ENDOCRINOLOGY

V O L U M E 76

JANUARY-JUNE, 1965

EDITOR-IN-CHIEF: FREDERICK G. HOFMANN

ASSOCIATE EDITORS: WILBUR H . SAWYER, DONALD F . TAPLEY

EDITORIAL BOARD

SAVINO A . D'ANCELO

HOWARD A . BERN

E . M . BOCDANOVE

H . LEON BRADLOW

JAMES O. DAVIS

LEWIS L . ENGEL

JOHN W . EVERETT

W I L L I A M F . GANONG

MONTE A . GREER

NICHOLAS S. H A L M I

A . BAIRD HASTINGS

LEON D . HELLMAN

ROY HERTZ

FREDERICK L . HISAW

SIDNEY H . INGBAR

DONALD C. JOHNSON

ERNST KNOBIL

MAURICE E . K R A H L

S. M . MCCANN

ANDREW V . NALBANDOV

DON H . NELSON

MAURICE S. RABEN

HOWARD RASMUSSEN

MURRAY SAFFRAN

LEO T . SAMUELS

KENNETH SAVARD

MINDEL C. SHEPS

SAMUEL SOLOMON

KENNETH STERLING

CLARA M. SZEGO

A L V I N TAUROG

ELEANOR H . VENNING

ALFRED E . W I L H E L M I

JAN WOLFF

IRA G. WOOL

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 U l i . -

M ü n o h & n

C O N T E N T S O F V O L U M E 76

No. 1, January 1965 O T A , K . , Y . S H I N D E , A N D A. Y O K O Y A M A :

Relationship Between Oxytocin and Prolactin Secretion i n Maintenance of Lactation in Rats 1

GOLDSMITH, R. E. , J. W U L S I N , A N D M . J. WiESTER: Effect of Thyroparathyroid-ectomy on the Clinical Course and the Serum Calcium and Phosphorus Con-centration in Dogs 9

SNOOK, R. B . , A N D H . H . C O L E : Bio-assay of Antisera Against Human Chorionic Gonadotropin 20

PARLOW, A. F., P. G. C O N D L I F F E , L . E. R E I C H E R T , JR. , A N D A. E . W I L H E L M I : Recovery and Partial Purification of FSH and L H During the Purification of T S H from Human Pi tui tary G lands . . . . 27

N A T A F , B . M . , E . M . R I V E R A , A N D I . L . C H A I K O F F : Role of Thyrotropic Hor-mone i n Iodine Metabolism of Em-bryonic Rat Thyro id Glands in Organ Culture 35

B L O C H , E., A N D K . B E N I R S C H K E : In Vitro Steroid Synthesis by Fetal, Newborn and Adul t Armadillo Adrenals and by Fetal Armadillo Testes 43

B L A I R , A. J., JR. , A N D T . U E T E : Effect of Triamcinolone Administration on Corticosteroid Metabolism in the Liver . 52

E R R A T U M 57 M O R I I , H . , T . F U J I T A , H . O R I M O , S. O K I -

N A K A , A N D K . N A K A O : Effect of Sym-pathetic Stimulation, Epinephrine and Phentolamine on Recovery from I n -duced Hypocalcemia 58

B I T M A N , J., H . C. C E C I L , M . L . M E N C H , A N D T . R. W R E N N : Kinetics of in Vivo Glycogen Synthesis i n the Estrogen-Stimulated Rat Uterus 63

SIPERSTEIN, E. R., A N D V. F. A L L I S O N : Fine Structure of the Cells Responsible for Secretion of Adrenocorticotrophin in the Adrenalectomized Rat 70

CLERMONT, Y . , A N D S. C. H A R V E Y : Dura-t ion of the Cycle of the Seminiferous Epithelium of Normal, Hypophysec-tomized and Hypophysectomized-Hor-mone Treated Albino Rats 80

M U N S I C K , R. A. , A N D S. C. JERONIMUS: Effects of Diethylstilbestrol and Mag-nesium on the Rat Oxytocic Potencies of Some Neurohypophysial Hormones and Analogues 90

EDGREN, R. A. , A. F. P A R L O W , D . L . PETERSON, A N D R. C. JONES: On the Mechanism of Ovarian Hypertrophy Following Hemicastration in Rats 97

T A L M A G E , R. V. , J . NEUENSCHWANDER, A N D L . K R A I N T Z : Evidence for the Existence of Thyrocalcitonin in the Rat 103

R Y A N , K . J., A N D R. V . SHORT: Formation of Estradiol by Granulosa and Theca Cells of the Equine Ovarian F o l l i c l e . . . . 108

B A R K E R , S. B. , M . S H I M A D A , A N D M . M A K I U C H I : Metabolic and Cardiac-Responses to Thyroxine Analogs 115

V E R N I K O S - D A N E L L I S , J.: Effect of Stress, Adrenalectomy, Hypophysectomy and Hydrocortisone on the Corticotropin-Releasing Ac t iv i t y of Rat Median Eminence 122

C L I T H E R O E , H . J., A N D J . H . L E A T H E M : Effect of Estradiol and Ethamoxytri-phetol (MER-25) on the Mouse Uterus 127

K A T Z M A N , P. A . : Bio-assay of Estrogens by In t ra vaginal Injection in Immature Rats 131

A L B E R T , A. , I . D E R N E R , E. ROSEMBERG, A N D W . B . L E W I S : Assay Characteristics of Tannate Complexes of Ovine Gonado-tropins 139

N O T E S A N D COMMENTS

L A N G E R , P., A N D V. STOLC: Goitro-genic A c t i v i t y of Allylisothiocyanate— A Widespread Natural Mustard O i l . . . . 151

N I C O L L , C. S., A N D H . A. B E R N : Pigeon Crop-Stimulating Ac t iv i ty (Prolactin) in the Adenohypophysis of Lungfish and Tetrapods 156

M U N S I C K , R. A. : Hen Oxytocic Activities of Oxytocin and 1-Deamino-oxytocin... 161

T O N G , W. : Thyrotropin Stimulation of Thyroxine Synthesis in Isolated Thyroid Cells Treated w i t h Puromycin 163

K E L L E R , P. J.: Some Observations on the Ovarian Ascorbic Acid Depletion Assay for Luteinizing Hormone Employing Immature Female Mice 165

No. 2, February 1965 B E G G , D . J., E . M . M C G I R R , A N D H . N .

