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Inhibition of collagen hydroxylysine formation by chelating agents
168
SHORT
BIOCHIMICA ET BIOPHYSICA ACTA
COMMUNICATIONS
BBA 33 O18
Inhibition of collagen hydroxylysine formation by chelating agents It has previously been shown that in the presence of metal chelating agents no collagen hydroxyproline is formed, whereas the amount of proline is doubled under these conditions z& In similar manner to the biosynthesis of hydroxyproline from proline, hydroxylysine, a second amino acid typical of collagen, is synthesized from lysine 3, i.e. hydroxylysine as such is not incorporated into the collagen molecule 3. Tile question arises: is it also possible to influence the formation of hydroxylysine with chelating substances?. This study deals with experiments aimed to clarify this question. Two g of skin slices from i4-i6-day-old chick embryos were incubated with 6.0-7.0/~C [14C~lysine and 20/,moles ATP (sodium salt) in IO ml Krebs-Ringer bicarbonate buffer. The concentration of chelating agents was always I raM. Incubation was carried out for 2 h at 37 ° in a Warburg apparatus, after which the samples were cooled and homogenized. The homogenates were first extracted with 0.2 M NaC1 (pH 7.4) and from the residue collagen was then extracted with trichloroacetic acid at 9 °° according to FITCH, HARKNESS AND HARKNESS4. This extract was dialysed against tap water. After hydrolysis of the samples (lO5°, 16 h, 6 M HC1) proline 5 and hydroxyproline 6 were determined. The residue from the trichloroacetic acid extraction contained non-collagenous proteins. This residue was washed with 5 % trichloroacetic acid, ethanol, ethanolether (I:4) and ether. The nitrogen content was determined 7, and aliquots were hydrolysed under the conditions mentioned above. The hydrolysates of both collagen and the non-collagenous proteins were evaporated on a flash evaporator. [14QLysine and E14C]hydroxylysine were separated on an Amberlite ICR 5o/AS column, 80 c m x 0. 9 cm, using citrate buffer (pH 7.0) as an eluent s. Fractions of 2 ml were collected. The amino acids were detected according to MOORE AND STEIN9. Radioactivity was measured with the Packard Tri-Carb liquid scintillation spectrometer 3003 (efficiency, 58.2-66.5 %), in a mixture of 4 ml ethanol, 5 ml scintillation solution and 0. 4 ml of sample. The collagen fraction was obtained as a comparatively pure preparation as shown by the molar ratios of proline to hydroxyproline and lysine to hydroxylysine. Collagen present in skin slices before incubation contains the normal amount of hydroxylysine. Collagen synthesized during incubation is labelled with both E14C~lysine and ~14CJhydroxylysine under control conditions; the radioactivity ratio of these amino acids (lysine 14300 disint./min per mg collagen, hydroxylysine 2500 disint./min per mg collagen) corresponds very closely to their stoichiometric ratio in the collagen molecule. In the presence of ~,~'-dipyridyl, however, hydroxylysine formation is inhibited (Table I). The inhibitory effect of other chelating substances is also demonstrated in Table I. Biochim. Biophys. Acta, 14o (1967) 168-17 °
169
SHORT COMMUNICATIONS
We also followed the incorporation of lysine into non-collagenous protein fractions in order to estimate the specificity of the blocking effect examined. These proteins contain approx. 8-IO % lysine. Therefore, the small difference in specific activities (Table I) per/,mole collagen lysine and hydroxylysine, respectively, could be due to some non-collagenous impurity with a comparatively high lysine content. Furthermore, comparison of lysine incorporation into collagen and unspecific proteins, respectively, confirms former findings TM on the higher metabolic turnover rate of noncollagenous proteins (Table I). TABLE
I
EFFECT OF CHELATING AGENTS ON THE SPECIFIC ACTIVITY OF LYSINE AND HYDROXYLYSINE IN COLLAGENOUS AND NON-COLLAGENOUS PROTEINS T w o g of s k i n slices w e r e i n c u b a t e d in i o m l m e d i a w i t h 6 . i 6 8 p C [14Cllysine h a v i n g a specific a c t i v i t y of 209 p C ( p m o l e ) . T h e c h e l a t i n g a g e n t s w e r e u s e d in 1 m M c o n c e n t r a t i o n s .
