ENDOCRINOLOGY
VOLUME 76
JANUARY-JUNE, 1965
EDITOR-IN-CHIEF: FREDERICK G. HOFMANN
ASSOCIATE EDITORS: WILBUR H. SAWYER, DONALD F. TAPLEY
EDITORIAL BOARD
SAVINO A. D'ANCELO NICHOLAS S. HALMI S. M. MCCANN SAMUEL SOLOMON HOWARD A. BERN A. BAIRD HASTINGS ANDREW V. NALBANDOV KENNETH STERLING E. M. BOCDANOVE LEON D. HELLMAN DON H. NELSON CLARA M. SZEGO H. LEON BRADLOW ROY HERTZ MAURICE S. RABEN ALVIN TAUROG JAMES O. DAVIS FREDERICK L. HISAW HOWARD RASMUSSEN ELEANOR H. VENNING LEWIS L. ENGEL SIDNEY H. INGBAR MURRAY SAFFRAN ALFRED E. WILHELM I JOHN W. EVERETT DONALD C. JOHNSON LEO T. SAMUELS JAN WOLFF WILLIAM F. GANONG ERNST KNOBIL KENNETH SAVARD IRA G. WOOL MONTE A. GREER MAURICE E. KRAHL MINDEL C. SHEPS
FOREICN CONSULTING EDITORS Derek A. Denton Egon R. Diczfalusy Roberto A. Mancini Luciano Martini Rosalind Pitt-Rivers Kazno Shizume
COPYRIGHT © 1965 BY THE ENDOCRINE SOCIETY tu Uli.- M ü n o h & n
CONTENTS OF VOLUME 76
No. 1, January 1965 BARKER, S. B., M. SHIMADA, AND M. MAKIUCHI: Metabolic and Cardiac- OTA, K., Y. SHINDE, AND A. YOKOYAMA: Responses to Thyroxine Analogs 115 Relationship Between Oxytocin and VERNIKOS-DANELLIS, J.: Effect of Stress, Prolactin Secretion in Maintenance of Adrenalectomy, Hypophysectomy and Lactation in Rats 1 Hydrocortisone on the Corticotropin- GOLDSMITH, R. E., J. WULSIN, AND M. J. Releasing Activity of Rat Median WiESTER: Effect of Thyroparathyroid• Eminence 122 ectomy on the Clinical Course and the CLITHEROE, H. J., AND J. H. LEATHEM: Serum Calcium and Phosphorus Con• Effect of Estradiol and Ethamoxytri- centration in Dogs 9 phetol (MER-25) on the Mouse Uterus 127 SNOOK, R. B., AND H. H. COLE: Bio-assay KATZMAN, P. A.: Bio-assay of Estrogens by of Antisera Against Human Chorionic Intra vaginal Injection in Immature Gonadotropin 20 Rats 131 PARLOW, A. F., P. G. CONDLIFFE, L. E. ALBERT, A., I. DERNER, E. ROSEMBERG, REICHERT, JR., AND A. E. WILHELMI: AND W. B. LEWIS: Assay Characteristics Recovery and Partial Purification of of Tannate Complexes of Ovine Gonado• FSH and LH During the Purification of tropins 139 TSH from Human Pituitary Glands.... 27 NATAF, B. M., E. M. RIVERA, AND I. L. NOTES AND COMMENTS CHAIKOFF: Role of Thyrotropic Hor• LANGER, P., AND V. STOLC: Goitro• mone in Iodine Metabolism of Em• genic Activity of Allylisothiocyanate— bryonic Rat Thyroid Glands in Organ A Widespread Natural Mustard Oil.... 151 Culture 35 NICOLL, C. S., AND H. A. BERN: Pigeon BLOCH, E., AND K. BENIRSCHKE: In Vitro Crop-Stimulating Activity (Prolactin) in Steroid Synthesis by Fetal, Newborn the Adenohypophysis of Lungfish and and Adult Armadillo Adrenals and by Tetrapods 156 Fetal Armadillo Testes 43 MUNSICK, R. A.: Hen Oxytocic Activities BLAIR, A. J., JR., AND T. UETE: Effect of of Oxytocin and 1-Deamino-oxytocin... 161 Triamcinolone Administration on TONG, W.: Thyrotropin Stimulation of Corticosteroid Metabolism in the Liver. 52 Thyroxine Synthesis in Isolated Thyroid ERRATUM 57 Cells Treated with Puromycin 163 MORII, H., T. FUJITA, H. ORIMO, S. OKI- KELLER, P. J.: Some Observations on the NAKA, AND K. NAKAO: Effect of Sym• Ovarian Ascorbic Acid Depletion Assay pathetic Stimulation, Epinephrine and for Luteinizing Hormone Employing Phentolamine on Recovery from In• Immature Female Mice 165 duced Hypocalcemia 58 BITMAN, J., H. C. CECIL, M. L. MENCH, No. 2, February 1965 AND T. R. WRENN: Kinetics of in Vivo Glycogen Synthesis in the Estrogen- BEGG, D. J., E. M. MCGIRR, AND H. N. Stimulated Rat Uterus 63 MUNRO: Protein and Nucleic Acid Con• SIPERSTEIN, E. R., AND V. F. ALLISON: tent of the Thyroid Glands of Different Fine Structure of the Cells Responsible Mammals 171 for Secretion of Adrenocorticotrophin in ABRAHAM, S., L. KOPELOVICH, P. R. the Adrenalectomized Rat 70 KERKOF, AND I. L. CHAIKOFF: Metabolic CLERMONT, Y., AND S. C. HARVEY: Dura• Characteristics of Preparations of Iso• tion of the Cycle of the Seminiferous lated Sheep Thyroid Gland Cells. I. Epithelium of Normal, Hypophysec- Activity Levels of Enzymes Concerned tomized and Hypophysectomized-Hor- with Glycolysis and the Tricarboxylic mone Treated Albino Rats 80 Acid Cycle 178 MUNSICK, R. A., AND S. C. JERONIMUS: EISENSTEIN, A. B., M. RON A, AND Effects of Diethylstilbestrol and Mag• B. BRUMMER: In Vitro Effects of nesium on the Rat Oxytocic Potencies of Puromycin Aminonucleoside on Liver Some Neurohypophysial Hormones and Carbohydrate Synthesis 191 Analogues 90 VAN TIENHOVEN, A., D. SIMKIN, J. WESKE, EDGREN, R. A., A. F. PARLOW, D. L. AND G. R. BARR: Effect of Thyroxine on PETERSON, AND R. C. JONES: On the the Gonadotrophin Dose Response Line Mechanism of Ovarian Hypertrophy Using the Chick's Testicular Response. 194 Following Hemicastration in Rats 97 JOHNSTON, C. C, JR., AND W. P. DEISS, TALMAGE, R. V., J. NEUENSCHWANDER, JR.: Some Effects of Hypophysectomy AND L. KRAINTZ: Evidence for the and Parathyroid Extract on Bone Ma• Existence of Thyrocalcitonin in the Rat 103 trix Biosynthesis 198 RYAN, K. J., AND R. V. SHORT: Formation OYAMA, J., AND W. T. PLATT: Metabolic of Estradiol by Granulosa and Theca Alterations in Rats Exposed to Acute Cells of the Equine Ovarian Follicle.... 108 Acceleration Stress 203 June 1965 VOLUME CONTENTS 111
