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Cell-free collagen biosynthesis and the hydroxylation of sRNA-proline
ARCHIVES
OF
Cell-Free
BIOCHEMISTRY
AND
Collagen
BIOPHYSICS
109,
(1965)
48o-‘i89
Biosynthesis
and
the
Hydroxylation
of sRNA-
Proline’ MORTON Medical
Research
URIVETZKY, Department
JOY M. E’REI,
of the Long Received
Island
Jewish
August
AND
EDWARD
Hospital,
New
MEILMAN Hyde
Park,
New
York
14, 1964
Cell-free incubation systems employing supernatant and ribosomal fractions prepared from chick-embryo homogenates were found to be active with respect to their ability to incorporate proline from free proline-Cl” or sRNA-prolineinto ribosomal protein. Aerobic conditions enhanced the formation of peptide-bound hydroxywas added. In either case, the inproline-Cl” particularly when sRNA-proline-Cl4 corporation of hydroxyproline lagged behind that of proline. In a separate series of experiments it was found that conditions similar to those which enhance the incorporation of hydroxyproline into ribosomal protein also favor the transformation of portions of sRNA-proline complexes to sRNA-hydroxyproline. Evidence was obtained that factors present in the microsomal supernates and which were associated with the microsomes themselves were involved in the hydroxylation of sRNA-proline. The microsomal-associated factor could be removed by treatment with deoxycholate in which case the RNP-particles were inactive.
The biosynthesis of collagen has been shown to involve microsomal mechanisms similar t,o those which have been established for other probeins (l-4). There are, however, special features connected with collagen synthesis, such as the formation of hydroxyproline, hydroxylysine, y-glutamyl (5) and ester linkages (6, 7) and their incorporation into protein, which require further elucidation. One of these, which may be used as an index of collagen synthesis, is the incorporation of hydroxyproline inbo peptide or protein linkages. Free hydroxyproline itself has been shown not to be a precursor of collagen hydroxyproline (2, 8, 9). Essentially two lines of opinion have developed with regard to the mechanisms involved: (a) that the hydroxylation of proline occurs prior to the formaGon of peptide linkages, in which case it is felt that “activated” or sRNA-proline complexes are 1 This project, was supported by grants from N.I.H., U. S. Public Health Service (A-1503), American Heart Association, and The Bernard Moncharch Foundation, Inc.
the immediate precursors (10-13) ; (b) that proline is hydroxylated after its incorporation into peptide or protein linkages (9, 14). In a previous paper (4) we reported that cell-free incubation systems containing microsomal fractions prepared from rabbitskins were able to incorporate proline-Cl4 into ribosomal protein. Although hydroxyproline incorporation was low and could not be conclusively demonstrated, it was shown that the ribosomal protein formed was suscept,ible t’o enzymic hydrolysis by a purified bacterial collagenase. This enzyme is specific for xigly-pro, a characteristic sequence seen predominantly in collagens. Peptides yielded by the enzymic reaction were separated by column and paper chromatography. Two of these, containing radioactivit,y, were identified as gly-pro-hypro and gly-pro-ala, both sequences known to occur in collagen. This indicated that proline had been incorporated into collagen precursors. Due to difficulty in obtaining sufficient quantity of starting material and the inability to demonstrate hydroxyproline
the the J. 480
CELL-FREE
COLLAGEN
incorporation with skin systems, it was decided to employ chick-embryo homogenate fractions for studies of cell-free collagen synthesis. In the first series of experiments described below evidence was obtained which suggested the participation of sRr\‘A-hydroxyproline in ribosomal collagen synthesis. As a result, studies were t,hen undertaken to determine the conditions most favorable for the formation of sRNAhydroxyproline with the ultimate aim of determining its effects on collagen synthesis, i.e., the incorporation of hydroxyproline into ribosomally synthesized protein. EXPERIMENTAL Embryos. Nine-day-old embryonated chicken eggs were obtained from Shamrock Farms and Hatchery (New Brunswick, New Jersey). Immediately following delivery, the embryos were removed, decapitated, washed with medium A (less glutathione), blotted t,o remove excess solution, and frozen at -40°C overnight. They were homogenized, while frozen in 135 volumes (v/w) of medium il in a \‘irtis homogenizer for 30 seconds at low speed. (Medium A consisted of 0.35 M sucrose, 0.025 M KCl, 0.004 M magnesium acetate, 0.05 M tris-HCl buffer, pH 7.8, and 0.005 M glutathione.) Preparation of supernatant and microsomal fractions. The homogenate was centrifuged at 15,000g for 30 minutes in a Spinco model L ultracentrifuge, yielding a residue which was discarded and supernatant fraction S15. The latter was centrifuged at 105,000g for 90 minutes and supernatant fraction $05, and the microsomal pellet &&I was isolated. In those experiments in which the conversion of sRNA”-proline to sRNA-hydroxproline was investigated, S,: was lyophilized and used whole (SISL) or it was centrifuged (after reconst,itution) at 105,000g 90 minutes, for the isolation of supernatant fraction SloaL and Sl~eL?vl (microsomal pellets). A scheme describing the procedures employed to obtain chick-embryo homogenate fractions is shon,n in Fig. 1. Ribonucleoprotein (RXP) particles were prepared from the microsomal pellets which w-ere re2 Abbreviations used: sRNA, soluble (transfer) ribonucleic acid; ATP, adenosine triphosphate; GTP, guanosine triphosphate; DPNH, reduced diphosphopyridine nucleotide; TPNH, reduced triphosphopyridine nucleotide; tris, tris (hydroxymethyl) aminomethane; TCA, trichloroacetic acid; DOC, deoxycholate; cpm, counts per minute.
BIOSYNTHESIS
481
suspended in medium A according to the procedure of Kirsch et al. (15). The yield of RNP was 25-30 mg of particulate protein for each 50 gm of blotted and decapitated embryos. The deoxycholatesoluble supernatant fraction, prepared from the microsomes, was dialyzed for 3 hours, at 2-5”C, against several changes of medium A (16): the retentate is further referred to as fraction DOCS. Preparation of chick-embryo sRNA. Chick embryo sRNA was prepared from fraction Slob by extraction with an equal volume of 90% phenol at 2”-4°C for 30 minutes (17). The mixture was centrifuged at 15,000g for 10 minutes, the aqueous phase was collected by aspiration, and the phenol layer was re-extracted with an equal volume of distilled water and recentrifuged. The combined aqueous phases were extracted three times with ethyl ether. Residual ether was removed by bubbling Nz through the solutions at O”C, and thesolutions were lyophilized. The residues were dissolved in 0.01 M tris-acetate buffer at pH 7.4 and dialyzed for 2 hours against two changes of the same buffer. After dialysis, NaCl was added to a final concentration of 0.1 M and sRNA was precipitated with three volumes of ethyl alcohol at - 10°C. The precipitated sRNA was dissolved in 0.01 M trisHCl buffer, pH 7.8, and dialyzed against the same buffer for 2 hours prior to its use for the formation of sRNA-proline-U1. The yield of sRNA by this procedure was 0.3-0.6 mg per gram of embryo. Preparation of sRXA-proline-C14. Three to 5 mg of chick-embryo sRNA was incubated in a reaction mixture with a pH 5 enzyme fraction containing 20 mg protein, prepared from fraction Slob (15), pyruvate kinase (Mann), 0.2 mg; and the following additives in micromoles: ATP (dipotassium, Mann), 50; phosphoenolpyruvic acid (sodium salt, CalBiochem.), 100; magnesium acetate, 50; KCl, 500, tris-HCI buffer, pH 7.8, 500; and 5.0 pmoles of proline-U-C14 (New England Nuclear or Nuclear-Chicago), containing 2 ~c (520,000 cpm). The reaction mixtures in final volume of 15 ml were incubated in Parafilm covered beakers at 37°C for 30 minutes with shaking. Aminoacyl sRNA, hereafter referred to as sRNA-proline, was isolated by phenol extraction, as described above, and purified by 2 cycles of successive dialysis (vs. tris-acetate buffer) and precipitation. About 7070 of the sRNA originally added was recovered with a specific activity of 450-520 cpm per milligram sRNA. Cl”-activity was determined using aliquots of dissolved sRNA-proline-C14 which were plated on stainless steel planchets. The samples were analyzed for radioactivity in a nuclear-Chicago gasflow counter with Micromil end window. Incubation of RNP particles with free proline04. The basal system for these studies contained 30-40 mg RNP protein; fraction DOCS, 6 mg
482
URIVETZKY,
FREI,
CHICK
AND
EMBRYO
HOMOGENATE
Centrifugation 15,000 X g -30
RESIDUE
(discard)
MEILMAN
SUPERNATE
min.
