Biosynthesis of abnormal collagens with amino acid analogues

I56

BIOCttlM1CA ET t3IOPHVSI(A A(TA

BBA 3 5 3 2 4

B I O S Y N T H E S I S OF ABNORMAL COLLAGENS W I T H AMINO ACID ANALOGUES II. I N A B I L I T Y OF C A R T I L A G E CELLS TO E X T R U D E COLLAGEN P O L Y P E P T I D E S C O N T A I N I N G L-AZETIDINE-2-CARBOXYLIC ACID OR

cis-4-FLUORO-I.-PROLINE TADASH1 TAKEUCHI',

J O E L R O S E N B L O O M AND D A R W I N J. P R O C K O P * "

Departments of Medicine and Biochemistry, University of Pennsylvania and Philadelphia General Hospital, Philadelphia, Pa. (U.S.A.) (Received A u g u s t 2nd, 1968)

SUMMARY

Autoradiographic studies demonstrated that the abnormal collagen polypeptides synthesized in the presence of L-azetidine-2-carboxylic acid or cis-4-fluoro-Lproline are retained intraeellularly, and they are not extruded into the extracellular matrix. The proline analogues did not in themselves prevent the extrusion of previously accumulated normal protocollagen after the protocollagen was hydroxylated to collagen, and the results indicated that the failure to extrude the abnormal collagens is directly related to the incorporation of the proline analogues into the polypeptide chains. In exploring the reasons why tile collagens containing either analogue are not extruded, it was found that the abnormal collagens not only have a decreased content of hydroxyproline, but also have a decreased content of hydroxylysine and glycosylated hydroxylysine.

INTRODUCTION

Previous reports have indicated that when isolated embryonic cartilage is incubated without 0 2 or in the presence of an iron chelator, the synthesis of protocollagen polypeptides continues at 4 ° 9o% of the control rate, but proline and lysine residues in protocollagen are not converted to hydroxyproline and hydroxylysine, because the hydroxylating enzymes require 0 2 and Fe 2+ (for reviews see refs. I, 2). * P r e s e n t a d d r e s s : National Cancer Center H o s p i t a l , Tsukiji 5-Chome, C h u o - K u , T o k y o , Japan. "" Please a d d r e s s r e p r i n t r e q u e s t s to D. J. p., T h e P h i l a d e l p h i a General Hospital, 34th Street a n d Curie A v e n u e , Philadelphia, Pa. 191o 4, U.S.A.

t~iochim. Bioph3,s..4ela, 175 (1969) 1 5 6 - t 6 4

BIOSYNTHESIS OF ABNORMAL COLLAGENS.

II

i57

Autoradiographic and chemical studies have demonstrated that under these conditions the protocollagen polypeptides accumulate in the ground cytoplasm and that the hydroxylation of protocollagen is an essential step for the extrusion of completed collagen molecules into the extracellular matrix 3. Recent evidence suggests that after protocollagen is hydroxylated, some of the hydroxylysine residues synthesized are glycosylated with galactose or glucosylgalactose 4. The available evidence does not, however, indicate whether the essential requirement for the extrusion of collagen molecules is the synthesis of hydroxyproline, the synthesis of hydroxylysine, the synthesis of glycosylated hydroxylysine, or all three of these reactions. As indicated in the accompanying paper s, incubation of embryonic cartilage with either L-azetidine-2-carboxylic acid or cis-4-fluoro-L-proline leads to the incorporation of these proline analogues in place of proline into an abnormal protocollagen and subsequently into an abnormal collagen. With the incorporation of these proline analogues, the hydroxyproline content of the collagen synthesized is reduced because the analogues substitute for proline residues which are normally converted to hydroxyproline, and because the proline residues still incorporated into protocollagen are not hydroxylated to the normal extent. In the present paper we report that the abnormal collagen molecules synthesized in the presence of L-azetidine-2-carboxylic acid or cis-4-fluoro-L-proline are retained intracellularly, and they are not extruded into the extracellular matrix. EXPERIMENTAL MATERIALS AND METHODS

Materials Radioisotopes, proline analogues, and other material were obtained from the sources indicated in the accompanying paper s. L-~3,4-3H21Proline , 5000 /zC/ctmole, was purchased from the New England Nuclear Corp.