LEWIS, U. J., E. V. CHEEVER, AND W. P. and Size of Thyroid Follicles in Guinea VANDERLAAN: Studies on the Growth Pigs of Different Ages 329 Hormone of Normal and Dwarf Mice. . 210 CALLANTINE, M. R., R. R. HUMPHREY, GOODMAN, H. M.: In Vitro Actions of AND S. LEE: Effect of Follicle-Stimulat• Growth Hormone on Glucose Metab• ing Hormone on Ovarian Nucleic Acid olism in Adipose Tissue 216 Content 332 GORSKI, R. A., AND J. W. WAGNER: HOCH, F. L.: Synergism Between Calori- Gonadal Activity and Sexual Differ• genic Effects: L-Thyroxine and 2,4- entiation of the Hypothalamus 226 Dinitrophenol or Sodium Salicylate in VERNIKOS-DANELLIS, J.: Effect of Rat Euthyroid Rats 335 Median Eminence Extracts on Pituitary GROSVENOR, C. E.: Evidence that Extero• ACTH Content in Normal and Adrenal- ceptive Stimuli Can Release Prolactin ectomized Rats 240 from the Pituitary Gland of the Lactat- ARMSTRONG, D. T., AND R. O. GREEP: ing Rat 340 Failure of Deciduomal Response to KUNIN, A. S., AND S. M. KRANE: Inhibi• Uterine Trauma, and Effects of LH tion by Puromycin of the Calcium- Upon Estrogen Secretion in Rats with Mobilizing Activity of Parathyroid Ex• Ovaries Luteinized by Exogenous tract 343 Gonadotropins 246 ORIMO, H., T. FUJITA, H. MORII, AND K. NAKAO: Inactivation in Vitro of No. 3, March 1965 Parathyroid Hormone Activity by Kid• ney Slices 255 IBAYASHI, H., M. NAKAMURA, T. UCHI- ALBERT, A., D. BENNETT, G. CARL, E. KAWA, S. MURAKAWA, S. YOSHIDA, K. NAKAO, AND S. OKINAKA: CI Ste• ROSEMBERG, P. KELLER, AND W. B. 9 LEWIS: Duration of Response to Free roids in Canine Spermatic Venous Blood and Tannated Luteinizing Hormone. . . 259 Following Gonadotropin Administra• SEGAL, S., S. MILKOVIC, AND L. E. tion 347 ROSENBERG: Amino Acid Accumulation VAN ZYL, A., K. SCHULZ, B. WILSON, AND by Kidney Cortex Slices and Intestinal D. PANSEGROUW: Thyroidal Iodine and Segments of Rats Bearing a Transplant• Enzymatic Defects in Cattle with Con• able Pituitary Tumor 267 genital Goiter 353 NALLAR, R., ANDS. M. MCCANN: Luteiniz• LEWIS, U. J., E. V. CHEEVER, AND W. P. ing Hormone-Releasing Activity in VANDERLAAN: Alteration of the Proteins Plasma of Hypophysectomized Rats. . . 272 of the Pituitary Gland of the Rat by LAWTON, I. E., AND N. B. SCHWARTZ: Estradiol and Cortisol 362 Effect of LH on Ovarian Ascorbic Acid FRIESEN, H.: Purification of a Placental Over a 24-Hour Period 276 Factor with Immunological and Chem• RAMIREZ, V. D., AND C. H. SAWYER: ical Similarity to Human Growth Hor• Fluctuations in Hypothalamic LH-RF mone 369 RUBIN, B. L., AND H. W. DEANE: Effects (Luteinizing Hormone-Releasing Fac• r> tor) During the Rat Estrous Cycle 282 of Superovulation on Ovarian A -3/^-Hy• DHARIWAL, A. P. S., R. NALLAR, M. BATT, droxy steroid Dehydrogenase Activity in AND S. M. MCCANN: Separation of Rats of Different Ages 382 Follicle-Stimulating Hormone-Releasing STANSFIELD, D. A., AND J. W. ROBINSON: Factor from Luteinizing Hormone-Re• Glycogen and Phosphorylase in Bovine leasing Factor 290 and Rat Corpora Lutea, and the Effect TISHLER, P. V., AND S. H. INGBAR: Cor• of Leuteinizing Hormone 390 relative Effects of Puromycin on ulI HALL, P. F.: Influence of Temperature Metabolism and Amino Acid Incorpora• Upon the Biosynthesis of Testosterone tion by Calf Thyroid Slices 295 by Rabbit Testis in Vitro 396 KLEBANOFF, S. J.: Inactivation of Estro• KOIDE, S. S., AND S. M. MITSUDO: Histo- gen by Rat Uterine Preparations 301 chemical Study of 3/3- and 17/3-Hydroxy- JORGENSEN, E. C, AND J. A. W. REID: steroid Dehydrogenases in Human Term Thyroxine Analogs. Synthesis and Anti- Placenta 403 goitrogenic Activity of 3'-Chloro- and FABIANEK, J., A. HERP, AND W. PIGMAN: 3 ^5'-Dichloro-3,5-diiodo-L-thyronines in Permeability of Dermal Connective the Rat 312 Tissue in Adrenalectomized and Hy• HELLERSTROM, C, I.-B. TÄLJEDAL, AND pophysectomized Rats 408 B. HELLMAN: Quantitative Studies on RAMIREZ, V. D., AND S. M. MCCANN: In• Isolated Pancreatic Islets of Mammals. hibitory Effect of Testosterone on 5-Nucleotidase and Adenosine Triphos• Luteinizing Hormone Secretion in Im• phatase Activities in Normal and Corti• mature and Adult Rats 412 sone-Treated Rats 315 YATVIN, M. B., AND R. W. WANNE- BRAY, G. A., AND H. M. GOODMAN: Studies MACHER, JR.: Action of Adrenal Corti- on the Early Effects of Thyroid Hor• coids on Protein Metabolism in the mones 323 Thiouracil-Treated Rat 418 CLARK, A. F., J. I. RAESIDE, AND S. SOLO• NOTES AND COMMENTS MON: Studies on the Urinary Excretion RIES, N., AND N. ALLEGRETTI: Number of 17-Ketosteroids by the Domestic Pig 427 iv VOLUME CONTENTS Volume 76
JARRETT, R. J.: Effects and Mode of Ac• Human Serum During Agar Gel Elec• tion of Adrenocorticotrophic Hormone trophoresis at pH 7.4 547 Upon the Reproductive Tract of the FAIN, J. N.: Comparison of Glucocorticoid Female Mouse 434 Effects on Brown and White Adipose SCRANTON, J. R., AND N. S. HALMII Tissue of the Rat 549 Thyroidal Iodide Accumulation and HAGERMAN, D. D.: Early Effect of Estro• Loss in Vitro 441 gen on Phosphate Metabolism in Rat FISHER, C. J., AND J. H. LEATHEM: Effect Uterine Nuclei 553 of a Protein-Free Diet on Protein MALOOF, F., AND M. SOODAK: Nature of Metabolism in the Pregnant Rat 454 the Porphyrin Group of a Heme Protein MAEIR, D.M.: Species Variation in Testic• Fraction Isolated from Mammalian ular A5-3ß-Hydroxysteroid Dehydro• Thyroid Microsomes 555 genase Activity: Absence of Activity in Primate Leydig Cells 463 SPAZIANI, E., AND A. GUTMAN: Distribu• No. 4, April 1965 tion Volumes of Sugars in Rat Uterus, Ileum and Skeletal Muscle as Affected SHIKITA, M., AND B.