(S,,) I
r
I I
I
Centrifuge 105,000
LYOPHYLIZE
x g
(S,,L)
reconstitute centrifuge 105,000
r-----s MICROSOMES
(S,,,M)
SUPERNATE
(S,,,)
/
I
phenol ethanol
deoxycholate
I
M,,,OMkTE
(S,,L)
extraction precipitation
s-RNA l---l RIBONUCLEAR PROTEIN
xg
I
deoxycholate
’
I
SUPERNATE I RIBONUCLEAR (RNP-L)
PROTEIN
SUPERNATE
dialyze dialyze I RETENTATE
RETENTATE
(DOCS)
FIG 1. Scheme
of fractionation
protein; chick supernatant fraction &OS, 5 ml; pH 5 enzyme protein, 20 mg. The final reaction mixture was 20 ml and the following substances were added, in rmoles: proline-U-C14, 5.0 (2 PC); ATP, 50; GTP (sodium, Mann), 25; phosphoenolpyruvic (sodium), 100; pyruvate kinase, 0.2 mg.; a mixture of 16 unlabeled amino acids (4), 2.5 pmoles of each amino acid; reduced glutathione, 150, magnesium acetate, 40; tris-HCl buffer, pH 7.8, 500. The incubations were carried out at 37°C for periods of 30-90 minutes, in Parafilm sealed flasks or under a gas mixture of 95y0 02-5% COz, with shaking. At the end of the incubation periods the reac-
(DOCS
.L)
procedure.
tion mixtures were brought to 0” and centrifuged at 105,OOllg for 90 minutes. The ribosomal pellets were washed with cold medium A (less sucrose and glutathione) by resuspension and centrifugation at 105,000g for 1 hour. The washed pellets were suspended in water and aliquots removed for precipitation with cold TCA (4, 18) and subsequent W-analyses of the ribosomal protein precipitates. The remaining water suspensions were dialyzed vs. 3 changes of distilled water (20 hours total dialysis time), and the nondialyzed material was hydrolyzed in 6 N HCl in sealed vials at 110°C for 16 hours. The hydrolyzates were employed for
CELL-FREE
COLLAGEN
chromatographic separations of the amino acids as described above. Only carrier hydroxyproline was added prior to fractionation and the radioactivities of proline and hydroxyproline were determined after chromatography on a Moore and Stein column (19). Hydroxylation of sRNA-proline-C14. The basal system for the hydroxylation of sRNA-prolineCl4 cont,ained 3-4 mg sRNA-proline-Cl4 and fraction &hL, containing 60 mg protein dissolved in 15 ml of Ringer-phosphate-bicarbonate medium (20) to which 150 rmoles of glucose was added. The mixture was incubated at 37°C under 957, 02-5% COZ for 30 minutes, after which it was chilled to 0°C. Hibonucleic acid was extracted either from the whole incubates or from the 105,OOOg supernatants and pellet,s (individually), which were obtained by centrifugation of the chilled incubates at 105,OOOg for 90 minutes. The extracted RNA fractions (phenol procedure described above) a-ere purified, as indicated above, the imino acids were discharged, and their C14-activities were determined. Discharge and identification of imino acids from .sRS$ complexes. The amino acids were stripped from their sRNA4 complexes by incubation at pH 10 for 1 hour at 37°C (17,21). The reaction mixture was then adjusted to pH 7.0 and RNA was precipitated with ethanol. The supernatant was concentrated by flash evaporation and the amino acids were fractionated by chromatography on Dowex 50 W X4 columns by the method of Moore and Stein (19j. Unlabeled carrier proline and hydroxyproline were added prior to ion-exchange chromatography. Portions of the hydroxyproline and proline fractions eluted from the columns were plated and their C14-activities were determined. Other portions of the hydroxyproline and proline fractions eluted from the Moore and Stein column were applied with butanol: acetic acid: water (63:27:10, v/v) to thin-layer cellulose plates for chromatography (22). After development the plates were dried, and standard markers of proline and hydroxyproline were revealed by spraying with ninhydrin and isatin. The corresponding unsprayed areas were eluted with 10 ml water, twice, at 70°C. The filtered eluates were concentrated in z’acuo, dissolved in 2 ml Hyamine base, and counted in a Packard Tricarb scintillation counter following t,he addition of 3 ml toluene and 10 ml of scint,illator solution. The concentrated supernatant containing the discharged imino acids was also analyzed for proline and hydroxyproline-radioactivity by the method of Prockop and Peterkofsky (23). Each vial used for counting contained 5 ml of the toluene extracts and 10 ml of scintillator solution.
483
BIOSYNTHESIS
The scintillator solution used for counting was composed of reagent-grade toluene containing 8 gm of 2,5-diphenyloxazole and 500 mg of 1,4-bis2-(5 phenyloxazolyl).benzene per liter. TolueneCl4 (New England Nuclear) was employed as an internal standard. Bssays. Protein was determined by the method of Lowry et al. (24) and correlated with microKjeldahl analyses of analagous fractions. Ribonucleic acid was analyzed spectrophotometrically at 260 m,~ (E. coli RNA was used as reference standard), or by the procedure of Schneider (25). Proline wa4 assayed for by the method of Piez et al. (26) and hydroxyproline by the method of Stegemann (27) or the Woessner modification of this procedure (28). RESULTS
Incorporation of Proline-Cl4 and Hydroxyproline-C14 into RNPParticles Studies with free proline-C’4. Incubation of chick-embryo RNP-particles with free proline-Cl4 in the basal system given above resulted in the incorporation of the iminoacid into ribosomal-bound protein or peptide fractions, the maximum incorporation of proline occurring between 30 and 60 minutes. Only relatively low levels of hydroxyproline-Cl4 were found to be present in the same fractions, but the maximum incorporation of the imino acid was found at 90 minutes (the longest incubation). When the reactions were carried out under 95% 02-5% coz , the incorporation of proline was slightly diminished, but that of hydroxyproline was enhanced, i.e., the ratio of incorporated hydroxyproline-C14: prolineCl4 increased. The addition of reduced pyridine nucleotides and ascorbic acid to the aerobic incubation reactions did not produce any significant effects on hydroxyproline-Cl4 incorporation. The detailed results are given in Table I. Treatment of ribosomal suspensions (after incubation) with 0.01 M KOH at pH 10 for 1 hour at 37°C resulted in the release of less than 20% of the proline-Cl4 activity and about 10 % of the hydroxyproline-CL4 activity associated with the ribosomal protein fraction. These results along with hydrolysistime studies (4) indicated that the major portions of the incorporated imino acids
484
URI\‘ETZKY,
INC~KPORATION OF PROLINEAY PARTICLES IN INCUBATION
Incubath
system
A. Parafilm covered flasks Basal” Basal0 Basal” Basala Basala Basal0 Basala Basala B. Aeration with 95% 02-57, co2 Basal Basal Basal Basal Basal Basal
Time (min)
AND
FREI,
AND
TABLE
I
MEILiVAN
HYDROXYPROLINE-Cl4 SYSTEMS CONTAINING
R-UP Incorporated Protein
30 30 GO 60 90 90 60 60
2970 2410 3550 3960 2505 2834 3246 2945
30 30 60 60 90 90
1864 1568 2612 2442 1804 1960
ISTO
ADDED Cl”-activity* Proline
CHICK-EMBRYO
RiVP
PROLINE-Cl’ (cpmjb Hydroxyproline
2745 3042 1550 1726 2510 1948
34 25 90 96 102 108 75 80
0.03 0.02 0.03 0.03 0.06 0.06 0.03 0.04
860 620 1206 1264 900 1065
45 50 140 160 190 210
0.0.5 0.08 0.12 0.13 0.21 0.19
(1 The basal incubation systems, as described in Experimental, contained the following amounts of RNP-particle protein: 34 mg, Experiment 1; 30 mg, Experiment 2; 38 mg, Experiments 3 and 4. The incubations were carried out (2.0 PC proline-C 14, 520,000 cpm added) for the time periods indicated; cold-TCA precipitated ribosomal protein, proline, and hydroxyproline fractions were prepared and their V-activities were determined (see Experimental). * Total C14-activity incorporated into total RNP protein, proline, and hypro fractions.
were present in peptide linkages bound to the RNP-particles. Studies with sRNA-proline-C14. When sRNA-proline-Cl4 (prepared as described in Experimental) was incubated with RNP particles, maximum incorporation of proline occurred by the end of 30 minutes. Maximum hydroxyproline incorporation occurred at 60 minutes under aerobic conditions (Table IIB) or in Parafilm covered flasks (Table HA). The ratios of incorporated hydroxyproline-Cl4 to proline-Cl4 were higher than for those obtained using free prolineCl4 especially when aerobic conditions were employed. Therefore, the use of sRNAproline and/or aerobic incubation conditions stimulated the incorporation of hydroxyproline into RNP particles. The most marked enhancement was obtained when both factors were employed simultaneously. The omission of DOCS, ATP, and the energy-generating system, or GTP, resulted
in a decrease in the incorporation imino acids (Table IIB). Conversion of sRNA-I’roline-Cl” sRNA-Hyclroxyp~oline-P
of both to
Other investigat.ors have proposed that sRNA-hydroxyproline is an intermediate in microsomal collagen synthesis (10-13). The result,s described above could be interpreted in accord with this hypothesis. The remaining st)udies described here were then carried out to determine whether sRNA-hydroxyproline could be formed under condibions similar to those favoring the incorporation of hydroxyproline into ribosomal protein and t)o evaluate those circumst,ances favoring the hydroxylation of sRNA-proline. For if sRKA-hydroxyproline was involved in collagen synt,hesis it would have to be formed from a species of sRKA-proline since free hydroxyproline itself is not incorporatjed into collagen.