Conditions for incubation of isolated cartilage Tibiae from Io-days-old chick embryos which consisted primarily of cartilage were incubated as described in the accompanying paper 5.

Autoradiographs of tritium-labeled cartilage At the end of the incubation, the tissues were fixed immediately in a chilled mixture of ethanol-acetic acid (3 :i, v/v) for 3 h, and they were transferred to a solution of 2% glutaraldehyde and o.oi M sodium phosphate (pH 7.4) for storage. The glutaraldehyde-fixed samples were dehydrated and embedded in parafilm blocks, and 5 # thick sections were prepared. The sections were mounted on glass slides, they were de-parafinized, and then they were coated with Kodak Nuclear Track Emulsion, Type NTB 3. After exposure for 4-5 days, the autoradiographs were developed for 5 min with Kodak Type D-I 9 developer. Sections were then stained with toluidine blue for microscopic examination.

Assays of [l*Cllysine and [l*C]hydroxylysine After incubation, the tissues were homogenized in 4.0 ml water and dialyzed against running tap water 5. In order to assay total [14C]lysine and El*Clhydroxylysine t?iochim. Biophys. ,4cta, 175 (i969) J56 164

I5~

T. TAKEUCHI el a / .

content, the dialyzed homogenates were hydrolyzed directly in 4 vol. of 3 times distilled 6 M HC1 at lO8 °/'or 16 h in tubes sealed under N 2. In order to assay nonglycosylated and glycosylated !t4Cjhydroxylysine, collagenase-solubilized peptides were prepared by incubating the dialyzed homogenates at 37 ° for 15 h with 3o IOO #g/ml bacterial collagenase (Worthington Biochemical Corp.) in I mM CaC12 and 5o mM Tris-HC1 buffer (pH 7.6). The samples were heated at 6o ° for 2o min, and the insoluble proteins were removed by centrifuging at 15 ooo × g for 15 rain. Aliquots of the collagenase-solubilized peptides in the supernatant fractions were then hydrolyzed under acidic or alkaline conditions as described by ROSENBLOOM, BLUMENKRANTZAND PROCKOP4. Acid hydrolysis was carried out as described above. Alkaline hydrolysis was carried out by adding NaOH to a final concentration of 2 M, and heating the samples at lO8 ° for 16 h in alkali-resistant tubes (Coming 7282) sealed under N 2. As reported previously 4, the conditions for acid hydrolysis did not produce any destruction of hydroxylysine, and the conditions for alkaline hydrolysis completely hydrolyzed the collagenase-solubilized peptides. The acid-hydrolyzed samples were evaporated under reduced pressure, and the [14Clhydroxylysine and [14Cllysine in the hydrolysates were separated by ion-exchange chromatography on a Beckman Model 116 amino acid analyzer. Aliquots of the colunm eluates were counted in a liquid scintillation counter. The alkali-hydrolyzed samples were desalted on a Dowex-5o column (H+ form) before chromatography. RESULTS

Intracellular accumulation of the collagen synthesized with azetidine-carboxylic acid or eis-fluoroproline Autoradiographs of embryonic cartilage were prepared u n d e r the same conditions used previously to demonstrate the reversible intracellular accumulation of protocollagen 1-a. In samples incubated with 200 /~g/ml azetidine-carboxylic acid, most of the tritium was retained in the cells, and considerably less tritium was found over the matrix than in the control samples (compare Figs. I and 2). Similar results

