1960
BRINK,BOXER,JELINEII, KUEHL,RICHTERAND FOLKERS [CONTRIBUTION FROM
THE
RESEARCH LABORATORIES, MERCK& Co.,
VOl. 75
INC.]
Pituitary Hormones. VII. The Nature of Corticotropin-B BY NORMAN G. BRINK,GEORGEE. BOXER,V. C. JELINEK, FREDERICK A. KUEHI,,JR., AND
JOHN
W. RICHTER
KARLFOLKERS
RECEIVED SEPTEMBER 41, 1932 Corticotropin-B appears to be a' peptide of molecular weight in the range of 5000 to 7000. It contains fourteen common amino acids in amounts corresponding to a chain of some sixty amino acid units. The preponderance of basic amino acids reflects the known basic nature of corticotropin-B. The purity of corticotropin-B and the question of the presence or absence of a prosthetic group are discussed. Corticotropin-B is highly active clinically in rheumatoid arthritis. It possesses adrenal weight-increasing activity and causes melanophore expansion in frog skins.
Corticotropin-B, a substance of high adrenocorticotropic activity, was isolated from pepsin-digested concentrates of hog pituitary gland extracts by the methods described in preceding papers of this series.' The final steps in the isolation procedure involved the use of ion-exchange resins and then countercurrent distribution and yielded material of 250-300 u./mg. activity. As judged by its behavior in countercurrent distributions, the corticotropin-B so obtained behaved as an essentially homogeneous substance. Not more than 5% of foreign material was detected by this method. There were indications, however, of some additional loss of biological activity during the later stages of the distribution. Evidence obtained during studies of corticotropin-B in the ultracentrifuge, as described below, add further weight to the belief that corticotropin-B has been obtained in essentially pure form. The acetate salt of pure corticotropin-B is a white, amorphous solid which is soluble in water and methanol, and insoluble in acetone, benzene, ether and other similar solvents. There can be no doubt of its gross polypeptide nature. Its infrared spectrum discloses only absorption of groups associated with peptides. In aqueous solution, the ultraviolet absorption spectrum exhibits a single maximum at about 2770 A., 19.9. This absorption may be ascribed to the presence of amino acids having aromatic nuclei. The acetate salt of corticotropin-B contains 17.6y0 of nitrogen, and less than 0.5% of sulfur. Corticotropin-B contains no phosphorus and no carbohydrate. It gives a very weak ninhydrin test. The amino acid composition of a sample of corticotropin-B is summarized in Table I; this sample was from a countercurrent distribution. The values recorded, with the exception of that for tryptophan, were obtained by fractionation of an acid hydrolyzate on ion-exchange columns according to the method of Stein and Moore.2 The tryptophan was determined separately by the method of Shaw and M ~ F a r l a n e . ~Fourteen common amino acids were present; the corticotropin-B hydrolyzate contained no leucine, isoleucine, threonine or cystine. It is of interest that 99.5y0 of the total nitro(1) (a) N. G. Brink, F. A. Kuehl, Jr., J. W. Richter, A. W. Bazemore, M. A. P. Meisinger, D. E. Ayer and K. Folkers, THIS JOURNAL, 74, 2120 (1952); (b) A. d.Bazemore, J. W. Richter, D. E. Ayer, J. Finnerty, N. G. Brink and K. Folkers, ibid., '76, 1945 (1953); (c) J. W. Richter, D. E. Ayer, A. W. Bazemore, N. G. Brink and K. Folkers, ibid., 76, 1952 (1953); (d) F. A. Kuehl, Jr., M. A. P. Meisinger, N. G. Brink and K. Folkers. ibid., 7S, 1955 (1953). (2) S. Moore and W. H. Stein, J. B i d . Chcm.. 182, 663 (1951). (3) J. L. D. Shaw and W. D. McFarlane, Can. J ,Research, 16B. 361 (1938).