CELL-FREE
COLLAGEN TABLE
INCORPORATION PARTICLES
OF PROLINE-Cl4 IN INCUBATION
II
BND HYDROXYPROLINE~~ SYSTEMS CONTMNIN~ RhWIncorporated
hp.
Incubation
1 1
A. Parafilm flasks Basalt Basalt Basalt
system
c The particle particle
C14-activity system
(min)
Protein
INTO
ADDED
CHICK-EMBRYO
Cl4 activity Proline
RNP
sRNA-PROLINE-Cl4 (cpm)” Hydroxyproline
C’a-Hypro C’4-Pro
covered
13. Aeration with 95yo 02-5% CO? Basal Basal Basal Basal Basal less DOCS Basal less GTP Basal less ATP and energy systemh a Total 5 Energy
Time
485
BIOSYNTHESIS
30 60 90
724 6G0 610
520 440 -400
30 GO 90 60 GO GO 60
804 720 636 780 695 465 310
462 395 340 435 310 285 160
58 86 82
92 142 135 172 90 80 36
0.11 0.19 0.20
0.20 0.36 0.40 0.40 0.29 0.28 0.22
RNP protein, proline, and hydrosyproline fractions. acid and pyruvate kinase. basal incubation systems, as described in Experimental, contained: Experiment 1,26 mg RNPprotein and 3.5 mg sRNA-proline-Cl4 (460 cpm/mg sRN-4) ; Experiments 2 and 3, 28 mg RNPprotein and 3.2 mg sRNA-proline-Cl4 (485 cpm/mg sRNA). The preparation of sRNA-proline-
Cl4 is described
incorporated into = phosphoenolpyruvic
in the text.
Presumably the species of sRNA-proline involved would be present in the complex which we refer to as sRn’A-proline. Aft,er the incubation of sRNA-prolineCl4 in the basal system described in Experilfzental t,here was a significant increase in the hydroxyproline-C14 content of the sRNA complexes which were isolated. When lyophilized fraction SlsL was omitted or the incubations were carried out under Nz , there was no increase in sRNA-hydroxyproline-Cl4 over t,hat. originally present in the sRn’A complexes which were added t,o the reaction mixtures (see Table III). When fraction S& was replaced by Sl,,L or S&I, the former had about 60% and the latter 40% of t,he activity of the whole lyophilized l,?,OOO g supernat’ant fraction. Ninety % of t’he activity of Sl,L was recovered when the two subfractions were recombined and added (Table III). The decrease in proline activity in the posbincubation RNA fractions was due lnost likely t,o one or all of four factors: (a) dilution with sR,KA present in S15L (0.75 mg
sRNA in SlsL); (b) conversion of proline to hydroxyproline; (c) incorporat,ion of proline into microsomes; (d) degradation of some sRNA-proline during incubat,ion and isolation procedures. Ribonucleic acid isolated from the postincubation microsomal fractions, when treated at pH 10, did yield small amounts of free proline and hydroxyproline with C14-activities (Table III), most of which was very likely due t)o sRNA fract]ions which remained bound to the microsomal pellets. However, since it was found I hat the activit,y in t)he phenol extracted microsomal RNA which was released by incubation at pH 10 did not have any significant influence on the interpretation of Ihe results, in subsequent experiments Ihe postjincubation mixtures were extract’ed without prior separation of microsomal and supernatant fractions. The data obtained in further studies on the hydroxylation of sRNA-proline are presented in Tables IV and V. Essentially, the findings may be summarized as follows: (a) the addition of ascorbic:
acid
and/or
pyridiue
nuclcotides
486
URIVETZKY,
FREI,
AXD
TABLE CONVERSION
MEILMAN
III
OF sRNA-PROLINE-04
TO sRNA-HYDROXYPROLINE-Cl4 Postincubation
Incubation
systema
P10lille Basal Basal Basal Basal Basal
less &L Incubation under but S,,L replaced
SK& Basal but, 81jL replaced
fractions (cpm/mg sRNA)
105,000 g supernatant
Time (min)
Hydrow proline
Microsomal C’h-Kypro ~~ C”-Pro
Proline
Hydroxyproline
fractions C”-Hypro W-Pro
Nz by
30 GO 30 30 30
285 228 410 406 340
75 60 30 22 54
0.26 0.26 0.07 0.05 0.16
62 75 -
10 18 -
0.16 0.24 -
by
30
380
48
0.13
40
8
0.20
by
30
315
72
0.23
56
15
0.27
S,asLM Basal but SlbL replaced &o;LM plus S,asL
a Incubation reactions were carried out by using the basal system for these studies, as described in Erperimental, under a gas mixture of 95’$$ OZ-5% CO2 except where otherwise indicated. RNA was isolated from supernatant and microsomal fractions by phenol extraction after incubation, and the free imino acids were released from their RNA complexes by incubation at pH 10 for 60 minutes at 37°C. The details of the procedures employed are given in the text. The sRNA-proline-C14 complex added to the incubation mixtures contained 3 mg sRNA with 520 cpm proline-Cld/mg sRNA and 24 cpm hydroxyproline-C14/mg sRNA (hydroxyproline-C14/proline-C14 = 0.05).
had at the most a slight stimulatory effect on the conversion (Table IV) ; and (b) replacement of the microsomal port,ions of S16L by RNP particles (RNP-L) prepared from it produced a marked decrease in sRNA-hydroxyproline-Cl* formation, most of which was restored when fractions DOCSL (dialyzed deoxycholate soluble) were added (Table V). The formation of sRNA-hydroxyproline was substantiated by the following evidence obtained from the analyses of t’he amino acids which were discharged from their sRNA complexes. (a) C14-activity (Tables III-V) was obtained in the hydroxyproline fraction after ion-exchange chromatography. (b) This fraction, when subject’ed to chromatography on cellulose plates, demonstrated U4-activity on the plate area corresponding to the position of hydroxyproline. At least 60% of the radioactivity present in the hydroxyproline fractions from the Moore and Stein column could be accounted for in the hydroxyproline areas eluted from the thin-layer chromatography plates. (c) The imino acids were stripped from sRNA and then analyzed by t.he procedure of Pet’erkofsky and Prockop (2) in which proline and hydroxyproline are converted to their
respective pyrroline and pyrrole products prior to extraction with toluene. The results of these studies are given in Table VI. DISCUSSION
In agreement with the studies of other investigat’ors (3, 12, 14), cell-free systems containing supernatant and ribosomal chickembryo homogenate fractions are able to activate proline to form sRNA-proline complexes from which the imino acid can be incorporated into ribosomally synthesized protein. It is in regard bo the ability of such systems to synthesize collagen, requiring the hydroxylation of proline at some given step, where there is need of further evaluation. Manner and Gould (12) and Daughaday and Mariz (11) have suggested on the basis of their studies that sRNA-hydroxyproline, formed by the hydroxylation of a species of sRNA-proline, is an intermediate in the incorporation of hydroxyproline into collagen. The studies of Green and Lowther with granuloma slices (29) and those of Stone and Meister (granuloma minces) (10) also favor the involvement of activated proline molecules as opposed to proline-rich peptide precursors. On the other hand, the recent report of Peterkofsky
CELL-FREE TABLE EFFECT OF NUCLEOTIDES
TABLE
ACID THE
AND
EFFECT
PYRIDINE
HYDROXYLATION
OF
sRNA-PROLINE-Cl4
Experiment Basal Basal + acid Basal + acid + Basal + acid + Basdl + Experiment Basal Basal + acid + Basal + acid + Basal + acid + DPNH
system’
proline
OF
(cpm/mg
HYdFYprolme
V
RIBOSOMAL
AND
DEOXCHOL.~TE-SOLUBLE
DIALYZED
FRXTIONS
HYDROXYL~TION
Postincubation Incubation
487
BIOSYNTHESIS
IV
ASCORBIC ON
COLLAGEN
OF
ON
RNA)
Postincubation (cpm,‘mg RNA)
C”-Hypro -GTE-
Incubation
THE
sRNA-PROLINE-Cl4
syste#
Cl’-Hypro C~“.pro
HydroxyXYDTO-
Proline
1 ascorbic ascorbic DPNH ascorbic TPNH DPNH 2 ascorbic DPN ascorbic DPNH ascorbic DPN +
312 295
84 95
0.28 0.32
305
102
0.33
286
102
0.36
324
98
0.30
321 290
78 75
0.24 0.25
305
88
0.28
320
92
0.29
a The conditions of incubation employed in the studies given here are the same as those described in Table III. All incubations were carried out for 30 minutes under 95% 02-5% COZ. However, for the reasons explained in text, RNA was extracted from the whole postincubat.ion mixtures. The sRNA-proline-Cl4 added contained: Experiment 1, 4 mg sRNA with 480 cpm proline-U4/mg sRNA and 28 cpm hydroxyproline-C14/mg sRNA; Experiment 2, 3.0 mg sRNA, 460 cpm proline-C14/mg sRNA and 20 cpm hydroxyproline/mg sRNA.