Addition to incubation medium
Collagen Lys*
Non-collagenous proteins Hyl*
Activity as % of activity in the control
Lys Control a,a'-Dipyridyl i,io-Phenanthroline 8-Hydroxyquinoline E D T A ( d i s o d i u m salt) Diethyldithiocarbamate ( s o d i u m salt)
37 36 5° 12 26 49 35
8oo 5oo ooo ioo 900 ooo ioo
32 3° o o o 39 4
ooo ioo
Activity as % of activity in the control
ttyl
8oo ooo 134 32 72 131 94
Lys*
o o o 124 13
96 91 74 24 55 ioi 87
ooo 6oo ooo ooo 4°0 300 5° 0
--79 25 58 lO8 93
* A c t i v i t y g i v e n in d i s i n t . / m i n per pmole.
The efficiency of the chelating substances used is markedly different, o~,£Dipyridyl completely inhibits hydroxylysine formation ; at the same time the content of lysine in collagen is increased (Table I). This effect has already been demonstrated by analogous experiments using [14Qprolinel,2 and apparently represents the biosynthesis of a protein similar to collagen in many respects, but enriched in proline (or lysine) and free of or poor in the corresponding hydroxy amino acids ('atypical collagen'). After addition of mmolar concentrations of I,io-phenanthroline and 8hydroxyquinoline, respectively, not only the conversion of lysine to hydroxylysine but also the formation of collagen and non-collagenous proteins is inhibited (Table I). EDTA does not influence hydroxylysine formation at all, though in vitro it is able to form Fe 2+ chelates with dissociation constants comparable to those of the corresponding dipyridyl complexes. However, EDTA molecules do not penetrate the cell membrane, as was shown previously*. The effect of diethyldithiocarbamate is in agreement with a slighter ability of this substance to sequester Fe z+. Hydroxylation of lysine is not completely inhibited (87 %), and protein biosynthesis occurs practically normally. Another series of experiments, in which the data were expressed as disint./min per mg collagen, confirmed all results obtained. * J . VoST•L, results.
V. NIKODI~MOVX, M. CHVAPIL, V. KOBRLE AND E . EHRLICHOVA, u n p u b l i s h e d
Biochim. Biophys. Acta,
14o (1967) 1 6 8 - 1 7 o
17o
SHORT COMMUNICATIONS
To summarize our findings, these results appear completely analogous to those reported on collagen hydroxyproline formation1, 2. Therefore, it m a y be assumed that the mechanisms involved in the hydroxylation of proline to hydroxyproline and of lysine to hydroxylysine are essentially the same, especially with regard to metal ion participation. The authors wish to thank Prof. W. GRASSMANN and Dr. M. CHVAPIL for valuable advice. The technical assistance of Miss H. RENZ is gratefully acknowledged. J. HURYCH was supported b y a fellowship of the Max-Planck-Gesellschaft zur F6rderung der Wissenschaften e.V.
Max-Planck-Institut fi~r Eiweiss und Lederforschung, Miinchen (Germany)
JOSEF HURYCH* ARNOLD N O R D W I G
I J. HURYCH AND M. CHVAHL, Biochim. Biophys. Acta, 97 (1965) 361. 2 M. CHVAPIL, J. HURYCH, E. EHRLICHOV~ AND B. ~MUCHALOV,6., Biochim. Biophys. Acta, in t h e press. 3 F. M. SINEX, D. D. VAN SLYKE AND D. R. CHRISTMAN, J. Biol. Chem., 234 (1959) 918. 4 S. M. FITCH, M. L. R. HARKNESS AND R. D. HARKNESS, Nature, 176 (1955) 163. 5 W. TROLL AND J. LI~IDSLEY, J. Biol. Chem., 215 (1955) 655. 6 H. STEGEMANN, Z. Physiol. Chem., 311 (1958) 41. 7 L. STRAUCH, Z. Klin. Chem., 3 (1965) 165. 8 K. HANNIG, Clin. Chim. Acta, 4 (1959) 51. 9 S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. io J. HURYCH AND M. CHVAPIL, Bioehem. J., 97 (1965) 236.
