient of 10 to 34% sucrose containing 25 mM KCl, 5 mM MgCl , and 10 mM.Tris- HCl buffer (pH 7.5). The gradients were cegtrifuged for 1.5 hours at 39,000 rpm in a SW 39 rotor at 4°C. The contents of the centrifuge tubes were then analyzed for ribosomal and polyribosomal content (see section on Methods). 107 EFFECT OF AMINO ACID INCORPORATION ON' RIBOSOMAL PATTERNS -O.5O ABSORBANCE J I 2 3 4 EFFLUENT VOLUME (m0 108 bacterial cell-free protein synthesizing systems indicated that protein synthesis was performed by aggregates of ribO- somes complexed with messenger RNA (129, 140, 141). Simi- larly the function of polyribosomes in the incorporation of C1“ leucine was investigated by means of the next two eXperi- ments. The complete system was incubated for O and 5 minutes. Aliquots of the mixtures were then subjected to the usual Sucrose density gradient centrifugation; equal volume frac- tions were collected and the radioactivity in each was deter- mined. The 0 time ribosomal pattern and the amount of radio- activity in each fraction of the gradients which contained an aliquot of the 0 and 5 minute incubation mixture are pre- sented in Figure 20. NO 0]"+ leucine was bound to the ribo- somes or polyribosomes in the non-incubated system. After 14 incubation for 5 minutes, C leucine became attached to the polyribosomes. In this test insignificant radioactivity was observed in the fractions containing the monomeric ribosomes. The 0 time and 5 minute samples had incorporated 87 and 7,361 cpm, reSpectively, when assayed by the usual methods. These data suggest that the mammary gland polyribo- somes rather than ribosomes are most active in protein syn- thesiS. Also, these data emphasize the importance ofABbtain- ing microsome preparations for amino acid incorporation. studies which contain the highest possible proportion of polyribosomes. The next experiment was also designed to study the 109 4 Figure 20. Labeling of ribosomes and polyribosomes with Cl leucine. The standard assay mixture contained microsomes (0.4 mg), pH 5 enzyme (0. 8 mg), and L- leucine-U-c1 1“ (0. 7 no, 225 uc/umole), and the usual amounts of the other constituents. Three-tenths ml of the samples incubated for 0 and 5 minutes were layered onto a linear sucrose gradient of 10 to 34% sucrose containing 10 mM Tris-HCl buffer (pH 7. 5), 25 mM KCl, and 5 mM MgCl (final volume 12. 0 ml). The gradientsO were centrifuged at 40, 000 rpm for 1 hour and 45 minutes at 4° C in a Model SB-283 rotor with a B- 60 International Preparative Ultracentrifuge. Fractions were collected (16 drops) and the absorbance (260 mm) and radioactivity were determined in each fraction. 110 LABELLING OF RIBOSOMES WITH 0'4 LEUCINE — II 700— H II 5 MIN EI '|‘_———— LABELLING FéL -06 I 600- l A "‘ E O. - 3 500— C)MIN ABSORBANCE 0 MIN LABELLJNG 300- . I I00— / "LA.I "n“ I tL.u.Lu.+.udu.uiu’ 1 L 3 7 II l5 I9 23 FRACTION NUMBER 111 function of polyribosomes in amino acid incorporation. Microsomes were layered onto a usual sucrose gradient which was subsequently centrifuged and divided into ten equal frac- tions. An aliquot of each fraction was utilized as the source of microsomes in the complete protein synthesizing system. Incorporation was assayed by the method previously described. The results of this eXperiment are presented in Table 10. The polyribosomes were 2-3 times more efficient in C11+ leucine incorporation than the ribosomes whose peak was located between fraction 6 and 7. Henshaw 23, 3;. (141) obtained similar results with a rat liver cell-free system. The rat liver polyribosomes were 2.5 times more efficient in synthesizing protein than ribosomes. When poly U was added to their ribosome prepara- tion, phenylalanine incorporation by the ribosomes equalled that of the polyribosomes; this suggests that messenger RNA limited synthesis by the ribosomes. The behavior of mammary gland ribosomes with respect to synthetic messenger RNA is unknown. The Effect of the Amount of Microsomes on Cl)+ Leucine IncorporatiOn--Many investigators (1, 2, 3) have determined the dependence of amino acid incorporation upon the amount of ribosomal fraction added to the cell-free system. For example, Allen and Schweet (58) observed a linear increase in incorporation as the reticulocyte ribosome and polyribo- some concentration was increased to 8 mg per ml. The purpose 112 TABLE 10 Amino acid incorporation by gradient-fractionated microSOmes One and nine—tenths mg of normally prepared microsomes (0.3 ml) were subjected to sucrose gradient centrifugation in the Model B-60 International Ultracentrifuge as described in the Methods section. Fractions containing 1.33 ml were col- lected by uncturing the bottom of the centrifuge tube. (Fraction 1 is nearest the bottom). Three-tenths ml of each fraction was utilized in the standard assay mixturelgontain- ing 1.2 mg Of pH 5 enzyme and 1 uc of L-leucine-U-C (251 uc/umole) for measuring amino acid incorporation. This mix- ture was incubated 40 minutes and the amount of amino acid incorporation was measured by the usual procedures. These assays of amino acid incorporation were conducted in dupli- cate. Seventy-five percent of the microsomes layered onto the gradient were recovered in the fractions collected. cpm Incorporatedi/ mg Ribosome (x 102)§/ cpm/mg Fraction 1 32 2.32 1378 2 60 3.56 1087 3 80 3.40 2350 4 84 3.37 2490 5 94 4.34 2168 6 72 6.57 1095 7 58 6.10 952 8 11 2.20 500 9 8 1.46 546 10 0 -- 0 Control 20 —— _- i/Average of two determinations and corrected for the control which contained no ribosomes. 3/ Based on 11.3 absorbancy units (260 mg) for 1.0 mg ribo- somes per ml (116). 