M U N R O : Protein and Nucleic Acid Con-tent of the Thyro id Glands of Different Mammals 171

A B R A H A M , S., L . K O P E L O V I C H , P. R. K E R K O F , A N D I . L . C H A I K O F F : Metabolic Characteristics of Preparations of Iso-lated Sheep Thyro id Gland Cells. I . Ac t iv i t y Levels of Enzymes Concerned wi th Glycolysis and the Tricarboxylic Acid Cycle 178

E I S E N S T E I N , A. B . , M . R O N A, A N D B . B R U M M E R : In Vitro Effects of Puromycin Aminonucleoside on Liver Carbohydrate Synthesis 191

V A N T I E N H O V E N , A. , D . S I M K I N , J . W E S K E , A N D G. R. B A R R : Effect of Thyroxine on the Gonadotrophin Dose Response Line Using the Chick's Testicular Response. 194

JOHNSTON, C. C , JR. , A N D W. P. DEISS , JR. : Some Effects of Hypophysectomy and Parathyroid Extract on Bone Ma-t r ix Biosynthesis 198

O Y A M A , J., A N D W . T . P L A T T : Metabolic Alterations in Rats Exposed to Acute Acceleration Stress 203

June 1965 V O L U M E C O N T E N T S 111

L E W I S , U . J., E . V . C H E E V E R , A N D W . P. V A N D E R L A A N : Studies on the Growth Hormone of Normal and Dwar f Mice . . 210

G O O D M A N , H . M . : In Vitro Actions of Growth Hormone on Glucose Metab-olism i n Adipose Tissue 216

G O R S K I , R. A . , A N D J . W . W A G N E R : Gonadal A c t i v i t y and Sexual Differ-entiation of the Hypothalamus 226

V E R N I K O S - D A N E L L I S , J.: Effect of Rat Median Eminence Extracts on Pi tui tary A C T H Content i n Normal and Adrenal-ectomized Rats 240

ARMSTRONG, D . T. , A N D R. O. G R E E P : Failure of Deciduomal Response to Uterine Trauma, and Effects of L H Upon Estrogen Secretion i n Rats w i th Ovaries Luteinized by Exogenous Gonadotropins 246

O R I M O , H . , T . F U J I T A , H . M O R I I , A N D K . N A K A O : Inactivation in Vitro of Parathyroid Hormone Ac t iv i t y by K i d -ney Slices 255

A L B E R T , A . , D . B E N N E T T , G. C A R L , E. ROSEMBERG, P. K E L L E R , A N D W . B . L E W I S : Durat ion of Response to Free and Tannated Luteinizing Hormone. . . 259

SEGAL, S., S. M I L K O V I C , A N D L . E . ROSENBERG: Amino Acid Accumulation by Kidney Cortex Slices and Intestinal Segments of Rats Bearing a Transplant-able Pi tu i ta ry Tumor 267

N A L L A R , R., A N D S . M . M C C A N N : Luteiniz-ing Hormone-Releasing Ac t iv i ty in Plasma of Hypophysectomized Rats. . . 272

L A W T O N , I . E., A N D N . B . SCHWARTZ: Effect of L H on Ovarian Ascorbic Acid Over a 24-Hour Period 276

R A M I R E Z , V . D . , A N D C. H . SAWYER: Fluctuations in Hypothalamic L H - R F (Luteinizing Hormone-Releasing Fac-tor) Dur ing the Rat Estrous Cycle 282

D H A R I W A L , A. P. S., R. N A L L A R , M . B A T T , A N D S. M . M C C A N N : Separation of Follicle-Stimulating Hormone-Releasing Factor from Luteinizing Hormone-Re-leasing Factor 290

T I S H L E R , P. V. , A N D S. H . I N G B A R : Cor-relative Effects of Puromycin on u l I Metabolism and Amino Acid Incorpora-t ion by Calf Thyroid Slices 295

K L E B A N O F F , S. J.: Inactivation of Estro-gen by Rat Uterine Preparations 301

JORGENSEN, E . C , A N D J . A . W . R E I D : Thyroxine Analogs. Synthesis and A n t i -goitrogenic Ac t iv i ty of 3'-Chloro- and 3 ̂ 5'-Dichloro-3,5-diiodo-L-thyronines in the Rat 312

H E L L E R S T R O M , C , I . - B . T Ä L J E D A L , A N D B. H E L L M A N : Quantitative Studies on Isolated Pancreatic Islets of Mammals. 5-Nucleotidase and Adenosine Triphos-phatase Activities i n Normal and Cort i-sone-Treated Rats 315

B R A Y , G. A. , A N D H . M . GOODMAN: Studies on the Early Effects of Thyroid Hor-mones 323

N O T E S A N D COMMENTS

R I E S , N . , A N D N . A L L E G R E T T I : Number

and Size of Thyroid Follicles i n Guinea Pigs of Different Ages 329

C A L L A N T I N E , M . R., R. R. H U M P H R E Y , A N D S. L E E : Effect of Follicle-Stimulat-ing Hormone on Ovarian Nucleic Acid Content 332

H O C H , F. L . : Synergism Between Calori-genic Effects: L-Thyroxine and 2,4-Dinitrophenol or Sodium Salicylate i n Euthyroid Rats 335

GROSVENOR, C. E. : Evidence that Extero-ceptive Stimuli Can Release Prolactin from the Pitui tary Gland of the Lactat-ing Rat 340

K U N I N , A. S., A N D S. M . K R A N E : Inhibi -t ion by Puromycin of the Calcium-Mobilizing Ac t iv i ty of Parathyroid Ex-tract 343

No. 3, March 1965 I B A Y A S H I , H . , M . N A K A M U R A , T . U C H I -

K A W A , S. M U R A K A W A , S. Y O S H I D A , K . N A K A O , A N D S. O K I N A K A : C I 9 Ste-roids i n Canine Spermatic Venous Blood Following Gonadotropin Administra-t ion 347

V A N Z Y L , A. , K . SCHULZ, B . W I L S O N , A N D D . PANSEGROUW: Thyroidal Iodine and Enzymatic Defects i n Cattle wi th Con-genital Goiter 353

L E W I S , U . J., E . V . C H E E V E R , A N D W . P. V A N D E R L A A N : Alteration of the Proteins of the Pi tui tary Gland of the Rat by Estradiol and Cortisol 362

F R I E S E N , H . : Purification of a Placental Factor w i t h Immunological and Chem-ical Similarity to Human Growth Hor-mone 369

R U B I N , B . L . , A N D H . W . D E A N E : Effects of Superovulation on Ovarian A r >-3/^-Hy-droxy steroid Dehydrogenase Ac t iv i t y in Rats of Different Ages 382

STANSFIELD, D . A. , A N D J . W . R O B I N S O N : Glycogen and Phosphorylase i n Bovine and Rat Corpora Lutea, and the Effect of Leuteinizing Hormone 390

H A L L , P. F.: Influence of Temperature Upon the Biosynthesis of Testosterone by Rabbit Testis in Vitro 396

K O I D E , S. S., A N D S. M . M I T S U D O : Histo-chemical Study of 3/3- and 17/3-Hydroxy-steroid Dehydrogenases i n Human Term Placenta 403

F A B I A N E K , J., A. H E R P , A N D W . P I G M A N : Permeability of Dermal Connective Tissue i n Adrenalectomized and H y -pophysectomized Rats 408

R A M I R E Z , V . D . , A N D S. M . M C C A N N : I n -hibitory Effect of Testosterone on Luteinizing Hormone Secretion in I m -mature and Adul t Rats 412

Y A T V I N , M . B . , A N D R. W . W A N N E -MACHER, JR . : Action of Adrenal Corti-coids on Protein Metabolism i n the Thiouracil-Treated Rat 418

C L A R K , A. F., J . I . R A E S I D E , A N D S. SOLO-M O N : Studies on the Urinary Excretion of 17-Ketosteroids by the Domestic Pig 427

i v V O L U M E C O N T E N T S Volume 76

JARRETT, R. J.: Effects and Mode of Ac-tion of Adrenocorticotrophic Hormone Upon the Reproductive Tract of the Female Mouse 434

SCRANTON, J. R., A N D N . S. H A L M I I Thyroidal Iodide Accumulation and Loss in Vitro 441