Fig. I. I n c o r p o r a t i g n ~ o f L-[3,4-aHz]proline~by e m b r y o n i c c a r t i l a g e u n d e r c o n t r o l c o n d i t i o n s . Two t i b i a e were i n c u b a t e d in 2. 5 mI m e d i u m w i t h io/~C L-E3,4-SH2]pro!ine for 2 h. The g r a i n s are a b o u t e q u a l l y d i s t r i b u t e d over t h e c e l l u l a r spaces a n d t h e m a t r i x . Fig. 2. Effect of L - a z e t i d i n e - 2 - c a r b o x y l i c acid on t h e i n c o r p o r a t i o n of L-~3,4-aH2]proline. Conditions were t h e s a m e as in Fig. i e x c e p t t h a t t h e m e d i u m c o n t a i n e d 200 t*g/ml L-azetidine-2c a r b o x y l i c acid, a n d 5 ° / z C L-E3,4-aH2]proline were used. Some of t h e r a d i o a c t i v i t y ha s been inc o r p o r a t e d i n t o t h e m a t r i x , b u t m o s t of t h e g r a i n s are ove r c e l l ul a r spaces.

Biochim. Biophys. ~4cta, I75 (1969) I50 164

B I O S Y N T H E S I S OF ABNORMAL COLLAGENS.

II

159

Fig. 3. E f f e c t o f cis-4-fluoro-L-proline o n t h e i n c o r p o r a t i o n ' o f L-F3,4-3H2~proline. C o n d i t i o n s w e r e t h e s a m e as i n Fig. 2 e x c e p t t h a t t h e m e d i u m c o n t a i n e d 200 iig/nll cis-4-fluoro-L-proline. S e c t i o n s w e r e i n d i s t i n g u i s h a b l e f r o m t h o s e o b t a i n e d w i t h L - a z e t i d i n e - 2 - c a r b o x y l i c a c i d (Fig. 2). Fig. 4. E f f e c t of trans-4-fluoro-L-proline o n t h e i n c o r p o r a t i o n of L-I3,4-aH21proline. C o n d i t i o n s w e r e t h e s a m e a s in Fig. 2 e x c e p t t h a t t h e m e d i u m c o n t a i n e d 200 t~g/ml trans-4-fluoro-L-proline. S e c t i o n s w e r e i n d i s t i n g u i s h a b l e f r o m c o n t r o l s (Fig. i).

were obtained with tissues incubated with 20o/~g/ml cis-fluoroproline (Fig. 3), and the autoradiographs were indistinguishable from those obtained by incubating the tissues without 02 or with a,E-dipyridyl a. The anak)gues did not significantly affect the relative distribution of radioactive proline among collagenous and noncollagenous proteins 5, and therefore the grains seen over the matrix in Figs. 2 and 3 probably reflect [aHlproline incorporated into the protein of extracellular mucopolysaccharides (see refs. I 3)- Incubation of the tissue with 200 #g/ml tralts-4-fluoro-L-proline (Fig. 4) or DL-pipecolic acid (2-piperdine carboxylic acid) (not shown) did not affect the incorporation of tritium in the matrix. In order to demonstrate that the proline analogues did not in themselves prevent the extrusion of normal collagen, tissues were pulse-labeled with IaH !proline in the presence of a,a'-dipyridyl so that normal [3H!protocollagen was synthesized. The inhibition of protocollagen proline hydroxylase was then reversed by transferring