Basal Basal less Basal but by &o&L Basal but by RNP-L Basal but by S,oiL Basal but by RNP-L Basal but by &,,jL DOCS-L
SljL SlsL replaced + SlojLM S,jL replaced SlsL replaced + RNP-L S,jL replaced + DOCS-L SljL replaced + RNP-L +
80 32 74
0.28 0.06 0.25
305
25
0.08
315
52
0.16
342
46
0.13
305
70
0.22
a The sRNA-proline-Cl4 complex added contained 3.5 mg sRNA with 512 cpm proline-C’*/mg sRNA and 22 cpm hydroxyproline-Cld/mg sRNA. Fractions S,ojLM, S,ojL, RNP-L, and DOCS-L were prepared from S,,L as described in Ezperimental (see also Fig. 1). When S1oQLM, SlojL, and RNP-L were used, the entire fractions prepared from SlsL (60 mg protein) were added. DOCS-L added contained 2 mg protein. All incubations for 30 minutes under 95y0 02-57, COz. TABLE INCREASE
IN
T-I
SRNA-HYDROXYPROLINE
INCIJBATION
OF
-4FTER
sRN~PRoLINE
I
,
DPM/mg
and Udenfriend (14) would rule out sRNAhydroxyproline as an intermediate, and instead a proline-rich peptide fraction associated with microsomal RNA would be the substrate for hydroxylation. Studies utilizing cell-free chick-embryo homogenate fractions in our laboratories indicated that the incorporation of proline and hydroxyproline into protein (or peptides) synthesized in the ribosomes was better than that obtained previously with rabbit-skin preparations (4). However, the incorporabion of hydroxyproline in systems containing free proline-Cl4 and incubated under relatively anaerobic conditions (see Table I) suggested that only a very limited synthesis of collagen molecules had occurred.
282 465 296
Prolin@ EXP.
1 2 3
C’J-Hypro
/ Hydroxyproline I
I
3864 3720 4200
RNA
2016 2430 2800
50 58 45
537 582 815
“‘-“’ I
/
0.01 0.02 0.01
0.27 0.24 0.29
a sRNA-proline was incubated with the basal system described in the text. The amino acids were discharged at pH 10 and the radioactivities of proline and hydroxyproline were determined by the oxidation and toluene extraction procedures as described by Peterkofsky and Prockop (23) ; these procedures were modified for use with 10 ml of the liquid scintillator solution described in text.
In such syst,enls, the ittcorporation of hydroxyproline-Cl4 ittt’o RNP particles could be itmeased by aerobic incubation under 95% 02-5 70 CO? ) particularly when the react’ions were carried out up to 90 ntinutes. hlaxitnutn hydroxyproline incorporation was found t)o lag behind that of proline (Table I). Incubation with sRNA-proline-C14 under aerobic conditions showed the highest activit,ies of hydroxyprolinc ittcorporatcd with protein, in which case it,s tnaxitnutn irtcorporation agaitt lagged behind that of prolinc-Cl4 (Table II). In subsequent experiments evidence for hhc formation of sRNAhydroxyproline was obtained as described above. These findings suggested t)he possibi1it.y that Ihe hydroxylatiott of a species of sRNA-prolitte was necessary prior to the incorporation of hydroxyproline into RSP particles. This pat,hway would be favored by aerobic conditions (10, 30, 31) and would also account for the lag period observed in hydroxyproline incorporation. Additional st)udies were carried out in order t)o determine the conditions most favorable for the hydroxylation of sRNA-proline-Cl”. The results (Tables III-V) indicate that) a portion of sRKX-proline can be converted to sRNA-hydroxyproline under aerobic conditions in incubation systems containing sypernatant and/or microsotnal fractions prepared from embryo homogenates. Maximum conversion was obtained with whole lyophilized 15,000g (StsL) supernatant fractions, but tnicrosomal (Sl,LM) and 105,OOOg superrtatant (&L) fractions prepared from it also showed significant activities. Ribonucleoprotein particles (RX-L) had no effect] on the hydroxylation of sltNAprolinc, but, most of the activity was restored when dialyzed deoxycholate-soluble material (DOCS-L) was added to RNP-L incubation mixtures. Therefore a fact,or or factors present in the 105,OOOg superttatatlt and in t.he intact microsomes is involved itt the conversion. The slight enhancement obtained with ascorbic acid and pyridine nucleotides would be in agreement, with the proposal of Prockop et al. (28) t,hat, an oxygenase rnechattistn was involved itt the hydroxylation of proline. The lack of a nlore marked effect can be due to already adequate arn0unt.s of these factors present in
the homogenates (14). In fact, the reduced conversions obtained wit,h tnicrosomal fractions might, be due to a lack of these cofactors. That the increased ratio of sEWAhydroxyprolitre/sR?A-prolinc is indeed evidence of formation of sRNA-hydroxyprolitte and not due to a greater instability of sRKAproline is support’ed by the data from cont,rol experiments. For example, RKA isolated from t)he basal system (tttinus Sl,L) after incubation (Tables III and V) shows a low ratio of hydroxyproline/prolirte Cl”-act)ivit,y, which is essentially t)he same as that. of the starting materials. Our results which indicate that sRNAhydroxyproline is formed are in agreement with those of other investigators (12,30-33). At least t,wo irnport’ant questions remain for further study: (a) Is sRKA-hydroxyprolitte a precursor of collagen hydroxyproline or is it sitnply an artifact or a by-product? Our results thus far do not’ contradict t,he fittdings of Peterkofsky and Udenfriend (14) and could very easily be interpreted in accord with their conclusions, especially if sRK\‘Ahydroxyproline will be found to be ineffective in the eventual incorporat’iott of the itnino acid into collagen. (b) Are t,he tnechanisms involved in t)he hydroxylation of sRNAproline as repot%ed here the same as t,hose involved in Pet,erkofsky and Udenfriend’s studies (14)? If t,hey are the same, then the nlechanistns involved would appear to be relatively nonspecific if both sRKA-prolitte and proline-rich peptides could act as subst’rates. In either case, the findings will have to be explained in the future interpretation of the tnechanisms involved in collagen synthesis and the formation of hydroxyproline in vim. REFERENCES 1. LOWTHER, D. A., GREEN, X. AI., AND CHAPMAN, J. A., J. Biophys. Biochem. Cytol. 10, 373 (1961). 2. PROCKOP, D. J., PETERICOFSKY, B., AND UDENFRIEND, S., J. Biol. Chem. 237, 1581 (1962). 3. PETERKOFSKY, B., AND UDENFRIEND, S., Biothem. Biophys. Res. Commun. 6, 184 (1961). 4. URIVETZKY, RI., KRANZ, V., AND MEILMAN, E., Arch. Biochem. Biophys. 100, 478 (1963). 5. FRANZBLAU, C., SEIFTER, S., AND GALLOP, P. Xl., Biopolymers 1, 79 (1963).
CELL-FREE 6. CALLOP, P. E., Nakre i.
hI.,
SEIFTEI~,
183, lF59
BLUMENFELD,
0.
Biochemistry 8. STETTEN,
Biol.
>I.
9. STETTEN,
11.
,\l.
DAUGHADA~,
BioZ. 12.
153, Ii.,
AND
AND
AND
GALLOP,
J. Biol.
P.
M.,
Chem. A.,
MANNER, ROBEKTSON,
L.
I.,
231,241
AND
ZAMECNIK,
P. C.,
(1958).
I)., AND JACKSON, I). S., Anal. Bio6, 193 (1963). 23. PE,I~ERKOFSKY, B., AND PRO~KOP, 1). J., Anal. Hiochem. 4,400 (1962). 24. LOWILY, 0. H., ROSEBROUGH, H. J., FARIL, A. L., AND RANDALL, Ii. J., J. Biol. Chew 193, 265 (1951). 25. SCHXEIDER, W. C., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, 22.
RlYHILL,
them.
AND SCHOENHE~MER, 113 (1943).
~IEISTER,
J. F., HECHT, J. Biol. Chem.
~IEILMAS,
(1962).
489
BIOSYNTHESIS
(1959).
R.,
J.
181, 31 (1949). 194, 555
:l’atlcre
W. H., AND 1\2;11uz, I. K., J. 237, 2831 (1962). C., AND GELD, B. S., Hiochem. Acta 72, 243 (1963). W. VAN B., KiophTys. J. 4,
Chem.
Biophys. 13.
R.,
Chem.