113 of the following eXperiment was to determine the dependency of C11+ leucine incorporation on the microsome concentration in the mammary gland cell-free system. The microsome con- centration was varied from 0 to 1.2 mg per ml while the concentration of the other components in the system was held constant. The mixtures were incubated for 40 minutes and the amount of incorporation determined by the usual method. In the standard assay system, C1“ leucine was incor- porated at a level which was linearly dependent on the micro- some concentration up to about 0.5 mg per ml. NO incorpora- tion occurred when the microsomes were omitted from the sys- tem. As Figure 21 indicates, incorporation was not signifi- cantly stimulated by further addition of microsomes suggesting that incorporation was limited by an insufficient amount of some other component in the system. This preparation of microsomes contained bound ribo- nuclease (0.23 units/mg microsomes) whose activity may have been partly responsible for the non-linearity of incorpora- tion with respect to microsome concentration. Elson (142) studied this phenomenon in relation to E, ggli. protein bio- synthesis and suggested that ribosomal-bound ribonuclease may play a role in controlling the synthesis of proteins in the cell-free system. Dependence of Incorporation on the Source of Aminoacyl- sRNASynthetases--Aminoacyl-SRNA synthetases catalyze the activation of amino acids and the subsequent esterification 114 .Amoonpmz so noapomw momv mmasomooaa Hammz on» an dopammma mm: soapmaoaaoosd daom osaam .mpsmsomaoo aospo one mo mp2§oam deans on» was .mmaomoaoaa no mHO>OH madmam> .Aoaoan\on OHN .01 mN.HV DIDImsaosmHIq .Awa N.Hv Oaxaca m ma dosampaoo campxaa zmmmm damosmpm one .Ho>ma maomOHOHs m> godpmaoaaoosa msaodma :Ho .HN maswdm 115 seen: muzomomoi 0.. mo mo ed No o 0006 000.0. oood. _ _ O . _ a A oooo~ ._m>m._ mzomomoi m> 20_._.m.._ 42mm m> 20_._.mI_ in_mwzmud—z 20_._. m... e.6 _ 000.0 _ 000.0N 3NI003'I ,.0 (Hide) caivaOdaOONI 132 of incubation, the amount of C14 leucine incorporation was assayed as usual. Figure 26 demonstrates that leucine was not incorpor- ated in the complete system without added magnesium chloride. Incorporation increased linearly as the magnesium chloride concentration was raised to 4 mmolar. No significant increase in leucine incorporation occurred when the magnesium ion con- centration was greater than 4 mmolar. The previous sucrose density gradient centrifugation data obtained with the same 0.5 M_KCl-treated ribosomes indi- cated that the predominant particle was a subunit of the normal ribosome or a "swelled" ribosome as previously described. The addition of magnesium chloride resulted in recombination of the subunits or in an alteration of the ribosome conforma- tion to form normal-Sized ribosomes and polyribosomes. Corre- lation of these results with the above incorporation studies suggest that the "shocked" microsomes are incapable of synthe- sis in a complete system. As magnesium ion concentration in the complete system was increased, normal ribosomes and poly- ribosomes were reformed and incorporation occurred. When the magnesium ion concentration in the complete system was 4 mmolar, the KCl-treated microsomes incorporated leucine 74% as well as the normal microsomes from which the treated microsomes were cierived; that is, 29, 850 Cpm incorporated per mg of treated Inicrosomes compared to 40,600 cpm per mg of normal microsomes. 14 In summary, the maximum C leucine incorporation c>ccurred at 4 to 5 mmolar magnesium chloride concentration. 133 Figure 26. C1“ leucine incorporation vs magnesium level using "shocked" microsomes. The standard assay mixture contained "shocked" microsomes (0.5 mg) as described by 14 Miller pp. al. (145), pH 5 enzyme (2.2 mg), L-leucine-U-C (1.0 no, ZSEch/umole), varying levels of MgCl , and the usual amounts of the other constituents. Amino acid incor- poration for the 40 minute incubation was determined by the usual procedures (see section on Methods). INCORPORATE D (0pm) C'4 LEUCINE I I I c'4 LEUCINE INCORPORATION vs MAGNESIUM LEVEL 20,000 l- I0,000 O 2 4 6 134 MgC I 2 CONCENTRATION (pmoles/ml) 8 135 Magnesium chloride functions to maintain the integrity of the ribosomes and polyribosomes in the incubation mixture and this is necessary in order to realize amino acid incorpora- tiOno Dependence of Incorporation on Amino Acids-~Allen and Schweet (58) reported that the reticulocyte cell-free system incorporated 50% less Cl)+ leucine into protein when the amino acid mixture was omitted. In contrast, Turba and Hilpert (109) and Singh‘gp..§;. (139) demonstrated that the mammary gland and thyroid gland systems incorporated less labeled amino acid into protein when an amino acid mixture was included in the complete system. No explanations for this discrepancy were reported. It is possible, however, that these results are a function of the level of free amino acids contaminating the cellular fractions involved. The effect of the amount Of the amino acid mixture present in the complete amino acid incorporating system on amino acid incorporation was investigated in the present study. A number of concentrations of amino acids minus leucine were added to complete systems. The samples were incubated for 40 minutes and the level of incorporation was determined. As the concentration of the amino acid mixture was increased, the quantity of C14 leucine which became incorpor- .ated in protein decreased (Figure 27). 'Because this observa- 'tion suggested dilution of the Specific activity of the jlabeled leucine with unlabeled leucine, an amino acid analysis zofiqmoamooz 2.03.. :0 . p _ 000.0. 000.0N 000.0 m 000.0.» 3NI0n3'I ,.0 (We) GBiVBOdHOONI 138 amino acid mixture which supposedly was leucine-free con- tained trace amounts of leucine. Nevertheless, to assure that an individual non-labeled amino acid would not limit incorporation of a Gin-containing amino acid, all assays were conducted with 0.05 ml of the 0.02 M amino acid mix- ture minus the labeled amino acid under study. The question of dilution of the Specific activity of the labeled leucine was investigated by adding non-radioac- tive leucine to the standard assay system. The addition of 1 umole of unlabeled leucine to the complete system decreased incorporation by 96%. The addition of 1 umole of leucine 14 lowered the specific activity of the C leucine by 250 fold. Higher levels of leucine permitted only trace amounts in CI“ leucine incorporation. Thus, it is likely that diminished C11+ leucine incorporation with increasing amino acid mixture results from the trace amounts of leucine present in the other amino acid preparations. Comparison of the Incorporation of Different Labeled Amino Acids-~The purpose of the following eXperiment was to determine the rate of incorporation of different amino acids. Separate complete systems which lacked the amino acid under study were incubated in the presence Of either L-glutamate U_C14 _Ci4 14 , L-lysine U-C , L-leucine U-Clu, or L-phenylalanine U for 40 minutes. The level Of incorporation of each labeled amino acid was determined as before. Table 12 illustrates that a higher percentage of the Homo.o nos.a 0.: om.a naououocaamaoaaaormua mom.o mmo.ma mm.a oo.oam :HOIDIoaaosoQIA namo.o ANA o.m ma.a aflospsoeanaqsa smao.o mum o.H om.ma naouoIoamampaaouq O, oopoaoaaoosH oopoaomaoosH, ooooo odoo Aoaoa1\01v MU odom osaam R ago no: osaam mo 01 apabap04 0ama0oam 0a0¢ caaad .mmm mos maapQSOo Co mosoaoaguo one .Hoaaopoa oHQopHmaoon odoo 0apo0oHOH£0aHp one oped oopoaoa900CH mos pozp odoo osaao