Fig.l[5 . EffectTof L - a z e t i d i n e c a r b o x y l i c a c i d o n t h e e x t r u s i o n o f a c c u m u l a t e d ? H ] p r o t o e o l l a g e n . F i v e t i b i a e w e r e i n c u b a t e d w i t h i m M a , a ' - d i p y r i d y l for 20 rain, a n d t h e n 2 0 / J C L-E3,4-aH2~ p r o l i n e ~ w e r e a d d e d for a 3 o - m i n l a b e l i n g p e r i o d . I n o r d e r t o c o n v e r t t h e ? H ~ p r o t o c o l l a g e n s y n t h e s i z e d i n t h e p r e s e n c e o f a , a ' - d i p y r i d y l t o [ a H ] c o l l a g e n l - a , t h e t i s s u e s w e r e i n c u b a t e d for 60 m i n i n t h r e e s u c c e s s i v e c h a n g e s o f m e d i u m c o n t a i n i n g n o a , a ' - d i p y r i d y l , o. 1 m M F e S O 4, I m M a s e o r b i c a c i d , a n d 200 # g / m l L - a z e t i d i n e - 2 - c a r b o x y l i c acid. T h e g r a i n s are a b o u t e q u a l l y d i s t r i b u t e d o v e r the cellular spaces and the matrix, and the sections are indistinguishable from controls (not s h o w n ) i n w h i c h n o L - a z e t i d i n e - 2 - c a r b o x y l i e a c i d w a s p r e s e n t in t h e m e d i u m u s e d t o r e v e r s e inh i b i t i o n of t h e h y d r o x y l a s e .

Biochim. Biophys. Acta,

I75 (1969) I 5 6 164

I()o

"1". TAKEU(HI d t a l .

the tissues to medium containing FeSO 4. tinder these circumstances appropriate proline residues in the accunmlated protocollagen are converted to hydroxyproline, and a portion of the lysine residues in the accunmlated protocollagen are converted to hydroxylysine and glycosylated hydroxylysine. The presence of 2oo #g/ml azetidinecarboxylic acid in the medium used to reverse the inhibition of the hydroxylase did not affect extrusion of the accuumlated collagen into the matrix (Fig. 5), and the autoradiographs were indistinguishable from those obtained when azetidine-carboxylic acid was not added to the medium used to reverse the inhibition of the hydroxylase (not shown).

Hydroxylysine content of collagen containing L-azetidine-2-carboxvlic acid or cis-fluoroproline Since hydroxylysine or glycosylated hydroxylysine residues may be essential features for the extrusion of collagen, the content of nonglycosylated and glycosylated hydroxylysine in collagen synthesized in the presence of the proline analogues was TABLE I EFFECT

OF PROLINE

COLLAGENOUS

ANALOGUES

ON THE

INCORPORATION

OF

[14(;ILYNINE

INTO

COLLAGEN

AND

NON ~

PROTEINS

Each sample contained two tibiae, and cross-matched pairs of tibiae tYom two xo-days-old emb r y o s were used as control and test samples. Samples were pre-incubated for 2 h, and the test samples were incubated for 2o min in m e d i u m containing 2oo ~¢g/ml of the proline analogues indicated. 5 #C E~4Cilysine were then added for a f u r t h e r incubation period of 2 h. Dialyzed homogenates of the tissues were incubated with purified collagenase, and the a4C-labeled peptides released by collagenase were assayed as described in t e x t and in the a c c o m p a n y i n g p a p e r ~.

Incubation conditio'7

(L~C)Lysine incorporation (disinl./ mi~ × zo ~)

Fractio~t dialyzable a[ter collagenase lrealment

(%)

Control L-A zetidine- 2-carboxylie acid

24.9 i6.6

20 22

Control

23.5 15.0

35 34

24.8 25.2

36 34

cis-4-Fluoro-a-proline Control

trans-4-Fluoro-L-proline

examined with isotopic techniques. Tissues were incubated with [aaCIlysine, and the relative fraction of the total 14C incorporated into collagen was measured by treating dialyzed homogenates of the tissue with collagenase. In control samples, 26-36% of the protein-bound 14C was digested by the collagenase (Table I). Incubation of the tissue with 2oo/~g/ml L-azetidine-2-carboxylic acid, cis- or trans-fluoroproline reduced these values o n l y slightly, indicating that the analogues did not markedly affect the fraction of [14Cllysine incorporated into collagen as distinct from other protein synthesized by the tissue. Incubation of the tissues with azetidine-2-carboxylic acid, however, produced a marked decrease in the conversion of protein-bound E14C!lysine to [14C!hydroxylysine l~iochim. Bioph3's. Acla, 175 (t969) 150 164