FISHER, C. J., A N D J. H . L E A T H E M : Effect of a Protein-Free Diet on Protein Metabolism i n the Pregnant Rat 454

M A E I R , D . M . : Species Variation i n Testic-ular A 5-3ß-Hydroxysteroid Dehydro-genase Ac t iv i t y : Absence of Ac t iv i t y in Primate Leydig Cells 463

S P A Z I A N I , E., A N D A. G U T M A N : Distr ibu-t ion Volumes of Sugars in Rat Uterus, Ileum and Skeletal Muscle as Affected by Estradiol 470

GALTON, V . A. , S. H . I N G B A R , A N D S. VON DER H E Y D E : Role of Catalases and Peroxidases i n the Deiodination of Thyroxine in Frog Liver 479

R A P P , J. P.: Alteration of Juxtaglomerular Index by Selective Inbreeding 486

SCHANE, H . P.: Estrogen-like Effect of Chelating Agents on Rat Uterine Phos-phorylase Ac t iv i ty 491

A L B E R T , A. , D . B E N N E T T , G. C A R L , E. ROSEMBERG, P. K E L L E R , A N D W . B . L E W I S : Assay Characteristics of Tan-nate Complexes of Human Chorionic Gonadotropin 499

A L B E R T , A. , D . B E N N E T T , G. C A R L , E. ROSEMBERG, P. K E L L E R , A N D W . B . L E W I S : Assay Characteristics of Tan-nate Complexes of Pregnant Mare Serum Gonadotropin 506

N I C O L I S , G. L . , A N D S. U L I C K : Role of 18-Hydroxylation i n the Biosynthesis of Aldosterone 514

M I L L S , J. B . , A N D A. E. W I L H E L M E : Sul-fitolysis of Bovine Growth Hormone. . . 522

GANONG, W . F., B . L . W I S E , R. S H A C K L E -FORD, A. T. , BORYCZKA, A N D B . Z I P F : Site at Which a-Ethyltryptamine Acts To Inh ib i t the Secretion of A C T H 526

NOTES A N D COMMENTS

GOODMAN, H . M . : Effects of Insulin on Water Uptake by Adipose Tissue Dur-ing Incubation in Vitro 531

R E N N I E , P., A N D J. D A V I E S : Implantation in the Rabbit Following Administration of 20a-Hydroxypregnen-3-one and 20/3-Hydroxypregnen-3-one 535

B A U M A N , T . R., G. W . PIPES, A N D C. W . T U R N E R : Relative Potency of Several Analogues When Substituted for L -Thyroxine in the Estimation of Thyro id Secretion Rate in Rats 537

LERNER, L . J., A N D A. R. T U R K H E I M E R : Effect of the Lack of Dietary Protein on the Uterine Response to Estrogen i n the Mouse 539

M O U N I B , M . S., A N D M . C. C H A N G : Me-tabolism of Endometrium and Fallopian Tube i n the Estrous and the Pseudo-pregnant Rabbit 542

B R A V E R M A N , L . E., A N D S. H . I N G B A R : Binding of 3,5,3'-L-Triiodothyronine in

Human Serum Dur ing Agar Gel Elec-trophoresis at p H 7.4 547

F A I N , J . N . : Comparison of Glucocorticoid Effects on Brown and White Adipose Tissue of the Rat 549

H A G E R M A N , D . D . : Early Effect of Estro-gen on Phosphate Metabolism in Rat Uterine Nuclei 553

M A L O O F , F. , A N D M . SOODAK: Nature of the Porphyrin Group of a Heme Protein Fraction Isolated from Mammalian Thyro id Microsomes 555

No. 4, April 1965 S H I K I T A , M . , A N D B . - I . T A M A O K I : Testos-

terone Formation by Subcellular Parti-cles of Rat Testes 563

S H I M A O K A , K . , A N D B . D . THOMPSON: Ra-dioiodine Labeling of Serum Albumin in Vivo 570

JONES, A. E. , J . N . F I S H E R , U . J . L E W I S , A N D W . P. V A N D E R L A A N : Electro-phoretic Comparison of Pitui tary Glands from Male and Female Rats 578

W O E B E R , K . A. , A N D S. H . I N G B A R : A n t i -thyroid Effect of Noncalorigenic Con-geners of Salicylate, w i t h Observations on the Influence of Serum Proteins on the Potency of Ant i thyro id A g e n t s . . . . 584

E L L I S , L E G . C., A N D D . L . B E R L I N E R : Se-quential Biotransformation of 5-Preg-nenolone^ a - 3 H and Progesterone-4-uC Into Androgens by Mouse Testes 591

L A Y N E , D . S.: Identification i n Rabbit Urine of the 3-Glucuronoside-17-N-acetylglucosaminide of 17«-Estradiol . . 600

KOSTYO, J . L . : In Vitro Effects of Adrenal Steroid Hormones on Amino Acid Transport i n Muscle 604

K U R O S H I M A , A. , Y . I S H I D A , C. Y . BOWERS, A N D A. V . S C H A L L Y : Stimulation of Re-lease of Follicle-Stimulating Hormone by Hypothalamic Extracts in Vitro and in Vivo 614

H O F F M A N N , J . C , A N D N . B . SCHWARTZ: Timing of Post-partum Ovulation i n the Rat 620

H O F F M A N N , J . C , A N D N . B . SCHWARTZ: Timing of Ovulation Following Pro-gesterone Withdrawal i n the Ra t . . . . 626

D E G R O O T , L . J., J . E. THOMPSON, A N D A. D . D U N N : Studies on an Iodinating Enzyme from Calf Thyroid 632

CHRISTENSEN, A . K . , A N D N . R. M A S O N : Comparative Ab i l i t y of Seminiferous Tubules and Interst i t ial Tissue of Rat Testes To Synthesize Androgens from Progesterone-4-14C in Vitro 646

M A R I E B , N . J., A N D P. J . M U L R O W : Role of the Renin-Angiotensin System in the Regulation of Aldosterone Secretion in the Rat 657

S W I S L O C K I , N . I . , A N D C M . SZEGO: Acute Reduction of Plasma Nonesterified Fa t ty Acid by Growth Hormone in Hypophysectomized and Houssay Rats 665

O P E L , H . : Oviposition in Chickens After Removal of the Posterior Lobe of the Pi tui tary by an Improved M e t h o d . . . . 673

June 1965 V O L U M E C O N T E N T S v

C A T T , K . , A N D B . M O F F A T : Fractionation of Rat Pi tu i ta ry Extract by Starch Gel Electrophoresis and Identification of Growth Hormone and Prolactin 678

S C H A N E , H . P . , A N D S. L . L E O N A R D : Rate of Glycogenolysis in the Estrogen-Primed Rat Uterus in Vitro 686

K A N E M A T S U , S., A N D C . H . S A W Y E R : Blockade of Ovulation i n Rabbits by Hypothalamic Implants of Norethin-drone 691

CHOWERS, L , A N D S. M . M C C A N N : Con-tent of Luteinizing Hormone-Releasing Factor and Luteinizing Hormone Dur-ing the Estrous Cycle and After Changes i n Gonadal Steroid Titers 700

A N T O N I A D E S , H . N . , A . M . H U B E R , B . R . B O S H E L L , C . A . SARAVIS, A N D S. N . GERSHOFF: Studies on the State of Insu-l in i n Blood: Properties of Circulating "Free" and "Bound" Insulin 709

