- Email: [email protected]
Hydroxylation of proline in collagen model peptide
I54
SHORT CO?d~UNICATiONS
original, unfractionated chondroitin sulphate, which was calculated by the present. method (Fig. ~). An ;~w value of ~5 3oo and aa ;~n value of ~o 8oo were obtained. They are in good agreement with recent data on the mo!ect~lar weight of chondrc;itin sulphate prepared b y papain digest~on~, 1~. This application of analytical gel chro-matography can serve as a valuabte tool for rapid and inexpensive molecular weight determinations on microgram specimens of chondroitin sulphate. Moiecutar weight distribution analysis on chondroitin sulphates from tissues ir~ various physiologicai and pathological states are in progress.
i~stitute of MedicaZ Chemistc¢, U~iversi~y of Uppsala, Uppsala (Sweden.)
~I~ W~s~sox
~ T. C. L~SHR~T, in G. Q~LWT_~R~LL~, The Chemical ~hysio~ogy of ;ldr~,c@olysaccha~,ides, Little, Brown, Boston, ~968, p. ~53. ~ T. C. LAURENT,Federa~iov¢ Proc., ~5 (~966) ~ 8 . 3 H. DETERh~ANN, Gdchromatogr@hie, Springer, Beriin, ~967. 4 T. C. L~UR~NT AND J. K~LLAND~R, ~. Chromatog., ~4 (1964) 3~75 T. C. LAURENT, B. 0BRINK, K. HELLSING ANn A. ~IASTGSON,in T. OERRITSEN, Modemz S@aration Methods of Macromo~ec~des a~d Particles, Wiley, N e w York, in ~che wess~ 6 K. A. GRANATH A~-D B. E. KWS% ~. Chromatog., ~8 (~967) 69. 7 J. E. SCOTT, in D. GL~CK, Methods of B¢ochemical A~Xysis, Vol. 8, Interscience, New York~ ~96o, p. ~46. 8 T. C. LAURENT AND K. A. @NANATH, Biochi~. B~ophys. dcta, ~36 (1967) I 9 r , 9 N. V. B. MARSDEN, A ~ z . N . Y . Acgd. Sci., I25 (I965) 428. xo E. A. BAL.~ZS, K. O. BERNTS~N, J. K,~ROSSA AN'D ~ . A. SWANX, d~zaL Biochem., ~2 (z965) 547~I E. B~DDECKE, XY. K~OZ AXD E. L_~NKA, Z. Physiol. Chem., 33x (~963) ~96. I2 ~I. LUSCOMBE AND C. F. PHELPS, Biochem. ,f., ~o 3 (~967) Io 3.
Received October zSth, z968 Biochim. ]3i@hyso Acta, ~77 (1969) 152-~54
BBA ~3475
Hydroxylation of proline in collagen model peptide Recent studies show that the hydroxyproline and hydroxylysine in collagen are synthesized b y hydroxylation reactions ~vhich occur after proline and lvsine trove been incorporated into some polypeptide precursor of collagen1, ~-. EIVIRIKKO AND PROCKOP~ suggested that the protocollagen obtained from embryonic tissues, in whicl~ the hydroxylations of proline and lvsine are inhibited, is comparable in size ro the x-chain of collagen and it serves as a substrate in the synthesis of hydroxyproline and hydroxylysine b y the hydroxylase of chick embryos. However, i~ is uncertain whether ~ vivo the hydroxylation process begins after the complete synthesis of_ proline- and tysine-rich polypeptides similar in size to the ~-chain of collagen. Recently, polypeptide models of collagen have been used in studies on the s~ructnre of collagen ~ and the mechanism of proline hydroxylationa-L This paper reports investigations on proline hydroxylations of synthetic model peptides consisting of repeating umrs of a tripeptide, P r o - P r o - E l y . These raodels of crystal!the regions of native collagen were synthesized b y Dr. S. SAKAKIBARA(The Institute for Protein Research of this University) by stepwise polymerization of P r o - P r o - G t v with chlorobenzy!polystyrene resin as a supporting materiaI. Details of the preparation of these model ~diochim. Bio_Ohys. AcXc~, ~77 (1969 z54-~56