-I. TAMAOKI: Testos• by Estradiol 470 terone Formation by Subcellular Parti• cles of Rat Testes 563 GALTON, V. A., S. H. INGBAR, AND S. VON DER HEYDE: Role of Catalases and SHIMAOKA, K., AND B. D. THOMPSON: Ra- Peroxidases in the Deiodination of dioiodine Labeling of Serum Albumin in Thyroxine in Frog Liver 479 Vivo 570 RAPP, J. P.: Alteration of Juxtaglomerular JONES, A. E., J. N. FISHER, U. J. LEWIS, Index by Selective Inbreeding 486 AND W. P. VANDERLAAN: Electro- phoretic Comparison of Pituitary Glands SCHANE, H. P.: Estrogen-like Effect of from Male and Female Rats 578 Chelating Agents on Rat Uterine Phos• phorylase Activity 491 WOEBER, K. A., AND S. H. INGBAR: Anti• thyroid Effect of Noncalorigenic Con• ALBERT, A., D. BENNETT, G. CARL, geners of Salicylate, with Observations E. ROSEMBERG, P. KELLER, AND W. B. on the Influence of Serum Proteins on LEWIS: Assay Characteristics of Tan- the Potency of Antithyroid Agents.... 584 nate Complexes of Human Chorionic Gonadotropin 499 ELLIS, LEG. C., AND D. L. BERLINER: Se• quential Biotransformation of 5-Preg• ALBERT, A., D. BENNETT, G. CARL, E. nenolone^ a-3H and Progesterone-4-uC ROSEMBERG, P. KELLER, AND W. B. Into Androgens by Mouse Testes 591 LEWIS: Assay Characteristics of Tan- nate Complexes of Pregnant Mare LAYNE, D. S.: Identification in Rabbit Serum Gonadotropin 506 Urine of the 3-Glucuronoside-17-N- acetylglucosaminide of 17«-Estradiol. . 600 NICOLIS, G. L., AND S. ULICK: Role of 18- KOSTYO, J. L.: In Vitro Effects of Adrenal Hydroxylation in the Biosynthesis of Steroid Hormones on Amino Acid Aldosterone 514 Transport in Muscle 604 MILLS, J. B., AND A. E. WILHELME: Sul- fitolysis of Bovine Growth Hormone. . . 522 KUROSHIMA, A., Y. ISHIDA, C. Y. BOWERS, AND A. V. SCHALLY: Stimulation of Re• GANONG, W. F., B. L. WISE, R. SHACKLE- lease of Follicle-Stimulating Hormone FORD, A. T., BORYCZKA, AND B. ZIPF: by Hypothalamic Extracts in Vitro and Site at Which a-Ethyltryptamine Acts in Vivo 614 To Inhibit the Secretion of ACTH 526 HOFFMANN, J. C, AND N. B. SCHWARTZ: Timing of Post-partum Ovulation in the NOTES AND COMMENTS Rat 620 GOODMAN, H. M.: Effects of Insulin on HOFFMANN, J. C, AND N. B. SCHWARTZ: Water Uptake by Adipose Tissue Dur• Timing of Ovulation Following Pro• ing Incubation in Vitro 531 gesterone Withdrawal in the Rat. . . . 626 RENNIE, P., AND J. DAVIES: Implantation DEGROOT, L. J., J. E. THOMPSON, AND in the Rabbit Following Administration A. D. DUNN: Studies on an Iodinating of 20a-Hydroxypregnen-3-one and 20/3- Enzyme from Calf Thyroid 632 Hydroxypregnen-3-one 535 CHRISTENSEN, A. K., AND N. R. MASON: BAUMAN, T. R., G. W. PIPES, AND C. W. Comparative Ability of Seminiferous TURNER: Relative Potency of Several Tubules and Interstitial Tissue of Rat Analogues When Substituted for L- Testes To Synthesize Androgens from Thyroxine in the Estimation of Thyroid Progesterone-4-14C in Vitro 646 Secretion Rate in Rats 537 MARIEB, N. J., AND P. J. MULROW: Role LERNER, L. J., AND A. R. TURKHEIMER: of the Renin-Angiotensin System in the Effect of the Lack of Dietary Protein on Regulation of Aldosterone Secretion in the Uterine Response to Estrogen in the the Rat 657 Mouse 539 SWISLOCKI, N. I., AND CM. SZEGO: Acute MOUNIB, M. S., AND M. C. CHANG: Me• Reduction of Plasma Nonesterified tabolism of Endometrium and Fallopian Fatty Acid by Growth Hormone in Tube in the Estrous and the Pseudo- Hypophysectomized and Houssay Rats 665 pregnant Rabbit 542 OPEL, H.: Oviposition in Chickens After BRAVERMAN, L. E., AND S. H. INGBAR: Removal of the Posterior Lobe of the Binding of 3,5,3'-L-Triiodothyronine in Pituitary by an Improved Method.... 673 June 1965 VOLUME CONTENTS v
CATT, K., AND B. MOFFAT: Fractionation Extracts Which Lower Serum Cal• of Rat Pituitary Extract by Starch Gel cium and Raise Serum Citrate Levels 783 Electrophoresis and Identification of FRIEDMAN, R. C, AND S. REICHLIN: Growth Hormone and Prolactin 678 Growth Hormone Content of the Pi• SCHANE, H. P., AND S. L. LEONARD: Rate tuitary Gland of Starved Rats 787 of Glycogenolysis in the Estrogen- HOSHINO, K.: Development and Func• Primed Rat Uterus in Vitro 686 tion of Mammary Glands of Mice KANEMATSU, S., AND C. H. SAWYER: Prenatally Exposed to Testosterone Blockade of Ovulation in Rabbits by Propionate 789 Hypothalamic Implants of Norethin- FARESE, R. V.: Quantitative Compari• drone 691 son of the Effects of ACTH Adminis• CHOWERS, L, AND S. M. MCCANN: Con• tration on the Activities of Soluble tent of Luteinizing Hormone-Releasing Cell Fraction and Microsomes for In• Factor and Luteinizing Hormone Dur• corporation of Amino Acid Into Pro• ing the Estrous Cycle and After Changes tein 795 in Gonadal Steroid Titers 700 WURTMAN, R. J., J. AXELROD, S. H. ANTONIADES, H. N., A. M. HUBER, B. R. SNYDER, AND E. W. CHU: Changes in BOSHELL, C. A. SARAVIS, AND S. N. the Enzymatic Synthesis of Mela• GERSHOFF: Studies on the State of Insu• tonin in the Pineal During the Estrous lin in Blood: Properties of Circulating Cycle 798 "Free" and "Bound" Insulin 709 HALKERSTON, I. D. K., M. FEINSTEIN, Erratum 721 AND O. HECHTER: Further Observa• KATZ, S. R., J. DANCIS, AND M. LEVITZ: tions on the Inhibition of Adrenal Relative Transfer of Estriol and Its Protein Synthesis by ACTH in Vitro 801 Conjugates Across the Fetal Mem• branes in Vitro 722 LINDSAY, R. H., H. NAKAGAWA, AND P. P. No. 5, May 1965 COHEN: Thiouracil-Pyrimidine Rela• tionships in Thyroid and Other Tissues 728 CORBIN, A., G. MANGILI, M. MOTTA, AND L. MARTINI: Effect of Hypothalamic LINDSAY, R. H., AND P. P. COHEN: Nu• cleic Acid Synthesis in Normal and and Mesencephalic Steroid Implanta• Goitrous Rat Thyroid 737 tions on ACTH Feedback Mechanisms 811 BIRMINGHAM, M. K., G. ROCHEFORT, AND SCRIBA, P. C, AND W. J. REDDY: Adreno- corticotrophin and Adrenal Protein H. TRAIKOV: Steroid Fractions from Synthesis 745 Incubated Normal and Regenerated Adrenal Glands of Male and Female BURSTEIN, S., B. R. BHAVNANI, AND Rats 819 H. L. KIMBALL: Genetic Aspects of HEIDEMAN, M. L., JR., R. P. LEVY, W. L. Cortisol Hydroxylation in Guinea Pigs: l3l Urinary Excretion and Production MCGUIRE, AND R. A. SHIPLEY: I- Rates of Cortisol and 2a-HydroxyCorti• Labeled Bovine Thyrotropin: Prepara• sol 753 tion Through a New Micro Technique of Iodine Distillation and Studies of Ad• REICHERT, L. E., JR., AND A. E. WIL- sorption to Glass and Polyethylene.... 