coaonoa ooooo 0:0 00 psooaoa ozp mm 020 as Hog oopoaoaaoosa op:500 mo oommoamxo oao mpHdmoa ose .mooSPoz no soap Ioom one 2H donaaomoo mos soapoaoaaoosa mo mommo 0cm Soa00950za mo mmoapaosoo one .aopmmm moap0950sd opoaaaoo Sooo op condo mos R0300 moons oaoo oafiao o£p manna oHSpMaa oaoo osaao one .Awa N.Hv oahuao m me one Awe .o. moaomoa0aa meaosaosa mpsodpapmsoo amono whoosopm ozp oosdopsoo aopmam opoH 800 one 0doo oaaao UoHoQoH mo omhu mP Codpoaomnoosa daoo osaa4 NH mqmde 140 added C14 leucine was incorporated than of the other three amino acids. Specifically, nine-tenths percent of the Cl“ leucine was incorporated while only 0.026% of the C14 phenylalanine and 0.02% of the glutamate and lysine were incorporated into protein. Based on the amino acid compo- sition of whole casein, the amount of incorporation of the different amino acids was expected to be as follows (in order of decreasing amounts): glutamate, leucine, lysine, and phenylalanine. This deviation from the expected may be due to 1) the limiting amount of aminoacyl-sRNA synthetase activity for a particular amino acid, 2) a limiting quantity of an sRNA Specific for a particular amino acid, 3) differ- ent rates of utilization of the amino acids for processes other than protein synthesis, and 4) synthesis of higher- than-normal amounts of non-casein proteins.g Since the high- est possible amount of incorporation was desired, C14 leucine was the labeled amino acid utilized in most eXperi- ments reported in this thesis. Inhibition of C14 Leucine Incorporation--Understand- ing the mechanism of protein synthesis has been increased by studying inhibition of synthesis by certain substances. In order to compare the mechanism of milk protein synthesis in a cell-free system with that observed in systems derived from other organisms, the effect of many known inhibitors of protein synthesis was examined. Puromycin inhibits protein synthesis in many mammalian 141 vsystems such as those derived from reticulocytes (58), spleen (61), liver (60), and the thyroid gland (139). The effect of puromycin on the mammary gland system was deter- mined by adding 1 umole of puromycin to complete amino acid incorporating systems containing either the pH 5 enzyme or the A570 enzyme as a source of aminoacyl-sRNA synthetases, 014 leucine incorporation was determined as usual. The results of this eXperiment are presented in Table 13. Incorporation by the systems containing either enzyme prep- aration was inhibited 95% by the addition of 1 umole of puromycin per ml. Based on the accepted mechanism of puromycin inhibi- tion, these data suggest that milk proteins were indeed synthesized through an aminoacyl-sRNA intermediate. Chloramphenicol inhibits protein synthesis by bac- terial cell-free systems but does not affect protein syn- thesis in similar systems derived from mammalian tissues (2). The effect of this antibiotic on inhibition of leucine incorporation was tested at a concentration of 50 mg per ml. The level of incorporation was determined as usual after the 40 minute incubation of the standard assay mixture. Chlor- amphenicol addition exerted no effect on incorporation by the system (Table 14). The standard complete system incor- porated 30,838 Cpm of C1“ leucine while 31,506 Cpm were incorporated by the mixture containing chloramphenicol. In another test, levels of chloramphenicol ranging from 0 to 200 pg per ml of incubation mixture did not alter the level of incorporation. 142 TABLE 13 Inhibition of Cl” leucine incorporation by puromycin Standard assay systems containing microsomes (0.4 mg), p¥u5 enzyme (1.2 mg) or AS 0 enzyme (3.6 mg), L-leucine-U- C (1.25 no, 210 uc/umoleg, and the usual amounts of the other constituents were tested for incorporation by the usual methods. Puromycin was added at a concentration of 10""3 M|to the test systems. The results are eXpressed as Cpm incorporated per ml of reaction mixture. System cpm Incorporated pH 5 Enzyme Complete system 19,183 + Puromycin 1,956 0 Time 158 AS Enzyme 70 Complete system 2,806 + Puromycin 389 0 Time 137 143 TABLE 14 Effect of L-chloramphenicol addition on C14 leucine incorporation The complete system contained the usual constituents including micrasomes (1.5 mg), pH 5 enzyme (2.2 mg), and -leucine-U-C (1.5 0c, 210 uc/umole). Chloramphenicol was added at a concentration of 50 pg per ml of incubation mixture and the incorporation was measured by the usual methods. Results are eXpressed as cpm incorporated per ml of incubation mixture. System ' cpm Incorporated Complete System 30,838 + Chloramphenicol 31,506 0 Time 220 144 Several workers (31, 33, 34) have suggested that chloramphenicol inhibits protein synthesis $p_y;ppp either by inhibiting the attachment of messenger RNA (mRNA) to ribosomes or by directly inactivating the mRNA. These mechanisms of action suggest two eXplanations for the absence of chloramphenicol inhibition of amino acid incor- poration in the mammary gland cell-free system. First, the amino acid incorporation was not dependent on the ;p_y;ppp attachment of an RNA to the ribosomes after the completion of a protein molecule. Secondly, the mammary gland mRNA was not deactivated by the chloramphenicol. Without further study, either eXplanation appears possible. According to previous studies (2), low levels of ribonuclease inhibit protein synthesis in cell-free systems derived from many organisms. Protein synthesis was inhib- ited to the extent of 90% by as little as 0.1 ug of pancre- atic ribonuclease in the reticulocyte cell-free system (68). Experiments with M, pp}; and rat liver ribosomes indicated that the nuclease destroyed the intact messenger RNA causing a degradation of polyribosomes to monomeric ribosomes (2, 63). Analogous studies of ribonuclease inhibition were performed with the mammary gland system. Pancreatic ribonuclease was added at a concentration of 1 ug per ml to a complete system containing either the A870 enzyme or the pH 5 enzyme. In a second eXperiment the level of ribonuclease was varied from 0 to 10 pg per ml of the reaction mixture. Inhibition of incorporation was 145 determined by incubating the system for 40 minutes and mea- suring the radioactivity associated with the washed tri- chloroacetic acid precipitate. When either the pH 5 enzyme or the AS enzyme was 70 utilized as a source of aminoacyl-sRNA synthetase activity, no 014 leucine was incorporated into protein when only 1 ug of ribonuclease was added to the complete system (Table 15). In the eXperiment where the concentration of ribonuclease was varied, as little as 0.1 ug of the inhibitor resulted in 