SHORT COMMUNICATIONS
155
peptides were published elsewhereS,9. Various sizes of the synthetic (Pro-Pro-Gly)n were incubated with a fraction of granuloma (see below) and various cofactors such as Fe 2+, ascorbate and e-ketoglutarate, under oxygen. Granuloma were produced by the subcutaneous injection of 1% carageenan solution into guinea pigs, as described by ROBERTSON AN~ SC~IWARTZt°. After 8 days, the animals were decapitated. The granuloma were removed, minced and homogenized with medium M by the method of MANNING AND MEISTERn. The homogenate was centrifuged at lO5OOO × g for 3o rain and the supernatant was used as the enzyme source. All polypeptides were used as aqueous solutions, except (Pro-Pro-Gly)2o which, being poorly soluble, was added as a suspension in buffer solution. After incubation, samples were extracted with 5 % trichloroacetic acid at 9 °o for 2 h and hydrolyzed with 6 M HC1 at I i o ° for 115 h. Then, proline and hydroxyproline were assayed. Table I shows that when the collagen model peptides were incubated with granuloma extracts, there was significant formation of hydroxyproline with (ProPro-Gly) a, (Pro-Pro-Gly)~ and (Pro-Pro-Gly)~0 but not with (Pro-Pro-Gly)~ and the hydroxylation activity increased in parallel with the molecular size of the peptide. However, the activity with (Pro-Pro-Gly)20 was considerably lower than that with (Pro-Pro-Gly)~ o. This low activity with (Pro-Pro-Gly)20 may be due to the low solubility of (Pro-Pro-Gly)20 at neutral pH. Polypeptides smaller than (Pro-ProGly)~ 0 are soluble at physiological pH values, while (Pro-Pro-Gly)~0 is not. (ProPro-Gly) 20 is soluble in IO % acetic acid or 5o % ethanol. From the solubilities of P r o - P r o - G l y polymers it seems that larger polytripeptides than (Pro-Pro-Gly)2o cannot be incubated as solutions at physiological pH values. However, KATCI~ALSKI and lhis associates reported that (Pro-Gly-Pro)n with molecular weights of up to I2 ooo were water-soluble ~. These apparently conflicting results need further research. On the basis of molecular weight, the (Pro-Pro-Gly)n used in these experiments had sharper thermal transitions in aqueous solutions than did KATCHALSKI'S (Pro-GlyPro)m as indicated by a decrease in laevorotation s& It should be noted that the solubility of many poly-~-amino acids is markedly affected by their molecular weight distribution, as well as b y the procedure employed in their synthesis. The experiments described in this paper show that larger polytripeptides than (Pro-Pro-Gly) ~ can serve as the substrate for proline hydroxylation in vitro. Recently, TABLE I HYDROXYLATION OF MOD]~L PEPTIDES T h e reaction m i x t u r e contained: ascorbate, 9/~moles; FeSO~, 2.4/zmoles; a-ketoglutarate, 2. 4 /zmoles; model peptide, i o rag; g r a n u l o m a fraction, 2 ml; m e d i u m M (ref. I I ) . The t o t a l v o l u m e w a s 3 ml. Mixtures were i n c u b a t e d for 2 h at 37 ° w i t h s h a k i n g u n d e r O 2 - C O ~ (95:5, v/v). After incubation, collagen was extracted and hydrolyzed as described in t h e text. Proline and h y d r o x y proline were assayed b y the procedures of TROLL AND LINDSLEY 1~ a n d ~X~EUMAN AND LOGAN 13, respectively. Degree of h y d r o x y l a t i o n is defined as 2 H y p × I o o / P r o + H y p .