Erra tum 721 K A T Z , S. R . , J. D A N C I S , A N D M . L E V I T Z :

Relative Transfer of Estriol and I ts Conjugates Across the Fetal Mem-branes in Vitro 722

L I N D S A Y , R . H . , H . N A K A G A W A , A N D P . P . C O H E N : Thiouracil-Pyrimidine Rela-tionships i n Thyro id and Other Tissues 728

L I N D S A Y , R . H . , A N D P . P . C O H E N : N u -cleic Acid Synthesis i n Normal and Goitrous Rat Thyro id 737

SCRIBA, P . C , A N D W . J. R E D D Y : Adreno-corticotrophin and Adrenal Protein Synthesis 745

B U R S T E I N , S., B . R . B H A V N A N I , A N D H . L . K I M B A L L : Genetic Aspects of Cortisol Hydroxylat ion in Guinea Pigs: Urinary Excretion and Production Rates of Cortisol and 2a-HydroxyCorti-sol 753

R E I C H E R T , L . E., JR. , A N D A. E. W I L -H E L M I : Preparation of Equine Luteiniz-ing Hormone 762

N O T E S A N D COMMENTS

U C H I D A , K . , M . K A D O W A K I , A N D T . M I Y A K E : Failure of Estrogen To Pro-duce a Hypocholesterolemic Effect in Immature Rats 766

H A Y S , R . L . , E. W . H A H N , AND K . A. K E N D A L L : Evidence for Decreased Steroidogenesis in Pregnant Rats Fed a Sucrose Diet 771

SWEAT, M . L . , A N D M . J. BRYSON: Ster-oid 11/3-Oxygenation, Ascorbic Acid, Epinephrine and Andrenochrome I n -terrelations in the Adrenal G l a n d . . . . 773

S W I G ART, R . H . , F . K . H I L T O N , M . M . D I C K I E , A N D B . J. FOSTER: Effect of Gonadal Hormones on Submandibu-lar Gland Amylase Act iv i ty i n Male and Female C57BL/6J Mice 776

I B A Y A S H I , H . , T . Y A M A J I , T . T A J I M A , A N D K . N A K A O : Antigranulomatous Act iv i ty of Vi tamin K i and I ts Sum-mation w i t h That of Prednisolone. . . 780

PINCUS, J. H . , R . G. F E L D M A N , G. R A N N A Z Z I S I , A N D S. N A T E L S O N : Elec-tromyographic Studies w i t h Pituitary

Extracts Which Lower Serum Cal-cium and Raise Serum Citrate Levels 783

F R I E D M A N , R. C , A N D S. R E I C H L I N : Growth Hormone Content of the Pi-tui tary Gland of Starved Rats 787

H O S H I N O , K . : Development and Func-tion of Mammary Glands of Mice Prenatally Exposed to Testosterone Propionate 789

FARESE, R. V. : Quantitative Compari-son of the Effects of A C T H Adminis-trat ion on the Activities of Soluble Cell Fraction and Microsomes for I n -corporation of Amino Acid In to Pro-tein 795

W U R T M A N , R. J., J. A X E L R O D , S. H . SNYDER, A N D E. W . C H U : Changes in the Enzymatic Synthesis of Mela-tonin in the Pineal During the Estrous Cycle 798

H A L K E R S T O N , I . D . K . , M . F E I N S T E I N , A N D O. H E C H T E R : Further Observa-tions on the Inhibi t ion of Adrenal Protein Synthesis by A C T H in Vitro 801

No. 5, May 1965 C O R B I N , A. , G. M A N G I L I , M . M O T T A , A N D

L . M A R T I N I : Effect of Hypothalamic and Mesencephalic Steroid Implanta-tions on A C T H Feedback Mechanisms 811

B I R M I N G H A M , M . K . , G. ROCHEFORT, A N D H . T R A I K O V : Steroid Fractions from Incubated Normal and Regenerated Adrenal Glands of Male and Female Rats 819

H E I D E M A N , M . L . , JR. , R. P. L E V Y , W . L . M C G U I R E , A N D R. A. S H I P L E Y : l 3 l I -Labeled Bovine Thyrotropin: Prepara-t ion Through a New Micro Technique of Iodine Disti l lation and Studies of Ad-sorption to Glass and Polyethylene . . . . 828

A S S A Y K E E N , T . A., A N D J. A . T H O M A S : Endogenous Histamine in Male Organs of Reproduction 839

A B E , T. , A N D C. V I L L E E : A Uterine Growth-Promoting Substance from Hu-man and Bovine Uterine Muscle 814

B U H L E R , D . R., R. C. T H O M A S , JR. , A N D C. A. SCHLÄGEL: Absorption, Metab-olism and Excretion of 6e*-Methy]pred-nisolone- 3H, 21-acetate Following Oral and Intramuscular Administrations in the Dog 852

COPPOLA, J. A. , A N D J. W. P E R R I N E : I n -Influence of Two Nonsteroidal Antiös-trogens on Vaginal Opening and PMS-Induced Ovulation i n Rats 865

L E O N A R D , S. L . , A N D H . P. SCHANE: Hor-monal Effects on Phosphorylase Ac-t i v i t y and Glycogen Levels i n the Rat Ductus Deferens 870

STEINETZ, B . G., J. P. M A N N I N G , M . B U T -LER, A N D V . B E A C H : Relationships of Growth Hormone, Steroids and Relaxin i n the Transformation of Pubic Joint Cartilage to Ligament i n Hypophysec-tomized Mice 876

GROSVENOR, C. E., S. M . M C C A N N , A N D R. N A L L A R : Inhibi t ion of Nursing-In-

v i

duced and Stress-Induced Fall i n Pi tu i -tary Prolactin Concentration i n Lactat-ing Rats by Injection of Acid Extracts of Bovine Hypothalamus 883

L E V Y , R. P., W. L . M C G U I R E , R. K . SHAW, A N D G . E . BARTSCH: Effect of Species Differences of Mice on the Bio-assay of Thyrotropin 890

G A L I C I C H , J . H . , F. H A L B E R G , L . A. FRENCH, A N D F. U N G A R : Effect of Cere-bral Ablation on a Circadian Pi tui tary Adrenocorticotropic Rhy thm in C Mice 895

W E B E R , G., R. L . S I N G H A L , S. K . SRIVAS-TAVA, H . J . H I R D , A N D J . F U R T H : Be-havior of Hepatic Gluconeogenic En-zymes, R N A Metabolism and Amino Acid Level i n Rats Carrying Trans-plantable ACTH-Secreting Pitui tary Tumors 902

T A N A Y A M A, S., A N D M . U i : Decrease in the Concentrations of Ketone Bodies and Free Fat ty Acids i n Rat Blood i n Re-sponse to a Glucose Load: I t s Possible Relation to Endogenous Insulin Act iv-i ty 910

R U H M A N N , A. G., A N D D . L . B E R L I N E R : Effect of Steroids on Growth of Mouse Fibroblasts in Vitro 916

PARKER, M . L . , L . JARETT, D . S. SCHALCH, A N D D . M . K I P N I S : Rat Growth Hor-mone: Immunofluorescent and Radio-immunologic Studies 928