BIOSYNTHESIS OF ABNORMAL COLLAGENS. 11

1

'_o

6o

4(

I6I

~

o_

~0~ =: o

2,

tu

=

8.~~== 1 I00

I i~O0

~ ~o

o ,~a

:500

~a

Co____

L-AZETIDINE- 2 - CARBOXYLIC AClO [ / x g / m l )

I

l

I00 200 cis- FLUOROPROLINE (/.zg/ml)

,

3OO

o~

Fig. 6. Effect of L - a z e t i d i n e - 2 - c a r b o x y l i c acid on the i n c o r p o r a t i o n of [14C]lysine a n d t he s ynthesis of [x4C]hydroxylysine. Two t i b i a e were i n c u b a t e d for 2 h w i t h 5 1~C [14C]ly sine u n d e r control c o n d i t i o n s or w i t h t h e c o n c e n t r a t i o n s of L - a z e t i d i n e - 2 - c a r b o x y l i c acid i n d i c a t e d . The s a m p l e s were h o m o g e n i z e d a n d d i a l y z e d , a n d t h e t o t a l t4C a n d [ t 4 C l h y d r o x y l y s i n e c o n t e n t of t h e d i a l y z e d homog e n a t e s were a s s a y e d as described in t e x t . Fig. 7. Effect of cis-4-fluoro-L-proline on t h e i n c o r p o r a t i o n of [~4C]lysine a nd t he s y n t h e s i s of f n C ? h y d r o x y l y s i n e . C o n d i t i o n s as d e s c r i b e d in Fig. 6 a n d in t e x t .

(Fig. 6). The decrease of the synthesis of [14C]hydroxylysine was apparent with only zo #g/ml of the proline analogue, and the effect appeared at concentrations where there was no significant inhibition of the total incorporation of 14C. cis-Fluoroproline also inhibited the synthesis of [14Clhydroxylysine, but the inhibition was not as marked as the effect observed with L-azetidine-carboxylic acid, and it was not as distinct from the inhibition of the incorporation of total ~4C (Fig. 7). With concentrations of IOO #g/ml or more, however, the synthesis of [14C]hydroxylysine was consistently depressed to a greater extent than the incorporation of ~4C. Similar T A B L E 11 FAILURE XYLATION

OF L - A Z E T I D I N E - 2 - C A R B O X Y L I C OF L Y S I N E - L A B E L E D

ACID OR C g S - 4 - F L U O R O - L - P R O L I N E

TO A F F E C T T H E H Y D R O -

PROTOCOLLAGEN IN ISOLATED CARTILAGE

Two t i b i a e were i n c u b a t e d in m e d i u m c o n t a i n i n g i mM a,(z'-dipyridyl for 2o min, a n d 5/~C [laCllysine was a d d e d for a l a b e l i n g period of 2o rain. The l a be l w a s c ha s e d w i t h t h r e e successive 5-rain i n c u b a t i o n s in m e d i u m c o n t a i n i n g I mM a,a'-dipyridyl a n d 2o/~g/ml u n l a b e l e d lysine. The i n h i b i t i o n of p r o t o c o l l a g e n h y d r o x y l a s e was t h e n r e v e r s e d b y five successive 5-min i n c u b a t i o n s in m e d i u m c o n t a i n i n g o.i mM FeSO 4 a n d l o mM ascorbic acid, a n d finally a 12o-min i n c u b a t i o n in this medium.