828 HELMI: Preparation of Equine Luteiniz• ing Hormone 762 ASSAYKEEN, T. A., AND J. A. THOMAS: Endogenous Histamine in Male Organs of Reproduction 839 NOTES AND COMMENTS ABE, T., AND C. VILLEE: A Uterine UCHIDA, K., M. KADOWAKI, AND T. Growth-Promoting Substance from Hu• MIYAKE: Failure of Estrogen To Pro• man and Bovine Uterine Muscle 814 duce a Hypocholesterolemic Effect in BUHLER, D. R., R. C. THOMAS, JR., AND Immature Rats 766 C. A. SCHLÄGEL: Absorption, Metab• HAYS, R. L., E. W. HAHN, AND K. A. olism and Excretion of 6e*-Methy]pred- KENDALL: Evidence for Decreased nisolone-3H, 21-acetate Following Oral Steroidogenesis in Pregnant Rats Fed and Intramuscular Administrations in a Sucrose Diet 771 the Dog 852 SWEAT, M. L., AND M. J. BRYSON: Ster• COPPOLA, J. A., AND J. W. PERRINE: In- oid 11/3-Oxygenation, Ascorbic Acid, Influence of Two Nonsteroidal Antiös• Epinephrine and Andrenochrome In• trogens on Vaginal Opening and PMS- terrelations in the Adrenal Gland.... 773 Induced Ovulation in Rats 865 SWIG ART, R. H., F. K. HILTON, M. M. LEONARD, S. L., AND H. P. SCHANE: Hor• DICKIE, AND B. J. FOSTER: Effect of monal Effects on Phosphorylase Ac• Gonadal Hormones on Submandibu• tivity and Glycogen Levels in the Rat lar Gland Amylase Activity in Male Ductus Deferens 870 and Female C57BL/6J Mice 776 STEINETZ, B. G., J. P. MANNING, M. BUT• IBAYASHI, H., T. YAMAJI, T. TAJIMA, LER, AND V. BEACH: Relationships of AND K. NAKAO: Antigranulomatous Growth Hormone, Steroids and Relaxin Activity of Vitamin Ki and Its Sum• in the Transformation of Pubic Joint mation with That of Prednisolone. . . 780 Cartilage to Ligament in Hypophysec- PINCUS, J. H., R. G. FELDMAN, G. tomized Mice 876 RANNAZZISI, AND S. NATELSON: Elec• GROSVENOR, C. E., S. M. MCCANN, AND tromyographic Studies with Pituitary R. NALLAR: Inhibition of Nursing-In- vi VOLUME CONTENTS Volume 76
duced and Stress-Induced Fall in Pitui• of Rats. A Histochemical Study 996 tary Prolactin Concentration in Lactat- SAWYER, W. H., AND H. VALTIN: Inhibi• ing Rats by Injection of Acid Extracts of tion of Vasopressin Antidiuresis by Bovine Hypothalamus 883 Extracts of Pituitaries from Rats LEVY, R. P., W. L. MCGUIRE, R. K. with Hereditary Hypothalamic Dia• SHAW, AND G. E. BARTSCH: Effect of betes Insipidus and by Oxytocin .... 999 Species Differences of Mice on the Bio- assay of Thyrotropin 890 No. 6, June 1965 GALICICH, J. H., F. HALBERG, L. A. FRENCH, AND F. UNGAR: Effect of Cere• GREEP, R. O.: Roy Graham Hoskins (1880- bral Ablation on a Circadian Pituitary 1964) 1007 Adrenocorticotropic Rhythm in C Mice 895 WEBER, G., R. L. SINGHAL, S. K. SRIVAS- MATSUDA, K., AND M. A. GREER: Nature TAVA, H. J. HIRD, AND J. FURTH: Be• of Thyroid Secretion in the Rat and the havior of Hepatic Gluconeogenic En• Manner in Which It Is Altered by Thy• zymes, RNA Metabolism and Amino rotropin 1012 Acid Level in Rats Carrying Trans• GOODNER, C. J.: Metabolism of the Anter• plantable ACTH-Secreting Pituitary ior Pituitary Gland in Vitro: Effects of Tumors 902 Fasting in Male and Female Rats. . . . 1022 TAN A YAM A, S., AND M. Ui: Decrease in the GOODALL, C. M.: Restoration by Iodide Concentrations of Ketone Bodies and of Hepatic Reactions to 2-Aminofluor- Free Fatty Acids in Rat Blood in Re• ene in Thyroidectomized Rats 1027 sponse to a Glucose Load: Its Possible YASUDA, M., AND D. C. JOHNSON: Effects Relation to Endogenous Insulin Activ• of Exogenous Androgen and Gonado• ity 910 tropins on the Testes and Hypophysial RUHMANN, A. G., AND D. L. BERLINER: Follicle-Stimulating Hormone Content Effect of Steroids on Growth of Mouse of the Immature Male Rat 1033 Fibroblasts in Vitro 916 ADAMS, W. C, AND J. H. LEATHEM: In• PARKER, M. L., L. JARETT, D. S. SCHALCH, fluence of Thyroid Hormones on Choles• AND D. M. KIPNIS: Rat Growth Hor• terol Metabolism in Rats with Induced mone: Immunofluorescent and Radio- Ovarian Cysts 1041 immunologic Studies 928 BOWERS, C. Y., AND A. V. SCHALLY: Ef• IMURA, H., S. MATSUKURA, H. MATSU- fects of Age, Sex, Castration and the YAMA, T. SETSUDA, AND T. MlYAKE: Administration of Testosterone and Adrenal Steroidogenic Effect of Adeno• Estradiol on Levels of Serum Choles- sine 3'-5'-Monophosphate and Its De• teryl Ester Fatty Acids in Rats 1047 rivatives in Vivo 933 SCHENKMAN, J. B., D. A. RICHERT, AND OSORIO, C, AND N. B. MYANT: Relation W. W. WESTERFELD: «-Glycerophos- Between the Biliary Excretion of Thy• phate Dehydrogenase Activity in Rat roxine and Its Binding by the Serum Spermatozoa 1055 Proteins in Monkeys 938 BORIS, A.: Endocrine Studies of a Non• WOLFF, J., AND J. E. RALL: Thyroidal Io• steroid Anti-androgen and Progestin. . . 