SOL UBLE COLLAGEN PROLINE HYDROX YLASE AND ITS SUBSTRA TES IN SEVERAL ANIMAL TISSUES BY JOHN J. HUTTON, JR., AND SIDNEY UDENFRIEND
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SOL UBLE COLLAGEN PROLINE HYDROX YLASE AND ITS SUBSTRA TES
IN SEVERAL ANIMAL TISSUES
BY JOHN J. HUTTON, JR., AND SIDNEY UDENFRIEND
NATIONAL HEART INSTITUTE, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND
Communicated by James A. Shannon, May 13, 1966
It has been shown that certain proline residues in peptide linkage can serve as
precursors of collagen hydroxyproline.1 In the intact cell, hydroxylation probably
occurs during the synthesis of the peptide chain before the latter is released from the
microsome.' The mechanisms for translation of the collagen messengers to form
polypeptides appear to function independently of the mechanisms for hydroxylation
so that hydroxyproline-deficient, collagenase-degradable polypeptides and proteins
accumulate when hydroxylation of proline is impaired (translation without hy-
droxylation).2' These peptides and proteins can be hydroxylated subsequently by
the enzyme, proline hydroxylase, in the absence of protein synthesis.4' Thus far
there have been no reports of proline hydroxylase activity in cell-free systems other
than the one derived from chick embryos,4' although most animal tissues synthe-
size collagen. It is the purpose of this paper to show that cell-free systems derived
from a variety of animal sources contain proline hydroxylase activity. In addition,
under appropriate conditions each of these tissues can synthesize hydroxyproline-
deficient collagen-like protein which can be solubilized and subsequently hydrox-
ylated by chick embryo proline hydroxylase.6 Conclusions drawn from the chick
embryo system'-' concerning proline hydroxylase and its substrates therefore appear
to be generally valid.
Materials and Methods.-The sources of most materials including fetal rat skin, carrageenan
granuloma, and embryonated eggs have been described previously.3 The tripeptides, glycyl-
prolylproline and glycylprolylhydroxyproline, were obtained from Cyclo Chemical Corporation
and were over 90% homogeneous when chromatographed on the "neutrals and acidics" column
of a Beckman amino acid analyzer.
Unless otherwise noted, all operations were carried out at 3VC. Preparation of homogenates
of 8-day-old chick embryos has been described.7 Granuloma and fetal rat skin were homogenized
in 1 vol of 0.25 M sucrose, adult rat livers in 2 vol, in a Servall Omni-Mixer at 90 v for 30 sec.
The crude homogenates were centrifuged at 15,000 X g for 10 min, and the supernatant fractions
(S-15) were collected. In some instances the S-15 fractions were respun at 105,000 X g, 60 min
to sediment the microsomes. This supernatant fluid is referred to as S-105 fraction. Approximate
yields were: 6 dozen 8-day-old chick embryos, 25 ml S-105, protein 10 mg/ml; 6 guinea pigs,
25 gm granuloma, 40 ml S-105, protein 7 mg/ml; 6 fetal rats, 6 gm skin, 10 ml S-105, protein 4
mg/ml; 30 gm adult rat liver, 60 ml S-105, protein 25 mg/ml. Cells of Pseudomonas sp. (ATCC
11299a), which had been broken in a French press in 4 vol of 0.005 M Tris-HCl, pH 7.3, were ob-
tained from Dr. G. Guroff.8 A 105,000 X g supernatant fraction containing 33 mg/ml protein
was prepared. Enzyme preparations stored at -20'C retained activity for several weeks.
Proline-C'4-labeled substrates of proline hydroxylase were prepared from minces of 8-day-old
decapitated chick embryos, guinea pig granuloma, and fetal rat skin. In each case the incubation
mixture contained 3.6 gm minced tissue, 6 mi modified Krebs-Ringer buffer," 1 mM aa'-di-
pyridyl and 25 juc uniformly labeled proline-C'4.10, 1 Incubation was for 60 min at 370C in air.
At the completion of incubation, the mixture was chilled and centrifuged. Collagen and newly
formed proline-C'4-labeled substrate in the pellet were solubilized and extracted by incubating
overnight at 4VC in 7 ml 0.5 N acetic acid per 3.6 gm tissue.'0 The supernatant solution, con-
taining collagen and proline hydroxylase substrate,6 was obtained by centrifugation and thoroughly
dialyzed against water. The solution generally contained 1-3 mg/ml protein of specific activity
10,000-50,000 cpm/mg, counting efficiency 70% in Bray's solution.'2 Fewer than 1% of the total
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1!9SVOL. 56, 1966 BIOCHEMISTRY: HUTTON AND UDENFRIEND 199
C14 counts were present as hydroxyproline. Collagenase degradation to determine the proportion
of proline-C'4 in unhydroxylated collagen-like protein was carried out as previously described.2' 3
The substrate solutions could be stored at -20'C for several weeks with no loss in activity.
