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The isolation and characterzation of growth hormone from sheep pituitary glands
VOL, 2 9 ( 1 9 5 8 )
BIOCHIMICA ET BIOPHY$ICA ACTA
I45
THE ISOLATION AND CHARACTERIZATION OF GROWTH HORMONE FROM S H E E P PITUITARY GLANDS HAROLD
PAPKOFF
AND C H O H H A O L I
Hormone Research Laboratory and the Department o] Biochemistry, University of California, Berkeley, Calif. (U.S.A .)
The isolation of growth hormones (somatotropins) from beef, human, monkey and whale pituitary glands has been described in previous reports from this laboratory1,2,3, 4. This communication describes the isolation and properties of growth hormone from yet another mammalian species, the sheep. ISOLATION PROCEDURE*
Starting material T h e s h e e p p i t u i t a r i e s u s e d in t h e s e s t u d i e s were o b t a i n e d frozen f r o m c o m m e r c i a l sources a n d w e r e s t o r e d a t - - I 5 ° u n t i l used. B o t h anterior lobes a n d whole p i t u i t a r i e s were e m p l o y e d w i t h e q u a l success.
Ca(OHt) s extraaion and (NHa)sSO 4 /raaionation T h e initial s t e p s e m p l o y e d in t h e purification of ovine s o m a t o t r o p i n were identical to t h o s e described p r e v i o u s l y b y o n e of u s 6 for t h e b o v i n e g r o w t h h o r m o n e . Briefly, t h e frozen g l a n d s are g r o u n d a n d stirred w i t h C a ( O H ) t solution a t p H zo. 5. T h e C a ( O H ) s e x t r a c t is a d j u s t e d to p H 6.8 a n d a n e q u a l v o l u m e of s a t u r a t e d a m m o n i u m sulfate solution is a d d e d ; t h e r e s u l t i n g p r e c i p i t a t e con_~ya_lug t h e g r o w t h - h o r m o n e a c t i v i t y . T h i s p r e c i p i t a t e is dissolved, a n d t h e solution is dialyzed a n d f u r t h e r ftactionateqi w i t h a m m o n i u m sulfate; t h e f r a c t i o n t h a t precipitates a t a c o n c e n t r a t i o n of s a t u r a t e d (NH4)sSO ~ b e t w e e n 0.2 a n d 0. 4 is left for f u r t h e r purification. I n a t y p i c a l e x p e r i m e n t , a yield of z8 g of protein, on t h e average, is o b t a i n e d a t t h i s s t a g e f r o m i kilo of whole sheep pituitaries.
Chromatography on the cation-exchange resin Amberlite IRC-5o F u r t h e r purification of t h e p r o t e i n o b t a i n e d b y t h e s t e p s outlined a b o v e is effected b y m e a n s of t h e c h r o m a t o g r a p h i c t e c h n i q u e p r e v i o u s l y described I. S h o w n in Fig. I is a t y p i c a l p a t t e r n o b t a i n e d b y c h r o m a t o g r a p h y o n t h e c a r b o x y l c a t i o n - e x c h a n g e resin, A m b e r l i t e IRC-5o (XE-97). i o o nag of t h e f r a c t i o n m e n t i o n e d a b o v e t h a t p r e c i p i t a t e d a t a c o n c e n t r a t i o n of o.2-o. 4 s a t u r a t e d a m m o n i u m s u l f a t e w a s e x t r a c t e d w i t h I o inl of p H 5. i buffer* * a n d t h e clear s u p e r n a t a n t fluid w a s applied t o a c o l u m n o.9 c m in d i a m e t e r , c o n t a i n i n g 2o m l of t h e resin, w h i c h h a d a l r e a d y been equilibrated t o t h e s a m e buffer. F r a c t i o n s of 3 m l p e r t u b e were collected w i t h t h e aid of a T e c h n i c o n d r o p - c o u n t i n g fraction collector, a n d t h e p r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y m e a s u r e m e n t of t h e optical d e n s i t y a t 275 m/~ in a B e c k m a n Model D U S p e c t r o p h o t o m e t e r . A p p r o x i m a t e l y 25 % of t h e m a t e r i a l is e l u t e d w i t h t h e buffer of p H 6.o; b i o a s s a y * * * of t h e v a r i o u s f r a c t i o n s o b t a i n e d i n d i c a t e s t h a t t h e g r o w t h - h o r m o n e a c t i v i t y is f o u n d solely in t h i s eluate. T h e p r o t e i n in t h i s e l u a t e is p r e c i p i t a t e d b y t h e a d d i t i o n of a n equal v o l u m e of s a t u r a t e d a m m o n i u m sulfate s o l u t i o n ; t h e p r e c i p i t a t e is dissolved in w a t e r , a n d t h e solution is dialyzed a g a i n s t distilled w a t e r o v e r n i g h t a n d lyophylized. F o r p r e p a r a t i v e p u r p o s e s , a larger c o l u m n c o n t a i n i n g 2 1 of • All s t e p s are p e r f o r m e d a t o - 2 ° w i t h t h e e x c e p t i o n of t h e c h r o m a t o g r a p h i c p r o c e d u r e w h i c h is c o n d u c t e d a t r o o m t e m p e r a t u r e . • * T h e c o m p o s i t i o n of buffers u s e d in t h e c h r o m a t o g r a p h y w a s as follows: (a) p H 5.I buffer: 0.052 M N a H s P O 4 , 0.0025 M N a s H P O ~ , a n d 0.45 M (NH4)zSO 4. (b) p H 6.0 buffer: o.I8 M N a H s P O 4 , o.o85 M N a s H P O o a n d o.45 M (NH4)sSO 4. • ** G r o w t h - h o r m o n e a c t i v i t y w a s a s s a y e d b y m e a n s of t h e tibia t e s t in female L o n g - E v a n s r a t s h y p o p h y s e c t o m i z e d at 28 d a y s of age a n d u s e d 14 d a y s postoperativelye, 7.
Relerences p. zSX.
146
H. PAPKOFF, C. H. LI
VOL. 2 9
(1958)
IRC-5 o resin, and capable of handling 3o-4o g of the o.2-0. 4 saturated ammonium sulfate fraction, was used with results identical to those obtained with the analytical column. The fraction isolated from the column is subjected to the final purification steps indicated below.