1062 dide Transport. VII. Acute Stimulation LICHTON, I. J.: Survival and Endocrine of Thyroidal Iodide Concentrating Function of Rat Placenta Implanted to Ability by Cystamine and Cysteamine 949 the Spleen 1068 WAGNER, J. W., AND K. BROWN-GRANT: GALA, R. R., AND U. WESTPHAL: Cortico- Studies on the Time of Luteinizing Hor• steroid-Binding Globulin in the Rat: mone Release in Gonadotrophin-Treated Possible Role in the Initiation of Lacta• Immature Rats 958 tion 1079 SHARMA, D. C, R. I. DORFMAN, AND A. L. KOWALEWSKI, K.: Changes in the Growth SOUTHREN: Steroid Biosynthesis in and Body Composition of Suckling Rats Vitro by Feminizing Testes 966 Caused by the Treatment of Lactating TASHJIAN, A. H., JR., L. LEVINE, AND Mothers with Steroid Hormones: Its P. L. MUNSON: Partial Hydrolysis of Relation to Litter Size 1089 Bovine Parathyroid Hormone by Dilute GENUTH, S., AND H. E. LEBOVITZ: Stimu• Acid and Proteolytic Enzymes: Effects lation of Insulin Release by Cortico• on Immunological and Biological Ac• tropin 1093 tivities 979 STEVENS, W., C. COLESSIDES, AND T. F. DOUGHERTY: Effects of Cortisol on the Incorporation of Thymidine-2-l4C Into NOTES AND COMMENTS Nucleic Acids of Lymphatic Tissue from PENHOS, J. C, B. A. HOUSSAY, AND Adrenalectomized CBA Mice 1100 M. A. LUJAN: Total Pancreatectomy MANNS, J. G., AND J. M. BODA: Effects of in Lizards. Effects of Several Hor• Ovine Growth Hormone and Prolactin mones 989 on Blood Glucose, Serum Insulin, RENNELS, E. G.: Sialic Acid Concen• Plasma Nonesterified Fatty Acids and trations in the Pituitary Glands of Amino Nitrogen in Sheep 1109 Normal and Castrated Rats 994 GLENN, E. M., AND J. GRAY: Effect of DELONG, M., AND K. BALOGH, JR.: Glu- Various Hormones on the Growth and cose-6-phosphate Dehydrogenase Ac• Histology of the Gerbil (Meriones un- tivity in the Subcommissural Organ guiculatus) Abdominal Sebaceous Gland, June 1965 VOLUME CONTENTS vii
Pad. 1115 Effect of Estrogen or Testosterone on CREEK, R. O.: Effect of Thyrotropin on Initiation and Maintenance of Sper• the Weight, Protein, Ribonucleic Acid, matogenesis in the Rat 1184 and the Radioactive Phosphorus Up• NOMURA, J.: Effect of Stress and Psycho- take of Chick Thyroids 1124 tropic Drugs on Rat Liver Tryptophan GOODMAN, H. M.: Early and Late Effects Pyrrolase 1190 of Growth Hormone on the Metabolism EVERETT, J. W.: Ovulation in Rats from of Glucose in Adipose Tissue 1134 Preoptic Stimulation Through Platinum GANONG, W. F., T. C. LEE, E. E. VAN Electrodes. Importance of Duration and BRUNT, AND E. G. BIGLIERI: Aldoster• Spread of Stimulus 1195 one Secretion in Dogs Immunized with Hog Renin 1141 NOTES AND COMMENTS ROSEMBERG, E., P. KELLER, W. B. LEWIS, FUJITA, T., H. ORIMO, M. YOSHIKAWA, A. ALBERT, G. CARL, AND D. BENNETT: H. MORII, AND K. NAKAO: Effect of Influence of Follice-Stimulating Hor• Acidosis and Alkalosis on Recovery mone on the Estimation of Luteinizing from Hypocalcemia 1202 Hormone in the Ventral Prostate and SUZUKI, M., S. WATANABE, AND M. Ovarian Ascorbic Acid Depletion As• HOSHII: Effect of Estrogen on Cop- says 1150 per-Induced Ovulation in the Rabbit 1205 RAMIREZ, V. D., AND C. H. SAWYER: Ad• BACA, Z. V., AND H. CHIODI: Develop• vancement of Puberty in the Female mental Changes in the Size and As- Rat by Estrogen 1158 corbie Acid Content of the Adrenals KRAGT, C. L., AND J. MEITES: Stimulation of White Rats 1208 of Pigeon Pituitary Prolactin Release GREENWALD, G. S.: Luteolytic Effect of by Pigeon Hypothalamic Extract in Estrogen on the Corpora Lutea of Vitro 1169 Pregnancy of the Hamster 1213 HOPKINS, T. F., AND G. PINCUS: Effects of MEITES, J.: Maintenance of the Mam• Rat Hypothalamic and Cerebral Tissue mary Lobulo-Alveolar System in Rats on PMS-Induced Ovulation 1177 After Adreno-Orchidectomy by Pro• STEINBERGER, E., AND G. E. DUCKETT: lactin and Growth Hormone 1220 Adrenocorticotrophin and Adrenal Protein Synthesis1
PETER C. SCRIBA- AND WILLIAM J. REDDY Departments of Biological Chemistry and Medicine, Harvard Medical School, and the Department of Medicine, Peter Bent Brigham Hospital, Boston 15, Massachusetts
ABSTRACT. In vitro protein synthesis by a 14 XlO1, and was inactivated by incubation at cell-free preparation (15,000 Xg supernatant 55 C and also by incubation with trypsin. It fraction) of pig adrenals was studied, measur• was not inactivated by ribonuclease. Nucleic ing glycine-1'C and /-leucine-14C incorporation acids prepared by several methods were inac• into protein. Adrenocorticotrophin adminis• tive. The "supernatant factor" could be puri• tered to rats in vivo caused a significant in• fied by diethylaminoethyl cellulose chromatog• crease in the total activity of nondialyzable raphy, and chemical analysis revealed the pro• "supernatant factor" stimulating amino acid tein nature of the material with virtually no incorporation into protein. The specificity of ribonucleic acid present in the preparation. It the observed effect for adrenocorticotrophin is tentatively concluded that this material is was confirmed by experiments with both a one of the transfer enzymes involved in pro• synthetic tricosapeptide and a highly purified tein synthesis, several other possibilities hav• preparation obtained from natural sources. ing been excluded. (Endocrinology 76: 745, The "supernatant factor" was nondialyzable, 1965) had an approximate molecular weight of 10-
HE ADMINISTRATION of adre• Materials Tnocorticotrophin in vivo (1) increased The preparations of adrenocorticotrophin the activity of a nondialyzable factor in were generously donated as follows: Corti- cotrophin-gel, porcine (80 USP units/ml) the 105,000 Xg supernatant of rat adre• lots SB 543; SB 849, PR 583, by Dr. F. G. nals that stimulated the incorporation of McMahon, The Upjohn Co.; Oxycel adreno• glycine-l4C into protein when added to a corticotrophin, bovine (86 USP units/mg) cell-free preparation from the adrenals of batch CN 145 by Dr. E. A. Laso-Wasem, control rats. Evidence has been pre• Wilson Co.; and N-terminal tricosapeptide synthetic (109 USP units/mg) by Dr. K. sented (1) indicating that this stimu• Hofmann, University of Pittsburgh.