gen in the acid hydro1yza;e is accounted for by the thirteen amino acids and ammonia; the contribution of the tryptophan nitrogen in the acid hydrolyzate is of course included in the value for ammonia. On a weight basis, 86.5% of the preparation, not corrected for moisture or anion (acetate) content, was recovered as weights of the amino acids. The known basic character of corticotropin-B is clearly reflected in the preponderance of the basic amino acids, as indicated in Table I. TABLE I AMINOACID COMPOSITION OF CORTICOTROPIN-B ISOLATED BY COUNTERCURRENT DISTRIBUTION Amino acid
% of Weight of prepma
% Total N in the acid hydrolyzate
Best calcd. molar ratio
Glycine 4.4 6.4 6.0 Alanine 1.9 2.3 2.2 Valine 6.7 6.3 5.9 .. ... Leucine None Isoleucine h'one .. ... Proline 5.8 5.5 5.1 Serine 4.5 4.7 4.3 Threonine None .. ... Phenylalanine 4.7 3.1 2.9 Tyrosine 6.9 4.2 3.9 Cystine None .. ... Methionine 2.5 1.9 1.7 Aspartic acid 5.3 4.4 4.1 Glutamic acid 8.1 6.0 5.7 Histidine 3.1 6.6 2.0 Lysine 11.4 17.0 8.0 Arginine 13.4 28.0 6.5 Ammonia 0.5 3.1 2.9 7.3b 3.7 Tryptophan Weight of preparation uncorrected for moisture. * Determined separately.
Table I1 presents the results of amino acid analyses of hydrolyzates of corticotropin-B purified to the 300-u./mg. potency level by oxycellulose (sample 1) and Amberlite IRC-50 (sample 2) columns. In general, the compositions of these fractions were similar to each other and to that of corticotropin-B which had been purified by countercurrent distribution. Small discrepancies may be seen in the values for serine, phenylalanine, and glutamic acid, and perhaps others. This is not unexpected since, as has been noted, Id countercurrent distribution revealed the presence of other components in corticotropin-B samples isolated even a t this activity level by the column techniques. Corticotropin-B appears to contain little or no cysteine. Analysis of an acid hydrolyzate which
THENATURE OF CORTICOTROPIN-B
April 20, 1953
1961
The agrement between the molecular weights of corticotropin-B observed in the ultracentrifuge and calculated from the amino acid composition could COLUMN TECHNIQUES be cited as additional evidence of purity. Despite % of total N Best calcd. in the hydrolyzate molar ratio all this, i t is clear that if other components having Sample 1 Sample 2 Sample 1 Sample 2 Amino acid the same or nearly the same amino acid composi6.9 6.9 Glycine 6.5 5.9 tions, molecular weight and solvent solubility (Le., 2.4 2.6 2.1 Alanine 2.1 distribution coefficient) were present, they might Valine 5.4 6.7 4.8 5.6 well remain undetected. Inactivation products of Leucine corticotropin-B, altered from the parent structive .. ... .*. ... Isoleucine in some subtle way, could well fit these specificaProline 7.0 6.2 6.4 5.2 tions; a sug estion that this may be the case has 3.1 3.1 2.9, 2.6 Serine been noted.'! Further study and the application .. ... ... ... Threonine of other criteria of purity or new fractionation tech2.2 2.8 2.0 2.1 Phenylalanine niques may help answer this question. 4.4 4.9 Tyrosine 4.0 4.1 The possible presence of a small active prosthetic .. ... ... ... Cystine group in corticotropin or corticotropin-B has long 1.8 1.6 Methionine 1.6 1.5 been of interest. No proof has been found to dem4.3 . 