Analogue added d~¢ring reversa! period

Non e L - A z e t i d i n e - 2 - c a r b o x y l i c acid, 200 ~ g / m l cis-4-Fluoro-L-proline, 2 0 0 / t g / m l None**

Total 14C* (disint./ rain x Io a)

[l*C]Hyl* (disint./ rain x zo a)

[14C]Hvl >~ IOO Total 14C

7t.6 77.6 92.4 80. 5

8. 5 8.2 lO.2 < 1.o

~ 1.8 io.6 I 1.o < I

* Values o b s e r v e d w i t h t h e a p p l i c a t i o n of a l i q u o t s c o n t a i n i n g 75 ooo-95 ooo d i s i n t . / m i n 14C to t h e i o n - e x c h a n g e c o l u m n for t h e a s s a y of [14Cllysine a n d [14C]hydroxylysine (see t e x t ) , a n d t h e y do n o t n e c e s s a r i l y reflect t o t a l p r o t e i n - b o u n d nC or r14C]hydroxylysine in t h e s a mpl e s . ** S a m p l e r e m o v e d a f t e r i n c u b a t i o n w i t h cl,a'-dipyridyl a n d n o t s u b j e c t e d t o r e v e r s a l period b y i n c u b a t i o n in m e d i u m c o n t a i n i n g FeSO 4.

Biochim. 13iophvs. Acta, I75 (7969) ~56 I64

T. TAKEUCH1 e{ a[.

162

effects of L-azetidine-carboxylic acid and cis-fluoroproline on the conversion of [14Cllysine to 1114Cihydroxylysine were seen in three other experiments (see "Fable III). Control experiments ('Fable [I) indicated that the effects of these two proline analogues on the synthesis of [laC hydroxylysine were not explained by any direct effect on hydroxylase required for the synthesis of hydroxylysine. Tissues were pulselabeled with I14C!lysine in the presence of a,/~'-dipyridyl so that lysine-labeled protocollagen accunmlated within the cells. The samples were then transferred to medium containing FeSO 4 so that [~4C~lysine in the accunmlated protocollagen was hydroxylated to !14Clhydroxylysine. The presence of 20o/,g/ml azetidine-carboxylic acid or cis-fluoroproline in the medium used to reverse inhibition of the hydroxylase did not affect hydroxylation of the [a4C!lysine in the accunmlated protocollagen.

Glycosylated hydroxvlysine in collagen containing azetidine-carboxvlic acid or cis-fluoroproline Hydroxylysine substituted either with galactose or glycosylgalactose is not converted to free hydroxylysine by hydrolysis under mild alkaline conditions4,6, 7. Accordingly, the amounts of nonglycosylated and glyeosylated ia4C)hydroxylysine were measured by incubating tissues with I~4Cllysine and comparing the amounts of El~Cihydroxylysine released by acid hydrolysis and mild basic hydrolysis. As reported elsewherO, the alkaline-resistant I~4Clhydroxylysine synthesized by embryonic cartilage under the conditions used here consisted of galactosyl-hydroxylysine and glucosylgalactosyl-hydroxylysine. Assay of the nonglycosylated and glycosylated I14C!hydroxylysine in collagenase peptides from the tissues indicated that there was also a decrease in the glyeosylated TABLE

11 l

EFFECT OF AZETIDINE-CARBOXYLIC ACID AND CiS-FLUOROPROLINE ON THE SYNTHESIS OF GLYCOSYLATED [14Cj HYDROXYLYSINE S a m p l e s c o n t a i n i n g i o t i b i a e e a c h w e r e p r e - i n c u b a t e d for 2 h, a n d t h e n t h e y w e r e i n c u b a t e d w i t h 5 or i o !tC [l*Cllysine a n d 200 p g / m l L - a z e t i d i n e - 2 - c a r b o x y l i e a c i d or cis-4-fluoro-L-proline as d e s c r i b e d in T a b l e 1. T h e c o l l a g e n a s e - s o l u b i l i z e d p e p t i d e s w e r e p r e p a r e d , a n d t h e ~14Cihydroxvl y s i n e w a s a s s a y e d a s d e s c r i b e d i n t e x t . G l y c o s y l a t e d ~ l * C ! h y d r o x y l y s i n e w a s m e a s u r e d as t h e d i f f e r e n c e b e t w e e n t h e a m o u n t of [ 1 4 C l h y d r o x y l y s i n e r e l e a s e d b y a c i d i c a n d m i l d a l k a l i n e h y d r o l y s i s (see t e x t ) . A l i q u o t s u s e d for t h e c h r o m a t o g r a p h i c a s s a y s c o n t a i n e d .25 o o o - i o o ooo d i s i n t . / rain, a n d t h e o b s e r v e d c o u n t s / m i n [ 1 4 C ] h y d r o x y l y s i n e w e r e a t l e a s t 50 t i m e s t h e b a c k g r o u n d o f the counting system.