Modal peptide
Proline (l~g)
Hydroxyproline formed (l'g)
Degreeof hydroxyl~tion
(Pr°-l~r°-Gly) 1 (Pro-Pro-Gly) 3 (Pro-Pro-Gly) s (Pro-]Pro-Gly) 10 ( P r o - P r o - G l y ) 20
675o 7 °00 6600 6500 6930
6.9 19-8 21.4 32.6 19.8
0.20 o.57 o.65 I.OO 0.57
Biochim. Biophys. Acta, 177 (1969) 1 5 4 - - 1 5 6
150
SHORT CO?eF.~iUXICATI0 N ):
BEK~OR AND BAV~ETTAsuggested that collagen-proline hydroxylation occurs on a: polypeptide of a too]ocular weighl of !ass than IOOOO in the rabbit einbryo ski:i m. Therefore, it seems ]ikely that dae hydroxylation of proline i~¢ vivo may occur before the completion of the synthesis of collagen ~-chains. KIVIRIKKOAND PROGKOPreported. that when the hydroxylations of proline and lysine are inhibited, the pro/ocoliagen molecules synthesized are comparable in size to the ~-chain of coiiagen and that they serve as substrates for the cel!-free hydroxylation reaction. However, it is not entJre)y clear whether a collagen precursor as large as the a-chain of coi]agen is the true substrata for the hydroxylation under normal conditions, because d~e protoco]lagen in their experiment was prepared in the presence of an inhibitor of the hydroxyia~ion. The model peptides used in this experiment consisted of oniy non-polar unit< and it seems ~hat the hydroxyla/ion system requires polypeptides wid~ a repeating structure of polar and non-polar peptides. Indeed, it is genera!1y d~ought that in [t~e polypeptide chain of coliagen, amorphous regions, where polar amino acids are abundant, alternate with crystalline regions consisting of - G l y - P r o - X - units~< i t wou!d be interesting to see whether collagen model peptides in which crystailine regions (non-polar peptides) alternate with amorphous regions (polar peptides) are a better substrata %r proline hydroxyia{ion than simple non-poiar peptides, (Pro:Pro-Gly)n. The former copolymer type of peptide is more ]ike native collagen and should also be more soluble than simple non-polar peptides, because it contaias polar amino acids. The precise nature of the polypeptide precursors %r t~e 5ydroxylado> stil! requires further research. This work was supported in part by a ~und from the Takeda Science Foundation. The authors wish to thank Professor Y. TAKEDA (Department o~ Biochemistry, Dental School, Osaka University) %r his valuable advice and encouragement. Tha=ks are also due to Dr. S. S.aKaK~>ARa (The institute tot Protein Research, Osaka University) for providing synthetic model peptides.
Depar¢me~t of Biochemistry, Dental SchooZ, Osaka University, Osaka (Japm¢)
Fuj~o SuzuKI EIZO
KOYAMA
I B. P ~ E R K O F S K ¥ ~ND S. UDENFRIEND, J. Biol. Chem., 238 (I963) 3966. 9_ D. J. P R o c K o P , E . WEINSTEIN AND T. ~{ULVENY Biochem. Bi@hys. Res. Commu¢~a. 22 ~966) 124 . 3 K. I. KIVlRIKNO AXD D. J. PROCKOP, Biochem. J., ~o2 (I967) 432. 4 J. E > , ~ L , J. N v ~ z , E . KATCHALSKI AND A. BgRGER J. Mol. BioL. ~7 (~966) 255. 5 D. J . P a o c K o e , K. J u v . ~ AX> J. E ~ ¢ E L , Z ~hysiol. Chem., 348 (~967) 553. 6 K. t. ~IVlRIKKO AND D. J. P~OCKOP, J. BioL Chem, 242 (I967) 4007 . 7 ~ " I. ~IVIRIKKO, H . J. BRIGHT AND D. J. PROCKOP, Biochim. ~iophys. Acta, IhZ (I968) 558. 8 Y . KISKtDA, S. SAKAKIBARA AND Y. KIKUCHI Proc. 5Xh Syrup. Peptide Chemistry, Kyoto, ~962, p. 3 o. 9 S. SAKAK~B~R*, Y . KISHIDA. Y. KIK~CHI. R. S&KAI AX-D E . K A ~ U C H I . B~H. Chem. Soc. ] @ ~ , 4~ (~968) ~273. xo W . VA~ RO~ER~SOX AND B. Scm~ARTz, J. Biol. Chem., 2o~ I 9 5 3 689. ~ J. M. M~NN~N~ AND A. M~ISTER. B{ochemistvy, 5 (~966) i 1 5 4 . ~2 ~V. TROLL AND J. LINDSL~Y. ~. Biol, C]zem. 2~5 (~953) 655, ~3 R . E . NEUMAN AND NI. A. LOGAN f . ~iol. Chem.. I 8 4 /~95o ) 299. ~4 I. BXK~OR A~-D L. A. BAVXTTA. PfOC. Natl. Acad. Sci. U,S., 58 {~967} Z35L 15 P. M. GALLOP, ~i@hys. ]. S~@pl., ~ (~964) 79.