Final purification Further purification is achieved by dissolving the material obtained from the column in distilled water to make a io mg/ml solution. The solution is adjusted to p H 7.7, the precipitate formed being removed by centrifugation; the resultant supernatant fluid is adjusted to pH 4.2, and the precipitate t h a t forms is again removed by centrifugation. The clear supernatant fluid is adjusted to p H 6.5~5.6, and the suspension is centrifuged. The supernatant fluid is set aside, and the precipitate is dissolved in 1/3 the volume of the original solution. The pH is adjusted to 5.1, any precipitate t h a t forms is removed by centrifugation, and the supernatant fluid is once again adjusted to p H 6.5-6.6; the resulting precipitate* is dissolved in distilled water and lyophylized. This fraction represents 2o % of the yield obtained from the column. According to criteria discussed below, it was shown to be a highly purified protein possessing growth-promoting activity comparable to the bovine hormone.
Chromatography
CHARACTERIZATION
The purified protein hormone was submitted to chromatography on Amberlite I R C - 5 0 resin, u n d e r t h e s a m e c o n d i t i o n s u s e d in t h e c h r o m a t o g r a p h i c s t e p of t h e p u r i f i c a t i o n p r o c e d u r e . A s i n d i c a t e d i n t h e l o w e r c h r o m a t o g r a m of F i g . I, t h e h o r m o n e exhibits chromatographic homogeneity. 7 pH 5.1, 0.057 M No+ 0.45 M (NI'14)2S04 O.D.=2.00
0.~
~0.D.=127 pH 6.0, 0.35 M No'jO.45 M (NH4)2SO4
F-... 0.4
i
,~
K02 M NoOH
~ o2 ~ 0.2 >, .~ oJ ~) o.o
r,,
50
IO0
I~O
0~- o.5 o.3
o°-:o 0
, 20
40
60 80 I00 140 140 :CoO Tube Number
Fig. x. upper: Chromatography on I1~C-5oresin of 0.2-0. 4 saturated (NH~)tSO 4 precipitate; 0.9 era diameter column containing 20 ml of resin; 58 mg of protein in IO ml of p H 5.1 buffer applied to column; 3.0 ml/tube collected. Lower: Chromatography on IRC-5o resin of purified ovine growth hormone; o, 9 cm diameter column containing 25 ml resin; 80 mg protein in ioo ml buffer p H 5. r applied to column; arrows indicate same buffer sequence as upper diagram; 7.0 ml/tube collected,
. . . . . . . . . . . . . Fig. 2. Electrophoretic patterns of ovine growth hormone. Conditions of electrophoresis: pH 4.0, 0.03 ionic strength acetate buffer; 0. 4 % protein solution;potentialgradient of 3.75V/cm; picture taken after 18,o6o sec of electrophoresis ; upper pattern represents the ascending boundary, and lower, the descending boundary.
* Material comparable to the isoelectric precipitate amounting to 25 % of the starting fraction is obtained by combining t h e isoelectric supernatant fluids (pH 6.5-6.6 ), and adding ice-cold 4 ° % (v/v) ethanol v e r y s l o w l y until a final concentration of 20 % ethanol (v/v) is reached. The precip i t a t e obtained is dissolved in water, small precipitates are r e m o v e d b y adjustment to p H ' s 4.8 and 8. 4, and the solution is lyophylized.
Re/erences p. zSz.
VOL. 2 9 ( I 9 5 8 )
GROWTH HORMONE FROM SHEEP PITUITARY GLANDS
I47
Elearophoresis All the electrophoretic experiments were performed in the Spinco Model H electrophoresis-diffusion apparatus, at I °. In buffers of 0.03 and o.I ionic strengths, and of pH's ranging from 4.0 to 9-5, the protein hormone behaved as a single component in all cases. Fig. 2 represents a typical electrophoretic pattern, obtained with a solution containing 4 mg protein per ml in an acetate buffer of pH 4.0 and 0.03 ionic strength, and with a potential gradient of 3.75 V/cm for 18,o6o. The isoelectric point of ovine somatotropin was determined by a series of electrophoretic experiments performed in various buffers of o.I ionic strength. Table I presents the electrophoretic mobilities obtained at various pH's; from a plot of mobility as function of pH, the isoelectric point was located at pH 6.80. TABLE I ELECTROPHORETIC
MOBILITIES
pH
4.0 5.1 5.6 6.1 7.2 8. 5 9.4 9-5
OF
OVINE
GROWTH
HORMONE
BuOer
Acetic acid- N a O H Acetic a c i d - N a O H Acetic a c i d - N a O H Cacodylic a c i d - N a O H TRIS-HCI** Glycine- N a O H Glycine-NaOH Glycine-NaOH
IN BUFFERS
OF O.I
IONIC
STRENGTH
Mobili,~y* (× zo 5 ont/sec/volt)
+ + + + -----
6.00 1.56 o.98 o.28 o.15 o.8o 2.26 3.22
* + indicates migration t o w a r d cathode. -indicates migration t o w a r d anode. * * Tris (hydroxymethyl) a m i n o m e t h a n e .
Sedimentation Ultracentrifugation experiments were carried out in a Spinco Model E centrifuge at a speed of 59,78o r.p.m, and at room temperature (2o-23°). The solvent employed was the borate buffer of pH 9-93 and o.2 ionic strength used by LI AND PEDERSEN8 for the sedimentation of bovine growth hormone. A single, symmetrical sedimenting boundary, indicating a high degree of homogeneity, was observed in all instances. A typical series of patterns may be seen in Fig. 3. The sedimentation constants ($20, w) determined at a number of different concentrations, ranging from o.15 % to 2.o %, are shown in Table II. Analysis of the data by the method of least squares
Fig. 3. Sehlieren p a t t e r n s of ovine g r o w t h h o r m o n e obtained in the ultracentrifuge at 59,780 r . p . m . a n d 21.3°; 2o m g of p r o t e i n / m l of solvent, b o r a t e buffer of p H 9.93; s e d i m e n t a t i o n f r o m right to left; pictures t a k e n e v e r y i6 rain beginning 19 min after a t t a i n m e n t of full speed; b a r angle 7 o°. R e / e v e n c e s p. x 5 x .