4.0 4.8 4.7 Aspartic acid onstrate that such a group exists. Should such a 6.0 6.1 Glutamic acid 5.4 5.1 prosthetic group occur in corticotropin-B, it would 6.6 Histidine 1.8 1.8 5.9 have to be of low molecular weight and contain so 16.0 7.6 6.9 16.7 Lysine little nitrogen and so little characteristic ultraviolet 29.2 28.1 6.6 6.0 Arginine or infrared absorption that these properties would 3.3 3.0 3.3 Ammonia 3.9 be obscured by those of the gross peptide. Other98.8 100.2 Total wise it would not have escaped detection. There is no clear explanation in chemical terms had been treated with performic acid4 showed only for the reversible inactivation and reactivation of a minute amount of cysteic acid, indicating a cor- corticotropin-B by mild oxidation and reduction, responding absence of cysteine in the original prepa- respectively. A sulfhydryl disulfide oxidation-reration. duction equilibrium was early considered. The 0.n the basis of the amino acid analyses of cortico- failure to demonstrate the presence of sulfhydryl tropin-B, molar ratios were calculated for the amino groups in the molecule makes this unlikely. One acids found to be present (Table I). From these might speculate on the pbssible role of an elusive, values, which are gratifyingly near to whole number reactive prosthetic group in this connection, but ratios, it may be inferred that the corticotropin-B again without any direct evidence. molecule contains about sixty amino acid units, or The most highly purified preparatiops of corticoan integral multiple thereof, and has a minimum tropin-B have shown activities of about 250 to 300 molecular weight of the order of 6,000-7,000. u./mg. in the adrenal ascorbic acid depletion assay A sample of highly purified corticotropin-B was in hypophysectomized rats. A sample of corticoexamined for its behavior in the ultracentrifuge. tropin-B was tested clinically in rheumatoid arthriThe material appeared to be of high purity, and tis by Dr. Charles Ragana6 It was found when gave a sedimentation constant (35') of 5.3 X administered by continuous intravenous injection cm./sec./dyne and a diffusion constant of 9.0 X in a dose of 0.1 mg., to be equivalent in antiarthritic lo-' cm.2/sec. The partial specific volume was effect to 20 mg. of Armour Standard La-1-A indetermined to be 0.70, a value significantly lower injected in the same fashion. than that of 0.75 usually assumed for proteins. Discrepancies have been noted in the assay of The calculated molecular weight, using these val- adrenocorticotropic preparations by adrenal ascorues, is 5,200. This appears to be in good agree- bic acid depletion and adrenal weight gain in hypoment with the figure suggested as a minimum molec- physectomized rats, suggesting that these two efular weight by the analytical findings. fects may be mediated by different hormone^.^^^** In materials of the nature of corticotropin and Samples of corticotropin-B which had been purified corticotropin-B, the establishment of absolute pur- to activities of about 250 u./mg. (adrenal ascorbic ity is a task of large proportions. Corticotropin- acid depletion assay) by ion-exchange treatment B appeared to be of high purity in its ultracentrifu- and countercurrent distribution were highly active gal behavior. It has behaved as a single substance in increasing adrenal weight and causing thymus in countercurrent distribution. Harfenist and atrophy in hypophysectomized rats. The identity Craig in their recent work on insulinbhave demon- or close relationship between corticotropin and the strated that a compound of this size can be purified melanophore-expanding hormone has been postuand characterized by countercurrent distribution ; lated by Sulmanlo and independently confirmed by and in one case they were able to show the hetero(6) Columbia University College of Physicians and Surgeons, Presgeneity of an insulin preparation which had ap- byterian Hospital, New York City. (7) W. 0. Reinhardt and C. H. Li,Proc. Soc. E x p f l . Biol. Mcd., 77, peared homogeneous by the criteria of the solubility method, electrophoresis and the ultracentrifuge. 229(8)(1951). A. W. Moyer, J. Van der Scheer. H. Ritter, W. C. Tesar, J. B. TABLEI1
AMINO ACID
COMF'OSJTIONS OF SAMPLES
PURIFIED
BY
(4) J. M. Mueller, J. G. Pierce, Helen Davoll and V. du Vigneaud, J . Biol. Chcm., 191, 309 (1951). (5) E. J. Harfenist and L. C. Craig, THISJOURNAL, 74, 3083
(1952).