lation occurs at a step after the forma• 14 1 Glycine-U- C and Ueucine-U- 'C were tion of amino acyl-sRNA. Since adrenal purchased from Nuclear-Chicago, and the growth is such an integral part of pitui• specific activity of each was diluted to 2.79 tary-adrenal interrelationships, it was juc/jLimole by the addition of carrier. Other felt of interest to characterize further materials were obtained as follows: phos- phoenolpyruvate (purchased as a barium salt this "supernatant factor.'' and converted to the potassium salt), di- sodium-adenosine triphosphate, adenosine• Received October 15, 1964. s'^'-cyclic phosphoric acid, trypsin, and 1 This study was supported by Grant A-4328 diethylaminoethyl cellulose (capacity: 0.9 from the NIH, USPHS, Bethesda 14, Maryland. mEq/g; mesh: medium, 19t D 62-13-240) Presented in part at the 47th Annual Meeting of from Sigma Chemical Co.; trisodium guano- the Federation of American Societies for Ex- sine triphosphate, crystalline lactic dehydro• parimental Biology, Atlantic City, New Jersey, genase (2 mg/ml) and pyruvate kinase (10 April, 1963 (48). 2 mg/ml) from Calbiochem.; ribonuclease from Postdoctoral Research Fellow FF-428, 1962- 63, of the NIH, USPHS. Present address: II. Worthington Biochemical Co.; dialysis tub• Medizinische Klinik der Universitaet, Muenchen, ing (No. 4465-A2) from A. H. Thomas Co.; Ziemssenstrasse 1, Germany. pig adrenals from Northeast Packing Co., 745 746 SCRIBA AND REDDY Volume 76
TABLE 1. Effect of addition of different 0.4 ml of the 15,000 Xg supernatant of pig substances to the assay system on U adrenals, 0.005M Mg++, 0.017M K+ 0.039M the glycine- C incorporation Tris-HCl buffer, pH 7.5, 0.17M sucrose, and Addition* % Change 0.4 ml of the test solution to be assayed. These mixtures were incubated at 37 C for Amino acid mixture 0 60 min (unless otherwise stated). The pro• Adenosine-3 ',5 '-cyclic phosphoric 4 tein concentration was measured (5), and acid 10~ M 0 Sodium ascorbate 1.6 X10 ~2M 0 the radioactivity of the residue insoluble in 57 fig PEP kinase 0 hot trichloroacetic acid was determined by 114 jug PEP kinase, 20 ^moles means of a liquid scintillation counter PEP and 2 moles ATP 0 M (Nuclear-Chicago) as previously described .25 Mmole GTP 0 7X10~5M Glutathione -12 (1). Differences between groups of values 7 XIO-'M Glutathione -33 were analyzed for significance by the t 7X10-3M Glutathione -66 test (6). 0.4 ml 15,000 X g supernatant Sucrose density gradients containing of pig adrenals + 100
0.025M KCl, 0.005M MgCl2, 0.05M Tris-HCl * All additions were added in 0.4 ml of Tris- buffer, pH 7.5, were prepared by a modifica• HC1 buffer containing sucrose and ions at the tion3 of the method of Britten and Roberts concentrations noted in the text. The pH of the (7). The gradients were centrifuged for 1-2 solution was checked and adjusted to 7.5 when necessary. hr at 37,500 rpm using the swinging bucket rotor SW-39 and the Spinco Model L ultra- Somerville, Mass.; male Sprague-Dawley centrifuge. The linearity of gradients pre• rats (250-300 g) from Charles River Breed• pared in this manner was checked by the ing Co. addition of a dye to the sucrose solution with Methods the higher concentration. Two hundred to 400 //I of test solutions was layered very care• The 15,000 Xg supernatants obtained from fully on top of such gradients. After cen• pig adrenals were employed as the basic trifugation, the tubes were punctured at the system for testing the stimulation of amino bottom and fractions were collected dropwise acid incorporation into proteins. The adrenals in the conventional manner. were placed in ice-cold 0.9% NaCl approxi• mately 20 min after death, dissected free of adherent fat tissue, and frozen (—10 C). Results Prior to each incubation, pig adrenals were Omission of adenosine triphosphate homogenized in medium A (200 mg/ml) of and the regenerating system resulted in
Littlefield and Keller (2) for 20 sec in a 14 Virtis blender and then for 3-5 min in a glass only 11% (22 cpm) of the glycine- C homogenizer equipped with a motor-driven incorporation obtained in the complete Teflon pestle. The supernatant fluids ob• system (192 cpm). Dialysis of the pig tained after 15 minutes' centrifugation of the adrenal 15,000 Xg supernatant for 18 homogenates at 15,000 Xg and 5 C contained hours vs. medium A resulted in the loss of microsomes and the soluble fraction (3, 4). Amino acid incorporation into protein did ability to incorporate amino acids into not change after storage of the adrenals at protein, but this ability was restored by — 10C for more than 2 months. the addition of 1 jumole of each of 19 Rat adrenals were removed immediately amino acids4 to the incubation mixture. after death, adherent fat tissue was removed, and the adrenals were homogenized in 3 We wish to thank Mr. S. Slapikoff for intro• medium A in a glass homogenizer. The 120 ducing us to the unpublished modification of min X 105,000 Xg supernatant was obtained Slapikoff, S., J. M. Fessenden, and K. Moldave, as previously described (1). of the method for the preparation of sucrose gradients of Britten and Roberts (12). The complete incubation mixtures con• 4 Ala, try, lys, val, his, ser, thr, arg, ileu, phe, tained in a total volume of 1.25 ml the fol• glu-NHo, leu, asp, cySH, cyS, glu, met, pro, 14 lowing: 1 ßc (0.36 /xmole) glycine- C, 10 tyr were dissolved in O.OIN HCl (10 /zmoles/ml) /rnioles phosphoenolpyruvate, 1 fxmole adeno• and neutralized immediately before the incuba• sine triphosphate, 0.25 pinole guanosine tri• tions. Under these conditions all amino acids, phosphate, 57 fig dialyzed pyruvate kinase, except tyr and cyS, were completely soluble. \pril 1965 "SUPERNATANT FACTOR" IN PROTEIN SYNTHESIS 747
The effects of various additions to the TABLE 3. Effect of infusion of synthetic tricosapeptide on supernatant factor in complete assay system are presented in adrenals of hypophysectomized rats Table 1. Test solution cpm in The addition of the 105,000 Xg super• protein ± SE natant from adrenals of control rats 0 .4 ml of medium A 183 + 3 increased the incorporation of glycine 0.35 ml of 105,000 X g into protein (Table 2) compared with the supernatant of control rats 356 ± 10 .7 0.35 ml of 105,000 X g addition of an equal volume of buffer supernatant of treated rats 527 ±19 .5 (p <0.001). The addition of the 105,000 Xg supernatant from an equal weight of Hypophysectomized animals were provided with 0.9% NaCl, water and Purina laboratory adrenals from rats treated with adreno• chow ad lib. for 10 days. Five days prior to the corticotrophin (7 X20 USP units adreno- infusion, im treatment with 0.1 mg/day desoxy- corticosterone acetate (4-pregnene-21-ol-3,20- corticotrophin-gel, Upjohn, intramuscu• dione) for suppression of the zona glomerulosa larly over 3 days) results in a signifi• of the adrenal (26, 27) and with 0.01 mg/day Decadron phosphate (16a-methyl-9
measured upon addition of 0.4 ml of each fraction of one density gradient as test solution to the assay system. The protein content of consecutive 0.1 ml fractions of the other gradients containing pyruvate kinase or lactic dehydrogenase was determined. Calculations were carried out as described by Martin and Ames using data from the literature (10-12) and the data obtained in the present experiments. The results indicate a molecular weight of 1.0-1.4 XlO5 and an