I M U R A , H . , S. M A T S U K U R A , H . M A T S U -YAMA, T . S E T S U D A , A N D T . M l Y A K E : Adrenal Steroidogenic Effect of Adeno-sine 3'-5'-Monophosphate and I ts De-rivatives in Vivo 933

OSORIO, C , A N D N . B . M Y A N T : Relation Between the Bil iary Excretion of Thy-roxine and I t s Binding by the Serum Proteins i n Monkeys 938

W O L F F , J., A N D J . E. R A L L : Thyroidal Io-dide Transport. V I I . Acute Stimulation of Thyroidal Iodide Concentrating Abi l i ty by Cystamine and Cysteamine 949

W A G N E R , J . W . , A N D K . B R O W N - G R A N T : Studies on the Time of Luteinizing Hor-mone Release i n Gonadotrophin-Treated Immature Rats 958

SHARMA, D . C , R. I . D O R F M A N , A N D A. L . SOUTHREN: Steroid Biosynthesis in Vitro by Feminizing Testes 966

T A S H J I A N , A. H . , JR. , L . L E V I N E , A N D P. L . M U N S O N : Partial Hydrolysis of Bovine Parathyroid Hormone by Dilute Acid and Proteolytic Enzymes: Effects on Immunological and Biological Ac-tivities 979

NOTES A N D COMMENTS

PENHOS, J . C , B . A. HOUSSAY, A N D M . A. L U J A N : Tota l Pancreatectomy in Lizards. Effects of Several Hor-mones 989

R E N N E L S , E . G.: Sialic Acid Concen-trations i n the Pi tui tary Glands of Normal and Castrated Rats 994

D E L O N G , M . , A N D K . B A L O G H , JR. : Glu-cose-6-phosphate Dehydrogenase Ac-t i v i t y i n the Subcommissural Organ

Volume 76

of Rats. A Histochemical Study 996 SAWYER, W. H . , A N D H . V A L T I N : Inhibi -

t ion of Vasopressin Antidiuresis by Extracts of Pituitaries from Rats w i t h Hereditary Hypothalamic Dia-betes Insipidus and by Oxytocin . . . . 999

No. 6, June 1965 GREEP, R. O.: Roy Graham Hoskins (1880-

1964) 1007

M A T S U D A , K . , A N D M . A. GREER: Nature of Thyro id Secretion in the Rat and the Manner i n Which I t Is Altered by Thy-rotropin 1012

GOODNER, C. J.: Metabolism of the Anter-ior Pi tui tary Gland in Vitro: Effects of Fasting in Male and Female Rats. . . . 1022

GOODALL, C. M . : Restoration by Iodide of Hepatic Reactions to 2-Aminofluor-ene i n Thyroidectomized Rats 1027

Y A S U D A , M . , A N D D . C. JOHNSON: Effects of Exogenous Androgen and Gonado-tropins on the Testes and Hypophysial Follicle-Stimulating Hormone Content of the Immature Male Rat 1033

A D A M S , W . C , A N D J . H . L E A T H E M : I n -fluence of Thyroid Hormones on Choles-terol Metabolism in Rats w i th Induced Ovarian Cysts 1041

BOWERS, C. Y . , A N D A. V. SCHALLY: Ef-fects of Age, Sex, Castration and the Administration of Testosterone and Estradiol on Levels of Serum Choles-teryl Ester Fa t ty Acids in Rats 1047

S C H E N K M A N , J . B . , D . A. R I C H E R T , A N D W . W . W E S T E R F E L D : «-Glycerophos-phate Dehydrogenase Ac t iv i ty in Rat Spermatozoa 1055

BORIS , A. : Endocrine Studies of a Non-steroid Anti-androgen and Progestin. . . 1062

L I C H T O N , I . J.: Survival and Endocrine Function of Rat Placenta Implanted to the Spleen 1068

G A L A , R. R., A N D U . W E S T P H A L : Cortico-steroid-Binding Globulin in the Rat: Possible Role i n the Ini t ia t ion of Lacta-t ion 1079

K O W A L E W S K I , K . : Changes i n the Growth and Body Composition of Suckling Rats Caused by the Treatment of Lactating Mothers w i th Steroid Hormones: I ts Relation to Li t t e r Size 1089

G E N U T H , S., A N D H . E. L E B O V I T Z : Stimu-lation of Insulin Release by Cortico-tropin 1093

STEVENS, W. , C. COLESSIDES, A N D T . F. D O U G H E R T Y : Effects of Cortisol on the Incorporation of Thymidine-2- l 4C Into Nucleic Acids of Lymphatic Tissue from Adrenalectomized CBA Mice 1100

M A N N S , J . G., A N D J . M . B O D A : Effects of Ovine Growth Hormone and Prolactin on Blood Glucose, Serum Insulin, Plasma Nonesterified Fat ty Acids and Amino Nitrogen in Sheep 1109

G L E N N , E . M . , A N D J . G R A Y : Effect of Various Hormones on the Growth and Histology of the Gerbil (Meriones un-guiculatus) Abdominal Sebaceous Gland,

V O L U M E C O N T E N T S

June 1965 V O L U M E C O N T E N T S v i i

Pad. C R E E K , R. O.: Effect of Thyrotropin on

the Weight, Protein, Ribonucleic Acid, and the Radioactive Phosphorus Up-take of Chick Thyroids

G O O D M A N , H . M . : Early and Late Effects of Growth Hormone on the Metabolism of Glucose i n Adipose Tissue

G A N O N G , W . F., T . C. L E E , E . E . V A N B R U N T , A N D E. G. B I G L I E R I : Aldoster-one Secretion in Dogs Immunized w i t h Hog Renin

ROSEMBERG, E., P. K E L L E R , W. B . L E W I S , A . A L B E R T , G. C A R L , A N D D . B E N N E T T : Influence of Follice-Stimulating Hor-mone on the Estimation of Luteinizing Hormone in the Ventral Prostate and Ovarian Ascorbic Acid Depletion As-says

R A M I R E Z , V . D . , A N D C. H . S A W Y E R : A d -vancement of Puberty in the Female Rat by Estrogen

K R A G T , C. L . , A N D J. M E I T E S : Stimulation of Pigeon Pi tui tary Prolactin Release by Pigeon Hypothalamic Extract in Vitro

H O P K I N S , T . F., A N D G. P INCUS: Effects of Rat Hypothalamic and Cerebral Tissue on PMS-Induced Ovulation

STEINBERGER, E., A N D G. E. D U C K E T T :

1115 Effect of Estrogen or Testosterone on Ini t ia t ion and Maintenance of Sper-matogenesis in the Rat 1184

N O M U R A , J.: Effect of Stress and Psycho-1124 tropic Drugs on Rat Liver Tryptophan

Pyrrolase 1190 E V E R E T T , J. W.: Ovulation in Rats from

1134 Preoptic Stimulation Through Platinum Electrodes. Importance of Durat ion and Spread of Stimulus 1195

1141 N O T E S A N D COMMENTS

F U J I T A , T. , H . O R I M O , M . Y O S H I K A W A , H . M O R I I , A N D K . N A K A O : Effect of Acidosis and Alkalosis on Recovery from Hypocalcemia 1202

S U Z U K I , M . , S. W A T A N A B E , A N D M . H O S H I I : Effect of Estrogen on Cop-