Logan, 5. J. Oleson and H. R. Cox, i b i d . , 79, 1 (1952). (9) H. B. F. Dixon, M. P. Stack-Dunne, F. G . Young and D. B. Cater, Noturc, 168, 1084 (1951). (10) F. G. Sulman, ibid., 169, 588 (1952).
113132
M. GOODMAN, D. F. BRADLEY AND M. CALVIN
Vol. 75
Johnsson and Hogsberg. l However, Geschwind, Acids.-Samples weighing 8 to 10 mg. were dissolved in about 0.05 ml. of double-distilled water, followed by the Keinhardt and Li12 do not accept this conclusion. addition of twice redistilled constant boiliug hydrochloric It has been observed here that, although quantita- acid. The mixtures were boiled under reflux for 18 hours tive values axe not available, highly purified in an atmosphere of nitrogea. Excess hydrochloric acid corticotropin-B caused melanophore expansion in was removed by repeated evaporation in vacuo under nitrogen, and the residues finally dried in mc%o. The dried resiexcised frog skin, even a t low concentrations where dues were taken up in the buffer of PH 3.42 described by crude corticotropin was ineffective. The details of Moore and Stein2 containing thiodiglycol and Versene . these studies will be reported elsewhere. One-half ml. aliquot portions containing the equivalent of 2.0 and 2.5 mg. of unhydrolyzed material were The recent preparations here’ and e l s e ~ h e r e l 3 ~between ~~ used for chromatography. Total nitrogen in the hydrolyu i unhydrolyzed corticotropin concentrates many zates was determined by a modification of the Nessler retimes more active than the protein described in action.17 1943 as the adrenocorticotropic h ~ r m o n e ’ ~have ~’~ Performic Acid Oxidation of Corticotropin-B.-A perinevitably raised questions as to the nature of the formic acid solution was prepared by adding 0.5 mi. of 30% hydrogen peroxide solution to 4.5 ml. of sS% formic acid reportedly pure protein preparation. The ease with and allowing the mixture to stand at room temperature for which corticotropin can be removed from the “pro- 25 minutes. A 10-mg. quantity of corticotropin-B was tein” can be interpreted by a hypothesis that the dissolved in 5 ml. of this solution and the reaction mixture “protein” consists of an aggregate of proteinaceous was kept a t -5’ for 45 minutes. It was then diluted with units having a more or less characteristic composi- 20 ml. of water and lyophilized. The solution in water and lyophilization were repeated. The product, which had a tion, but still capable of easy separation into the bioactivity of less than 25 u./mg. was hydrolyzed with acid component parts as, for example upon dialysis, ul- as described above. Only a trace of cysteic acid was found tra-filtration, acetic acid extraction or adsorption on in the hydrolyzate. oxycddose or resin. A second interpretation is Acknowledgment.-The authors are indebted t o that the “protein” preparation considered homo- Mr. R. N. Boos and his associates for the microgeneous actually contained lY0 or less of the active analyses, to Dr. N. R. Trenner and co-workers for principle as a trace constituent, an amount impos- the infrared spectra and to Mr. Arthur Rosenberg sible to detect in material of this nature by the for the ultracentrifuge determinations. They wish physical criteria which were used. to thank Dr. C. A. Winter of the Merck Imtitute for Therapeutic Research for his studies on the Experimental Acid Hydrolysis Prior to Analytical Separation of Amino adrenal weight and melanophore-expanding properties of corticotropin-B. The assistance of Messrs. (11) S.Johnason and B. Hogsberg, .Vuture, 169, 286 (1952). D. E. Ayer and M. A. P. Meisinger in the prepara(12) 1 . I. Geschwind, IT.D Reinharcit and C. XI. Li, ibid., 169, 1061 tion of samples of corticotropin-B for this work is (1952). (18) H. B. F. Dixon, S. Moore, 51. P. Stack-Dunneand F. G. Young, gratefully acknowledged. i t i d . , 168, 1044 !1961!. (14) E. B. Astwood, RI. S. Iiabcn, K. W. Papne and A . U. Grady, Tars JOURNAL, 73, 2969 (1951). (15) C.H. I,i,,H. 5f. Evans ani1 M . E. Simpson, J . B i d . Chem., 149, 413 (1943). (le) C. Saycrs, A. Whitc and C. S . 11. Lung, ibid., 149, 425 (1943).