90% inhibition of incorporation whereas the higher levels completely abolished incorporation (Figure 28). Since two types of RNA, messenger RNA and transfer RNA, are presumed to be required, this level of inhibition was to be eXpected, Deoxyribonuclease should inhibit protein synthesis if DNA is a required component of the cell-free system. How- ever, protein synthesis in most of the reported cell-free systems involves only translation of preformed RNA and not the transcription of DNA to form RNA. Therefore, the effect of deoxyribonuclease addition to the standard assay system was investigated. Fifty ugrams of deoxyribonuclease was added to the standard assay mixture containing either the pH 5 enzyme or the AS enzyme. This mixture was incubated for 40 minutes 70 and assayed for C1“ leucine incorporation by the usual methods, The addition of deoxyribonuclease did not alter the rate of synthesis by the complete system (Table 16). The result of this study demonstrates that incorporation in 146 TABLE 15 Inhibition of C14 leucine incopporation by ribonuclease The complete system contained the usual constituents including microsomes (0.4 mg), either pH 5 enzyme (1.2 mg) or AS enzyme (3.6 mg), and L--leucine-U--Cl’+ (1.25 no, 210 uc/ umgge). Crystalline pancreatic ribonuclease was added at concentrations of 1 pg per ml. Results are eXpressed as cpm incorporated per ml of the reaction mixture. System cpm Incorporated pH 5 Enzyme Complete System + Ribonuclease 0 Time A370 Enzyme Complete System + Ribonuclease 0 Time 19.183 316 158 2,806 222 137 147 .AmooSpoz no soapoom oom. monsooooaa Homo: one an nonaaaopoo mos Soapoaomaoozfi oaoo Osasm .mopssda o: oopoQS0sa oaoa £0H£z maopmmm oooaaaoo osp op mHoPoH madmaoP no condo oaos Ha\w1 OOH measampsoo soHpSHom omooaossonaa capooaosoa 0 mo mposvaad .mpaoSpHpmsoo Hospo one 00 mpsdoao H0505 one new .Aoaoa1\01 Hmm .01 o.H. HUIDIosfiosoHIQ .Awa ©.NV oaawao m ma .Awa w.ov moaomoaoaa 0o:aM0:o0 oaprHa mommo oaooaopm one .HoPoH omooaossonaa mp soapoaoaaoosa ocfiosoa 1H0 .mm oazwam 148 2.503 mmqoaonzoma ._ L0. 0.0 0.0 «.0 N0 0 Cull. ‘ JOI . + J 000.N I 000.0 .00Qo Jw>w4 mm 20....4m0dm002. wz.03mI. 20 _ _ _ 000.0 3NIOOB‘I ,.0 (MO) CBiVUOdHOONI 149 TABLE 16 Effect of deoxyribonuclease addition to the complete system The complete system containing the pH 5 enzyme (4.4 mg) was composed of the standard assay confitituents includ- ing microsomes (1.4 mg) and L-leucine-U-Cl (1.5 uc, 210 uc/umole). The complete system containing the AS 0 fflzyme (3.6 mg) included microsomes (0.4 mg), L-leucine- -C (1.25 no, 210 uc/umole), and the usual amounts of the other constituents. Crystalline deoxyribonuclease, dissolved in 0.05 M phOSphate buffer (pH 7.0), was added to the test systems at a concentration of 50 pg per ml of incubation mixture. Incorporation was assayed as described in the Methods section and the results are eXpressed as cpm incor- porated per ml of the complete systems. , __ r r System cpm Incorporated pH 5 Enzyme Complete System 30,838 + Deoxyribonuclease 29,726 0 Time 158 A870 Enzyme Complete System 2,806 + Deoxyribonuclease 2,825 0 Time 137 150 this system is a result of translation of preformed RNA rather than both transcription of DNA to form RNA and then translation of the RNA to synthesize protein. Sodium fluoride and cycloheximide inhibited protein synthesis in the reticulocyte cell-free system (54). Studies by Lin 23, pl. (54) illustrated that cycloheximide slows, but does not completely block, the extension of existing nascent peptide chains while sodium fluoride exerted no effect on this process. Both compounds inhibited the ip ypppg initiation of polypeptide chains on the ribo- somes. The effect of these inhibitors on C11+ leucine incor- poration was investigated in the mammary gland cell-free system. Complete systems were incubated for 40 minutes in the presence of varying levels of sodium fluoride (0 to 40 mmolar) and cycloheximide (0 to 0.8 mmolar). Amino acid incorpora- tion was determined as usual. 14 The data in Figure 27 indicates that C leucine incor- poration was partially inhibited by either compound. Increas- ing the concentration of cycloheximide beyond 0.4 mmolar did not significantly increase the degree of inhibition. Accord- ing to the accepted mechanism of action of these inhibitors, this residual level of incorporation reflects only that incorporation obtained by the continuation of existing poly- peptide chains, amounting to about 10% of the total incor- poration as in the uninhibited system. Similar reSults were obtained when sodium fluoride was used as an inhibitor except 151 .AmooSPoz so aoapoom oom. Homo: mo 0ocaaaopo0 mos myopapdssH neon mo mHoPoH wzazHoP osp Co oosomoaa on» ma Soaponixzefimo mopssda 0: Romeo soapoaoaaoosa oaoo osas< .AoHoa1\01 mmm .01 om.ov OIDIosdosoHIA 0:0 .Ama w.ov oSANSo m mm 020 .Ama 2.0. moaomoaoda msawwaosa meoSpapmsoo aopmmm opoamaoo Homo: ozp 0ozdopsoo oHpraa amono osmosopm one .ooaaosae azaoom 02o oofiaaxos Ioaomo an soapoaoaaoosd oaaosoa :Ho 00 soHpHDHSQH .mm oaswam causes... 323310100 00 0.0 v0 N0 ceases... 00:03... 2208 on ON 0. _ 1 l _ _ _ u _ A. _ 292282.002 . . . m2.03m.._ e_0 m0 20Em_rz_ . l . 1 0 O O 0 IO 00 0.0. 0006 . 000.0N 0006M NO ENIODEW OBlVHOdHOONI (de) 153 that one hundred times higher concentrations were required to produce inhibition equal to that of cycloheximide. This reduction in incorporation caused by these two compounds with their known modes of action indicates that incorpora- tion consists of both elongation and initiation of new chains in the mammary gland system. Inhibition by both compounds was of the same magnitude as found by Lin 2;, 3;. (54) in the reticulocyte cell-free system. In conclusion, these inhibition studies suggest that incorporation by this system is a result of 1) the continua- tion of existing polypeptide chains, and 2) the initiation and elongation of new polypeptide chains. Investigators have utilized the technique of adding low levels of sodium deoxycholate (DOC) to solutions of microsomes in order to solubulize the endoplasmic reticulum. However, Korner (60) utilizing the rat-liver cell-free Sys- tem, noted a 79% decrease in incorporation when the system contained 0.1% DOC. A level of 0.5% caused 97% inhibition. Complete removal of DOC from the ribosome and polyribosome preparations is difficult, so the level of inhibition by this detergent was examined by the following experiment. The complete system was adjusted to 0.1% with respect to DOC concentration and the incorporation was then measured as usual. When one mg of DOC was added to the complete sys- tem (0.1%), 6,614 cpm of C14 leucine were incorporated into protein. However, if the detergent was omitted, 19,183 Cpm were incorporated, thus the incorporation in the DOC-treated 154 system was reduced by 66%. Because of this inhibition, microsomes were not pretreated with DOC in any of the amino acid incorporation eXperiments. 