10. STOKE, N., (1962).
O.,
1, 947
S.,
COLLAGEN
93 (1964). 14. PETERKOFSKY, B., AND CDEXFMESD, S., J. Biol. Chem. 238, 3966 (1963). 15. KIRSCH, J. F., SIEKEVITZ, P., AXD PALADE, (:. E., J. Biol. Chem. 235,1419 (1960). 16. FESSEXDEN, J. RI., AND ~VIOLDA~E, I,., Hiochemistry 1, 485 (1962). 17. CIEKER, 8., AND RCHILAMM, C., ~\~ature 177, 782 (1956). 18. Vos DER DECKEN, -4., ASD H~:LTIN, T., Riothem. Actu 45, 139 (1960). 19. MOORE, S., APSD STEIN, W. H., .I. Hiol. Chem. 211, 893 (1954). 20. KILEBS, H. A., AND HENSELEIT, K., Z. Physiol. Chew 210,33 (1932). 21. HOAOLAND, RI. B., STEPHENSON, RI. L., Scow,
ed.), York
Vol III, (1957).
p.
680.
Academic
Press,
New
26. PIEZ. K. A., TRREVERRE, F., AND WOLF, H. J., J. Biol. Chem. 223, 687 (1956). 27. STEGEMANN, H., Z. Physiol. Chem. 311, 41 (1958). 28. WOESSNER, J. F. JR., Arch. Hiochem. Biophys. 93, 440 (1961). 29. GREE:N, N.&l., AND LOWTHER, D. A., Biochem. J. 71,55 (1959). 30. PROCKOP, I)., KAPLAN, .4., AND LDENFRIEND, S., Arch. Biochem. Biophys. 101, 499 (1962). 31. FUJIMOTO, L)., AND TAMIYA, IL’., Biochem. J. 84, 333 (1962). 32. CORONADO, A., MARDONES, E., AND ALLENDE, V. E., Hiochem. Biophys. Res. Commun. 13, 75 (1963). 33. JACKSON, D. S., WATKINS, l)., AND WINKLER, A., Biochem. Biophys. Actu 87, 152 (1964).
OF
Cell-Free
BIOCHEMISTRY
AND
Collagen
BIOPHYSICS
109,
(1965)
48o-‘i89
Biosynthesis
and
the
Hydroxylation
of sRNA-
Proline’ MORTON Medical
Research
URIVETZKY, Department
JOY M. E’REI,
of the Long Received
Island
Jewish
August
AND
EDWARD
Hospital,
New
MEILMAN Hyde
Park,
New
York
14, 1964
Cell-free incubation systems employing supernatant and ribosomal fractions prepared from chick-embryo homogenates were found to be active with respect to their ability to incorporate proline from free proline-Cl” or sRNA-prolineinto ribosomal protein. Aerobic conditions enhanced the formation of peptide-bound hydroxywas added. In either case, the inproline-Cl” particularly when sRNA-proline-Cl4 corporation of hydroxyproline lagged behind that of proline. In a separate series of experiments it was found that conditions similar to those which enhance the incorporation of hydroxyproline into ribosomal protein also favor the transformation of portions of sRNA-proline complexes to sRNA-hydroxyproline. Evidence was obtained that factors present in the microsomal supernates and which were associated with the microsomes themselves were involved in the hydroxylation of sRNA-proline. The microsomal-associated factor could be removed by treatment with deoxycholate in which case the RNP-particles were inactive.
The biosynthesis of collagen has been shown to involve microsomal mechanisms similar t,o those which have been established for other probeins (l-4). There are, however, special features connected with collagen synthesis, such as the formation of hydroxyproline, hydroxylysine, y-glutamyl (5) and ester linkages (6, 7) and their incorporation into protein, which require further elucidation. One of these, which may be used as an index of collagen synthesis, is the incorporation of hydroxyproline inbo peptide or protein linkages. Free hydroxyproline itself has been shown not to be a precursor of collagen hydroxyproline (2, 8, 9). Essentially two lines of opinion have developed with regard to the mechanisms involved: (a) that the hydroxylation of proline occurs prior to the formaGon of peptide linkages, in which case it is felt that “activated” or sRNA-proline complexes are 1 This project, was supported by grants from N.I.H., U. S. Public Health Service (A-1503), American Heart Association, and The Bernard Moncharch Foundation, Inc.
the immediate precursors (10-13) ; (b) that proline is hydroxylated after its incorporation into peptide or protein linkages (9, 14). In a previous paper (4) we reported that cell-free incubation systems containing microsomal fractions prepared from rabbitskins were able to incorporate proline-Cl4 into ribosomal protein. Although hydroxyproline incorporation was low and could not be conclusively demonstrated, it was shown that the ribosomal protein formed was suscept,ible t’o enzymic hydrolysis by a purified bacterial collagenase. This enzyme is specific for xigly-pro, a characteristic sequence seen predominantly in collagens. Peptides yielded by the enzymic reaction were separated by column and paper chromatography. Two of these, containing radioactivit,y, were identified as gly-pro-hypro and gly-pro-ala, both sequences known to occur in collagen. This indicated that proline had been incorporated into collagen precursors. Due to difficulty in obtaining sufficient quantity of starting material and the inability to demonstrate hydroxyproline
the the J. 480
CELL-FREE
COLLAGEN
incorporation with skin systems, it was decided to employ chick-embryo homogenate fractions for studies of cell-free collagen synthesis. In the first series of experiments described below evidence was obtained which suggested the participation of sRr\‘A-hydroxyproline in ribosomal collagen synthesis. As a result, studies were t,hen undertaken to determine the conditions most favorable for the formation of sRNAhydroxyproline with the ultimate aim of determining its effects on collagen synthesis, i.e., the incorporation of hydroxyproline into ribosomally synthesized protein. EXPERIMENTAL Embryos. Nine-day-old embryonated chicken eggs were obtained from Shamrock Farms and Hatchery (New Brunswick, New Jersey). Immediately following delivery, the embryos were removed, decapitated, washed with medium A (less glutathione), blotted t,o remove excess solution, and frozen at -40°C overnight. They were homogenized, while frozen in 135 volumes (v/w) of medium il in a \‘irtis homogenizer for 30 seconds at low speed. (Medium A consisted of 0.35 M sucrose, 0.025 M KCl, 0.004 M magnesium acetate, 0.05 M tris-HCl buffer, pH 7.8, and 0.005 M glutathione.) Preparation of supernatant and microsomal fractions. The homogenate was centrifuged at 15,000g for 30 minutes in a Spinco model L ultracentrifuge, yielding a residue which was discarded and supernatant fraction S15. The latter was centrifuged at 105,000g for 90 minutes and supernatant fraction $05, and the microsomal pellet &&I was isolated. In those experiments in which the conversion of sRNA”-proline to sRNA-hydroxproline was investigated, S,: was lyophilized and used whole (SISL) or it was centrifuged (after reconst,itution) at 105,000g 90 minutes, for the isolation of supernatant fraction SloaL and Sl~eL?vl (microsomal pellets). A scheme describing the procedures employed to obtain chick-embryo homogenate fractions is shon,n in Fig. 1. Ribonucleoprotein (RXP) particles were prepared from the microsomal pellets which w-ere re2 Abbreviations used: sRNA, soluble (transfer) ribonucleic acid; ATP, adenosine triphosphate; GTP, guanosine triphosphate; DPNH, reduced diphosphopyridine nucleotide; TPNH, reduced triphosphopyridine nucleotide; tris, tris (hydroxymethyl) aminomethane; TCA, trichloroacetic acid; DOC, deoxycholate; cpm, counts per minute.
BIOSYNTHESIS
481
suspended in medium A according to the procedure of Kirsch et al. (15). The yield of RNP was 25-30 mg of particulate protein for each 50 gm of blotted and decapitated embryos. The deoxycholatesoluble supernatant fraction, prepared from the microsomes, was dialyzed for 3 hours, at 2-5”C, against several changes of medium A (16): the retentate is further referred to as fraction DOCS. Preparation of chick-embryo sRNA. Chick embryo sRNA was prepared from fraction Slob by extraction with an equal volume of 90% phenol at 2”-4°C for 30 minutes (17). The mixture was centrifuged at 15,000g for 10 minutes, the aqueous phase was collected by aspiration, and the phenol layer was re-extracted with an equal volume of distilled water and recentrifuged. The combined aqueous phases were extracted three times with ethyl ether. Residual ether was removed by bubbling Nz through the solutions at O”C, and thesolutions were lyophilized. The residues were dissolved in 0.01 M tris-acetate buffer at pH 7.4 and dialyzed for 2 hours against two changes of the same buffer. After dialysis, NaCl was added to a final concentration of 0.1 M and sRNA was precipitated with three volumes of ethyl alcohol at - 10°C. The precipitated sRNA was dissolved in 0.01 M trisHCl buffer, pH 7.8, and dialyzed against the same buffer for 2 hours prior to its use for the formation of sRNA-proline-U1. The yield of sRNA by this procedure was 0.3-0.6 mg per gram of embryo. Preparation of sRXA-proline-C14. Three to 5 mg of chick-embryo sRNA was incubated in a reaction mixture with a pH 5 enzyme fraction containing 20 mg protein, prepared from fraction Slob (15), pyruvate kinase (Mann), 0.2 mg; and the following additives in micromoles: ATP (dipotassium, Mann), 50; phosphoenolpyruvic acid (sodium salt, CalBiochem.), 100; magnesium acetate, 50; KCl, 500, tris-HCI buffer, pH 7.8, 500; and 5.0 pmoles of proline-U-C14 (New England Nuclear or Nuclear-Chicago), containing 2 ~c (520,000 cpm). The reaction mixtures in final volume of 15 ml were incubated in Parafilm covered beakers at 37°C for 30 minutes with shaking. Aminoacyl sRNA, hereafter referred to as sRNA-proline, was isolated by phenol extraction, as described above, and purified by 2 cycles of successive dialysis (vs. tris-acetate buffer) and precipitation. About 7070 of the sRNA originally added was recovered with a specific activity of 450-520 cpm per milligram sRNA. Cl”-activity was determined using aliquots of dissolved sRNA-proline-C14 which were plated on stainless steel planchets. The samples were analyzed for radioactivity in a nuclear-Chicago gasflow counter with Micromil end window. Incubation of RNP particles with free proline04. The basal system for these studies contained 30-40 mg RNP protein; fraction DOCS, 6 mg
482
URIVETZKY,
FREI,
CHICK
AND
EMBRYO
HOMOGENATE
Centrifugation 15,000 X g -30
RESIDUE
(discard)
MEILMAN
SUPERNATE
min.