(17) W.W. Umbreit, R.H. Burris and S. F. Stauffer, “Maadmetric Techniques and Related Methods for the Study of Tissue Metabolism,” Burgess Publishing Co., Minneapolis, Minn., 1945, p. 103. RAHWAY,
SEW JERSEY
-
___I____
~ ~ O ~ l R I U I LKOM ~ l l ~t l b ~ RAI)IALIuP*‘ J ~
LABORATORY AND DEPARTMENT OF CHEMISIRY,
Phosphorus and Photosynthesis.
UNIVh.RbI1 Y OH C A L I I W R N I A ]
I. Differences in the Light and Dark Incorporation of Radiophosphat e’
BIT11.GOODMAP,-, D. F. BRADLEY AND 11.CALVIN RECEIVED SEPTEMBER 22, 1952 The distribution of radioactivity in metabolites of photosynthetic algae exposed to KHzPaeO~ in light and dark has been determined by paper chromatography and radioautography. The large relative increase of adenosine triphosphate in the dark and of 3-phosphoglyceric acid in the light is discussed in relation to the mechanism of phosphoflation of photosynthetic intermediates. Evidence is given indicating that adenosine triphosphate is the first isolable product formed from inorganic phosphate, and that uridine diphosphate glucose and adenosine diphosphate are active in phosphate metabolism.
Since the majority of significant photosynthetic intermediates are phosphorylated,?-j one might expect that there would be differences in the steadystate distribution of phosphate among intermediate metabolites in green plants depending on whether they are photosynthesizing or merely re( 1 ) T h e work described iu this paper was spousorad b y the U. S
Atomic Energy Commission. (2) M. Calvin and A. A. Benson, Science, 107, 476 (1948). ( 3 ) A . A. Benson, J. A. Bassham, M . Calvin, T .C. Goodale, V. A . rrdas i l l l d w. Stepka, ’ T I U S JOrJKNAL, 72, 1710 (195o). (4) J. G . Bochannn, e1 al., “Phosphorus hletabolisin,” 1‘01. 11, J o h n s Hopkins Press, Daltimorr, b l d . , 19.52. ,5: E. W. F a g v r , J . 1. l>ciilBei-ga n d IC. Chrffron, Fcderulirin l’rvc. 9 , ,73.j (l!?>D),
spiring. Furthermore, there might be differences in the relative r a t a at which entering phosphate is distributed among these same intermediates. However, fractionation methods6-11that have heretofore been employed in such studies have not per(6) M. D. Kamen and S. Spiegelman, Cold Spring Harbor Symposia on Quanfitotiae Biology, 19, I61 (1948). (7) R. L. Emerson, J. F. Stauffer and W. W.Umbteit, A m r . J . Bot., 81, 107 (1944). (8) W. W. Umbreit, R. H. Burris and J. F. Stauffer,“Manometric Techniques,” Eurgess t’iiblishing Co., Minneapolis, M i n n . . 1945 Chap. X V . (9) S. Aronoff and Rl. Calvin, Plant Physiology, Q8,351 (1948). (10) H. Gest and M. D. Kamen, J . B i d . Chcm., 176,299 (1948). (11) W. Sinlotiis and K. H. Grube, Z . ‘ V a l . , 7b, 194 (1952).