148
H. PAPKOFF, C. H. LI
VOL. 2 9 (1958)
produced the following equation: S~o,to = 2.76 + o.15 C, where C is the protein concentration in g per IOO ml solvent. Thus, at infinite dilution, the sedimentation constant is 2.76 S. TABLE n SEDIMENTATION*
OF O V I N E G R O W T H
Co~a~n (C)
HORMONE
AT
p H 9-93
$"
g/aoo ml 2.0 I.O I.O 0.8
S 3.08 2.83 2.72 3.oo
0.5 o.5 O.5 o. 4 o. 3 o.15
3.15 2.84 2.78 2.90 2.60 2.73
* All r u n s at 59,780 r.p.m., 2o-24°; S values corrected to 20 ° and H t O .
Digusion Diffusion experiments were carried out in the Spinco Model H electrophoresisdiffusion apparatus with a standard electrophoresis cell at I ° and with buffers of pH 9.93 and pH 2.3S. Prior to establishing the diffusion boundary, the protein solution was dialyzed against the buffer for 24 h; sharpening of the boundary was effected b y the method of KAHN AND POLSON9. Diffusion coefficients (D~o,w) were calculated by the maximal ordinate--area method a°. With a 0.4 % protein solution at pH 9.93, values for Dzo, to of 5.28. lO-7 cmS/sec and 5.22" lO-7 cmS/sec were obtained. At pH 2.3, a 0.5 % protein solution gave a value for Din, to of 5.25" lO -7 cm2/sec. The average of these three values is 5.25" lO-7 cmZ/sec. From the sedimentation and diffusion coefficients (S~o,to----2.76, and Dz0, to = 5.25" IO-7), together with an assumed value for partial specific volume of 0.73 ml]g, the molecular weight of ovine growth hormone may be calculated from the Svedberg equationn; a value of 47,400 was obtained in this manner.
Tyrosine and tryptophan content Tyrosine and tryptophan were determined quantitatively by the speetrophotometric procedure of GOODWIN AND MORTONas. Average values of 4-75 % tyrosine and 1.3o % tryptophan were obtained from analysis of four different preparations of ovine growth hormone (Table III). In addition, the ultraviolet absorption spectrum of the ovine hormone was examined in both acidic and alkaline solutions. The results were typical of proteins containing tyrosine and tryptophan as the only ultraviolet-absorbing moieties in the region examined aS. N-terminal residues The N-terminal residues of the ovine hormone were identified by paper chromatography, by means of the fluorodinitrobenzene (FDNB) method a4,an whereby a quantiRe/erences p. I51.
vOL. 29 (I958)
G R O W T H H O R M O N E FROM S H E E P P I T U I T A R Y GLANDS
149
TABLE III SPECTROPHOTOMETRIC DETERMINATION OF THE CONTENT OF TYROSINE AND TRYPTOPHAN IN OVINE GROWTH HORMONE l~e~rat~
Ty~osine
TrFptopban
%
%
I I II Ill IV M e a n -4- S . E .
4.74 4.89 4.67 4.75 4.7:t 4.75 -4- o.o 4
I.I3 I.I 9 1.27 1.43 1.47 1.3o -~- 0.07
tative determination of the yield of the dinitrophenyl (DNP-) derivatives of the amino acids in the acid hydrolysates of DNP-somatotropin is obtained. Determinations were performed on four different preparations of sheep growth hormone; in all cases, phenylalanine and alanine were found to be the N-terminal residues. In addition, the DNP-alanine and DNP-phenylalanine, in each instance, were found in very nearly a r : r ratio and in an amount consistent with one mole of each for a molecular weight of 47,000. Biological studies The tibia test6, 7 was employed for biological assay of the growth-promoting activity of the ovine hormone. The potency, as indicated in the results summarized in Table IV, is comparable to that of bovine growth hormone as determined b y this assay method. Thus, a total dose of o.o6 mg injected over a period of 4 days stimulated an increase of the uncalcified cartilage of the tibia in hypophysectomized rats (LongEvans, female, operated upon at 28 days of age and injected 14 days postoperatively), amounting to 74 V greater than the control. TABLE
IV
BIOASSAY OF OVINE SOMATOTROPIN ACCORDING TO TIBIAL RESPONSE IN HYPOPHYSECTOMIZED RATS Total dose*
No. ol rats
Tibia width
pg 20
6
micra I 9 9 i 2**
60 i2o
5 6
z3I q- 3 255 -4- 3
* Total dose injected over a period of 4 days. Mean q- standard error.
**
When the ovine hormone was tested for thyrotropic (TSH) contamination b y the method of BATES AND CORNFIELDTM, a value of o.023 units of TSH per mg of protein* was found. Other studies indicated a follicle-stimulating (FSH) activity of less than o.1%, an interstitial cell-stimulating (ICSH) activity of less than o.z %, and a lactogenic hormone activity of less than r.o %**. No corticotropic (ACTH) contamination * T h e a u t h o r s w i s h t o t h a n k D r . R . W . BATES of t h e N a t i o n a l I n s t i t u t e s of H e a l t h of t h e U.S. P u b l i c H e a l t h S e r v i c e for p e r f o r m i n g t h e T S H a s s a y . ** T h e f o l l o w i n g p r o c e d u r e s w e r e u s e d f o r t h e d e t e c t i o n of t h e s e a c t i v e s ; for F S H , t h a t of SXMPSON et al. 1~, f o r I C S H , t h a t of GREEP et a/. ls, a n d for p r o l a c t i n , t h a t of LYONS TM. W e w i s h t o
Dr. A. J. LOSTROHfor performing these Re/erences p. X5L
thank
assays.