(S,,) I
r
I I
I
Centrifuge 105,000
LYOPHYLIZE
x g
(S,,L)
reconstitute centrifuge 105,000
r-----s MICROSOMES
(S,,,M)
SUPERNATE
(S,,,)
/
I
phenol ethanol
deoxycholate
I
M,,,OMkTE
(S,,L)
extraction precipitation
s-RNA l---l RIBONUCLEAR PROTEIN
xg
I
deoxycholate
’
I
SUPERNATE I RIBONUCLEAR (RNP-L)
PROTEIN
SUPERNATE
dialyze dialyze I RETENTATE
RETENTATE
(DOCS)
FIG 1. Scheme
of fractionation
protein; chick supernatant fraction &OS, 5 ml; pH 5 enzyme protein, 20 mg. The final reaction mixture was 20 ml and the following substances were added, in rmoles: proline-U-C14, 5.0 (2 PC); ATP, 50; GTP (sodium, Mann), 25; phosphoenolpyruvic (sodium), 100; pyruvate kinase, 0.2 mg.; a mixture of 16 unlabeled amino acids (4), 2.5 pmoles of each amino acid; reduced glutathione, 150, magnesium acetate, 40; tris-HCl buffer, pH 7.8, 500. The incubations were carried out at 37°C for periods of 30-90 minutes, in Parafilm sealed flasks or under a gas mixture of 95y0 02-5% COz, with shaking. At the end of the incubation periods the reac-
(DOCS
.L)
procedure.
tion mixtures were brought to 0” and centrifuged at 105,OOllg for 90 minutes. The ribosomal pellets were washed with cold medium A (less sucrose and glutathione) by resuspension and centrifugation at 105,000g for 1 hour. The washed pellets were suspended in water and aliquots removed for precipitation with cold TCA (4, 18) and subsequent W-analyses of the ribosomal protein precipitates. The remaining water suspensions were dialyzed vs. 3 changes of distilled water (20 hours total dialysis time), and the nondialyzed material was hydrolyzed in 6 N HCl in sealed vials at 110°C for 16 hours. The hydrolyzates were employed for
CELL-FREE
COLLAGEN
chromatographic separations of the amino acids as described above. Only carrier hydroxyproline was added prior to fractionation and the radioactivities of proline and hydroxyproline were determined after chromatography on a Moore and Stein column (19). Hydroxylation of sRNA-proline-C14. The basal system for the hydroxylation of sRNA-prolineCl4 cont,ained 3-4 mg sRNA-proline-Cl4 and fraction &hL, containing 60 mg protein dissolved in 15 ml of Ringer-phosphate-bicarbonate medium (20) to which 150 rmoles of glucose was added. The mixture was incubated at 37°C under 957, 02-5% COZ for 30 minutes, after which it was chilled to 0°C. Hibonucleic acid was extracted either from the whole incubates or from the 105,OOOg supernatants and pellet,s (individually), which were obtained by centrifugation of the chilled incubates at 105,OOOg for 90 minutes. The extracted RNA fractions (phenol procedure described above) a-ere purified, as indicated above, the imino acids were discharged, and their C14-activities were determined. Discharge and identification of imino acids from .sRS$ complexes. The amino acids were stripped from their sRNA4 complexes by incubation at pH 10 for 1 hour at 37°C (17,21). The reaction mixture was then adjusted to pH 7.0 and RNA was precipitated with ethanol. The supernatant was concentrated by flash evaporation and the amino acids were fractionated by chromatography on Dowex 50 W X4 columns by the method of Moore and Stein (19j. Unlabeled carrier proline and hydroxyproline were added prior to ion-exchange chromatography. Portions of the hydroxyproline and proline fractions eluted from the columns were plated and their C14-activities were determined. Other portions of the hydroxyproline and proline fractions eluted from the Moore and Stein column were applied with butanol: acetic acid: water (63:27:10, v/v) to thin-layer cellulose plates for chromatography (22). After development the plates were dried, and standard markers of proline and hydroxyproline were revealed by spraying with ninhydrin and isatin. The corresponding unsprayed areas were eluted with 10 ml water, twice, at 70°C. The filtered eluates were concentrated in z’acuo, dissolved in 2 ml Hyamine base, and counted in a Packard Tricarb scintillation counter following t,he addition of 3 ml toluene and 10 ml of scint,illator solution. The concentrated supernatant containing the discharged imino acids was also analyzed for proline and hydroxyproline-radioactivity by the method of Prockop and Peterkofsky (23). Each vial used for counting contained 5 ml of the toluene extracts and 10 ml of scintillator solution.
483
BIOSYNTHESIS
The scintillator solution used for counting was composed of reagent-grade toluene containing 8 gm of 2,5-diphenyloxazole and 500 mg of 1,4-bis2-(5 phenyloxazolyl).benzene per liter. TolueneCl4 (New England Nuclear) was employed as an internal standard. Bssays. Protein was determined by the method of Lowry et al. (24) and correlated with microKjeldahl analyses of analagous fractions. Ribonucleic acid was analyzed spectrophotometrically at 260 m,~ (E. coli RNA was used as reference standard), or by the procedure of Schneider (25). Proline wa4 assayed for by the method of Piez et al. (26) and hydroxyproline by the method of Stegemann (27) or the Woessner modification of this procedure (28). RESULTS
Incorporation of Proline-Cl4 and Hydroxyproline-C14 into RNPParticles Studies with free proline-C’4. Incubation of chick-embryo RNP-particles with free proline-Cl4 in the basal system given above resulted in the incorporation of the iminoacid into ribosomal-bound protein or peptide fractions, the maximum incorporation of proline occurring between 30 and 60 minutes. Only relatively low levels of hydroxyproline-Cl4 were found to be present in the same fractions, but the maximum incorporation of the imino acid was found at 90 minutes (the longest incubation). When the reactions were carried out under 95% 02-5% coz , the incorporation of proline was slightly diminished, but that of hydroxyproline was enhanced, i.e., the ratio of incorporated hydroxyproline-C14: prolineCl4 increased. The addition of reduced pyridine nucleotides and ascorbic acid to the aerobic incubation reactions did not produce any significant effects on hydroxyproline-Cl4 incorporation. The detailed results are given in Table I. Treatment of ribosomal suspensions (after incubation) with 0.01 M KOH at pH 10 for 1 hour at 37°C resulted in the release of less than 20% of the proline-Cl4 activity and about 10 % of the hydroxyproline-CL4 activity associated with the ribosomal protein fraction. These results along with hydrolysistime studies (4) indicated that the major portions of the incorporated imino acids
484
URI\‘ETZKY,
INC~KPORATION OF PROLINEAY PARTICLES IN INCUBATION
Incubath
system
A. Parafilm covered flasks Basal” Basal0 Basal” Basala Basala Basal0 Basala Basala B. Aeration with 95% 02-57, co2 Basal Basal Basal Basal Basal Basal
Time (min)
AND
FREI,
AND
TABLE
I
MEILiVAN
HYDROXYPROLINE-Cl4 SYSTEMS CONTAINING
R-UP Incorporated Protein
30 30 GO 60 90 90 60 60
2970 2410 3550 3960 2505 2834 3246 2945
30 30 60 60 90 90
1864 1568 2612 2442 1804 1960
ISTO
ADDED Cl”-activity* Proline
CHICK-EMBRYO
RiVP
PROLINE-Cl’ (cpmjb Hydroxyproline
2745 3042 1550 1726 2510 1948
34 25 90 96 102 108 75 80
0.03 0.02 0.03 0.03 0.06 0.06 0.03 0.04
860 620 1206 1264 900 1065
45 50 140 160 190 210
0.0.5 0.08 0.12 0.13 0.21 0.19
(1 The basal incubation systems, as described in Experimental, contained the following amounts of RNP-particle protein: 34 mg, Experiment 1; 30 mg, Experiment 2; 38 mg, Experiments 3 and 4. The incubations were carried out (2.0 PC proline-C 14, 520,000 cpm added) for the time periods indicated; cold-TCA precipitated ribosomal protein, proline, and hydroxyproline fractions were prepared and their V-activities were determined (see Experimental). * Total C14-activity incorporated into total RNP protein, proline, and hypro fractions.