1150 per-Induced Ovulation i n the Rabbit 1205 B A C A , Z . V . , A N D H . C H I O D I : Develop-

mental Changes in the Size and As-1158 corbie Acid Content of the Adrenals

of White Rats 1208 G R E E N W A L D , G. S.: Luteolytic Effect of

Estrogen on the Corpora Lutea of 1169 Pregnancy of the Hamster 1213

M E I T E S , J.: Maintenance of the Mam-mary Lobulo-Alveolar System in Rats

1177 After Adreno-Orchidectomy by Pro-lactin and Growth Hormone 1220

Adrenocorticotrophin and Adrenal Protein Synthesis1

P E T E R C . S C R I B A - A N D W I L L I A M J . R E D D Y Departments of Biological Chemistry and Medicine, Harvard Medical School, and the Department of Medicine, Peter Bent Brigham Hospital, Boston 15, Massachusetts

A B S T R A C T . In vitro protein synthesis by a cell-free preparation (15,000 X g supernatant fraction) of pig adrenals was studied, measur-ing glycine- 1 'C and /-leucine-1 4C incorporation into protein. Adrenocorticotrophin adminis-tered to rats in vivo caused a significant in-crease in the total act ivi ty of nondialyzable "supernatant factor" stimulating amino acid incorporation into protein. The specificity of the observed effect for adrenocorticotrophin was confirmed by experiments w i t h both a synthetic tricosapeptide and a highly purified preparation obtained from natural sources. The "supernatant factor" was nondialyzable, had an approximate molecular weight of 10-

14 X l O 1 , and was inactivated by incubation at 55 C and also by incubation wi th trypsin. I t was not inactivated by ribonuclease. Nucleic acids prepared by several methods were inac-tive. The "supernatant factor" could be puri-fied by diethylaminoethyl cellulose chromatog-raphy, and chemical analysis revealed the pro-tein nature of the material w i t h virtually no ribonucleic acid present in the preparation. I t is tentatively concluded that this material is one of the transfer enzymes involved in pro-tein synthesis, several other possibilities hav-ing been excluded. (Endocrinology 76: 745, 1965)

TH E A D M I N I S T R A T I O N of adre-nocorticotrophin in vivo (1) increased the activity of a nondialyzable factor in the 105,000 X g supernatant of rat adre-nals that stimulated the incorporation of glycine- l 4C into protein when added to a cell-free preparation from the adrenals of control rats. Evidence has been pre-sented (1) indicating that this stimu-lation occurs at a step after the forma-tion of amino acyl - sRNA. Since adrenal growth is such an integral part of pitui-tary-adrenal interrelationships, it was felt of interest to characterize further this "supernatant factor.''

Received October 15, 1964. 1 This study was supported by Grant A-4328

from the N I H , USPHS, Bethesda 14, Maryland. Presented in part at the 47th Annual Meeting of the Federation of American Societies for Ex-parimental Biology, Atlant ic Ci ty , New Jersey, April , 1963 (48).

2 Postdoctoral Research Fellow FF-428, 1962-63, of the N I H , USPHS. Present address: I I . Medizinische K l i n i k der Universitaet, Muenchen, Ziemssenstrasse 1, Germany.

Materials T h e preparations of adrenocorticotrophin

were generously donated as follows: Corti-cotrophin-gel, porcine (80 U S P units/ml) lots S B 543 ; S B 849, P R 583, by D r . F . G . McMahon, The Upjohn Co.; Oxycel adreno-corticotrophin, bovine (86 U S P units/mg) batch C N 145 by D r . E . A . Laso-Wasem, Wilson Co.; and N-terminal tricosapeptide synthetic (109 U S P units/mg) by Dr . K . Hofmann, University of Pittsburgh.

G l y c i n e - U - 1 4 C and Ueuc ine-U- 1 'C were purchased from Nuclear-Chicago, and the specific activity of each was diluted to 2.79 juc/jLimole by the addition of carrier. Other materials were obtained as follows: phos-phoenolpyruvate (purchased as a barium salt and converted to the potassium salt), di-sodium-adenosine triphosphate, adenosine-s'^'-cyclic phosphoric acid, trypsin, and diethylaminoethyl cellulose (capacity: 0.9 m E q / g ; mesh: medium, 19t D 62-13-240) from Sigma Chemical Co.; trisodium guano-sine triphosphate, crystalline lactic dehydro-genase (2 mg/ml) and pyruvate kinase (10 mg/ml) from Calbiochem.; ribonuclease from Worthington Biochemical Co.; dialysis tub-ing (No. 4465-A2) from A. H . Thomas Co.; pig adrenals from Northeast Packing Co. ,

745

746 S C R I B A A N D R E D D Y Volume 76

T A B L E 1 . Effect of addition of different substances to the assay system on

the glycine- UC incorporation

Addit ion* % Change

Amino acid mixture 0 Adenosine-3 ' ,5 '-cyclic phosphoric

acid 1 0 ~ 4 M 0 Sodium ascorbate 1.6 X 1 0 ~ 2 M 0 5 7 fig PEP kinase 0 1 1 4 jug PEP kinase, 2 0 ^moles

PEP and 2 Mmoles A T P 0 . 25 Mmole GTP 7 X 1 0 ~ 5 M Glutathione

0 . 25 Mmole GTP 7 X 1 0 ~ 5 M Glutathione - 1 2 7 X I O - ' M Glutathione - 3 3 7 X 1 0 - 3 M Glutathione - 6 6 0.4 ml 1 5 , 0 0 0 X g supernatant

of pig adrenals + 1 0 0

* A l l additions were added in 0 .4 ml of Tris-H C 1 buffer containing sucrose and ions at the concentrations noted in the text. The p H of the solution was checked and adjusted to 7.5 when necessary.

Somerville, Mass.; male Sprague-Dawley rats (250-300 g) from Charles River Breed-ing Co.

Methods The 15,000 X g supernatants obtained from

pig adrenals were employed as the basic system for testing the stimulation of amino acid incorporation into proteins. The adrenals were placed in ice-cold 0.9% N a C l approxi-mately 20 min after death, dissected free of adherent fat tissue, and frozen (—10 C ) . Prior to each incubation, pig adrenals were homogenized in medium A (200 mg/ml) of Littlefield and Keller (2) for 20 sec in a Virtis blender and then for 3-5 min in a glass homogenizer equipped with a motor-driven Teflon pestle. T h e supernatant fluids ob-tained after 15 minutes' centrifugation of the homogenates at 15,000 Xg and 5 C contained microsomes and the soluble fraction (3, 4). Amino acid incorporation into protein did not change after storage of the adrenals at — 1 0 C for more than 2 months.

R a t adrenals were removed immediately after death, adherent fat tissue was removed, and the adrenals were homogenized in medium A in a glass homogenizer. The 120 min X 105,000 Xg supernatant was obtained as previously described (1).

The complete incubation mixtures con-tained in a total volume of 1.25 ml the fol-lowing: 1 ßc (0.36 /xmole) glycine- 1 4 C, 10 /rnioles phosphoenolpyruvate, 1 fxmole adeno-sine triphosphate, 0.25 pinole guanosine tri-phosphate, 57 fig dialyzed pyruvate kinase,

0.4 ml of the 15,000 Xg supernatant of pig adrenals, 0.005M Mg++, 0.017M K + 0.039M T r i s - H C l buffer, p H 7.5, 0.17M sucrose, and 0.4 ml of the test solution to be assayed. These mixtures were incubated at 37 C for 60 min (unless otherwise stated). T h e pro-tein concentration was measured (5), and the radioactivity of the residue insoluble in hot trichloroacetic acid was determined by means of a liquid scintillation counter (Nuclear-Chicago) as previously described (1). Differences between groups of values were analyzed for significance by the t test (6).