I50
H. PAPKOFF, C. H. LI
VOL. 2 9 (I958)
was detected when the ovine hormone was assayed by the procedure of SAYERSet al. 2° at a dose level of I mg iniected intravenously into hypophysectomized rats*. DISCUSSION
Very tittle work has been reported in the literature on the purification of growth hormone from sheep glands. WILHELM121reported the preparation of a sheep growthhormone concentrate, but from the few studies performed, it was inferred to be an inhomogeneous product. In this investigation, we have employed either whole glands or anterior lobes of sheep pituitaries; from I kg of fresh whole glands, we were able to isolate 2.o g of growth hormone. The isolation procedure involved ammonium sulfate fractionation, chromatography on Amberlite IRC-5o resin, and isoelectric fractionation. It is of interest that the steps employed up to and including the chromatography are identical with those that have been employed in this laboratory for the isolation of several other species of growth hormones ~,8. The purified ovine somatotropin has been studied by means of chromatography, electrophoresis, ultracentrifugation, and N-terminalresidue analysis. These techniques were all consistent in indicating a high degree of homogeneity for the protein hormone. The molecular weight, as determined by molecular kinetic data, was found to be very similar to that of bovine somatotropin 8. It is also notable that the isoelectric point is very close to that of the bovine hormonO, and the N-terminal residues of the two species are identical 2~. Also, preliminary investigations of the C-terminus by means of digestion with carboxypeptidase have indicated that like bovine growth hormone ~3, ptienylalanine is the sole terminal residue of the sheep somatotropin molecule. In spite of these similarities, there is evidence indicating that the protein hormones isolated from beef and sheep pituitaries are not identical. Thus, preliminary amino acid analyses show differences in composition; in addition, ovine growth hormone has been observed to be a more soluble protein than the bovine product when tested in various solvents; the ovine hormone also appears to be more labile than the bovinO 4. It is of interest that the mammalian somatotropins thus far isolated (beef, sheep, whale, monkey, human) 1, 2,3,4 have all exhibited characteristic differences in their various properties. It may not be unreasonable to expect every species to produce and secrete its own characteristic somatotropin, with the hormones from closely related species perhaps manifesting the closest resemblance to one another. SUMMARY I. Highly purified ovine growth hormone has been prepared from sheep pituitary glands by conventional extraction procedures, chromatography on Amberlite IRC-5o resin, and ffactionation by adjustment of pH. 2. The preparation is homogeneous according to the criteria of electrophoresis, ultracentrifugation, chromatography, and terminal-group analysis. 3. The isoelectric point determined by free electrophoresis was found to be at pH 6.80, and the molecular weight determined from molecular-kinetic data is 47,4oo. 4-The N-terminal amino acids disclosed by reaction with FDNB are phenylalanine and alanine.
5- The tibial response of hypophysectomized rats to ovine growth hormone is comparable to
that obtained with the bovine hormone.
* We wish to thank Dr. S. HXERof Wilson Laboratories for performing the ACTH assay. Re]erences p. x 5z.
VOL. 2 9 ( 1 9 5 8 )
GROWTH HORMONE FROM SHEEP PITUITARY GLANDS
151
REFERENCES 1 C. H. LI, H. M. EVANS AND M. E. SIMPSON, J. Biol. Chem., 159 (1945) 353. 2 C. H. LI AND H. PAPKOFF, Science, 124 (1956) 1293. 3 H . PAPKOFF AND C. H . LI, J. Biol. Chem., in t h e press. 4 C. H. LI, Federation Proc., 16 (1957) 775. 5 C. H. LI, J. Biol. Chem., 211 (1954) 555. 6 F. S. GREENSPAN,C. H. LI, M. E. SIMPSON, AND H. M. EVANS,Emlocrinology, 45 (1949) 455I. I. GESCHWlND AND C. S . LI, in R. W. SMITH JR., O. H. GAEELER AND C. N. H. LONG. The Hypophyseal Growth Hormone, Nature and Actions, B l a k i s t o n , N e w Y o r k , 1955, P. 28. s C. H. LI AND K. O. PEDERSEN, J. Biol. Chem., 2Ol (1953) 595. 8 D. S. KAHN AND A. J. POLSON, J. Phys. ~ Colloid Chem., 51 (1947) 816. 10 H. NEURATH,Chem. Revs., 3 ° (I942) 357. 11 T. SVEDBERG AND K. O. PEDERSEN, The Ultracentri/uge, Oxford, C l a r e n d o n Press, 194 o. zz T. W. GOODWlN AND R. A. MORTON, Biochem. J., 4 ° (1948) 620. 13 G. H. HEAVEN AND E. R. HOLIDAY, Advances in Protein Chem., 7 (1952) 319. 14 F. SANGER, Biochem. J., 39 (1945) 507 • 15 A. L. LEVY, Nature, 174 (1954) 126. 16 R. W. BATES AND J. J. CORNEFIELD, J. Clin. Endocrinol. and Metabolism, 15 (1955) 838. iT M. E. SIMPSON, G. VAN WAGENEN AND V. CARTER,Proc. Soc. Exptl. Biol. Med., 91 (1956) 6. 18 R. O. GREEP, M. B. VAN DYKE AND g . F. CHOW, Proc. Soc. Exptl. Biol. Med., 46 (1941) 644. z9 W. R. LYoNs, Cold Spring Harbor Symposia on Quant. Biol., 5 (1937) 198. 20 M. A. SAYERS,G. SAYERS AND L. A. WOODBURY, Endocrinology, 42 (1948) 37921 A. E. WILHELMI, in R. W. SMITH, JR., O. H. GAEBLER AND C. N. n . LONG, The Hypophyseal Growth Hormone, Nature and Actions, B l a k i s t o n , N e w Y ork, 1955, P- 59~2 C. H. LI AND L. ASH, J. Biol. Chem., 2o 3 (1953) 419 • J. I. HARRIS, C. H. LI, P. G. CONDLIFFE AND N. G. PON, J. Biol. Chem., 209 (1954) 133. C. H. LI AND H. PAPKOFF, J. Biol. Chem., 2o 4 (1953) 391.
Received March Ist, 1958
VITESSE DE RESPIRATION DE B. S U B T I L I S
DANS SON M I L I E U D E C U L T U R E I N F L U E N C E DE CO s
P. CHAIX, A. LORTHIOIS ET I. ISSALY Laboratoire de Chimie biologique de la Facult~ des Sciences, Paris (France)
La vitesse de la respiration (Qo2) de B.subtilis n'a 6t6 6tudi6e jusqu'ici 1-5 qu'en utilisant cette bact6rie ~ l'6tat de masses non prolif6rantes (resting cells) et en mesurant l'oxyg~ne consomm6 par la m6thode manom6trique, d'une fa~on directe, en pr6sence de potasse qui fixe continuellement le gaz carbonique contenu dans l'atmosph~re des essais. Les valeurs de Qo2 ainsi trouv6es sont r6unies dans le Tableau I: en presence de glucose le Qo2 est voisin de IOO (88 ~ 95); en pr6sence de glyc6rol il atteint 198. I1 6tait int6ressant de rechercher si la vitesse de respiration de B.subtilis en pr6sence de glucose, ou de quelques autres substrats, n'6tait pas susceptible de varlet suivant que ce microorganisme pr6sente un spectre cytochromique caract6ris6, k la temp6rature de l'azote liquide soit par 3 bandes a: (aa(599-6o2 mt~) + ba(56I m/z) + ca(548-549 m~)) soit par 5 bandes a: (aa(599-6o2 mp), ba(56I m~), ya(556--557 m~), Bibliographie p. z6o.