Received July 8th, 1966 Revised manuscript received November 23rd, 1966 * P e r m a n e n t a d d r e s s : D e p a r t m e n t of E x p e r i m e n t a l Biology, I n s t i t u t e of I n d u s t r i a l H y g i e n e a n d O c c u p a t i o n a l Diseases, Prague, Czechoslovakia.
Note added in proof (Received April 27th, 1967) PROCKOP, WEINSTEIN AND MULVEN¥ (Biochem. Biophys. Res. Commun., 22 (1966) 124) c a m e to t h e s a m e conclusions r e p o r t e d here, u s i n g a different s y s t e m .
Biochim. Biophys. :4cta, 14o (1967) 168-17o
BBA 33022
Ultracentrifugal analysis of seminal ribonuclease Seminal ribonuclease is an enzyme, isolated from bull seminal plasma, whose general properties are similar to those of the pancreatic ribonuclease isolated b y Kunitzl, 2. This note presents results obtained in a comparative study of the behaviour in the ultracentrifuge of the seminal and pancreatic ribonucleases. Seminal ribonuclease was prepared according to D'ALESSlO et al. (refs. I, 2 and manuscript in preparation). Five different preparations were analyzed, whose specific activity varied between 18 and 33 Kunitz units. In fact the highest specific activity values were obtained in the last two preparations in which a new purification step was added. Pancreatic ribonuclease was the commercially available Sigma 5 × crystalBiochim. Biophys. Acta, 14o (1967) 17o-173
SHORT
BIOCHIMICA ET BIOPHYSICA ACTA
COMMUNICATIONS
BBA 33 O18
Inhibition of collagen hydroxylysine formation by chelating agents It has previously been shown that in the presence of metal chelating agents no collagen hydroxyproline is formed, whereas the amount of proline is doubled under these conditions z& In similar manner to the biosynthesis of hydroxyproline from proline, hydroxylysine, a second amino acid typical of collagen, is synthesized from lysine 3, i.e. hydroxylysine as such is not incorporated into the collagen molecule 3. Tile question arises: is it also possible to influence the formation of hydroxylysine with chelating substances?. This study deals with experiments aimed to clarify this question. Two g of skin slices from i4-i6-day-old chick embryos were incubated with 6.0-7.0/~C [14C~lysine and 20/,moles ATP (sodium salt) in IO ml Krebs-Ringer bicarbonate buffer. The concentration of chelating agents was always I raM. Incubation was carried out for 2 h at 37 ° in a Warburg apparatus, after which the samples were cooled and homogenized. The homogenates were first extracted with 0.2 M NaC1 (pH 7.4) and from the residue collagen was then extracted with trichloroacetic acid at 9 °° according to FITCH, HARKNESS AND HARKNESS4. This extract was dialysed against tap water. After hydrolysis of the samples (lO5°, 16 h, 6 M HC1) proline 5 and hydroxyproline 6 were determined. The residue from the trichloroacetic acid extraction contained non-collagenous proteins. This residue was washed with 5 % trichloroacetic acid, ethanol, ethanolether (I:4) and ether. The nitrogen content was determined 7, and aliquots were hydrolysed under the conditions mentioned above. The hydrolysates of both collagen and the non-collagenous proteins were evaporated on a flash evaporator. [14QLysine and E14C]hydroxylysine were separated on an Amberlite ICR 5o/AS column, 80 c m x 0. 9 cm, using citrate buffer (pH 7.0) as an eluent s. Fractions of 2 ml were collected. The amino acids were detected according to MOORE AND STEIN9. Radioactivity was measured with the Packard Tri-Carb liquid scintillation spectrometer 3003 (efficiency, 58.2-66.5 %), in a mixture of 4 ml ethanol, 5 ml scintillation solution and 0. 