Expt. incubation condition

Total [a4C]Hyl" ( disint./ rain X zo ~)

Glvcosylated [l~CjHyl" ( disint./ rain × zo -3)

Ratio gIycosylated to total F14C]Hyl

Control L-Azetidine-2-carboxylic acid

21, 4 0. I

i t .4 2.1

53 34

Control

21.4 I2.6

I 1.5 6. 5

53 52

No.

cis-4-Fluoro-L-proline

* V a l u e s b a s e d o n i o o ooo d i s i n t . / m i n of p e p t i d e - b o u n d 14C i n c o l l a g e n a s e - s o l u b i l i z e d p e p t i d e s , A c c o r d i n g l y , v a l u e s d o n o t r e f l e c t t h e f a c t t h a t t o t a l i n c o r p o r a t i o n o f [t4C!lysine i n t o p r o t e i n w a s i n h i b i t e d a b o u t l O % i n t h e p r e s e n c e of 2 o o / ~ g / m l L - a z e t i d i n e - 2 - c a r b o x y l i c a c i d (Fig. 6), a n d a b o u t 2 6 % i n t h e p r e s e n c e of 2oo p g / m l cis-4-fluoro-L-proline (Fig. 7).

Biochim. Biophys. Acta, 175 (1969) 1 5 6 - 1 6 4

BIOSYNTHESIS

OF ABNORMAL COLLAGENS.

II

163

!~4Clhydroxylysine in the collagen synthesized in the presence of the two analogues (Table III). In the collagen containing azetidine-carboxylic acid, the ratio of glycosylated E14Clhydroxylysine to total E14C!hydroxylysine was decreased, suggesting that the presence of this analogue also interfered with the glycosylating reactions, cisFluoroproline did not produce this effect, and the decrease in glycosylated [14C~hydroxylysine could be accounted for by the glycosylations decreasing in the same proportion as the synthesis of [14Cqhydroxylysine. DISCUSSION

In previous studies l-a, the hydroxylation of proline and lysine in protocollagen were shown to be essential for the extrusion of the protein by incubating isolated cartilage under anaerobic conditions or with the iron chelator a,a'-dipyridyl. Extensive control experimentsa,8, 9 suggested that the effects of anaerobiosis and a,a'-dipyridyl on extrusion were specifically related to the fact that 02 was an essential cosubstrate and iron was an essential cofactor for the hydroxylation of proline and lysine in protocollagen. The results presented here offer an independent technique by which connective tissues can be made to synthesize a protein related to collagen without extruding it. Incubation of embryonic cartilage with either L-azetidine-2-carboxylic acid or cis4-fluoro-L-proline results in the synthesis of abnormal collagen containing these proline analogues ~, and autoradiographs prepared with EaHlproline indicated that the abnormal collagen containing either analogue is not extruded into the extracellular matrix. The [14Cllysine incorporation 5 indicated that protein synthesis continues at rates comparable to the control rate, and the analogues did not in themselves prevent extrusion of previously accumulated protocollagen after the protocollagen was hydroxylated to collagen. The results indicate, therefore, that the failure to extrude the abnormal collagens is directly related to the incorporation of the proline analogues into the protein. In exploring the reasons why the abnormal collagens are not extruded, it was found that these collagens not only have a decreased content of hydroxyproline, but also a decreased content of hydroxylysine and glycosylated hydroxylysine. In addition, incorporation of the analogue in place of proline probably decreases the proline content of the collagens, but because the analogues decrease the cellular uptake of [14ClprolineS, and because quantitative measurements could not be made on the small amounts of newly synthesized collagen in the tissue, it was not possible to demonstrate a decrease in proline content. Accordingly, the failure of the abnormal collagens to be extruded could be explained by the presence of the proline analogue, or perhaps by their low content of proline. Previous studies on conditions which limit 1-a or facilitate extrusion 1°, however, suggest that the critical features of the abnormal collagens are their low contents of hydroxyproline, hydroxylysine, or glycosylated hydroxylysinell, 12. The low hydroxylysine content of the collagens containing L-azetidine-carboxylic acid or cis-fluoroproline is probably related to the decreased hydroxylation of the proline still incorporated into protocollagen in the presence of the analogues 5. The analogues did not in themselves inhibit the hydroxylation of lysine in normal protocollagen previously accumulated in the tissue, and therefore the decreased conversion of protein-bound [14Cllysine to [14Clhydroxylysine is probably explained by the