14 Effect of Polynucleotides on C Leucine Incorpora- plppg-There has been a number of studies concerned with the interaction between ribosomes and polyribonucleotides aimed at elucidation of the role of this complex in protein syn- thesis. Polyadenylic acid (poly A) and polyuridylic acid (poly U) have been shown to stimulate lysine and phenyla- lanine incorporation, reSpectively (59). For example, HenshaW'pp,‘§;. (141) observed a 3-fold increase in phenyla- lanine incorporation in the rat liver system by the addition of 500 ug of poly U per ml while Florini and Breuer (143) found a 5-fold increase by adding 200 ug of poly U per ml to their rat muscle system. In order to investigate the possibility that various artificial messengers would stimu- late amino acid incorporation by the mammary gland cell-free system, denatured DNA, poly A, and poly U additions were examined. DNA was isolated from mammary tissue by following the procedures of Chargaff (124) as described in the section on Methods. This DNA was then denatured to single strands by maintaining the temperature of the solution at 90°C until the 260 mu absorbance reached a maximum. At this time the solution was cooled rapidly to prevent reassociation of the DNA strands. One and one-half mg of this DNA preparation 155 was added to a complete system and the incorporation of C14 leucine was measured. Addition of the DNA preparation to the standard assay mixture resulted in a 36% increase in leucine incorporation when compared to the system containing no DNA (Table 17). This stimulation of incorporation was minimal when compared to the 5-fold increase in phenylalanine incorporation caused by the addition of poly U to a muscle cell-free system (143). The results obtained from the mammary gland cell-free system indicate that mammary gland microsomes were unable to utilize the strands of DNA as messages for protein synthesis. Possible stimulation of incorporation was examined also by adding poly A (70 mg per ml) or poly U (20 pg per ml) to the system. Two uc of L-lysine-Uaclu (1.7 00 per umole) were added to a system containing poly A and 4 no of L-phenyl- alanine-U-Clu (1.30 no per umole) were added to the system containing poly U. Incorporation of the amino acids was determined as usual. Results of this study are illustrated in Table 17. No stimulatory effect was observed by adding the synthetic poly- nucleotides. Under the assay conditions for amino acid incor- poration either the mammary gland ribosomes could not bind to the polynucleotides or the genetic message was not translated even though ribosomal binding had occurred. Without further study under different assay conditions neither possibility can be verified. Miller 23. 3;. (145) in a reticulocyte system have postulated that an initiation factor is necessary 156 TABLE 17 Effect of addition of single-stranded DNA, olypA, and poly U to the complete system The complete systems contained the usual constituents including pH 5 enzyme (1. 4 mg) and microsomes (1. 1 mg). The DNA, poly A, and the poly U were dissolved in 0. 05 M phos- phate buffer (pH 7. 0) and were added at concentrations of 150 mg, 70 0g, and 20 pg per ml of the incubation mixture, reSpectively. The following labeled amino acids were added to the respective1 systems: singlenstranded DNA system-- L-leucine-U- C1 One, 254 uc/umole), the poly A system-- DL-lysine-4, 5-H3(( (518 uc /umole), and the poly U system-- L-phenylalanine-U- C 4.0 uc/umole). The amount of incor- poration was assayed by procedures described in the section on Methods. Results are eXpressed as cpm incorporated per ml of incubation mixture. System cpm Incorporated Complete System 3,378 + DNA (single stranded) 4,641 Complete System 17,469 + Poly A 16,452 Complete System 1,611 + Poly U 1,197 157 for amino acid incorporation to be stimulated by poly A or poly U additions. Since this mammary system did not require an initiation factor (next section), the lack of this compo- nent did not prevent a possible stimulation of amino acid incorporation by poly A and poly U addition. The results also suggest a structural peculiarity of the mammary gland ribosomes contained in the microsome fraction. According to Redman (144) proteins which are synthesized on ribosomes attached to the membranes of the endoplasmic reticulum are transferred into its cisternae. Possibly, the microsomes which are present in the mammary gland cell-free system possess this structural arrangement. Perhaps this feature restricts the binding of the ribosomes to the synthetic messages added to the system. Conceivably, the leucine concentration in the stand- ard assay system limited amino acid incorporation; therefore, a stimulation of incorporation by the synthetic polynucleo- tides was impossible. 0n the other hand, a limitation of C11+ leucine incorporation by insufficient leucine was unlikely because of the following reasons: 1) the microsomes may have contained free amino acids within their cisternae, 2) the sRNA probably had leucine already esterified to it, and 3) a high level of C14 leucine incorporation was observed when no amino acids were added to the complete amino acid incorporation system. Effect of Initiation Factor on C11+ Leucine Incorpora- tion--Miller 23. 2;. (145) have isolated an unidentified 158 factor from reticulocyte ribosomes which is necessary for polypeptide chain initiation in the cell-free synthesis of hemoglobin. This initiation factor (IF) was solubulized when the ribosomes were washed with a 0.5 M_KCl solution. In the following study an attempt was made to demonstrate the role of a similar factor bonnd to mammary gland ribo- somes. Since Miller 23, 2;. (145) noted that the initiation factor altered the dependence of the reticulocyte complete system on magnesium ion concentration, the following eXperiment was conducted with the mammary gland cell-free system. The microsomes which were suspended in 0.5 M KCl were added to the standard incubation mixtures which con- tained varying levels of magnesium chloride (0 to 8 mmolar). An aliquot of the fraction, presumably containing the initia- tion factor which was prepared by the procedure of Miller pp.