BIOCHIMICA ET BIOPHY$ICA ACTA
I45
THE ISOLATION AND CHARACTERIZATION OF GROWTH HORMONE FROM S H E E P PITUITARY GLANDS HAROLD
PAPKOFF
AND C H O H H A O L I
Hormone Research Laboratory and the Department o] Biochemistry, University of California, Berkeley, Calif. (U.S.A .)
The isolation of growth hormones (somatotropins) from beef, human, monkey and whale pituitary glands has been described in previous reports from this laboratory1,2,3, 4. This communication describes the isolation and properties of growth hormone from yet another mammalian species, the sheep. ISOLATION PROCEDURE*
Starting material T h e s h e e p p i t u i t a r i e s u s e d in t h e s e s t u d i e s were o b t a i n e d frozen f r o m c o m m e r c i a l sources a n d w e r e s t o r e d a t - - I 5 ° u n t i l used. B o t h anterior lobes a n d whole p i t u i t a r i e s were e m p l o y e d w i t h e q u a l success.
Ca(OHt) s extraaion and (NHa)sSO 4 /raaionation T h e initial s t e p s e m p l o y e d in t h e purification of ovine s o m a t o t r o p i n were identical to t h o s e described p r e v i o u s l y b y o n e of u s 6 for t h e b o v i n e g r o w t h h o r m o n e . Briefly, t h e frozen g l a n d s are g r o u n d a n d stirred w i t h C a ( O H ) t solution a t p H zo. 5. T h e C a ( O H ) s e x t r a c t is a d j u s t e d to p H 6.8 a n d a n e q u a l v o l u m e of s a t u r a t e d a m m o n i u m sulfate solution is a d d e d ; t h e r e s u l t i n g p r e c i p i t a t e con_~ya_lug t h e g r o w t h - h o r m o n e a c t i v i t y . T h i s p r e c i p i t a t e is dissolved, a n d t h e solution is dialyzed a n d f u r t h e r ftactionateqi w i t h a m m o n i u m sulfate; t h e f r a c t i o n t h a t precipitates a t a c o n c e n t r a t i o n of s a t u r a t e d (NH4)sSO ~ b e t w e e n 0.2 a n d 0. 4 is left for f u r t h e r purification. I n a t y p i c a l e x p e r i m e n t , a yield of z8 g of protein, on t h e average, is o b t a i n e d a t t h i s s t a g e f r o m i kilo of whole sheep pituitaries.
Chromatography on the cation-exchange resin Amberlite IRC-5o F u r t h e r purification of t h e p r o t e i n o b t a i n e d b y t h e s t e p s outlined a b o v e is effected b y m e a n s of t h e c h r o m a t o g r a p h i c t e c h n i q u e p r e v i o u s l y described I. S h o w n in Fig. I is a t y p i c a l p a t t e r n o b t a i n e d b y c h r o m a t o g r a p h y o n t h e c a r b o x y l c a t i o n - e x c h a n g e resin, A m b e r l i t e IRC-5o (XE-97). i o o nag of t h e f r a c t i o n m e n t i o n e d a b o v e t h a t p r e c i p i t a t e d a t a c o n c e n t r a t i o n of o.2-o. 4 s a t u r a t e d a m m o n i u m s u l f a t e w a s e x t r a c t e d w i t h I o inl of p H 5. i buffer* * a n d t h e clear s u p e r n a t a n t fluid w a s applied t o a c o l u m n o.9 c m in d i a m e t e r , c o n t a i n i n g 2o m l of t h e resin, w h i c h h a d a l r e a d y been equilibrated t o t h e s a m e buffer. F r a c t i o n s of 3 m l p e r t u b e were collected w i t h t h e aid of a T e c h n i c o n d r o p - c o u n t i n g fraction collector, a n d t h e p r o t e i n c o n c e n t r a t i o n w a s d e t e r m i n e d b y m e a s u r e m e n t of t h e optical d e n s i t y a t 275 m/~ in a B e c k m a n Model D U S p e c t r o p h o t o m e t e r . A p p r o x i m a t e l y 25 % of t h e m a t e r i a l is e l u t e d w i t h t h e buffer of p H 6.o; b i o a s s a y * * * of t h e v a r i o u s f r a c t i o n s o b t a i n e d i n d i c a t e s t h a t t h e g r o w t h - h o r m o n e a c t i v i t y is f o u n d solely in t h i s eluate. T h e p r o t e i n in t h i s e l u a t e is p r e c i p i t a t e d b y t h e a d d i t i o n of a n equal v o l u m e of s a t u r a t e d a m m o n i u m sulfate s o l u t i o n ; t h e p r e c i p i t a t e is dissolved in w a t e r , a n d t h e solution is dialyzed a g a i n s t distilled w a t e r o v e r n i g h t a n d lyophylized. F o r p r e p a r a t i v e p u r p o s e s , a larger c o l u m n c o n t a i n i n g 2 1 of • All s t e p s are p e r f o r m e d a t o - 2 ° w i t h t h e e x c e p t i o n of t h e c h r o m a t o g r a p h i c p r o c e d u r e w h i c h is c o n d u c t e d a t r o o m t e m p e r a t u r e . • * T h e c o m p o s i t i o n of buffers u s e d in t h e c h r o m a t o g r a p h y w a s as follows: (a) p H 5.I buffer: 0.052 M N a H s P O 4 , 0.0025 M N a s H P O ~ , a n d 0.45 M (NH4)zSO 4. (b) p H 6.0 buffer: o.I8 M N a H s P O 4 , o.o85 M N a s H P O o a n d o.45 M (NH4)sSO 4. • ** G r o w t h - h o r m o n e a c t i v i t y w a s a s s a y e d b y m e a n s of t h e tibia t e s t in female L o n g - E v a n s r a t s h y p o p h y s e c t o m i z e d at 28 d a y s of age a n d u s e d 14 d a y s postoperativelye, 7.
Relerences p. zSX.
146
H. PAPKOFF, C. H. LI
VOL. 2 9
(1958)
IRC-5 o resin, and capable of handling 3o-4o g of the o.2-0. 4 saturated ammonium sulfate fraction, was used with results identical to those obtained with the analytical column. The fraction isolated from the column is subjected to the final purification steps indicated below.