Biochirn. Biophys. Acta, ~75 (I969) ~56-T64

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presence of the analogue in the polypeptide chains of tile abnormal protocollagen. Prelinfinary observations (see refs. i, 2) suggested that the same protocollagen hydroxylase is involved in the synthesis of both hydroxyproline and hydroxylysine, but more recent results (E. WEINSTEIN AND I). J . PRO('KOP, in preparation) suggest that a separate enzyme is required in the synthesis of hydroxylysine. The results obtained here indicate that if two separate enzymes are involved, substitution of L-azetidinecarboxylic acid or cis-fluoroproline into protocollagen adversely affects its substrate activity with both hydroxylases. ACKNOWLEDGEMENTS

The authors gratefully acknowledge the expert technical assistance of Mrs. A. CYWlNSKI, Miss E. YEZDIMIR,and Mrs. L. MURPHY. The work was supported in part by research grants FR-Io7, HD-I83, and GM14583 from the National Institutes of Health, U.S. Public Health Service. REFERENCES I ]). J. PROCKOP AND I{. ][. KIVIRIKKO, Ann. Intern. Med., 66 (I967) 1243. 2 J. ROSENBLOOM AND D. J. PROCKOP, in J. E. DUNPHY AND VC VAN V~rlNKLE, JR., Repair and regeneration: the scientific basis of surgical practive, McGraw-Hill, New York, 1968, in the press. 3 G. \V. COOPER AND D. J. PROCKOP, J. Cell Biol., 38 (1968) 537. 4 J. ROSENBLOOM, N. BLUMENKRANTZ AND D. J. PROCKOP, Biochem. Biophys. Res. Commun., 31 (1968) 792. 5 T. TAKEUCttI AND D. J. PROCKOP, Biochim. Biophys. ,4cta, 175 (1969) I42. 0 W. T. BUTLER AND L. \¥. CUNNINGHAM, J. Biol. Chem., 241 (1966) 3882. 7 R. G. SPIRO, J. Biol. Chem., 242 (I967) 4813 . 8 K. JUVA, D. J. PROCKOP, F. VV. COOPER AND J. W. LASH, Science, 152 (r966) 92. 9 R. S. BHATNAGAR AND D. J. PROCKOP, Biochim. Biophys. Acta, 13o (1966) 383 . IO R. S. BHATNAGAR, I{. l. t{IVIRIKKO, J. ROSENBLOOM AND D. J. PROCKOP, Proc. Natl. Acad. Sci., U.S., 58 (1967) 248. ix H. B. BOSMAN AND E. H. EYLAR, Biochem. Biophys. Res. Commun., 30 (1968) 89. 12 I{. G. SPIRO AND M. J. SPIRO, Federation Proc., 27 (1968) 345-

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