were present in peptide linkages bound to the RNP-particles. Studies with sRNA-proline-C14. When sRNA-proline-Cl4 (prepared as described in Experimental) was incubated with RNP particles, maximum incorporation of proline occurred by the end of 30 minutes. Maximum hydroxyproline incorporation occurred at 60 minutes under aerobic conditions (Table IIB) or in Parafilm covered flasks (Table HA). The ratios of incorporated hydroxyproline-Cl4 to proline-Cl4 were higher than for those obtained using free prolineCl4 especially when aerobic conditions were employed. Therefore, the use of sRNAproline and/or aerobic incubation conditions stimulated the incorporation of hydroxyproline into RNP particles. The most marked enhancement was obtained when both factors were employed simultaneously. The omission of DOCS, ATP, and the energy-generating system, or GTP, resulted
in a decrease in the incorporation imino acids (Table IIB). Conversion of sRNA-I’roline-Cl” sRNA-Hyclroxyp~oline-P
of both to
Other investigat.ors have proposed that sRNA-hydroxyproline is an intermediate in microsomal collagen synthesis (10-13). The result,s described above could be interpreted in accord with this hypothesis. The remaining st)udies described here were then carried out to determine whether sRNA-hydroxyproline could be formed under condibions similar to those favoring the incorporation of hydroxyproline into ribosomal protein and t)o evaluate those circumst,ances favoring the hydroxylation of sRNA-proline. For if sRKA-hydroxyproline was involved in collagen synt,hesis it would have to be formed from a species of sRKA-proline since free hydroxyproline itself is not incorporatjed into collagen.
CELL-FREE
COLLAGEN TABLE
INCORPORATION PARTICLES
OF PROLINE-Cl4 IN INCUBATION
II
BND HYDROXYPROLINE~~ SYSTEMS CONTMNIN~ RhWIncorporated
hp.
Incubation
1 1
A. Parafilm flasks Basalt Basalt Basalt
system
c The particle particle
C14-activity system
(min)
Protein
INTO
ADDED
CHICK-EMBRYO
Cl4 activity Proline
RNP
sRNA-PROLINE-Cl4 (cpm)” Hydroxyproline
C’a-Hypro C’4-Pro
covered
13. Aeration with 95yo 02-5% CO? Basal Basal Basal Basal Basal less DOCS Basal less GTP Basal less ATP and energy systemh a Total 5 Energy
Time
485
BIOSYNTHESIS
30 60 90
724 6G0 610
520 440 -400
30 GO 90 60 GO GO 60
804 720 636 780 695 465 310
462 395 340 435 310 285 160
58 86 82
92 142 135 172 90 80 36
0.11 0.19 0.20
0.20 0.36 0.40 0.40 0.29 0.28 0.22
RNP protein, proline, and hydrosyproline fractions. acid and pyruvate kinase. basal incubation systems, as described in Experimental, contained: Experiment 1,26 mg RNPprotein and 3.5 mg sRNA-proline-Cl4 (460 cpm/mg sRN-4) ; Experiments 2 and 3, 28 mg RNPprotein and 3.2 mg sRNA-proline-Cl4 (485 cpm/mg sRNA). The preparation of sRNA-proline-
Cl4 is described
incorporated into = phosphoenolpyruvic
in the text.
Presumably the species of sRNA-proline involved would be present in the complex which we refer to as sRn’A-proline. Aft,er the incubation of sRNA-prolineCl4 in the basal system described in Experilfzental t,here was a significant increase in the hydroxyproline-C14 content of the sRNA complexes which were isolated. When lyophilized fraction SlsL was omitted or the incubations were carried out under Nz , there was no increase in sRNA-hydroxyproline-Cl4 over t,hat. originally present in the sRn’A complexes which were added t,o the reaction mixtures (see Table III). When fraction S& was replaced by Sl,,L or S&I, the former had about 60% and the latter 40% of t,he activity of the whole lyophilized l,?,OOO g supernat’ant fraction. Ninety % of t’he activity of Sl,L was recovered when the two subfractions were recombined and added (Table III). The decrease in proline activity in the posbincubation RNA fractions was due lnost likely t,o one or all of four factors: (a) dilution with sR,KA present in S15L (0.75 mg
sRNA in SlsL); (b) conversion of proline to hydroxyproline; (c) incorporat,ion of proline into microsomes; (d) degradation of some sRNA-proline during incubat,ion and isolation procedures. Ribonucleic acid isolated from the postincubation microsomal fractions, when treated at pH 10, did yield small amounts of free proline and hydroxyproline with C14-activities (Table III), most of which was very likely due t)o sRNA fract]ions which remained bound to the microsomal pellets. However, since it was found I hat the activit,y in t)he phenol extracted microsomal RNA which was released by incubation at pH 10 did not have any significant influence on the interpretation of Ihe results, in subsequent experiments Ihe postjincubation mixtures were extract’ed without prior separation of microsomal and supernatant fractions. The data obtained in further studies on the hydroxylation of sRNA-proline are presented in Tables IV and V. Essentially, the findings may be summarized as follows: (a) the addition of ascorbic:
acid
and/or
pyridiue
nuclcotides
486
URIVETZKY,
FREI,
AXD
TABLE CONVERSION
MEILMAN
III
OF sRNA-PROLINE-04
TO sRNA-HYDROXYPROLINE-Cl4 Postincubation
Incubation
systema
P10lille Basal Basal Basal Basal Basal
less &L Incubation under but S,,L replaced
SK& Basal but, 81jL replaced
fractions (cpm/mg sRNA)
105,000 g supernatant
Time (min)
Hydrow proline
Microsomal C’h-Kypro ~~ C”-Pro
Proline
Hydroxyproline
fractions C”-Hypro W-Pro
Nz by
30 GO 30 30 30
285 228 410 406 340
75 60 30 22 54
0.26 0.26 0.07 0.05 0.16
62 75 -
10 18 -
0.16 0.24 -
by
30
380
48
0.13
40
8
0.20
by
30
315
72
0.23
56
15
0.27
S,asLM Basal but SlbL replaced &o;LM plus S,asL
a Incubation reactions were carried out by using the basal system for these studies, as described in Erperimental, under a gas mixture of 95’$$ OZ-5% CO2 except where otherwise indicated. RNA was isolated from supernatant and microsomal fractions by phenol extraction after incubation, and the free imino acids were released from their RNA complexes by incubation at pH 10 for 60 minutes at 37°C. The details of the procedures employed are given in the text. The sRNA-proline-C14 complex added to the incubation mixtures contained 3 mg sRNA with 520 cpm proline-Cld/mg sRNA and 24 cpm hydroxyproline-C14/mg sRNA (hydroxyproline-C14/proline-C14 = 0.05).
