15.5. 1976

Speciatia

were found to be p o l y p e p t i d e in nature, and t h e aminoacid compositions were d e t e r m i n e d b y the use of an a u t o m a t i c amino-acid analyzer. F r o m r a t heart, an active p o l y p e p t i d e of molecular weight a p p r o x i m a t e l y 700 was isolated, w i t h an aminoacid composition as follows, t h e figures in parenthesis indicating the n u m b e r of amino-acid residues per molecule of p o l y p e p t i d e : - alanine (1), aspartic acid (1), g l u t a m i c acid (1), glycine (3), lysine (1). F r o m ox plasma, an active p o l y p e p t i d e was isolated of molecular weight a p p r o x i m a t e l y 5000, witll an amino-acid composition as f o l l o w s : alanine (3), arginine (2), aspartic acid (3), cystine (2), g l u t a m i c acid (4), glycine (2), histidine (2), isoleucine (1), leucine (3), lysine (5), phenylalanine (2), proline (2), serine (2), threonine (2), tyrosine (1), valine (2). The p o l y p e p t i d e from blood p r o v e d to be a m u c h b e t t e r complexing agent t h a n the smaller cardiac polypeptide, and results indicate t h a t it is capable of, complexing app r o x i m a t e l y 50 sodium ions per molecule of polypeptide in 0.15 M sodium chloride solution. This is an o r d e r of m a g n i t u d e greater t h a n would be in accord w i t h t h e n u m ber of acidic side chains available, which seem to be the m o s t obvious source of complexing ability, b u t a possible m e c h a n i s m for the c o m p l e x i n g of relatively large n u m bers of alkali m e t a l cations was proposed b y MUELLER and RUDIN% This involves t h e r e p l a c e m e n t of w a t e r molecules in the cation h y d r a t i o n shell b y carbonyl oxygen atoms, which, of course, are plentiful ill polypeptide structures. I t was felt desirable to confirm the existence of sodium ion complex f o r m a t i o n w i t h an i n d e p e n d e n t m e t h o d . The studies of PALATu1~ indicated t h a t t h e presence, even in relatively small amounts, of a cation w i t h a high surface charge density inhibits t h e close a p p r o a c h of sodium

Is there a Recycling

555

ions to binding s i t e s , and t h a t l a n t h a n u m is the best available ion for this purpose. W e therefore i n v e s t i g a t e d the effect of low concentrations of l a n t h a n u m ion on the a p p a r e n t depression of sodium ion a c t i v i t y b y the isolated plasma polypeptide and found t h a t the presence of lant h a n u m ions did indeed p r e v e n t the plasma p o l y p e p t i d e from lowering sodium ion activity. I t seems possible t h a t one function of the polypeptides isolated b y us and r e p o r t e d here could be t h a t of a sodium ion buffer. The c o n c e n t r a t i o n of sodium ions in plasma is k n o w n to be precisely controlled. According to PITTS 1~, for m a m m a l i a n plasma the sodium ion c o n c e n t r a t i o n lies in t h e range 138-146 m M . The presence of an effective sodium ion buffer would lead to an even narrower range for the sodium ion activity. The precision of control which this represents m a y be appreciated from the calculation t h a t this sodium ion concentration range, w h e n expressed on a logarithmic scale, corresponds to a range of o n l y 0.02. B y comparison, t h e n o r m a l range of h y d r o g e n ion Concentration, expressed on t h e p H scale, is 0.10 (i.e. 7.35-7.45, cf. PITTSlS). Thus t h e precision of control exerted on the sodium ion c o n c e n t r a t i o n is r e m a r k a b l e ; b y implication, the presence of a sodium ion buffer m u s t exert an additional control w i t h a precision even more remarkable.

9 p. MUELLERand D. O. RUDIN,Biochem. biophys. Res. Commun. 96, 398 (1967). 10 V. PALATY,Nature, Lend. 211, 1177 (1966). 11 R. F. PITTS, Physiology o/ the Kidney and Body Fluids, 3rd edn. (Year Book Medical Publishers Inc., Chicago 1974), p. 19. 12 R. F. PITTS, Physiology o~ the Kidney and Body Fluids, 3rd edn. (Year Book Medical Publishers Inc., Chicago 1974), p. 178.

of Hydroxyproline?

A. RUIZ-TORRES and I. I~0RTEN

Bereioh Sto//wechsel W E 3, Freie Universitiit Berlin, Klinikum Charlottenburg, Spandauer Datum 730, D-7 Berlin 79 (German Federal Republic, BRD), 21 November 7975. Summary. R a t s can produce glycine from h y d r o x y p r o l i n e and vice versa. A f t e r the injection, h y d r o x y p r o l i n e is rapidly c o n v e r t e d into glycine and t h e n incorporated into collagen. L a t e r the labelled amino acids in collagen are h y d r o x y p r o line, proline, serine, threonine, alanine, and g l u t a m i c acid. W e suppose t h a t all these labelled amino acids come from glycine. I t is generally held t h a t hydroxyproline, in c o n t r a s t to the large a m o u n t s of proline, c a n n o t be recycled. This m e a n s t h a t h y d r o x y p r o l i n e liberated in collagen catabolism would h a v e to be eliminated 1, ~. On the other hand, we h a v e shown in previous experiments t h a t r e m a r k a b l e quantities of labelled glycine can be found in collagen after the application of 14C-proline 8. The best e x p l a n a t i o n for this is t h e assumption t h a t this glycine was derived from h y d r o x y p r o l i n e directly and not from g l u t a m i c acid. Therefore h y d r o x y p r o l i n e m u s t be catabolized in the rat's organism. Methods. L-4-hydroxyproline-aH from N E N (5 mCi/ 0,148 rag) w i t h a p u r i t y grade of > 98% (scan of t h i n layer c h r o m a t o g r a p h y in phenol (75 g): H~O (25 g): N a C N (20 mg)) was injected into the p e r i t o n e u m of male S p r a g u e - D a w l e y rats weighing ca. 100 g each (ca. 400 [xCi/animal). The animals were sacrificed 1 or 24 h later. Taft skin, and t e n d o n collagen was extracted, diaIyzed, and isolated in fibres b y dialysis against p h o s p h a t e buffer