4 ml of sample. The collagen fraction was obtained as a comparatively pure preparation as shown by the molar ratios of proline to hydroxyproline and lysine to hydroxylysine. Collagen present in skin slices before incubation contains the normal amount of hydroxylysine. Collagen synthesized during incubation is labelled with both E14C~lysine and ~14CJhydroxylysine under control conditions; the radioactivity ratio of these amino acids (lysine 14300 disint./min per mg collagen, hydroxylysine 2500 disint./min per mg collagen) corresponds very closely to their stoichiometric ratio in the collagen molecule. In the presence of ~,~'-dipyridyl, however, hydroxylysine formation is inhibited (Table I). The inhibitory effect of other chelating substances is also demonstrated in Table I. Biochim. Biophys. Acta, 14o (1967) 168-17 °
169
SHORT COMMUNICATIONS
We also followed the incorporation of lysine into non-collagenous protein fractions in order to estimate the specificity of the blocking effect examined. These proteins contain approx. 8-IO % lysine. Therefore, the small difference in specific activities (Table I) per/,mole collagen lysine and hydroxylysine, respectively, could be due to some non-collagenous impurity with a comparatively high lysine content. Furthermore, comparison of lysine incorporation into collagen and unspecific proteins, respectively, confirms former findings TM on the higher metabolic turnover rate of noncollagenous proteins (Table I). TABLE
I
EFFECT OF CHELATING AGENTS ON THE SPECIFIC ACTIVITY OF LYSINE AND HYDROXYLYSINE IN COLLAGENOUS AND NON-COLLAGENOUS PROTEINS T w o g of s k i n slices w e r e i n c u b a t e d in i o m l m e d i a w i t h 6 . i 6 8 p C [14Cllysine h a v i n g a specific a c t i v i t y of 209 p C ( p m o l e ) . T h e c h e l a t i n g a g e n t s w e r e u s e d in 1 m M c o n c e n t r a t i o n s .
Addition to incubation medium
Collagen Lys*
Non-collagenous proteins Hyl*
Activity as % of activity in the control
Lys Control a,a'-Dipyridyl i,io-Phenanthroline 8-Hydroxyquinoline E D T A ( d i s o d i u m salt) Diethyldithiocarbamate ( s o d i u m salt)
37 36 5° 12 26 49 35
8oo 5oo ooo ioo 900 ooo ioo
32 3° o o o 39 4
ooo ioo
Activity as % of activity in the control
ttyl
8oo ooo 134 32 72 131 94
Lys*
o o o 124 13
96 91 74 24 55 ioi 87
ooo 6oo ooo ooo 4°0 300 5° 0
--79 25 58 lO8 93
* A c t i v i t y g i v e n in d i s i n t . / m i n per pmole.
The efficiency of the chelating substances used is markedly different, o~,£Dipyridyl completely inhibits hydroxylysine formation ; at the same time the content of lysine in collagen is increased (Table I). This effect has already been demonstrated by analogous experiments using [14Qprolinel,2 and apparently represents the biosynthesis of a protein similar to collagen in many respects, but enriched in proline (or lysine) and free of or poor in the corresponding hydroxy amino acids ('atypical collagen'). After addition of mmolar concentrations of I,io-phenanthroline and 8hydroxyquinoline, respectively, not only the conversion of lysine to hydroxylysine but also the formation of collagen and non-collagenous proteins is inhibited (Table I). EDTA does not influence hydroxylysine formation at all, though in vitro it is able to form Fe 2+ chelates with dissociation constants comparable to those of the corresponding dipyridyl complexes. However, EDTA molecules do not penetrate the cell membrane, as was shown previously*. The effect of diethyldithiocarbamate is in agreement with a slighter ability of this substance to sequester Fe z+. Hydroxylation of lysine is not completely inhibited (87 %), and protein biosynthesis occurs practically normally. Another series of experiments, in which the data were expressed as disint./min per mg collagen, confirmed all results obtained. * J . VoST•L, results.