Received August i6th, m968 Biochim. Biophys. dcta, 177 (I969) I 5 4 - ~ 5 6
SHORT CO?d~UNICATiONS
original, unfractionated chondroitin sulphate, which was calculated by the present. method (Fig. ~). An ;~w value of ~5 3oo and aa ;~n value of ~o 8oo were obtained. They are in good agreement with recent data on the mo!ect~lar weight of chondrc;itin sulphate prepared b y papain digest~on~, 1~. This application of analytical gel chro-matography can serve as a valuabte tool for rapid and inexpensive molecular weight determinations on microgram specimens of chondroitin sulphate. Moiecutar weight distribution analysis on chondroitin sulphates from tissues ir~ various physiologicai and pathological states are in progress.
i~stitute of MedicaZ Chemistc¢, U~iversi~y of Uppsala, Uppsala (Sweden.)
~I~ W~s~sox
~ T. C. L~SHR~T, in G. Q~LWT_~R~LL~, The Chemical ~hysio~ogy of ;ldr~,c@olysaccha~,ides, Little, Brown, Boston, ~968, p. ~53. ~ T. C. LAURENT,Federa~iov¢ Proc., ~5 (~966) ~ 8 . 3 H. DETERh~ANN, Gdchromatogr@hie, Springer, Beriin, ~967. 4 T. C. L~UR~NT AND J. K~LLAND~R, ~. Chromatog., ~4 (1964) 3~75 T. C. LAURENT, B. 0BRINK, K. HELLSING ANn A. ~IASTGSON,in T. OERRITSEN, Modemz S@aration Methods of Macromo~ec~des a~d Particles, Wiley, N e w York, in ~che wess~ 6 K. A. GRANATH A~-D B. E. KWS% ~. Chromatog., ~8 (~967) 69. 7 J. E. SCOTT, in D. GL~CK, Methods of B¢ochemical A~Xysis, Vol. 8, Interscience, New York~ ~96o, p. ~46. 8 T. C. LAURENT AND K. A. @NANATH, Biochi~. B~ophys. dcta, ~36 (1967) I 9 r , 9 N. V. B. MARSDEN, A ~ z . N . Y . Acgd. Sci., I25 (I965) 428. xo E. A. BAL.~ZS, K. O. BERNTS~N, J. K,~ROSSA AN'D ~ . A. SWANX, d~zaL Biochem., ~2 (z965) 547~I E. B~DDECKE, XY. K~OZ AXD E. L_~NKA, Z. Physiol. Chem., 33x (~963) ~96. I2 ~I. LUSCOMBE AND C. F. PHELPS, Biochem. ,f., ~o 3 (~967) Io 3.
Received October zSth, z968 Biochim. ]3i@hyso Acta, ~77 (1969) 152-~54
BBA ~3475
Hydroxylation of proline in collagen model peptide Recent studies show that the hydroxyproline and hydroxylysine in collagen are synthesized b y hydroxylation reactions ~vhich occur after proline and lvsine trove been incorporated into some polypeptide precursor of collagen1, ~-. EIVIRIKKO AND PROCKOP~ suggested that the protocollagen obtained from embryonic tissues, in whicl~ the hydroxylations of proline and lvsine are inhibited, is comparable in size ro the x-chain of collagen and it serves as a substrate in the synthesis of hydroxyproline and hydroxylysine b y the hydroxylase of chick embryos. However, i~ is uncertain whether ~ vivo the hydroxylation process begins after the complete synthesis of_ proline- and tysine-rich polypeptides similar in size to the ~-chain of collagen. Recently, polypeptide models of collagen have been used in studies on the s~ructnre of collagen ~ and the mechanism of proline hydroxylationa-L This paper reports investigations on proline hydroxylations of synthetic model peptides consisting of repeating umrs of a tripeptide, P r o - P r o - E l y . These raodels of crystal!the regions of native collagen were synthesized b y Dr. S. SAKAKIBARA(The Institute for Protein Research of this University) by stepwise polymerization of P r o - P r o - G t v with chlorobenzy!polystyrene resin as a supporting materiaI. Details of the preparation of these model ~diochim. Bio_Ohys. AcXc~, ~77 (1969 z54-~56