Final purification Further purification is achieved by dissolving the material obtained from the column in distilled water to make a io mg/ml solution. The solution is adjusted to p H 7.7, the precipitate formed being removed by centrifugation; the resultant supernatant fluid is adjusted to pH 4.2, and the precipitate t h a t forms is again removed by centrifugation. The clear supernatant fluid is adjusted to p H 6.5~5.6, and the suspension is centrifuged. The supernatant fluid is set aside, and the precipitate is dissolved in 1/3 the volume of the original solution. The pH is adjusted to 5.1, any precipitate t h a t forms is removed by centrifugation, and the supernatant fluid is once again adjusted to p H 6.5-6.6; the resulting precipitate* is dissolved in distilled water and lyophylized. This fraction represents 2o % of the yield obtained from the column. According to criteria discussed below, it was shown to be a highly purified protein possessing growth-promoting activity comparable to the bovine hormone.
Chromatography
CHARACTERIZATION
The purified protein hormone was submitted to chromatography on Amberlite I R C - 5 0 resin, u n d e r t h e s a m e c o n d i t i o n s u s e d in t h e c h r o m a t o g r a p h i c s t e p of t h e p u r i f i c a t i o n p r o c e d u r e . A s i n d i c a t e d i n t h e l o w e r c h r o m a t o g r a m of F i g . I, t h e h o r m o n e exhibits chromatographic homogeneity. 7 pH 5.1, 0.057 M No+ 0.45 M (NI'14)2S04 O.D.=2.00
0.~
~0.D.=127 pH 6.0, 0.35 M No'jO.45 M (NH4)2SO4
F-... 0.4
i
,~
K02 M NoOH
~ o2 ~ 0.2 >, .~ oJ ~) o.o
r,,
50
IO0
I~O
0~- o.5 o.3
o°-:o 0
, 20
40
60 80 I00 140 140 :CoO Tube Number
Fig. x. upper: Chromatography on I1~C-5oresin of 0.2-0. 4 saturated (NH~)tSO 4 precipitate; 0.9 era diameter column containing 20 ml of resin; 58 mg of protein in IO ml of p H 5.1 buffer applied to column; 3.0 ml/tube collected. Lower: Chromatography on IRC-5o resin of purified ovine growth hormone; o, 9 cm diameter column containing 25 ml resin; 80 mg protein in ioo ml buffer p H 5. r applied to column; arrows indicate same buffer sequence as upper diagram; 7.0 ml/tube collected,
. . . . . . . . . . . . . Fig. 2. Electrophoretic patterns of ovine growth hormone. Conditions of electrophoresis: pH 4.0, 0.03 ionic strength acetate buffer; 0. 4 % protein solution;potentialgradient of 3.75V/cm; picture taken after 18,o6o sec of electrophoresis ; upper pattern represents the ascending boundary, and lower, the descending boundary.
* Material comparable to the isoelectric precipitate amounting to 25 % of the starting fraction is obtained by combining t h e isoelectric supernatant fluids (pH 6.5-6.6 ), and adding ice-cold 4 ° % (v/v) ethanol v e r y s l o w l y until a final concentration of 20 % ethanol (v/v) is reached. The precip i t a t e obtained is dissolved in water, small precipitates are r e m o v e d b y adjustment to p H ' s 4.8 and 8. 4, and the solution is lyophylized.
Re/erences p. zSz.
VOL. 2 9 ( I 9 5 8 )
GROWTH HORMONE FROM SHEEP PITUITARY GLANDS
I47
Elearophoresis All the electrophoretic experiments were performed in the Spinco Model H electrophoresis-diffusion apparatus, at I °. In buffers of 0.03 and o.I ionic strengths, and of pH's ranging from 4.0 to 9-5, the protein hormone behaved as a single component in all cases. Fig. 2 represents a typical electrophoretic pattern, obtained with a solution containing 4 mg protein per ml in an acetate buffer of pH 4.0 and 0.03 ionic strength, and with a potential gradient of 3.75 V/cm for 18,o6o. The isoelectric point of ovine somatotropin was determined by a series of electrophoretic experiments performed in various buffers of o.I ionic strength. Table I presents the electrophoretic mobilities obtained at various pH's; from a plot of mobility as function of pH, the isoelectric point was located at pH 6.80. TABLE I ELECTROPHORETIC
MOBILITIES
pH
4.0 5.1 5.6 6.1 7.2 8. 5 9.4 9-5
OF
OVINE
GROWTH
HORMONE
BuOer
Acetic acid- N a O H Acetic a c i d - N a O H Acetic a c i d - N a O H Cacodylic a c i d - N a O H TRIS-HCI** Glycine- N a O H Glycine-NaOH Glycine-NaOH
IN BUFFERS
OF O.I
IONIC
STRENGTH
Mobili,~y* (× zo 5 ont/sec/volt)
+ + + + -----
6.00 1.56 o.98 o.28 o.15 o.8o 2.26 3.22
* + indicates migration t o w a r d cathode. -indicates migration t o w a r d anode. * * Tris (hydroxymethyl) a m i n o m e t h a n e .
Sedimentation Ultracentrifugation experiments were carried out in a Spinco Model E centrifuge at a speed of 59,78o r.p.m, and at room temperature (2o-23°). The solvent employed was the borate buffer of pH 9-93 and o.2 ionic strength used by LI AND PEDERSEN8 for the sedimentation of bovine growth hormone. A single, symmetrical sedimenting boundary, indicating a high degree of homogeneity, was observed in all instances. A typical series of patterns may be seen in Fig. 3. The sedimentation constants ($20, w) determined at a number of different concentrations, ranging from o.15 % to 2.o %, are shown in Table II. Analysis of the data by the method of least squares
Fig. 3. Sehlieren p a t t e r n s of ovine g r o w t h h o r m o n e obtained in the ultracentrifuge at 59,780 r . p . m . a n d 21.3°; 2o m g of p r o t e i n / m l of solvent, b o r a t e buffer of p H 9.93; s e d i m e n t a t i o n f r o m right to left; pictures t a k e n e v e r y i6 rain beginning 19 min after a t t a i n m e n t of full speed; b a r angle 7 o°. R e / e v e n c e s p. x 5 x .