(0.02 M Na2HPO~)~. The fibres were also washed m a n y times. Purification was stopped w h e n there was no longer a n y r a d i o a c t i v i t y in the washing water. The collagen was t h e n h y d r o l y z e d and its amino acids isolated b y c o l u m n c h r o m a t o g r a p h y as described previously 6 (Figure). All the isolated 8H-amino acids again u n d e r w e n t c h r o m a t o g r a p h y and were identified separately. Results. 1 h after the injection of L-4-hydroxyprolineaH r a d i o a c t i v i t y can be d e t e c t e d in t h e collagen of skin. This r a d i o a c t i v i t y comes almost exclusively from glycine ( T a b l e ) . 2 4 h after application, the r a d i o a c t i v i t y of skin 1 S. H. JACKSONand J. A. HEINIGER, Biochim. biophys. Acta 387, 358 (1975). 2 K. I. KIVlRI~KO, Acta physiol, stand. Suppl. 60, 219 (1963). 8 A. R~JIz-ToRRES and I. K~)RTEN, Experientia 29, 968 (1973). 4 M. R. STETTed, J. biol. Chem. 153, 113 (1944). 5 p. M. GALLOP,Arch: Biochem. 5d, 486 (1955). A. RUIZ-ToRRESand I. K~RTEN, Z. Geront. 7, 133 (1974).

556

Specialia

EXPERIENTIA32/5

Labelled amino acids in collagen of tail skin, and tendon after aH-hydroxyproline injection to rats Amino acids

Hy-proline Threonine Serine Glut. ae. Proline Glyeine Alanine

Skin Total radioactivity (%)

cpm/mg

lb

24h

lh

-----80% --

8.84 8.65 5.37 5.30 8.95 53.55 9.34

-----106 --

24h 269 2.035 843 230 353 956 713

Tendon Total radioactivity (%)

cpm/mg

lh

24h

lh

n.J. n.J. n.J. n.i. n.i. n.i. n.i.

13.48 8.78 7.02 4.49 13.68 45.93 6.62

n.J. n.i. n.J. n.J. n.J. n.J. n.i.

24h 192 1,141 536 118 248 390 169

n.J., not investigated

cpm

61X

40

30

20. " 20

* 40

60

i 30

i 700

.., 120

|

|

140

160

clef/on volume {rnlsJ

GI/ 400.

300

§ Ser

200

100

i

20

t

t

40

6O

t

t

!

t

t

80

I00

120

140

780

collagen is distributed among 7 amino acids (Table). Here again glycine is predominant, accounting for more than 50% of radioactivity. Tail tendon collagen did not differ remarkably from this. Glycine also has the highest specific radioactivity, except for threonine. At 24 h the amount of radioactive hydroxyproline is approximately equal to t h a t of radioactive proline. Discussion. Our results have shown, in accordance with other investigations 4,~, t h a t hydroxyproline cannot be incorporated into collagen directly, b u t t h a t it is metabolized into glycine and then rapidly incorporated into collagen. The distribution of radioactivity in collagen amino acids 24 h after aH-hydroxyproline injection suggests the following assumptions concerning the metabolic p a t h w a y of hydroxyproline:

elut:e~ votumo trots)

Profile of eluted radioactivity from chromatography of hydrolysates of skin c o l lagen 1 h (above) and 24 h (bottom) after aH-hydroxyproline injection.

Hydroxyproline _~ Glycine ~ 4} Proline

Threonine Serine Alanine

4}

Glutamic acid Our previous experiments a with l~C(U)-proline injection i n t o r a t s h a v e s h o w n t h a t t h e d i s t r i b u t i o n of r a d i o a c t i v i t y i n c o l l a g e n a m i n o a c i d s is s i m i l a r t o t h e p r e s e n t r e s u l t s 24 h a f t e r a H - h y d r o x y p r o l i n e a p p l i c a t i o n . T h e r e c y c l i n g of h y d r o x y p r o l i n e v i a t h e g l y c i n e p a t h w a y is p r o b a b l e i n p l a n t s o r m i c r o o r g a n i s m s , b u t is n o t y e t determined in animals. 7 j . ROSENBLOOM, Arch. Biochem. Biophys. 1d2, 718 (1971).

Is there a recycling of hydroxyproline?

Rats can produce glycine from hydroxyproline and vice versa. After the injection, hydroxyproline is rapidly converted into glycine and then incorporat...
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