S H O R T C O M M U N I C A T I O N S 18 9

BBA 93204

The action of hydrocortisone on collagen formation with special reference to its action on ribosomal aggregation

Certain of the adrenal cortical hormones have proiound effects on the metab- olism of connective tissue and particularly on the formation of collagen. It is still not amply clear whether the hormone affects the hydroxylation of proline or the assembly of the amino acids into collagen. Recent work 1 has suggested that there is an effect on the mRNA-rRNA complex involving a reduction in total particulate RNA, particularly the large aggregates. KAJIKAWA ~ observed a reduction in ribosomes attached to the endoplasmic reticulum along with free particles dispersed through the system in hydrocortisone-treated fibroblasts. Other work s suggests that the hormone may actually stimulate liver protein synthesis.

Collagen formation in the chick embryo in vivo has been studied 4,5 by sucrose gradient analysis, and is found to be associated with very large ribosomal aggregates presumably held together by mRNA. This technique has been directed now toward determining whether the effect of hydrocortisone on collagen formation results from heavy polyribosomes disaggregation, or to impairment of a biosynthetic reaction such as hydroxylation and whether the hormone interacts directly with the protein-forming sites,

Materials and methods are described in detail elsewhere 5 and briefly in the legends to the figures.

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F ig . I. Sucrose-gradient analys is of chick embryo homogenates . Chick embryos , both normal and others treated for 18 h w i t h hydrocort i sone , were injected w i t h 5 - 8 / z C L- [14C]pro l ine and al lowed to incubate 20-30 rain before h o m o g e n i z a t i o n in Ringer-sa l ine buffer 5 at p H 7.4. A b o u t 1. 5 m l of the IO ooo × g pos tmi tochondr ia l supernatant was layered above 20 ml of a 1 5 - 3 o ~o l inear sucrose gradient and then centri fuged at 25 o o o revs . /min near 2 ° ill the Spinco L centrifuge for 12o rain. The absorbance, as wel l as the prol ine and hydroxypro l ine rad ioac t iv i ty of serial frac- t ions col lected after centr i fugat ion, are plotted. The absorbance peak corresponds to the single r ibosomes s ed iment ing at about 8oS.

Abbreviat ions: m R N A , messenger R N A ; r R N A , r i b o s o m a l R N A .

Biochim. Biophys. Acta, 138 (1967) 1 8 9 - 1 9 2

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SHORT COMMUNICATIONS 191

Results and discussion: The amount of radioactivity incorporated both as [14C]proline and as ~14C]hydroxyproline into trichloroacetic acid-insoluble material is inhibited 9 ° °/o in hydrocortisone-treated animals (Fig. I) whereas there is only a 50 % decrease in large polysomes. While the effect of I #g hydrocortisone on the distribution of polysomes is not far different from the effect of much higher doses, a more direct relationship exists between dosage and the uptake of [14Clproline into collagen as well as into non-collagenous protein (Table I). The change in proline pool size in itself has been shown not to account for the decrease in E14C]proline in- corporation. It appears, on the one hand, that hydrocortisone may interfere with normal mRNA activity and limit ribosomal aggregation for collagen and protein synthesis, and, on the other hand, may interfere with either polyribosomal function; the former very sensitive to hydrocortisone and the latter less sensitive.

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Fig. 2. R ibonuc lease t r e a t m e n t of h o m o g e n a t e of cor t i so l - t rea ted chick embryo . 8 -day chick e m b r y o s were t r ea t ed wi th 5oo/~g d ihydrocor t i sone and incuba t ion was con t inued for 18 h. H o m o g e n a t e s p repa red as in Fig. I were s e d i m e n t e d on a sucrose g rad ien t wi th and w i thou t ribo- nuc lease t r e a t m e n t (5/~g r ibonuclease /ml) and t he d i s t r ibu t ion of single r ibosomes , po ly r ibosomes and pa r t i cu la r ly of pel le table R N A - c o n t a i n i n g mater ia l , was compa red wi th t h a t f rom the homo- g e n a t e of n o r m a l u n t r e a t e d chick embryo . The 8o-S peaks cor respond to t he single r ibosomes ,

The large polysomes involved in the biosynthesis of collagen 4,5 are not com- pletely disaggregated by ribonuclease, suggesting it is not mRNA alone that is involved in ribosomal aggregation and it appears not to be due to intertwining collagen chains. The present data indicate that it is not the noncollagenous protein either which is involved. In addition hydrocortisone affects neither the ribonuclease- sensitive nor ribonuclease-resistant ribosomal aggregates preferentially (Fig. 2).

Direct binding of [14C]hydrocortisone to polysomes: Chick embryos were injected with 25 #C of [14C]hydrocortisone (N. E. Nuclear) and incubated for I h after which suitably prepared homogenates were subjected to sucrose gradient analysis without prior deoxycholate treatment. It is evident that the amounts of radioactivity associated with the trichloroacetic acid-precipitated fractions (Fig. 3)

Biochim. Biophys. Acta, 138 (1967) 189-192

192 SHORT COMMUNICATIONS

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Fig. 3. Sucrose-gradient analysis of chick embryos homogenate of an 8-day embryo injected with 25 #C of [14C]hydrocortisone i h before analysis. The fractions and the pellet were collected after 12o miD centrifugation of the post-mitochondrial homogenate which had not been treated with deoxycholate and the absorbance at 26o mff, as well as the hydrocortisone radioactivity, were plotted.

are extremely small. At most about I molecule of hydrocortisone is associated with every 20-4 ° ribosomes. The significance of such small amounts in a direct action of the hormone at the synthetic sites is questionable. DIGMAN AND SPOON 6 iound that subcellular fractions from liver also failed to bind hydrocortisone. As in the present study, the bulk of the radioactivity was found in the 'light' fraction of the homogenate.

This work was supported by U.S. Public Health Service Grant AM-oI27O from the National Insti tutes of Health, Bethesda, Yd.

Division o/Biochemistry, Department o/Biology, BERNARD S. GOULD Massachusetts Institute o/Technology, Cambridge, Mass. (U.S.A.) Albert Einstein School o/Medicine, New York, N.Y. (U.S.A.) GEORG MANNER

I J. D. GABOUREL AND E. K. FOX, Biochem. Biophys. Res. Commun., 18 (1964) 81. 2 F. T. KENNEY AND F. J. KULL, Proc. Natl. Acad. Sc. U.S., 5 ° (1963) 493. 3 K. KAJIKAWA, Acta Pathol. Jap. Suppl., 9 (1959) 7Ol. 4 R. H. KRETSINGER, G. MANNER, B. S. GOULD AND A. RICH, Nature, 2o2 (1964) 438. 5 G. MANNER, R. H. KRETSINGER, B. S, GOULD AND A. RICH, Biochim. Biophys. Acta, 95534. 6 C. W. DINGMAN AND M. B. SPOON, Science, 149 (1965) 1251.

Received September I5th, 1966

Biochim. Biophys. Acta, 138 (1967) 189-192

B B A 93208

The infrared spectra of some complexes of metal ions with nucleosides and nucleotides*

R e c e n t l y , m u c h a t t e n t i o n has b e e n g i v e n to t h e s t u d y of c o m p l e x f o r m a t i o n

b e t w e e n p o l y n u c l e o t i d e s a n d h e a v y m e t a l ions 1-a. S t u d i e s of t h e r e a c t i o n s of h e a v y

* Contribution No. 1233, Central Research Department, Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, Dela. 19898, U.S.A.

Biochim. Biophys. ,4eta, 138 (1967) 192-195