Proline hydroxylase activity was assayed in a system similar to that described by Prockop and
Juva.4 It contained: Tris-HCl, pH 7.5, 400 Mmoles; KCl, 160 /Amoles; ferrous ammonium sul
fate, 0.8 Mmole; ascorbic acid, 12 smoles; 0.5-2.5 mg proline-C'4 substrate totaling 20,000-
40,000 cpm; crude enzyme protein 10-50 mg; water to 8 ml final vol. Incubation was carried
out at 370C for 60 min in 50-ml beakers. The reaction was stopped by chilling and adding tri-
chloroacetic acid (TCA) to a final concentration of 5%. The precipitates were collected by cen-
trifugation and were washed once with cold 5% TCA. Collagen was extracted from the pre-
cipitates with hot 5% TCA and its content of proline-C'4 and hydroxyproline-C'4 was assayed
as described previously.7 Results are presented as the observed cpm, counting efficiency 70%.
Where hydroxyproline alone is presented, results were corrected for recovery of total radio-
activity (proline plus hydroxyproline) from each incubation. Variation in recovery of total
counts among the tubes of a single experiment was usually less than 20%. Protein concentration
was estimated by the method of Warburg and Christian.'3
Results.-Proline hydroxylase: Homogenates of chick embryo, guinea pig gran-
uloma, fetal rat skin, and adult rat liver were tested for proline hydroxylase activity
using the chick embryo substrate. Incubation of the substrate with supplemented
cell-free extracts from each of these tissues resulted in the conversion of peptidyl
proline to peptidyl hydroxyproline (Table 1). In each case the precursor proline
and the resulting hydroxyproline were in a fraction which was insoluble in cold 5
per cent TCA, nondialyzable, and soluble in hot 5 per cent TCA. Proline hydrox-
ylase activity was found in both soluble and microsomal fractions from the chick
embryo (Table 1). The specific activity of the S-105 fraction was several times as
great as that of the microsomes. Proline hydroxylase activity in homogenates of
fetal rat skin, guinea pig granuloma (Table 1), and adult rat liver was also found in
the S-105 fraction. Microsomes were not assayed directly in these tissues. The
specific activity of enzyme derived from fetal rat skin has consistently been the
highest of all systems tested, although it is obtained in low yield. In all cases hy-
droxylating activity of S-105 fractions was stimulated by the addition of ferrous ion
(Table 2). Ascorbic acid was stimulatory in all systems except that of fetal rat
skin. Chick microsomal fractions were stimulated slightly by the addition of iron
and ascorbic acid. The addition of boiled S-1055 did not enhance microsomal activ-
ity. Hydroxylation of peptidyl proline-C14 did not occur when enzyme was omitted,
boiled, or replaced with bovine serum albumin (Table 2). Extracts of Pseudomonas
sp. had essentially no proline hydroxylase activity (Table 2). Additional studies
TABLE 1
INThACELLULAR£i DISTRIBUTION OF PROLINE IIYDROXYLASE ACrTIVIrY
Hydroxyproline
Source of enzyme Cell fraction formed (cpm)
Chick embryo 8-15 430
8-105 390
Microsomes 100
Fetal rat skin 8-15 548
S-105 560
Guinea pig granuloma 8-15 192
8-105 291
Incubations were carried out in an 8-ml vol as described under Materials and Methods. Chick
embryo proline hydroxylase substrate added: 0.5 mg to the chick embryo fractions, 1.5 mg to
those of fetal rat skin and guinea pig granuloma. Total cpm proline plus hydroxyproline were
3500 in all cases. Omission of enzyme resulted in fewer than 20 cpm hydroxyproline. Total
protein added: chick embryo S-15, 44 mg; S-105, 21 mg; microsomes 30 mg. Fetal rat skin
S-15, 25 mg; S-105, 13 mg. Guinea pig granuloma S-15, 15 mg; S-105, 15 mg.