Sucrose density gradients containing 0.025M K C l , 0.005M M g C l 2 , 0.05M T r i s - H C l buffer, p H 7.5, were prepared by a modifica-t ion 3 of the method of Britten and Roberts (7). T h e gradients were centrifuged for 1-2 hr at 37,500 rpm using the swinging bucket rotor SW-39 and the Spinco Model L ultra-centrifuge. T h e linearity of gradients pre-pared in this manner was checked by the addition of a dye to the sucrose solution with the higher concentration. Two hundred to 400 //I of test solutions was layered very care-fully on top of such gradients. After cen-trifugation, the tubes were punctured at the bottom and fractions were collected dropwise in the conventional manner.

Results Omission of adenosine triphosphate

and the regenerating system resulted in only 11% (22 cpm) of the glycine- 1 4C incorporation obtained in the complete system (192 cpm). Dialysis of the pig adrenal 15,000 X g supernatant for 18 hours vs. medium A resulted in the loss of ability to incorporate amino acids into protein, but this ability was restored by the addition of 1 jumole of each of 19 amino acids 4 to the incubation mixture.

3 We wish to thank M r . S. Slapikoff for intro-ducing us to the unpublished modification of Slapikoff, S., J. M . Fessenden, and K . Moldave, of the method for the preparation of sucrose gradients of Br i t ten and Roberts ( 1 2 ) .

4 Ala, t ry , lys, val, his, ser, thr, arg, ileu, phe, glu-NHo, leu, asp, cySH, cyS, glu, met, pro, ty r were dissolved in O . O I N H C l ( 1 0 /zmoles/ml) and neutralized immediately before the incuba-tions. Under these conditions all amino acids, except ty r and cyS, were completely soluble.

\pril 1965 " S U P E R N A T A N T F A C T O R " I N P R O T E I N S Y N T H E S I S 747

T h e effects of various additions to the complete assay system are presented in Table 1.

The addition of the 105,000 X g super-natant from adrenals of control rats increased the incorporation of glycine into protein (Table 2) compared with the addition of an equal volume of buffer (p

748 S C R I B A A N D R E D D Y Volume 76

F I G . 1. Distribution of activity of 105,000 X g supernatant of adrenals from rats treated w i t h adrenocorticotrophin after centrifugation on a sucrose gradient. The supernatant from 200 mg of adrenal was layered on a 6-20% sucrose gradient and centrifuged for 19 hr at 37,500 rpm, t = - f l C. Triangles represent glycine- u C incorporation into protein upon addition of 0.3 ml of each fraction of the density gradients as test solution to the assay system. I n the upper half of the figure, the optical density of 0.05 ml of each density gradient fraction diluted 1:8 is shown. I n the lower half of the figure, no point is present for the upper area of the gradient. This fraction in several experiments decreased incorporation to variable levels below that ob-tained wi th the basal test solution.

Martin and Ames (9) have suggested the use of purified proteins of known molecular weights as references for the determination of an approximate molec-ular weight of an unknown substance. The 105,000 Xg supernatant of adrenals from rats treated with adrenocortico-trophin was centrifuged on sucrose gradients 5-20% and 5-13%. Pyruvate kinase and lactic dehydrogenase were centrifuged simultaneously on separate gradients. The latter gradient was em-ployed to improve the separation of the stimulating factor, but was without significant effect. Centrifugation was performed for 16.5 hours at 37,500 rpm and +1 C . Glycine- 1 4 C incorporation was

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 X l O 5 and an S 2oW 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-bation for 2 hours at 37 C and for 15 minutes at 45 C prior to addition to the assay system. The activity from both sources was completely destroyed upon preincubation for 15 minutes at 55 C or higher temperature.

The effect of trypsin on the "superna-tant factor'' was studied. Trypsin was shown to inhibit glycine incorporation into protein by the assay system, and it was necessary to remove the trypsin from the test solution after preincuba-tion with this enzyme. As shown in the upper part of Fig. 2, trypsin having a molecular weight of 23,700 (13) did not sediment upon density gradient centrifu-gation into the first four (counted from the bottom) fractions. Addition of 0.4 ml of fractions 3 and 4 as test solutions to the assay system allowed base-line incor-poration (188 cpm) of glycine- 1 4C into protein. Hence, the elimination of the "supernatant factor" in fraction 4 (lower part of Fig. 2) is ascribed to trypsin digestion during the preincubation. Pre-incubation with trypsin also inactivated the "supernatant factor" present in an equal weight of adrenals from control rats.

Preincubation with ribonuclease did not cause inactivation of the "superna-

April 1965 " S U P E R N A T A N T F A C T O R " I N P R O T E I N S Y N T H E S I S 749

tant factor." "Supernatant factor" from adrenals of (adrenocorticotrophin-gel, Upjolhn, 140 U S P units intramuscularly over 3 days)—treated rats was prepared by concentrating the activity from the 120 min X 105,000 Xg supernatant into a 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 37,500 rpm and +1 C on a 5-13% su-crose gradient, and aliquots of the frac-tions of both gradients were assayed for stimulation of glycine incorporation in the usual test system. Neither the total activity nor the pattern of sedimentation of the "supernatant factor" was affected by treatment with ribonuclease. This lack of effect was confirmed by experi-ments in which bentonite was used for the adsorption of ribonuclease.

I n similar experiments the effect of 0.5% sodium lauryl sulfate on the gly-c ine - U C incorporation was studied. No effect of sodium lauryl sulfate (14) on the sedimentation of the activity on su-crose gradients was observed, suggesting that the material is not a nucleoprotein.

Phenol extraction (15) of either 105,000 Xg supernatant of adrenals from rats treated with adrenocorticotrophin-gel or active fractions obtained after density gradient centrifugation resulted in complete loss of stimulation of gly-cine- 1 4 C incorporation. Care had to be taken to assure complete extraction of protein from the aqueous layer, and, when necessary, the phenol treatment was repeated (14). Ribonucleic acid prepared by the procedure of Sevag et al. (16) was inactive when added to the assay system.

The above results suggested the pro-tein nature of the "supernatant factor," and it was suspected that it might be a transfer enzyme. The dialyzed 105,000

200

CP

o 1— -- ' ' - '•• - 1 1 ' L—J 10 20 30 40 42

bottom mm 'op

F I G . 2 . Effect of trypsin on the factor stimulating glycine- 1 4C incorporation. The fractions of a density gradient ( 5 - 1 3 % sucrose) containing the "supernatant factor" (cf Fig. 1 ) were combined and the active material was concentrated into a loose pellet by centrifugation for 5 hr at 1 4 0 , 0 0 0 X g. This material was incubated for 6 0 min at 3 7 C in the absence and in the presence of trypsin, 2 mg in 0 .4 ml . The 2 incubation mix-tures and an equivalent amount of trypsin were each layered on top of a 5 - 1 3 % sucrose gradient. The 3 samples were centrifuged simultaneously for 1 6 hr at 3 7 , 5 0 0 rpm at + 1 C. Triangles are glycine- u C incorporated into protein by the assay system upon addition of 0 .4 ml of each fraction of the density gradients which contained the "supernatant factor." Points are pg of pro-tein per 0 . 1 ml of each fraction of the density gradient which contained trypsin alone.