148
H. PAPKOFF, C. H. LI
VOL. 2 9 (1958)
produced the following equation: S~o,to = 2.76 + o.15 C, where C is the protein concentration in g per IOO ml solvent. Thus, at infinite dilution, the sedimentation constant is 2.76 S. TABLE n SEDIMENTATION*
OF O V I N E G R O W T H
Co~a~n (C)
HORMONE
AT
p H 9-93
$"
g/aoo ml 2.0 I.O I.O 0.8
S 3.08 2.83 2.72 3.oo
0.5 o.5 O.5 o. 4 o. 3 o.15
3.15 2.84 2.78 2.90 2.60 2.73
* All r u n s at 59,780 r.p.m., 2o-24°; S values corrected to 20 ° and H t O .
Digusion Diffusion experiments were carried out in the Spinco Model H electrophoresisdiffusion apparatus with a standard electrophoresis cell at I ° and with buffers of pH 9.93 and pH 2.3S. Prior to establishing the diffusion boundary, the protein solution was dialyzed against the buffer for 24 h; sharpening of the boundary was effected b y the method of KAHN AND POLSON9. Diffusion coefficients (D~o,w) were calculated by the maximal ordinate--area method a°. With a 0.4 % protein solution at pH 9.93, values for Dzo, to of 5.28. lO-7 cmS/sec and 5.22" lO-7 cmS/sec were obtained. At pH 2.3, a 0.5 % protein solution gave a value for Din, to of 5.25" lO -7 cm2/sec. The average of these three values is 5.25" lO-7 cmZ/sec. From the sedimentation and diffusion coefficients (S~o,to----2.76, and Dz0, to = 5.25" IO-7), together with an assumed value for partial specific volume of 0.73 ml]g, the molecular weight of ovine growth hormone may be calculated from the Svedberg equationn; a value of 47,400 was obtained in this manner.
Tyrosine and tryptophan content Tyrosine and tryptophan were determined quantitatively by the speetrophotometric procedure of GOODWIN AND MORTONas. Average values of 4-75 % tyrosine and 1.3o % tryptophan were obtained from analysis of four different preparations of ovine growth hormone (Table III). In addition, the ultraviolet absorption spectrum of the ovine hormone was examined in both acidic and alkaline solutions. The results were typical of proteins containing tyrosine and tryptophan as the only ultraviolet-absorbing moieties in the region examined aS. N-terminal residues The N-terminal residues of the ovine hormone were identified by paper chromatography, by means of the fluorodinitrobenzene (FDNB) method a4,an whereby a quantiRe/erences p. I51.
vOL. 29 (I958)
G R O W T H H O R M O N E FROM S H E E P P I T U I T A R Y GLANDS
149
TABLE III SPECTROPHOTOMETRIC DETERMINATION OF THE CONTENT OF TYROSINE AND TRYPTOPHAN IN OVINE GROWTH HORMONE l~e~rat~
Ty~osine
TrFptopban
%
%
I I II Ill IV M e a n -4- S . E .
4.74 4.89 4.67 4.75 4.7:t 4.75 -4- o.o 4
I.I3 I.I 9 1.27 1.43 1.47 1.3o -~- 0.07
tative determination of the yield of the dinitrophenyl (DNP-) derivatives of the amino acids in the acid hydrolysates of DNP-somatotropin is obtained. Determinations were performed on four different preparations of sheep growth hormone; in all cases, phenylalanine and alanine were found to be the N-terminal residues. In addition, the DNP-alanine and DNP-phenylalanine, in each instance, were found in very nearly a r : r ratio and in an amount consistent with one mole of each for a molecular weight of 47,000. Biological studies The tibia test6, 7 was employed for biological assay of the growth-promoting activity of the ovine hormone. The potency, as indicated in the results summarized in Table IV, is comparable to that of bovine growth hormone as determined b y this assay method. Thus, a total dose of o.o6 mg injected over a period of 4 days stimulated an increase of the uncalcified cartilage of the tibia in hypophysectomized rats (LongEvans, female, operated upon at 28 days of age and injected 14 days postoperatively), amounting to 74 V greater than the control. TABLE
IV
BIOASSAY OF OVINE SOMATOTROPIN ACCORDING TO TIBIAL RESPONSE IN HYPOPHYSECTOMIZED RATS Total dose*
No. ol rats
Tibia width
pg 20
6
micra I 9 9 i 2**
60 i2o
5 6
z3I q- 3 255 -4- 3
* Total dose injected over a period of 4 days. Mean q- standard error.
**
When the ovine hormone was tested for thyrotropic (TSH) contamination b y the method of BATES AND CORNFIELDTM, a value of o.023 units of TSH per mg of protein* was found. Other studies indicated a follicle-stimulating (FSH) activity of less than o.1%, an interstitial cell-stimulating (ICSH) activity of less than o.z %, and a lactogenic hormone activity of less than r.o %**. No corticotropic (ACTH) contamination * T h e a u t h o r s w i s h t o t h a n k D r . R . W . BATES of t h e N a t i o n a l I n s t i t u t e s of H e a l t h of t h e U.S. P u b l i c H e a l t h S e r v i c e for p e r f o r m i n g t h e T S H a s s a y . ** T h e f o l l o w i n g p r o c e d u r e s w e r e u s e d f o r t h e d e t e c t i o n of t h e s e a c t i v e s ; for F S H , t h a t of SXMPSON et al. 1~, f o r I C S H , t h a t of GREEP et a/. ls, a n d for p r o l a c t i n , t h a t of LYONS TM. W e w i s h t o
Dr. A. J. LOSTROHfor performing these Re/erences p. X5L
thank
assays.