‘g;. (145) as described in the Methods section, was added to the test sample prior to the incubation. The 14 samples were incubated 40 minutes and the C leucine incor- poration was assayed as usual. The results of this study are presented in Figure 30. No differences in leucine incorporation at the various magnesium chloride concentrations were observed due to the addition of the fraction which presumably contained the initiation factor. Therefore, if active initiation factor was indeed present in the fraction obtained from the micro- sones, it exerted no effect on the dependence of incorpora- tion on magnesium concentration. 159 Figure 30. C1“ leucine incorporation in the presence of initiation factor. The complete incubation mixture con- tained the following: 0.47 mg of 0.5 M KCl-treated pierc- somes, 2.2 mg of pH 5 enzyme, 1 uc of L-leucine-U-C1 (254 uc/umole),varying levels of MgClZ, and the usual amounts of the other components. The presumed initiation factor equiva- lent to 0.4 mg of protein was added at the beginning of incu- bation. Amino acid incorporation after the 40 minute incuba- tion was assayed by the usual techniques (see section on Methods). INCORPORATE D (Cpm) C'4 LEUCINE 20,000 I 5,000 I0,0 00 5,000 160 I I I C" LEUCINE INCORPORATION IN PRESENCE OF INITIATION FACTOR *- + FACTOR O 2 4 6 8 MgCl2 CONCE NTRATION (umoles/ml) 161 The hypothesis that amino acid incorporation approaches a maximum at approximately 40 minutes due to a deficiency of active initiation factor was also tested. A normal time course eXperiment was conducted to serve as a control by measuring the incorporation of leucine at various time intervals. In duplicate incubation mixtures, initiation factor was added at 0, 10, and 40 minutes after the incuba- tion was started. In this experiment normal microsomes were utilized and the protein synthesis was assayed by the incor- poration of C1” leucine. The results of this eXperiment are presented in Figure 31. The initiation factor did not alter the level of leucine incorporation when it was added at 0 time, 10 minutes, or 40 minutes after the incubation. From these results, it may be concluded that the mammary gland micro- somes either required no initiation factor or that it was not removed from the mammary gland microsomes by the methods described for their preparation. Effect of Lactogenic Hormones on C1)+ Leucine Incor- poration--Turkington pp, 3;. (78, 79) have recently found that prolactin, insulin, and cortisone act synergistically to stimulate casein and whey protein synthesis in cultures of mammary tissue. But the effect of these hormones on cell-free preparations has not been investigated. To examine the effect of certain hormones on amino acid incorporation, the following eXperiment was performed. 162 Figurg 31. The effect of initiation factor on the time course of C1 leucine incorporation. The complete incubation mixture contained: 0.90 mg of normal microsomes, 2.2 mg of pH 5 enzyme, 1 he of L-leucine-U-C (254 uc/umole) and the usual amounts of the other components. The presumed initia- tion factor equivalent to 0.4 mg of protein was added at the beginning of incubation (0 0), 10 minutes after the initia- tion of incubation (:0 ° 0») and 40 minutes after the begin- ning of incubation (_ -). Amino acid incorporation was assayed by the usual tenchiques (see section on Methods). INCORPORATED (Cpm) C” LEUCINE 30,000 20,000 I0,000 163 I I I I C.4 LEUCINE INCORPORATION VS TIME AND INITIATION FACTOR ’ I “""u‘i 20 4O 60 80 TIME (minutes) IOO 164 Estrogen, prolactin, cortisone, growth hormone, and insulin were each suspended in a 0.01 M phosphate buffer, pH 7.0, at a concentration of 10 mg per ml. Various amounts of these solutions were then added to the standard assay system C1)+ leucine incorporation was determined and the amount of by the usual procedures. The results of this study are illustrated in Table 18. Addition of growth hormone, prolactin, insulin, or cortisone did not alter the amount of leucine incorporated. Various combinations of cortisone, insulin, and prolactin also exerted no effect. These results agree with the pre- viously published observations that growth hormone caused no stimulation in the amino acid incorporation by the rat liver system (72) and that estrogen and progesterone exerted no effect on protein synthesis in cell-free systems derived from accessory sexual tissues. The results of Florini (73) and Tata (71) obtained by studying protein synthesis in cell-free muscle and liver systems suggest that a hormone effect would be observed only if RNA was being synthesized in the system. As previously discussed in the experiment where poly U and poly A were added to the standard assay system, possibly the leucine concentration limited incorporation so as not to permit a stimulation by the lactogenic hormones. To summarize, various hormones which act on the mam- mary gland 12.2232 were assayed for their effect on cell- free amino acid incorporation. No effect was observed for 165 TABLE 18 C1“ leucine incorporation Effect of hormone additions on The standard assay mixture contaipfid microsomes (0.7 mg), pH 5 enzyme (2.6 mg), L-leucine-U-C (1.0 uc, 251 uc/ umole), and the usual amounts of the other constituents. The various hormones were suSpended in 0.01 M phosphate buffer, (pH 7.0) at concentrations of 10 mg per ml. The hormones were added individually or in combinations at con- centrations of 50 or 100 ug of each hormone per ml of incu- bation mixture. Activity of the hormones were as follows: insulin, 23.4 IU/mg; growth hormone, 1.0 USP unit/mg; and prolactin, 20 IU/mg. Amino acid incorporation was assayed by the usual procedures (see section on Methods). Radioactivity Amount of each hormone added (cpm) Hormone(s) 0 50 pg 200 pg added 0 Time 38 Control, 40 minutes 7,562 Growth Hormone 7.384 7.237 Cortisone 7.475 7.267 Insulin 7,169 7,866 Prolactin 8,971 7,469 Cortisone and Insulin -- 8,033 Cortisone and Prolactin -- 8,556 Insulin and Prolactin -- 7,941 Cortisone, Insulin, and Prolactin 7,940 8,966 166 individual hormones or various combinations of the hormones. The ;p_ylyp_effect of mammary gland sensitive hormones must then be at a level prior to the translation of RNA for pro- tein synthesis. Characterization of the Synthesized Protein The standard assay of protein synthesis used in the present study measures the amount of labeled amino acid which is incorporated into trichloroacetic acid-precipitable material. This is only an approximate estimate since amino acid incorporation may occur with either pOlypeptide chain initiation, further elongation of existing pOlypeptide Chains, or perhaps with random side chain reactions. There- fore, it was necessary to verify