X g supernatant of adrenals from rats treated with adrenocorticotrophin-gel was chromatographed on a diethylamino-ethyl cellulose column as described by Takanami (17). The diethylaminoethyl cellulose was prepared as previously de-scribed (18). More than 90-95% of the recovered activity was eluted by the K C l gradient into samples 5 and 6 (Fig. 3), i.e., between 0.15 and 0.275M potas-sium (approximately 0.17 and 0.295M chloride). The highest specific activity was in sample 6. The chromatography on diethylaminoethyl cellulose provided a total yield of 53, 58 and 70% of the stimulating activity. The pattern of elu-tion was identical with the one described by Takanami (17) for transfer enzyme.

7 5 0 S C R I B A A N D R E D D Y Volumie 76

Somple

F I G . 3. Dis t r ibu t ion of act ivi ty following (chro-matography on dietthyl-aminoethyl cellulosae of the 1 0 5 , 0 0 0 X g X 2 hr supernatant prepared from the homogenaite of 1 4 7 3 mg of adrcenals from rats treated wi th adrenocorticotrophim-gel. After determination of the optical density at 2 6 0 and 280 m M „ the fractions collected from the column were? re-combined into 8 sam-ples as indicated b>y the vertical bars. Tine ac-t iv i t y of aliquots was determined by true test system, and the total stimulatory ac t iv i ty (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 . 3 6 mg; 7, 0 . 2 9 mg; 8, 0.3,8 mg.

The amino acid-activating enzymes are eluted before this fraction. 5

Sucrose gradient centrifugation of the preparation with the highest specific ac-tivity (sample 6, Fig. 3) showed only partial sedimentation of the protein into the fraction where the "supernatant factor" moved, indicating further puri-fication. Upon storage at 3 C , 50% of the activity in sample 5 (Fig. 3) was re-covered after four weeks.

Aliquots of samples 5 and 6 (Fig. 3) were now assayed for their protein and R N A content. A preparation containing 2.5 mg protein and weighing 2.6 mg was dissolved in distilled water and pre-cipitated with cold 5% T C A , washed with ethanol-ethyl-acetate, extracted twice for 15 minutes at 90 C with 5% T C A and washed again with ethanol. The dry weight of the residue was 2.7 mg

5 Schatz, D . , and W. J. Reddy (unpublished observations).

and it contained 2.4 mg of proteiin. I n both the cold and the hot trichloroacetic acid extracts, chemical analysis for R N A was carried out as described by Schneider (19), using the colorimetric assay for ribose of Dische (20). Zero readings were obtained. From the limits of sensi-tivity of this technique, this preparation contained less than 1 % ribonucleic acid. The "supernatant factor" prepared by diethylaminoethyl cellulose chromato-graphy was shown (Table 4) to stimu-late the incorporation of both glycine-1 } C and leucine- 1 4C into protein.

Discussion I n this study, the 15,000 Xg super-

natant fraction of pig adrenals was chosen as the cell-free protein-synthesiz-ing system. I t had the obvious advan-tage that the small amount of rat adrenal tissue was not a problem. On an equal weight basis, pig adrenal super-natant appears to be approximately

April 1965 " S U P E R N A T A N T F A C T O R " I N P R O T E I N S Y N T H E S I S 751

twice as responsive to the "supernatant factor" from rat adrenals as the control rat adrenal 15,000 Xg supernatant (1).

T h e "supernatant factor" from rat adrenals was active in the 15,000 Xg supernatant from rat, pig, dog and rab-bit adrenals, whereas it did not stimu-late glycine incorporation in an analo-gous preparation from normal rat liver, indicating organ rather than species specificity.

The effect observed with commer-cial preparations of adrenocorticotrophin could have been caused by impurities. That this effect was due to adreno-corticotrophin has been demonstrated in experiments with a synthetic peptide. Infusion 6 of this compound into hypo-physectomized rats gave rise to material in the soluble fraction of the adrenals that stimulated protein synthesis in the cell-free system and sedimented on su-crose gradients exactly as the "super-natant factor" from the adrenals of rats treated with adrenocorticotrophin-gel. Although the synthetic peptide is only a portion (23 amino acids) of the "physiological'' nonatriacontapeptide, the assumption that the observed effect is caused by the physiological principle of adrenocorticotrophin seems justified.

The factor stimulating amino acid in-corporation has a molecular weight of approximately 1.0 to 1.4 X l O 5 (S value = 6-7) and has the properties of a pro-tein. Preincubation at 55 C or with trypsin destroyed the activity. I t was not inactivated by preincubation with ribonuclease. Nucleic acid fractions pre-pared by phenol extraction, in which ex-hai^tive extraction of protein material

6 We wish to express our gratitude to Dr . Philip Hirsch, Harvard Dental School, for i n -structing us in the technique and for providing equipment used in the infusion experiments w i t h hypophysectomized rats.

T A B L E 4. Incorporation of glycine- UC and leucine- l 4C into protein

Test cpm in Protein solution Glycine- 1 4C Leucine- 1 4C

I 162 24 I I 892 366

I I I 1331 648

The complete incubation mixtures (cf Meth-ods) contained either 1 /j,c =0.36 /xmole glycine-1 4C or leucine- 1 4C. Test solutions added were: I = medium A; 11=0.36 mg, and 111=0.72 mg of "supernatant factor" as prepared by diethyl-aminoethyl cellulose chromatography (cf Fig. 3) in 0.4 m l medium A. For incubation condi-tions and determination of the radioactivity cf Methods.

into the phenolic layers was assured, had no activity. Finally, more than 90% of the activity recovered after chromatog-raphy on diethylaminoethyl cellulose was shown to be of protein nature, with virtually no ribonucleic acid detectable upon chemical analysis.

Recent evidence (21-23) indicates that, in addition to amino acid activat-ing enzymes, s - R N A and ribosomes, two transfer enzymes are involved in protein synthesis. Earlier studies had located the stimulation of protein synthesis to a nondialyzable substance which acted be-yond the formation of amino acyl transfer ribonucleic acid (1). The pat-tern of elution of the activity from the diethylaminoethyl cellulose columns was identical with the one observed by Takanami (17) upon preparation of transfer enzyme from rat liver. This and the protein nature of the "supernatant factor" suggest that it is probably a transfer enzyme.

Ferguson (25) has reported that actinomycin markedly inhibits adrenal ribonucleic acid synthesis but does not affect adrenal steroidogenesis, which sug-gests that adrenocorticotrophin does not act through synthesis of messenger ribonucleic acid. I n previous work (24), puromycin was shown to block steroido-

752 S C R I B A A N D R E D D Y Volume 76

genesis, indicating a relationship be-tween adrenocorticotrophin-induced ste-roidogenesis and protein synthesis. Since it has been shown previously (1) that there is no increased activity of amino acid-activating enzymes or transfer ribo-nucleic acids and messenger ribonucleic acid appears to be excluded by the studies of Ferguson, the only reasonable explanation left is that adrenocortico-trophin increases the activity of a trans-fer enzyme.

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