I50
H. PAPKOFF, C. H. LI
VOL. 2 9 (I958)
was detected when the ovine hormone was assayed by the procedure of SAYERSet al. 2° at a dose level of I mg iniected intravenously into hypophysectomized rats*. DISCUSSION
Very tittle work has been reported in the literature on the purification of growth hormone from sheep glands. WILHELM121reported the preparation of a sheep growthhormone concentrate, but from the few studies performed, it was inferred to be an inhomogeneous product. In this investigation, we have employed either whole glands or anterior lobes of sheep pituitaries; from I kg of fresh whole glands, we were able to isolate 2.o g of growth hormone. The isolation procedure involved ammonium sulfate fractionation, chromatography on Amberlite IRC-5o resin, and isoelectric fractionation. It is of interest that the steps employed up to and including the chromatography are identical with those that have been employed in this laboratory for the isolation of several other species of growth hormones ~,8. The purified ovine somatotropin has been studied by means of chromatography, electrophoresis, ultracentrifugation, and N-terminalresidue analysis. These techniques were all consistent in indicating a high degree of homogeneity for the protein hormone. The molecular weight, as determined by molecular kinetic data, was found to be very similar to that of bovine somatotropin 8. It is also notable that the isoelectric point is very close to that of the bovine hormonO, and the N-terminal residues of the two species are identical 2~. Also, preliminary investigations of the C-terminus by means of digestion with carboxypeptidase have indicated that like bovine growth hormone ~3, ptienylalanine is the sole terminal residue of the sheep somatotropin molecule. In spite of these similarities, there is evidence indicating that the protein hormones isolated from beef and sheep pituitaries are not identical. Thus, preliminary amino acid analyses show differences in composition; in addition, ovine growth hormone has been observed to be a more soluble protein than the bovine product when tested in various solvents; the ovine hormone also appears to be more labile than the bovinO 4. It is of interest that the mammalian somatotropins thus far isolated (beef, sheep, whale, monkey, human) 1, 2,3,4 have all exhibited characteristic differences in their various properties. It may not be unreasonable to expect every species to produce and secrete its own characteristic somatotropin, with the hormones from closely related species perhaps manifesting the closest resemblance to one another. SUMMARY I. Highly purified ovine growth hormone has been prepared from sheep pituitary glands by conventional extraction procedures, chromatography on Amberlite IRC-5o resin, and ffactionation by adjustment of pH. 2. The preparation is homogeneous according to the criteria of electrophoresis, ultracentrifugation, chromatography, and terminal-group analysis. 3. The isoelectric point determined by free electrophoresis was found to be at pH 6.80, and the molecular weight determined from molecular-kinetic data is 47,4oo. 4-The N-terminal amino acids disclosed by reaction with FDNB are phenylalanine and alanine.
5- The tibial response of hypophysectomized rats to ovine growth hormone is comparable to
that obtained with the bovine hormone.
* We wish to thank Dr. S. HXERof Wilson Laboratories for performing the ACTH assay. Re]erences p. x 5z.
VOL. 2 9 ( 1 9 5 8 )
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151
REFERENCES 1 C. H. LI, H. M. EVANS AND M. E. SIMPSON, J. Biol. Chem., 159 (1945) 353. 2 C. H. LI AND H. PAPKOFF, Science, 124 (1956) 1293. 3 H . PAPKOFF AND C. H . LI, J. Biol. Chem., in t h e press. 4 C. H. LI, Federation Proc., 16 (1957) 775. 5 C. H. LI, J. Biol. Chem., 211 (1954) 555. 6 F. S. GREENSPAN,C. H. LI, M. E. SIMPSON, AND H. M. EVANS,Emlocrinology, 45 (1949) 455I. I. GESCHWlND AND C. S . LI, in R. W. SMITH JR., O. H. GAEELER AND C. N. H. LONG. The Hypophyseal Growth Hormone, Nature and Actions, B l a k i s t o n , N e w Y o r k , 1955, P. 28. s C. H. LI AND K. O. PEDERSEN, J. Biol. Chem., 2Ol (1953) 595. 8 D. S. KAHN AND A. J. POLSON, J. Phys. ~ Colloid Chem., 51 (1947) 816. 10 H. NEURATH,Chem. Revs., 3 ° (I942) 357. 11 T. SVEDBERG AND K. O. PEDERSEN, The Ultracentri/uge, Oxford, C l a r e n d o n Press, 194 o. zz T. W. GOODWlN AND R. A. MORTON, Biochem. J., 4 ° (1948) 620. 13 G. H. HEAVEN AND E. R. HOLIDAY, Advances in Protein Chem., 7 (1952) 319. 14 F. SANGER, Biochem. J., 39 (1945) 507 • 15 A. L. LEVY, Nature, 174 (1954) 126. 16 R. W. BATES AND J. J. CORNEFIELD, J. Clin. Endocrinol. and Metabolism, 15 (1955) 838. iT M. E. SIMPSON, G. VAN WAGENEN AND V. CARTER,Proc. Soc. Exptl. Biol. Med., 91 (1956) 6. 18 R. O. GREEP, M. B. VAN DYKE AND g . F. CHOW, Proc. Soc. Exptl. Biol. Med., 46 (1941) 644. z9 W. R. LYoNs, Cold Spring Harbor Symposia on Quant. Biol., 5 (1937) 198. 20 M. A. SAYERS,G. SAYERS AND L. A. WOODBURY, Endocrinology, 42 (1948) 37921 A. E. WILHELMI, in R. W. SMITH, JR., O. H. GAEBLER AND C. N. n . LONG, The Hypophyseal Growth Hormone, Nature and Actions, B l a k i s t o n , N e w Y ork, 1955, P- 59~2 C. H. LI AND L. ASH, J. Biol. Chem., 2o 3 (1953) 419 • J. I. HARRIS, C. H. LI, P. G. CONDLIFFE AND N. G. PON, J. Biol. Chem., 209 (1954) 133. C. H. LI AND H. PAPKOFF, J. Biol. Chem., 2o 4 (1953) 391.
Received March Ist, 1958
VITESSE DE RESPIRATION DE B. S U B T I L I S
DANS SON M I L I E U D E C U L T U R E I N F L U E N C E DE CO s
P. CHAIX, A. LORTHIOIS ET I. ISSALY Laboratoire de Chimie biologique de la Facult~ des Sciences, Paris (France)
La vitesse de la respiration (Qo2) de B.subtilis n'a 6t6 6tudi6e jusqu'ici 1-5 qu'en utilisant cette bact6rie ~ l'6tat de masses non prolif6rantes (resting cells) et en mesurant l'oxyg~ne consomm6 par la m6thode manom6trique, d'une fa~on directe, en pr6sence de potasse qui fixe continuellement le gaz carbonique contenu dans l'atmosph~re des essais. Les valeurs de Qo2 ainsi trouv6es sont r6unies dans le Tableau I: en presence de glucose le Qo2 est voisin de IOO (88 ~ 95); en pr6sence de glyc6rol il atteint 198. I1 6tait int6ressant de rechercher si la vitesse de respiration de B.subtilis en pr6sence de glucose, ou de quelques autres substrats, n'6tait pas susceptible de varlet suivant que ce microorganisme pr6sente un spectre cytochromique caract6ris6, k la temp6rature de l'azote liquide soit par 3 bandes a: (aa(599-6o2 mt~) + ba(56I m/z) + ca(548-549 m~)) soit par 5 bandes a: (aa(599-6o2 mp), ba(56I m~), ya(556--557 m~), Bibliographie p. z6o.