had at the most a slight stimulatory effect on the conversion (Table IV) ; and (b) replacement of the microsomal port,ions of S16L by RNP particles (RNP-L) prepared from it produced a marked decrease in sRNA-hydroxyproline-Cl* formation, most of which was restored when fractions DOCSL (dialyzed deoxycholate soluble) were added (Table V). The formation of sRNA-hydroxyproline was substantiated by the following evidence obtained from the analyses of t’he amino acids which were discharged from their sRNA complexes. (a) C14-activity (Tables III-V) was obtained in the hydroxyproline fraction after ion-exchange chromatography. (b) This fraction, when subject’ed to chromatography on cellulose plates, demonstrated U4-activity on the plate area corresponding to the position of hydroxyproline. At least 60% of the radioactivity present in the hydroxyproline fractions from the Moore and Stein column could be accounted for in the hydroxyproline areas eluted from the thin-layer chromatography plates. (c) The imino acids were stripped from sRNA and then analyzed by t.he procedure of Pet’erkofsky and Prockop (2) in which proline and hydroxyproline are converted to their
respective pyrroline and pyrrole products prior to extraction with toluene. The results of these studies are given in Table VI. DISCUSSION
In agreement with the studies of other investigat’ors (3, 12, 14), cell-free systems containing supernatant and ribosomal chickembryo homogenate fractions are able to activate proline to form sRNA-proline complexes from which the imino acid can be incorporated into ribosomally synthesized protein. It is in regard bo the ability of such systems to synthesize collagen, requiring the hydroxylation of proline at some given step, where there is need of further evaluation. Manner and Gould (12) and Daughaday and Mariz (11) have suggested on the basis of their studies that sRNA-hydroxyproline, formed by the hydroxylation of a species of sRNA-proline, is an intermediate in the incorporation of hydroxyproline into collagen. The studies of Green and Lowther with granuloma slices (29) and those of Stone and Meister (granuloma minces) (10) also favor the involvement of activated proline molecules as opposed to proline-rich peptide precursors. On the other hand, the recent report of Peterkofsky
CELL-FREE TABLE EFFECT OF NUCLEOTIDES
TABLE
ACID THE
AND
EFFECT
PYRIDINE
HYDROXYLATION
OF
sRNA-PROLINE-Cl4
Experiment Basal Basal + acid Basal + acid + Basal + acid + Basdl + Experiment Basal Basal + acid + Basal + acid + Basal + acid + DPNH
system’
proline
OF
(cpm/mg
HYdFYprolme
V
RIBOSOMAL
AND
DEOXCHOL.~TE-SOLUBLE
DIALYZED
FRXTIONS
HYDROXYL~TION
Postincubation Incubation
487
BIOSYNTHESIS
IV
ASCORBIC ON
COLLAGEN
OF
ON
RNA)
Postincubation (cpm,‘mg RNA)
C”-Hypro -GTE-
Incubation
THE
sRNA-PROLINE-Cl4
syste#
Cl’-Hypro C~“.pro
HydroxyXYDTO-
Proline
1 ascorbic ascorbic DPNH ascorbic TPNH DPNH 2 ascorbic DPN ascorbic DPNH ascorbic DPN +
312 295
84 95
0.28 0.32
305
102
0.33
286
102
0.36
324
98
0.30
321 290
78 75
0.24 0.25
305
88
0.28
320
92
0.29
a The conditions of incubation employed in the studies given here are the same as those described in Table III. All incubations were carried out for 30 minutes under 95% 02-5% COZ. However, for the reasons explained in text, RNA was extracted from the whole postincubat.ion mixtures. The sRNA-proline-Cl4 added contained: Experiment 1, 4 mg sRNA with 480 cpm proline-U4/mg sRNA and 28 cpm hydroxyproline-C14/mg sRNA; Experiment 2, 3.0 mg sRNA, 460 cpm proline-C14/mg sRNA and 20 cpm hydroxyproline/mg sRNA.
Basal Basal less Basal but by &o&L Basal but by RNP-L Basal but by S,oiL Basal but by RNP-L Basal but by &,,jL DOCS-L
SljL SlsL replaced + SlojLM S,jL replaced SlsL replaced + RNP-L S,jL replaced + DOCS-L SljL replaced + RNP-L +
80 32 74
0.28 0.06 0.25
305
25
0.08
315
52
0.16
342
46
0.13
305
70
0.22
a The sRNA-proline-Cl4 complex added contained 3.5 mg sRNA with 512 cpm proline-C’*/mg sRNA and 22 cpm hydroxyproline-Cld/mg sRNA. Fractions S,ojLM, S,ojL, RNP-L, and DOCS-L were prepared from S,,L as described in Ezperimental (see also Fig. 1). When S1oQLM, SlojL, and RNP-L were used, the entire fractions prepared from SlsL (60 mg protein) were added. DOCS-L added contained 2 mg protein. All incubations for 30 minutes under 95y0 02-57, COz. TABLE INCREASE
IN
T-I
SRNA-HYDROXYPROLINE
INCIJBATION
OF
-4FTER
sRN~PRoLINE
I
,
DPM/mg
and Udenfriend (14) would rule out sRNAhydroxyproline as an intermediate, and instead a proline-rich peptide fraction associated with microsomal RNA would be the substrate for hydroxylation. Studies utilizing cell-free chick-embryo homogenate fractions in our laboratories indicated that the incorporation of proline and hydroxyproline into protein (or peptides) synthesized in the ribosomes was better than that obtained previously with rabbit-skin preparations (4). However, the incorporabion of hydroxyproline in systems containing free proline-Cl4 and incubated under relatively anaerobic conditions (see Table I) suggested that only a very limited synthesis of collagen molecules had occurred.
282 465 296
Prolin@ EXP.
1 2 3
C’J-Hypro
/ Hydroxyproline I
I
3864 3720 4200
RNA
2016 2430 2800
50 58 45
537 582 815
“‘-“’ I
/
0.01 0.02 0.01
0.27 0.24 0.29
a sRNA-proline was incubated with the basal system described in the text. The amino acids were discharged at pH 10 and the radioactivities of proline and hydroxyproline were determined by the oxidation and toluene extraction procedures as described by Peterkofsky and Prockop (23) ; these procedures were modified for use with 10 ml of the liquid scintillator solution described in text.
In such syst,enls, the ittcorporation of hydroxyproline-Cl4 ittt’o RNP particles could be itmeased by aerobic incubation under 95% 02-5 70 CO? ) particularly when the react’ions were carried out up to 90 ntinutes. hlaxitnutn hydroxyproline incorporation was found t)o lag behind that of proline (Table I). Incubation with sRNA-proline-C14 under aerobic conditions showed the highest activit,ies of hydroxyprolinc ittcorporatcd with protein, in which case it,s tnaxitnutn irtcorporation agaitt lagged behind that of prolinc-Cl4 (Table II). In subsequent experiments evidence for hhc formation of sRNAhydroxyproline was obtained as described above. These findings suggested t)he possibi1it.y that Ihe hydroxylatiott of a species of sRNA-prolitte was necessary prior to the incorporation of hydroxyproline into RSP particles. This pat,hway would be favored by aerobic conditions (10, 30, 31) and would also account for the lag period observed in hydroxyproline incorporation. Additional st)udies were carried out in order t)o determine the conditions most favorable for the hydroxylation of sRNA-proline-Cl”. The results (Tables III-V) indicate that) a portion of sRKX-proline can be converted to sRNA-hydroxyproline under aerobic conditions in incubation systems containing sypernatant and/or microsotnal fractions prepared from embryo homogenates. Maximum conversion was obtained with whole lyophilized 15,000g (StsL) supernatant fractions, but tnicrosomal (Sl,LM) and 105,OOOg superrtatant (&L) fractions prepared from it also showed significant activities. Ribonucleoprotein particles (RX-L) had no effect] on the hydroxylation of sltNAprolinc, but, most of the activity was restored when dialyzed deoxycholate-soluble material (DOCS-L) was added to RNP-L incubation mixtures. Therefore a fact,or or factors present in the 105,OOOg superttatatlt and in t.he intact microsomes is involved itt the conversion. The slight enhancement obtained with ascorbic acid and pyridine nucleotides would be in agreement, with the proposal of Prockop et al. (28) t,hat, an oxygenase rnechattistn was involved itt the hydroxylation of proline. The lack of a nlore marked effect can be due to already adequate arn0unt.s of these factors present in
the homogenates (14). In fact, the reduced conversions obtained wit,h tnicrosomal fractions might, be due to a lack of these cofactors. That the increased ratio of sEWAhydroxyprolitre/sR?A-prolinc is indeed evidence of formation of sRNA-hydroxyprolitte and not due to a greater instability of sRKAproline is support’ed by the data from cont,rol experiments. For example, RKA isolated from t)he basal system (tttinus Sl,L) after incubation (Tables III and V) shows a low ratio of hydroxyproline/prolirte Cl”-act)ivit,y, which is essentially t)he same as that. of the starting materials. Our results which indicate that sRNAhydroxyproline is formed are in agreement with those of other investigators (12,30-33). At least t,wo irnport’ant questions remain for further study: (a) Is sRKA-hydroxyprolitte a precursor of collagen hydroxyproline or is it sitnply an artifact or a by-product? Our results thus far do not’ contradict t,he fittdings of Peterkofsky and Udenfriend (14) and could very easily be interpreted in accord with their conclusions, especially if sRK\‘Ahydroxyproline will be found to be ineffective in the eventual incorporat’iott of the itnino acid into collagen. (b) Are t,he tnechanisms involved in t)he hydroxylation of sRNAproline as repot%ed here the same as t,hose involved in Pet,erkofsky and Udenfriend’s studies (14)? If t,hey are the same, then the nlechanistns involved would appear to be relatively nonspecific if both sRKA-prolitte and proline-rich peptides could act as subst’rates. In either case, the findings will have to be explained in the future interpretation of the tnechanisms involved in collagen synthesis and the formation of hydroxyproline in vim. REFERENCES 1. LOWTHER, D. A., GREEN, X. AI., AND CHAPMAN, J. A., J. Biophys. Biochem. Cytol. 10, 373 (1961). 2. PROCKOP, D. J., PETERICOFSKY, B., AND UDENFRIEND, S., J. Biol. Chem. 237, 1581 (1962). 3. PETERKOFSKY, B., AND UDENFRIEND, S., Biothem. Biophys. Res. Commun. 6, 184 (1961). 4. URIVETZKY, RI., KRANZ, V., AND MEILMAN, E., Arch. Biochem. Biophys. 100, 478 (1963). 5. FRANZBLAU, C., SEIFTER, S., AND GALLOP, P. Xl., Biopolymers 1, 79 (1963).
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