S2oW of 6 to 7 for the stimulating factor. The "supernatant factor" of adrenals from control or rats treated with adreno• corticotrophin was stable upon preincu• FIG. 1. Distribution of activity of 105,000 X g supernatant of adrenals from rats treated with bation for 2 hours at 37 C and for 15 adrenocorticotrophin after centrifugation on a minutes at 45 C prior to addition to the sucrose gradient. The supernatant from 200 mg assay system. The activity from both of adrenal was layered on a 6-20% sucrose gradient and centrifuged for 19 hr at 37,500 sources was completely destroyed upon rpm, t= -fl C. Triangles represent glycine-uC preincubation for 15 minutes at 55 C or incorporation into protein upon addition of 0.3 higher temperature. ml of each fraction of the density gradients as test solution to the assay system. In the upper The effect of trypsin on the "superna• half of the figure, the optical density of 0.05 ml of tant factor'' was studied. Trypsin was each density gradient fraction diluted 1:8 is shown. In the lower half of the figure, no point shown to inhibit glycine incorporation is present for the upper area of the gradient. into protein by the assay system, and it This fraction in several experiments decreased was necessary to remove the trypsin incorporation to variable levels below that ob• tained with the basal test solution. from the test solution after preincuba• tion with this enzyme. As shown in the upper part of Fig. 2, trypsin having a Martin and Ames (9) have suggested molecular weight of 23,700 (13) did not the use of purified proteins of known sediment upon density gradient centrifu• molecular weights as references for the gation into the first four (counted from determination of an approximate molec• the bottom) fractions. Addition of 0.4 ml ular weight of an unknown substance. of fractions 3 and 4 as test solutions to The 105,000 Xg supernatant of adrenals the assay system allowed base-line incor• from rats treated with adrenocortico• poration (188 cpm) of glycine-14C into trophin was centrifuged on sucrose protein. Hence, the elimination of the gradients 5-20% and 5-13%. Pyruvate "supernatant factor" in fraction 4 (lower kinase and lactic dehydrogenase were part of Fig. 2) is ascribed to trypsin centrifuged simultaneously on separate digestion during the preincubation. Pre• gradients. The latter gradient was em• incubation with trypsin also inactivated ployed to improve the separation of the the "supernatant factor" present in an stimulating factor, but was without equal weight of adrenals from control significant effect. Centrifugation was rats. performed for 16.5 hours at 37,500 rpm Preincubation with ribonuclease did and +1 C. Glycine-14C incorporation was not cause inactivation of the "superna- April 1965 "SUPERNATANT FACTOR" IN PROTEIN SYNTHESIS 749 tant factor." "Supernatant factor" from 200 adrenals of (adrenocorticotrophin-gel, Upjolhn, 140 USP units intramuscularly over 3 days)—treated rats was prepared by concentrating the activity from the
120 min X 105,000 Xg supernatant into a CP loose pellet by centrifugation at 140,000 Xg for five hours and was preincubated for 30 minutes at 37 C with and without 20 jug ribonuclease. The incubation mix• tures were centrifuged for 15 hours at o1— --' ' - '•• - 1 1 ' L—J 37,500 rpm and +1 C on a 5-13% su• 10 20 30 40 42 bottom mm 'op crose gradient, and aliquots of the frac• FIG. 2. Effect of trypsin on the factor stimulating tions of both gradients were assayed for 14 glycine- C incorporation. The fractions of a stimulation of glycine incorporation in density gradient (5-13 % sucrose) containing the the usual test system. Neither the total "supernatant factor" (cf Fig. 1) were combined activity nor the pattern of sedimentation and the active material was concentrated into a loose pellet by centrifugation for 5 hr at of the "supernatant factor" was affected 140,000 X g. This material was incubated for 60 by treatment with ribonuclease. This min at 37 C in the absence and in the presence of lack of effect was confirmed by experi• trypsin, 2 mg in 0.4 ml. The 2 incubation mix• tures and an equivalent amount of trypsin were ments in which bentonite was used for each layered on top of a 5-13 % sucrose gradient. the adsorption of ribonuclease. The 3 samples were centrifuged simultaneously for 16 hr at 37,500 rpm at +1 C. Triangles are In similar experiments the effect of glycine-uC incorporated into protein by the 0.5% sodium lauryl sulfate on the gly• assay system upon addition of 0.4 ml of each cine-UC incorporation was studied. No fraction of the density gradients which contained the "supernatant factor." Points are pg of pro• effect of sodium lauryl sulfate (14) on tein per 0.1 ml of each fraction of the density the sedimentation of the activity on su• gradient which contained trypsin alone. crose gradients was observed, suggesting that the material is not a nucleoprotein. Phenol extraction (15) of either Xg supernatant of adrenals from rats 105,000 Xg supernatant of adrenals from treated with adrenocorticotrophin-gel rats treated with adrenocorticotrophin- was chromatographed on a diethylamino- gel or active fractions obtained after ethyl cellulose column as described by density gradient centrifugation resulted Takanami (17). The diethylaminoethyl in complete loss of stimulation of gly• cellulose was prepared as previously de• cine-14C incorporation. Care had to be scribed (18). More than 90-95% of the taken to assure complete extraction of recovered activity was eluted by the protein from the aqueous layer, and, KCl gradient into samples 5 and 6 (Fig. when necessary, the phenol treatment 3), i.e., between 0.15 and 0.275M potas• was repeated (14). Ribonucleic acid sium (approximately 0.17 and 0.295M prepared by the procedure of Sevag et al. chloride). The highest specific activity (16) was inactive when added to the was in sample 6. The chromatography assay system. on diethylaminoethyl cellulose provided The above results suggested the pro• a total yield of 53, 58 and 70% of the tein nature of the "supernatant factor," stimulating activity. The pattern of elu- and it was suspected that it might be a tion was identical with the one described transfer enzyme. The dialyzed 105,000 by Takanami (17) for transfer enzyme. 750 SCRIBA AND REDDY Volumie 76
FIG. 3. Distribution of activity following (chro• matography on dietthyl- aminoethyl cellulosae of the 105,000 X g X 2 hr supernatant prepared from the homogenaite of 1473 mg of adrcenals from rats treated with adrenocorticotrophim-gel. After determination of the optical density at
260 and 280 mM„ the fractions collected from the column were? re- combined into 8 sam• ples as indicated b>y the vertical bars. Tine ac• tivity of aliquots was determined by true test system, and the total stimulatory activity (columns) was (calcu• lated. Protein comtents of these samples were: 1, 5.7 mg; 2, 1.1 nng; 3, 0.52 mg; 4, 4.98> mg; 5, 5.0 mg; 6, 3.36 mg; Somple 7, 0.29 mg; 8, 0.3,8 mg.