V. NIKODI~MOVX, M. CHVAPIL, V. KOBRLE AND E . EHRLICHOVA, u n p u b l i s h e d
Biochim. Biophys. Acta,
14o (1967) 1 6 8 - 1 7 o
17o
SHORT COMMUNICATIONS
To summarize our findings, these results appear completely analogous to those reported on collagen hydroxyproline formation1, 2. Therefore, it m a y be assumed that the mechanisms involved in the hydroxylation of proline to hydroxyproline and of lysine to hydroxylysine are essentially the same, especially with regard to metal ion participation. The authors wish to thank Prof. W. GRASSMANN and Dr. M. CHVAPIL for valuable advice. The technical assistance of Miss H. RENZ is gratefully acknowledged. J. HURYCH was supported b y a fellowship of the Max-Planck-Gesellschaft zur F6rderung der Wissenschaften e.V.
Max-Planck-Institut fi~r Eiweiss und Lederforschung, Miinchen (Germany)
JOSEF HURYCH* ARNOLD N O R D W I G
I J. HURYCH AND M. CHVAHL, Biochim. Biophys. Acta, 97 (1965) 361. 2 M. CHVAPIL, J. HURYCH, E. EHRLICHOV~ AND B. ~MUCHALOV,6., Biochim. Biophys. Acta, in t h e press. 3 F. M. SINEX, D. D. VAN SLYKE AND D. R. CHRISTMAN, J. Biol. Chem., 234 (1959) 918. 4 S. M. FITCH, M. L. R. HARKNESS AND R. D. HARKNESS, Nature, 176 (1955) 163. 5 W. TROLL AND J. LI~IDSLEY, J. Biol. Chem., 215 (1955) 655. 6 H. STEGEMANN, Z. Physiol. Chem., 311 (1958) 41. 7 L. STRAUCH, Z. Klin. Chem., 3 (1965) 165. 8 K. HANNIG, Clin. Chim. Acta, 4 (1959) 51. 9 S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. io J. HURYCH AND M. CHVAPIL, Bioehem. J., 97 (1965) 236.
Received July 8th, 1966 Revised manuscript received November 23rd, 1966 * P e r m a n e n t a d d r e s s : D e p a r t m e n t of E x p e r i m e n t a l Biology, I n s t i t u t e of I n d u s t r i a l H y g i e n e a n d O c c u p a t i o n a l Diseases, Prague, Czechoslovakia.
Note added in proof (Received April 27th, 1967) PROCKOP, WEINSTEIN AND MULVEN¥ (Biochem. Biophys. Res. Commun., 22 (1966) 124) c a m e to t h e s a m e conclusions r e p o r t e d here, u s i n g a different s y s t e m .
Biochim. Biophys. :4cta, 14o (1967) 168-17o
BBA 33022
Ultracentrifugal analysis of seminal ribonuclease Seminal ribonuclease is an enzyme, isolated from bull seminal plasma, whose general properties are similar to those of the pancreatic ribonuclease isolated b y Kunitzl, 2. This note presents results obtained in a comparative study of the behaviour in the ultracentrifuge of the seminal and pancreatic ribonucleases. Seminal ribonuclease was prepared according to D'ALESSlO et al. (refs. I, 2 and manuscript in preparation). Five different preparations were analyzed, whose specific activity varied between 18 and 33 Kunitz units. In fact the highest specific activity values were obtained in the last two preparations in which a new purification step was added. Pancreatic ribonuclease was the commercially available Sigma 5 × crystalBiochim. Biophys. Acta, 14o (1967) 17o-173