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TABLE 2
DEPENDENCE OF PROLINE IYDROXYLASE ACTIVITY FROM VARIOUS SOURCES ON FERROUS
ION, ASCORBIC ACID, AND ENZYME
-Hydroxyproline Formed (cpm)-_
Minus Fe+ +
Complete Minus Minus and Boiled
Source of enzyme system Fe+ + ascorbate ascorbate enzyme
Chick embryo 331 29 274 62 0
Fetal rat skill 563 450 562 35) 0)
Adult rat liver 325 106 '2 56 -
Guinea pig granuloma 291 6 123 () 0
Pseudomonas sp. 37 12 1223 0
Bovine serum albumin 14
Incubations were carried out in an 8-ml vol as described under Mat i ds and JMethods with omissions
as indicated. Total extracted radioactive proline plus hydroxyproline was 3500 cpm in each case. To
each tube 1.5 mg chick embryo proline hydroxylase substrate was added. Total enzyme protein added
as S-105 fraction was: chick embryo, 13 mg; fetal rat skin, 12 mg; adult rat liver, 50 mg; guinea pig
granuloma, 15 mg; Pseudomonas sp., 65 mg. Bovine serum albumin, 25 mg, replaced enzyme in one
incubation. For inactivation, enzyme was heated in boiling water for 5 min.
with the chick embryo system have shown that hydroxylation is 80 per cent com-
plete in 30 min. Termination of the reaction appears to be due to enzyme destruc-
tion and not to exhaustion of substrate, ascorbate, or ferrous ion.
Proline hydroxylase substrate: Proline-C14 substrate was prepared from minces
of chick embryo, fetal rat skin, and guinea pig granuloma. When chick embryo
substrate preparations were extracted with hot TCA and the extracts incubated
with bacterial collagenase, about 17 per cent of the proline-C14 activity in the ex-
tracts was released in the form of soluble peptides. 14 Since collagenase releases only
half of the collagen as soluble peptides, these findings indicate that approximately
35 per cent of the hot TCA-extractable proline-C"4 is present in a hydroxyproline-
deficient collagen-like protein (further discussed in ref. 3). The chick embryo
substrate has been further characterized.4 10 Following incubation of chick em-
bryo substrate with the hydroxylase, 20 per cent of the proline-C14 and over 90 per
cent of the hydroxyproline-C"4 was extractable into hot 5 per cent TCA. The chick
embryo enzyme also hydroxylated 10-30 per cent of the proline-C14 in the hy-
droxyproline-deficient substrates prepared from each of the other tissues (Table 3).
It can be seen that the proline-C14 which disappeared in each case was comparable
to the hydroxyproline-C14 which appeared. However, stoichiometric comparisons
will require more highly purified substrate and enzyme and better isolation proce-
dures. There was no evidence of preference of the chick embryo enzyme for chick
embryo substrate. The higher hypro/pro ratios (Table 3) obtained with granuloma
and skin substrate probably reflect less contamination of these substrates by pro-
TABLE :3
HYDROXYLATION OF VARIOUS SUBSTRATES BY CHICK EMBRYO PROLINE ILYDROXYLASE
Radioactivity in Protein-Bound
Imino Acids (cpm)
Temperature Hydroxy- Hydroxyproline:
Substrate source (0C) Proline proline proline ratio
Chick embryo 0 3570 14VOL. 56, 1966 BIOCHEMISTRY: HUTTON AND UDENFRIEND 201
TABLE 4
EFFECT OF AMINO ACIDS ANDTRIPEPTIDES ON CHICK EMBRYO PROLINE
HYDROXYLASE
Final conc. Hydroxyproline
Addition (M) formed (cpm)
None 339
L-proline 2.5 X 10-2 329
L-Hydroxyproline 2.5 X 10-2 309
Gly-pro-pro 3.0 X 10-3 404
Gly-pro-hypro 3.0 X 10-3 387
(No enzyme) 6
Incubations were carried out in an 8-ml vol as described under Materials and Methods. Each
beaker contained 25 mg chick embryo S-105 and 0.8 mg substrate derived from chick embryo.
Total extracted radioactive proline plus hydroxyproline was 4500 cpm in all cases.
line-C 14-labeled noncollagenous materials. The total incorporation of proline-C14
into substrate by granuloma and skin was lower than in the chick embryo.
The hydroxylation of substrate was not inhibited by L-proline, L-hydroxyproline,
or by the tripeptides gly pro * pro and gly pro * hypro (Table 4).