The amino acid-activating enzymes are and it contained 2.4 mg of proteiin. In eluted before this fraction.5 both the cold and the hot trichloroacetic Sucrose gradient centrifugation of the acid extracts, chemical analysis for RNA preparation with the highest specific ac• was carried out as described by Schneider tivity (sample 6, Fig. 3) showed only (19), using the colorimetric assay for partial sedimentation of the protein into ribose of Dische (20). Zero readings the fraction where the "supernatant were obtained. From the limits of sensi• factor" moved, indicating further puri• tivity of this technique, this preparation fication. Upon storage at 3 C, 50% of contained less than 1 % ribonucleic acid. the activity in sample 5 (Fig. 3) was re• The "supernatant factor" prepared by covered after four weeks. diethylaminoethyl cellulose chromato• Aliquots of samples 5 and 6 (Fig. 3) graphy was shown (Table 4) to stimu• were now assayed for their protein and late the incorporation of both glycine- RNA content. A preparation containing 1 }C and leucine-14C into protein. 2.5 mg protein and weighing 2.6 mg was dissolved in distilled water and pre• Discussion cipitated with cold 5% TCA, washed In this study, the 15,000 Xg super• with ethanol-ethyl-acetate, extracted natant fraction of pig adrenals was twice for 15 minutes at 90 C with 5% chosen as the cell-free protein-synthesiz• TCA and washed again with ethanol. ing system. It had the obvious advan• The dry weight of the residue was 2.7 mg tage that the small amount of rat adrenal tissue was not a problem. On an
5 Schatz, D., and W. J. Reddy (unpublished equal weight basis, pig adrenal super• observations). natant appears to be approximately April 1965 "SUPERNATANT FACTOR" IN PROTEIN SYNTHESIS 751
U twice as responsive to the "supernatant TABLE 4. Incorporation of glycine- C and leucine-l4C into protein factor" from rat adrenals as the control rat adrenal 15,000 Xg supernatant (1). Test cpm in Protein The "supernatant factor" from rat solution Glycine-14C Leucine-14C adrenals was active in the 15,000 Xg I 162 24 supernatant from rat, pig, dog and rab• II 892 366 bit adrenals, whereas it did not stimu• III 1331 648 late glycine incorporation in an analo• The complete incubation mixtures (cf Meth• gous preparation from normal rat liver, ods) contained either 1 /j,c =0.36 /xmole glycine- 14 14 indicating organ rather than species C or leucine- C. Test solutions added were: I = medium A; 11=0.36 mg, and 111=0.72 mg specificity. of "supernatant factor" as prepared by diethyl• aminoethyl cellulose chromatography (cf Fig. The effect observed with commer• 3) in 0.4 ml medium A. For incubation condi• cial preparations of adrenocorticotrophin tions and determination of the radioactivity cf Methods. could have been caused by impurities. That this effect was due to adreno• corticotrophin has been demonstrated into the phenolic layers was assured, had in experiments with a synthetic peptide. no activity. Finally, more than 90% of Infusion6 of this compound into hypo- the activity recovered after chromatog• physectomized rats gave rise to material raphy on diethylaminoethyl cellulose in the soluble fraction of the adrenals was shown to be of protein nature, with that stimulated protein synthesis in the virtually no ribonucleic acid detectable cell-free system and sedimented on su• upon chemical analysis. crose gradients exactly as the "super• Recent evidence (21-23) indicates natant factor" from the adrenals of rats that, in addition to amino acid activat• treated with adrenocorticotrophin-gel. ing enzymes, s-RNA and ribosomes, two Although the synthetic peptide is only transfer enzymes are involved in protein a portion (23 amino acids) of the synthesis. Earlier studies had located the "physiological'' nonatriacontapeptide, stimulation of protein synthesis to a the assumption that the observed effect nondialyzable substance which acted be• is caused by the physiological principle yond the formation of amino acyl of adrenocorticotrophin seems justified. transfer ribonucleic acid (1). The pat• The factor stimulating amino acid in• tern of elution of the activity from the corporation has a molecular weight of diethylaminoethyl cellulose columns was approximately 1.0 to 1.4 XlO5 (S value identical with the one observed by = 6-7) and has the properties of a pro• Takanami (17) upon preparation of tein. Preincubation at 55 C or with transfer enzyme from rat liver. This and trypsin destroyed the activity. It was the protein nature of the "supernatant not inactivated by preincubation with factor" suggest that it is probably a ribonuclease. Nucleic acid fractions pre• transfer enzyme. pared by phenol extraction, in which ex- Ferguson (25) has reported that hai^tive extraction of protein material actinomycin markedly inhibits adrenal ribonucleic acid synthesis but does not affect adrenal steroidogenesis, which sug• 6 We wish to express our gratitude to Dr. gests that adrenocorticotrophin does not Philip Hirsch, Harvard Dental School, for in• act through synthesis of messenger structing us in the technique and for providing equipment used in the infusion experiments with ribonucleic acid. In previous work (24), hypophysectomized rats. puromycin was shown to block steroido- 752 SCRIBA AND REDDY Volume 76
genesis, indicating a relationship be• 12. Pesce, A., F. Stolzenbach, I. Freedberg, and N. O. Kaplan, Fed Proc 22: 486, 1963 tween adrenocorticotrophin-induced ste• (Abstract). roidogenesis and protein synthesis. Since 13. Tietze, F., J Biol Chem 204: 1, 1953. it has been shown previously (1) that 14. Gros, F., W. Gilbert, H. H. Hiatt, G. At- taroi, P. I. Spaur, and J. D. Watson, Cold there is no increased activity of amino Spring Harbor Symp Quant Biol 26: 111, acid-activating enzymes or transfer ribo• 1961. nucleic acids and messenger ribonucleic 15. Gierer, A., and G. Schramm, Nature (Lon• acid appears to be excluded by the don) 177: 702, 1956. 16. Sevag, M. G., D. B. Lackman, and studies of Ferguson, the only reasonable J. Smolens, J Biol Chem 124: 425, 1938. explanation left is that adrenocortico• 17. Takanami, M., Biochim Biophys Acta 51: trophin increases the activity of a trans• 85, 1961. 18. Peterson, E. A., and H. A. Sober, In Colo- fer enzyme. wick, S. P., and N. O. Kaplan, (eds.), References Methods in Enzymology, Academic Press, New York, 1962, vol. 5, p. 3. 1. Farese, R. V., and W. J. Reddy, Endo• 19. Schneider, W. C, J Biol Chem 161: 293, crinology 73: 294, 1963. 1945. 2. Littlefield, J. W., and E. B. Keller, J Biol 20. Dische, Z., In Chargaff, E., and J. N. Chem 224: 13, 1957. Davidson, (eds.), The Nucleic Acids, Aca• 3. Schneider, W. C, and G. H. Hogeboom, demic Press, New York, 1955, vol. I, p. 301. Cancer Res 11: 1, 1951. 21. Fessenden, J. M., and K. Moldave, J Biol 4. Palade, G. E., and P. J. Siekevitz, Biophys Chem 238: 1479, 1963. Biochem Cytol 2: 171, 1956. 22. Bishop, J., and R. Schweet, Biochim Biophys 5. Lowry, O. H., N. J. Rosebrough, A. L. Farr, Acta 65: 553, 1962. and R. J. Randall, J Biol Chem 193: 265, 1951. 23. Lipmann, F., Basic Problems in Neoplastic 6. Snedecor, G. W., Statistical Methods, ed. 5, Disease, Columbia University Press, 1962, Iowa State College Press, Ames, Iowa, 1956, p. 131. p. 85. 24. Ferguson, J. J., Jr., J Biol Chem 238: 2754, 7. Britten, R. J., and R. B. Roberts, Science 1963. 131:32,1960. 25. Ferguson, J. J., Jr., and Y. Morita, Biochim 8. Hofmann, K., and H. Yajima, Recent Progr Biophys Acta 87: 348, 1964. Hormone Res 18: 41, 1962. 26. Gross, F., P. Loustalot, and R. Meier, 9. Martin, R. G., and B. N. Ames, J Biol Chem Acta Endocr (Kobenhavn) 26: 417, 1957. 236: 1372, 1961. 27. Pfeiffer, E. F., W. E. Vaubel, K. Retienne, 10. Warner, R. C., Arch Biochem 78: 494, 1958. D. Berg, and H. Ditschuneit, Klin Wschr 11. Edsall, J. T., In Neurath, H., and K. Bailey, 38: 980, 1960. (eds.), The Proteins, Academic Press, New 28. Hodges, J. R., and J. Vernikos-Danellis, York, 1953, vol. I, B, p. 563. Acta Endocr (Kobenhavn) 39: 79, 1962.