SHORT COMMUNICATIONS
155
peptides were published elsewhereS,9. Various sizes of the synthetic (Pro-Pro-Gly)n were incubated with a fraction of granuloma (see below) and various cofactors such as Fe 2+, ascorbate and e-ketoglutarate, under oxygen. Granuloma were produced by the subcutaneous injection of 1% carageenan solution into guinea pigs, as described by ROBERTSON AN~ SC~IWARTZt°. After 8 days, the animals were decapitated. The granuloma were removed, minced and homogenized with medium M by the method of MANNING AND MEISTERn. The homogenate was centrifuged at lO5OOO × g for 3o rain and the supernatant was used as the enzyme source. All polypeptides were used as aqueous solutions, except (Pro-Pro-Gly)2o which, being poorly soluble, was added as a suspension in buffer solution. After incubation, samples were extracted with 5 % trichloroacetic acid at 9 °o for 2 h and hydrolyzed with 6 M HC1 at I i o ° for 115 h. Then, proline and hydroxyproline were assayed. Table I shows that when the collagen model peptides were incubated with granuloma extracts, there was significant formation of hydroxyproline with (ProPro-Gly) a, (Pro-Pro-Gly)~ and (Pro-Pro-Gly)~0 but not with (Pro-Pro-Gly)~ and the hydroxylation activity increased in parallel with the molecular size of the peptide. However, the activity with (Pro-Pro-Gly)20 was considerably lower than that with (Pro-Pro-Gly)~ o. This low activity with (Pro-Pro-Gly)20 may be due to the low solubility of (Pro-Pro-Gly)20 at neutral pH. Polypeptides smaller than (Pro-ProGly)~ 0 are soluble at physiological pH values, while (Pro-Pro-Gly)~0 is not. (ProPro-Gly) 20 is soluble in IO % acetic acid or 5o % ethanol. From the solubilities of P r o - P r o - G l y polymers it seems that larger polytripeptides than (Pro-Pro-Gly)2o cannot be incubated as solutions at physiological pH values. However, KATCI~ALSKI and lhis associates reported that (Pro-Gly-Pro)n with molecular weights of up to I2 ooo were water-soluble ~. These apparently conflicting results need further research. On the basis of molecular weight, the (Pro-Pro-Gly)n used in these experiments had sharper thermal transitions in aqueous solutions than did KATCHALSKI'S (Pro-GlyPro)m as indicated by a decrease in laevorotation s& It should be noted that the solubility of many poly-~-amino acids is markedly affected by their molecular weight distribution, as well as b y the procedure employed in their synthesis. The experiments described in this paper show that larger polytripeptides than (Pro-Pro-Gly) ~ can serve as the substrate for proline hydroxylation in vitro. Recently, TABLE I HYDROXYLATION OF MOD]~L PEPTIDES T h e reaction m i x t u r e contained: ascorbate, 9/~moles; FeSO~, 2.4/zmoles; a-ketoglutarate, 2. 4 /zmoles; model peptide, i o rag; g r a n u l o m a fraction, 2 ml; m e d i u m M (ref. I I ) . The t o t a l v o l u m e w a s 3 ml. Mixtures were i n c u b a t e d for 2 h at 37 ° w i t h s h a k i n g u n d e r O 2 - C O ~ (95:5, v/v). After incubation, collagen was extracted and hydrolyzed as described in t h e text. Proline and h y d r o x y proline were assayed b y the procedures of TROLL AND LINDSLEY 1~ a n d ~X~EUMAN AND LOGAN 13, respectively. Degree of h y d r o x y l a t i o n is defined as 2 H y p × I o o / P r o + H y p .