- -
Discussion.-For the past year we have been interested in the accumulation of
hydroxyproline-deficient, proline-rich, collagenase-degradable protein in systems
derived from tissues that synthesize collagen. We showed that such a protein
accumulates when hydroxylation is impaired in a variety of tissues.2 3 We believe
that this hydroxyproline-deficient protein does not represent an obligatory inter-
mediate in collagen biosynthesis, but an abnormal product formed under unusual
circumstances when translation occurs without hydroxylation. Until this report,
cell-free hydroxylation of microsomal-bound5 and solubilized4 hydroxyproline-
deficient material could be demonstrated only in extracts derived from chick em-
bryos. Prior attempts to demonstrate proline hydroxylase activity in cell-free sys-
tems derived from granuloma"5 or fetal rabbit skin'6 failed. In this paper we have
shown that soluble cell-free enzyme preparations derived from chick embryo, guinea
pig granuloma, fetal rat skin, and adult rat liver can hydroxylate proline-rich, hy-
droxyproline-deficient protein. As one would expect, a cell-free preparation derived
from bacteria was inactive. It remains to be seen whether the proline residues hy-
droxylated in this system are the same as those which are hydroxylated in vivo.
Proline hydroxylase is largely a soluble enzyme in the cell-free systems which we
have studied. Ferrous ion4 and ascorbic acid5 usually stimulate its activity. How-
ever, until purified preparations of the enzyme become available, the identification
of the natural cofactors will remain in doubt. The metal requirement of the present
system may be partly due to trace amounts of aa'-dipyridyl present in the sub-
strate preparations. Enzyme activity is rapidly lost during incubation in the
assay system. It should be noted, however, that the combination of oxygen,
ferrous ion, and ascorbate in the assay forms a powerful oxidizing system and a free
radical generator which may destroy the enzyme. Attempts to protect the en-
zyme have been only partially successful.
Several lines of evidence suggest that the natural substrate for proline hy-
droxylase is a unique polypeptide of high molecular weight." 4 The failure of high
concentrations of free proline, hydroxyproline, and the tripeptides, gly - proepro
and gly pro- hypro, to inhibit this enzyme supports the concept that the active
-
hydroxylating site interacts with specific peptidyl proline residues in high-molec-
ular-weight polypeptides. Such data eliminated the possibility of substrate break-
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down to very small units followed by proline hydroxylation and reincorporation
into high-molecular-weight material.
Summary.-It has been possible to obtain soluble proline hydroxylase prepara-
tions from fetal rat skin, adult rat liver, guinea pig granuloma, as well as chick
embryo. Proline-C14-rich, hydroxyproline-C'4-deficient proteins susceptible of
degradation by bacterial collagenase have also been prepared from each of these
tissues and have been shown to be hydroxylated by chick embryo proline hy-
droxylase. These studies represent the first demonstration of cell-free hydroxylation
of peptidyl proline in tissues other than those of chick embryo.
1 Evidence for the hydroxylation of proline residues after incorporation into peptide linkage
has been reviewed recently. [Udenfriend, S., Science, 152, 1335 (1966)].
2 Gottlieb, A. A., B. Peterkofsky, and S. Udenfriend, J. Biol. Chem., 240, 3099 (1965).
3 Gottlieb, A. A., A. Kaplan, and S. Udenfriend, J. Biol. Chem., 241, 1551 (1966).
4 Prockop, D. J., and K. Juva, these PROCEEDINGS, 53, 661 (1965).
6 Peterkofsky, B., and S. Udenfriend, these PROCEEDINGS, 53, 335 (1965).
6 Proline hydroxylase substrate is defined as newly formed, solubilized, hydroxyproline-defi-
cient, proline-C14-labeled, collagen-like protein. This material is extracted with pre-existing
collagen.
IPeterkofsky, B., and S. Udenfriend, J. Biol. Chem., 238, 3966 (1963).
8 Guroff, G., and T. Ito, J. Biol. Chem., 240, 1175
(1965).
9 Stone, N., and A. Meister, Nature, 194, 555 (1962).
0 Lukens, L. N., Federation Proc., 25, Abst. 2971 (1966).
11 Juva, K., and D. J. Prockop, in Abstracts, 150th Meeting of the American Chemical Society,
Atlantic City, N. J. (1965), p. liC.
12 Bray, G. A., Anal. Biochem., 1, 279 (1960).
13 Warburg, O., and W. Christian, Biochem. Z., 310, 384 (1941).
14 We should like to thank Dr. Arnold Kaplan for performing this degradation and for his
generous advice and assistance.
16 Manning, J. M., and A. Meister, Biochemistry, 5, 1154 (1966).
16 Urivetzky, M., V. Kranz, and E. Meilman, Arch. Biochem. Biophys., 100, 478 (1963).
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