whether the labeled products synthesized by the mammary gland cell-free system were, in fact, milk proteins. Gel Filtration StudieS--According to the recent review by Thompson pp. pl. (146) on the physical prOperties of milk proteins, those proteins which are synthesized by the mammary gland possess molecular weights in the range of 16,000 to 36,000. If the mammary gland cell-free system synthesized authentic milk proteins, the incorporated radioactivity would be associated with proteins with molecular weights in this region. This possibility was investigated by the following gel filtration study with Sephadex G-75. After incubating the standard assay mixture for O and 167 40 minutes, the reaction was stopped by rapid freezing. The 0 time and the 40 minute sample incorporated 57 Cpm and 18,458 cpm, respectively, when measured by the usual proce- dures. Aliquots of these incubated preparations together with horse radish peroxidase and cytochrome c were layered onto the Sephadex G-75 columns and fractionated according to the conditions described in the section on Methods. The amount of radioactivity in each fraction was determined by combining an aliquot with 1 M sodium hydroxide (0.5 ml) and suSpending the resultant solution in a thixotropic counting fluid as described by Casjens and Morris (65). The results of these gel filtration studies are pre- sented in Figure 32. Two major radioactive peaks in addi- tion to the peak of free 013 leucine were found for the sample incubated for 40 minutes. The first radioactive material to be eluted corresponded to material eluted with the void volume of the column and presumably consisted of labeled peptides which remain attached to the microsomes. The next radioactive material to be eluted represented pro- tein which had an average molecular weight of 32,000. This calculation was based on the method reported by Andrews (127) in which horse radish peroxidase and cytochrome c were used as protein markers of known molecular weights. None of these peaks were present when a zero time sample was chro- matographed in the same manner. Thus, the above radioactive peaks represent proteins that were synthesized in the cell- free system. In addition to demonstrating Mp vitro protein 168 Figure 32. Behavior of the incorporated leucine. Standard assay systems contained; microsomes (1.0 mg) PH 5 enzyme (0.8 mg), L-leucine-U-Cl (0.8 Iic, 251 Iic/Iimole)o and the usual amounts of the other constituents. Systems incubated for 0 and 40 minutes were subjected to gel fill" tration studies by placing 1 ml of the 0 time incubation mixture and 2.5 ml of the 40 minute mixture on SephacheX 0475 column. The specific details of this experiment; are presented in the Methods section. The radioactivity in fractions 10 to 40 for the 0 time sample coincided with the base line. RADIOACTIVITY (Cpm) I5,000 I0,000 5,000 169 GEL FILTRATION OF INCUBATED SYSTEM fl L3 IO 20 30 4O FRACTION NUMBER SO 170 synthesis, these results Show that synthesis was confined to a limited molecular weight range because most of the radioactivity appeared in a well-defined area of the column chromatograph. This suggests that the radioactive proteins could be milk proteins since most of the proteins synthe- sized by the bovine mammary gland possess molecular weights in the 16 to 36,000 range. Isotope Dilution Tests of the Incubated Standard Assay System-~Previous examination of the incubated assay system by column chromatography illustrated that radioac- tively labeled proteins were synthesized by the ip’ylppp_ system. Further identification of the synthesized proteins was conducted by isotope dilution tests. Of course, results derived from this experiment are based on the assumption that the synthesized product can be isolated by the same procedures as used for known milk proteins. This eXperi- ment was conducted as follows. At zero time and at 40 minutes of incubation, 1.0 and 10.8 ml aliquots, respectively, were withdrawn and each added to 100 ml of skimmed milk. The Specific milk proteins were then isolated from these solutions by the procedures described in the Methods section. The 0 time sample con- tained 220 cpm precipitated by the trichloroacetic acid method while the 40 minute incubated sample contained 102,751 cpm in the acid precipitate. The B-casein.)<-casein, dulactalbumin, B-lactoglobulin, 171 and cs-casein fractions were recovered by the purification procedure and extensive dialysis (see section on Methods) and each contained radioactivity (Table 19). The amount of radioactivity in each of the respective milk protein frac- tions was significantly increased over the 0 time value. The radioactivity in each protein fraction was roughly pro- portional to the amount of each protein usually present in skimmed milk (146). When the results were corrected to equilize the volumes of the original samples, the radio- activity in each of the protein fractions isolated from the 40 minute incubated sample was significantly increased over the 0 time control protein fractions. Based on this study, the complete system incubated for 40 minutes synthesized cS-casein, B-casein,)(-casein, c-lactalbumin, and a-lactoglobulin. A second isotope dilution test was conducted by Dr. B. L. Larson of the University of Illinois Dairy Science Department. Standard assay systems of 10 ml each, instead of the usual 1 ml, were incubated for 0, 5, and 40 minutes. A 1 ml aliquot of each system was assayed for 014 leucine incorporation by the usual methods. Four and one-half ml of the 0 and 5 minute incubated samples and 5 ml of the 40 minute incubated sample were frozen at the end of the incu- bation period and sent to Dr. Larson while in the frozen state. Each sample was thawed and combined with 16 ml of fresh skimmed milk by Dr. Larson who subsequently isolated, purified, and determined the radioactivity of the B-casein 172 TABLE 19 Identification of synthesized protein by isotope dilution The standard assay mixtures contaifled microsomes (0.7 mg), pH 5 enzyme (2.6 mg), ~leucine-U-C (1.0 no, 251 uc/ umole), and the usual amounts of the other constituents. The mixtures were incubated for 0 and 40 minutes. One ml of the 0 time sample and 10.8 ml of the 40 minute sample were each combined with 100 ml of fresh skimmed milk. Specific milk proteins were then isolated from each mixture according to the procedures described in the Methods section. Protein Total Increase % of Fraction Radioactivity per ml* Total 0 min 40 min (cpm) (Cpm) (cpm) B-Casein 248 38,610 3.327 35.0 dS-Casein 1,064 57,582 4,276 45.0 x-Casein 105 14,500 1,247 13.2 c—Lactalbumin 