Modal peptide
Proline (l~g)
Hydroxyproline formed (l'g)
Degreeof hydroxyl~tion
(Pr°-l~r°-Gly) 1 (Pro-Pro-Gly) 3 (Pro-Pro-Gly) s (Pro-]Pro-Gly) 10 ( P r o - P r o - G l y ) 20
675o 7 °00 6600 6500 6930
6.9 19-8 21.4 32.6 19.8
0.20 o.57 o.65 I.OO 0.57
Biochim. Biophys. Acta, 177 (1969) 1 5 4 - - 1 5 6
150
SHORT CO?eF.~iUXICATI0 N ):
BEK~OR AND BAV~ETTAsuggested that collagen-proline hydroxylation occurs on a: polypeptide of a too]ocular weighl of !ass than IOOOO in the rabbit einbryo ski:i m. Therefore, it seems ]ikely that dae hydroxylation of proline i~¢ vivo may occur before the completion of the synthesis of collagen ~-chains. KIVIRIKKOAND PROGKOPreported. that when the hydroxylations of proline and lysine are inhibited, the pro/ocoliagen molecules synthesized are comparable in size to the ~-chain of coiiagen and that they serve as substrates for the cel!-free hydroxylation reaction. However, it is not entJre)y clear whether a collagen precursor as large as the a-chain of coi]agen is the true substrata for the hydroxylation under normal conditions, because d~e protoco]lagen in their experiment was prepared in the presence of an inhibitor of the hydroxyia~ion. The model peptides used in this experiment consisted of oniy non-polar unit< and it seems ~hat the hydroxyla/ion system requires polypeptides wid~ a repeating structure of polar and non-polar peptides. Indeed, it is genera!1y d~ought that in [t~e polypeptide chain of coliagen, amorphous regions, where polar amino acids are abundant, alternate with crystalline regions consisting of - G l y - P r o - X - units~< i t wou!d be interesting to see whether collagen model peptides in which crystailine regions (non-polar peptides) alternate with amorphous regions (polar peptides) are a better substrata %r proline hydroxyia{ion than simple non-poiar peptides, (Pro:Pro-Gly)n. The former copolymer type of peptide is more ]ike native collagen and should also be more soluble than simple non-polar peptides, because it contaias polar amino acids. The precise nature of the polypeptide precursors %r t~e 5ydroxylado> stil! requires further research. This work was supported in part by a ~und from the Takeda Science Foundation. The authors wish to thank Professor Y. TAKEDA (Department o~ Biochemistry, Dental School, Osaka University) %r his valuable advice and encouragement. Tha=ks are also due to Dr. S. S.aKaK~>ARa (The institute tot Protein Research, Osaka University) for providing synthetic model peptides.
Depar¢me~t of Biochemistry, Dental SchooZ, Osaka University, Osaka (Japm¢)
Fuj~o SuzuKI EIZO
KOYAMA
I B. P ~ E R K O F S K ¥ ~ND S. UDENFRIEND, J. Biol. Chem., 238 (I963) 3966. 9_ D. J. P R o c K o P , E . WEINSTEIN AND T. ~{ULVENY Biochem. Bi@hys. Res. Commu¢~a. 22 ~966) 124 . 3 K. I. KIVlRIKNO AXD D. J. PROCKOP, Biochem. J., ~o2 (I967) 432. 4 J. E > , ~ L , J. N v ~ z , E . KATCHALSKI AND A. BgRGER J. Mol. BioL. ~7 (~966) 255. 5 D. J . P a o c K o e , K. J u v . ~ AX> J. E ~ ¢ E L , Z ~hysiol. Chem., 348 (~967) 553. 6 K. t. ~IVlRIKKO AND D. J. P~OCKOP, J. BioL Chem, 242 (I967) 4007 . 7 ~ " I. ~IVIRIKKO, H . J. BRIGHT AND D. J. PROCKOP, Biochim. ~iophys. Acta, IhZ (I968) 558. 8 Y . KISKtDA, S. SAKAKIBARA AND Y. KIKUCHI Proc. 5Xh Syrup. Peptide Chemistry, Kyoto, ~962, p. 3 o. 9 S. SAKAK~B~R*, Y . KISHIDA. Y. KIK~CHI. R. S&KAI AX-D E . K A ~ U C H I . B~H. Chem. Soc. ] @ ~ , 4~ (~968) ~273. xo W . VA~ RO~ER~SOX AND B. Scm~ARTz, J. Biol. Chem., 2o~ I 9 5 3 689. ~ J. M. M~NN~N~ AND A. M~ISTER. B{ochemistvy, 5 (~966) i 1 5 4 . ~2 ~V. TROLL AND J. LINDSL~Y. ~. Biol, C]zem. 2~5 (~953) 655, ~3 R . E . NEUMAN AND NI. A. LOGAN f . ~iol. Chem.. I 8 4 /~95o ) 299. ~4 I. BXK~OR A~-D L. A. BAVXTTA. PfOC. Natl. Acad. Sci. U,S., 58 {~967} Z35L 15 P. M. GALLOP, ~i@hys. ]. S~@pl., ~ (~964) 79.
Received August i6th, m968 Biochim. Biophys. dcta, 177 (I969) I 5 4 - ~ 5 6