357 7,259 317 3.2 B-Lactoglobulin 576 9.875 339 3.5 *EXpressed as the net increase in cpm per ml of incubation m1Xture o 173 and B-lactoglobulin. The B-casein and B-lactoglobulin were purified according to the methods of Aschaffenburg (147) and Aschaffenburg and Drewry (128), respectively. The isolated proteins were checked for purity by electrophoresis on cellulose acetate strips. The results, illustrated in Table 20, again Show that both B-Casein and B-lactoglobulin were radioactive and, As therefore, were synthesized by the cell-free system. expected, the synthesis of these proteins was time-dependent. The amount of dpm present in the trichloroacetic acid (TCA) precipitates which correSponds to actual milk protein syn- thesis may be estimated. In doing this one must assume that the same ratio exists Mp vitro as _.'I_.p_ vivo between the rela- tive amounts of milk proteins synthesized. The assumption was also made that B-lactoglobulin comprises about 10% and B-casein about 25% of the total milk proteins synthesized in the mammary gland. Further, an assumption was made that 17% and 31% of the leucine present in the total milk proteins normally synthesized _i_p_ vivo is represented by B-lactoglobulin and B-casein, respectively. These results are also presented in Table 20 and indicate that about 25% of the dpm found in the TCA precipitable protein are due to the synthesis of milk proteins with the remaining counts due to other reasons including the synthesis of other proteins perhaps both by the components from the secretory cell as well as from the other cells present in mammary tissue. Perhaps the _i_p vitro synthesis of these two milk proteins (B—casein and .sHomoOIm an downpaapsoo msaopoam xaaa couamoSpsam Hopop one Ca osaosod mo pcooaoa one maosoo Hm oaonz Ha\aa0 00am n HM\ooa N one AN .0aaanoaoopooHIm an oopsndnpcoo msaopoag Bade oouamonpamm Hopop on» ad osdosoa co pso0aoa one oaosoo 5H oaosz Ha\aao wood 4 mH\ooa x 2mm AH .oacaoxo Hom\M .HE\EQ0 mm .Hoapsoo oaap 0 Row oopooaaoo\m oom.w 0mm.m osm.m 0A0 000.Hm on 00a.m mo:.a one 2mm aam.s m o o o o o o :Homoolm pooHIm aaomooIm pooHIm .0ouamoSp:Am .0» one \IHa Hog adopoaa :Hopomm xaaa HopOp op oaapxaa soapoooa manopaaaooam ¢UB Asaav odd oaspxaa soapoooa mo as non and 2H ace oopooaaoo oaae ed as non Eco oopoaapmm 174 III III .Amoozpoz so soapoom oom. monsooooaa Henna on» an soaponoanoosa ofioo osdao sow oohomwo ooao oaoz wohfipxda soapopsocH on» mo oposoaad .maobapoocmon .Ammav masons one wasnsommosoms mo 0cm Audav wasnsommosome mo cospoa one an mops» Ixaa 0005p scam coamaasa 0cm oopoaoma oaos ceasnoawopooalm 0am saomoolm .MHHB coaaaxm gnome no as ea Spas condnaoo nooo oaos moRSpRHa so«90950s« ounces 0: on» no He m 0cm opssaa m 0cm 0 on» 00 H8 maosloco 0cm 930m .mpsocoaaoo nonpo one no mpssoao Homes on» 020 AoHoa1\01 mmm .01 m.o. NHOIDIosdosoHIA .Ama w.o. oahuco n ma .Awa 1.0. moaomoaoaa condopsoo oHprHa Comhdfl .A .m .HQ hp dmpoddfloo mm ommo oaoosopm one 175 Bibctoglobulin) is not a good index from which to extrapo- IMm the synthesis of the other milk proteins. In summary, the results indicate that milk proteins hmm been synthesized by the mammary gland cell-free system. Hmmver, the results are complicated by the presence of addi- thmal counts in the TCA precipitated protein. Identification Studies by Immunodiffusion--The tech- rnque of immunodiffusion has been utilized by investigators toidentify the products of protein synthesis in cell-free swstems. For example, Stenzel and Rubin (148), who studied protein synthesis in a rabbit Spleen system, identified one of the synthesized proteins as y-globulin by using a com- bination of immunodiffusion and radioautography. Because this technique permitted identification of minute quantities of labeled protein, it was utilized to identify the milk proteins which were synthesized in the mammary gland cell- free system. An aliquot (0.05 ml) of a complete system at 0 time enui after 40 minutes of incubation were utilized for the ilmnunodiffusion studies as described in the Methods section. TTuese samples had been stored at -20°C for 6 months. The 0 'tinue and 40 minute sample had incorporated 220 cpm and 19,183 cxpni per ml, respectively, into protein as assayed by the IIStual trichloroacetic acid precipitation procedure. The samples were placed in the center well of an agar plate and the antibodies against 0.3-casein, x-casein, proteose-peptone, 176 and whole casein were placed in wells equidistant from the center. The immunodiffusion was allowed to proceed for two days. The unprecipitated proteins were then leached from the agar gels, and the gels air-dried. Then the agar films were eXposed to x-ray film for visualization of the areas of radioactivity. Precipitin bands were observed in the agar gels for both test samples and each authentic antibody. However, after radioautography, a radioactive precipitin band was observed only between the sample incubated for 40 minutes and both the whole casein and the cS-casein antibodies (see Figure 33). The location of the band between the whole casein antibody and the 40 minute sample correSponded exactly with the position of the radioactive band between the 40 minute sample and the dS-casein antibody. In addition, these radioactive bands coincided with the respective pre- cipitin bands observed prior to the drying of the gel. No radioactive bands were observed for the xecasein and proteose- peptone antibodies probably because of inadequate sensitivity. Furthermore, no radioactive bands were formed in the 0 time control sample. Therefore, the synthesis of milk proteins by the mammary gland cell-free system was again verified. Specifically, this particular study demonstrated a synthesis of cS-casein. 177 .monon neeeaeooaa oeeeoooaooa enomoamoa AQIQIOV onHH o nee: ooeoonnoo moaoaeo oeaom one oHenz A. 0 av moaoae0 ceaom one an ooeooeone ohm mmnon ndeeaeooaa one .monoecoQIomooeoam .mIm 0am “neomoo .Ix .vxnneomooi 0 .m0 “neomoo oHonz ..0.s ”poms oaos mnoeeoeeoanno wne Isoaaom one .mooneoz no noeeoom one ne coneeomoo mo coeosonoo oaos mnemaw Ioesmoeooa one scammeeeooaae one .mHHoz aoeso one ne coooam oaos moon Ieeno nooo no HE oneooaondn obem .menoSeeemnoo aoneo one no mendoao HoSmS one end .Aoaoa1\01 cam .01 mm.H. OI IoneosoHIA .Awa N.H. oahmno m we .Awa $.00 moaomoaoea doneoenoo monsemwa amono oaoonoem .waoemmm oeoHaaoo coeo930ne e0 moeozem scammemeoonsaae mo oncoawoesoOHUom .mm oaswem 178 2:). 0.4 a... 2 . . o . o o 00 000 cc 00000. o o undidw 2:). 0 n.In. v. . . . .. . co. co co co 0 o co . 0.52% . .. .. . ...... . mt .. .. .. ...U.; Emhm>m wanna/.00 om...=>.. “.0 m:a