THE JOURNAL. OF B~~.

OGKAL CHEM~~RY
Vol. 254, No. 23, I.wue of December 10, pp. 1219%12203. 1979
Printed in U.S.A.

Cell-free Synthesis of Hyaluronic Acid in Marfan Syndrome*

(Received for publication, August 28, 1979)

Alan Appel,$ Allen L. Horwitz, and Albert Dorfman

From the Department of Pediatrics, Committee on Genetics, and the Joseph P. Kennedy, Jr. Mental Retardation Research
Center, Pritzker School of Medicine, University of Chicago, Chicago, Illinois 60637

The cell-free synthesis of hyaluronic acid has been from cultured human fibroblasts. The present work reports
demonstrated in extracts of cultured human fibro- the development of a cell-free system to study the enzymic
blasts. Preparations from fibroblasts of normal individ- reactions involved in the synthesis of hyaluronic acid. In the
uals as well as those from patients with Marfan syn- course of characterizing this system, a comparison was made
drome incorporate glucuronic acid and N-acetylgluco- of extracts derived from normal fibroblasts and from fibro-
samine from their UDP derivatives into hyaluronic blasts from patients with Marfan syndrome.
acid. Extracts from Marfan fibroblasts demonstrate 3
to 10 times more total and specific hyaluronic acid
EXPERIMENTAL PROCEDURES
synthetase activity than do preparations from normal
fibroblasts. All synthetic activity was found in partic- Materials-Dithiothreitol was obtained from Calbiochem; ATP,
ulate fractions with the bulk of activity localized in UDP-GlcNAc,’ and UDP-GlcUA from Sigma; UDP-[“H]GlcNAc
material sedimenting as large membrane fragments. (6.75 Ci/mmol), UDP-[“‘C]GlcUA (224 mCi/mmol), Scintisol, and
Aquasol from New England Nuclear; and Sepharose 4B from Phar-
Marfan and normal preparations exhibited similar macia. Umbilical cord hyaluronic acid was a gift of Dr. Martin B.
properties with respect to substrate, cofactor, pH re- Mathews (University of Chicago).
quirements, and heat stability. Neither the Marfan nor Streptococcal hyaluronidase was obtained as preparations of Var-
normal enzyme systems could be stimulated by exoge- idase (Lederle Laboratories) which have been shown to have this
nous acceptors, nor did either preparation contain a activity (16). Leech hyaluronidase was purchased from Biotrics Co.
soluble factor which stimulated or inhibited the en- Cell Culture-Fibroblasts were derived from skin biopsies of the
zymic activity of the other. The genetic defect in Marfan deltoid region of the upper arms of individuals. Marfan syndrome
syndrome appears to result in increased activity of fibroblasts were from patients exhibiting the full syndrome as de-
hyaluronic acid synthetase without demonstrable scribed previously (3). Normal skin fibroblasts were obtained from
biopsies of clinically normal patients of the same age. The fibroblasts
changes in properties of the particulate enzymes in-
were cultured in IOO-mm plastic culture dishes in a modified Eagle’s
volved. medium containing 10% fetal bovine serum and 10% calf serum
(GIBCO). The medium, used by Matalon et al. (2, 17) in previous
studies, was Dulbecco’s modified Eagle’s medium with 100 mg/liter
of ascorbic acid, 4.2 g/liter of NaHC03, 100 pg/ml of streptomycin,
Marfan’s syndrome is an autosomal dominant disorder of and 100 units/ml of penicillin G. Cultures were incubated at 37°C in
connective tissue characterized by skeletal, cardiovascular, a humidified atmosphere of 10% CO? in air.
and ocular abnormalities (1). Previous studies in this labora- Preparation and Assay of Enzymes-Preparative and assay pro-
tory (2, 3) have shown an accumulation of hyaluronic acid in cedures for the hyaluronic acid synthesizing system were adapted
fibroblast cultures from Marfan patients compared with those from methods reported by Hopwood et al. (16). Cell cultures were
grown to confluency (4 to 6 mg of cell protein/dish), washed with
from normal individuals. Since degradation of hyaluronic acid Hanks’ balanced salt solution, then scraped from the culture dish into
proceeds at the same rate in both normal and Marfan cultures, 50 mM Tris-HCl, pH 7.05, containing 0.24 M sucrose (Buffer A). Cells
a defect in the control of hyaluronic acid synthesis has been were disrupted using a Sonifier (Heat Systems) with a microtip at
suggested (3). It is not clear whether the alteration in hyalu- 100 watts output for 1 min. Sonicates were centrifuged at 600 X g for
ronic acid synthesis is the primary gene defect in Marfan’s 10 min at 4°C to remove large debris and unbroken cells. The resulting
syndrome, or is secondary to some other biochemical abnor- supernatant solution was centrifuged at 100,000 x g for 60 min at 4°C.
The 100,000 x g precipitate (designated as “membrane fraction”) was
mality. It was, therefore, of some interest to study the enzymic
resuspended in Buffer A and used as an enzyme source in most assays.
reactions in hyaluronic acid synthesis in order to search for Protein was measured by the method of Lowry et al. (18).
alterations in the synthetic system. To assay activity of the hyaluronic acid synthesizing system, incu-
Hyaluronic acid is the principal glycosaminoglycan synthe- bation mixtures contained 0.24 pmol of dithiothreitol, 0.50 pmol of
sized by human skin fibroblasts (4). It is thought to play a ATP, 2.4 pmol of NaHzP04 buffer, pH 6.7, 0.84 pmol of MgC12, 0.48
role in processes of cell aggregation and mobility (5-7). Al- nmol of UDP-[‘?]GlcUA, 10 nmol of UDP-GlcNAc, and the enzyme
suspension in a final volume of 60 ~1. Incubations were carried out at
though synthesis of this polysaccharide has been observed in 37°C.
cell-free systems derived from Group A streptococci (8-lo), Following incubation, samples were heated for 5 min at lOO’C!,
from hyalocytes (11)) and from virally transformed and other digested 15 to 18 h with 100 pg of Varidase in 50 ,ul of 0.01 M NaH2PO.J
established cell lines (12-15), there have been no reports of K2HP04 buffer, pH 6, containing 0.15 M NaCl. Digests were chroma-
cell-free synthesis of hyaluronic acid in preparations derived tographed on Whatman 3MM paper with ethanol:lM ammonium
acetate, pH 5 (65:35) for 16 h. The origin and the region corresponding
* This investigation was supported by National Institutes of Health to the unsaturated disaccharide produced by digestion of hyaluronic
Grants AM-05996, HD-09402 and HD-04583. The costs of publication acid by streptococcal hyaluronidase (Adi-HA) were cut out and radio-
of this article were defrayed in part by the payment of page charges. activity was measured by adding 1 ml of water and 10 ml of Aquasol
This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Present address, Division of Human Genetics, Department of ’ The abbreviations used are: GlcNAc, N-acetylglucosamine;
Pediatrics, State University of New York at Buffalo, Buffalo, NY GlcUA, glucuronic acid; Adi-HA, 2-acetamido-2-deoxy-3-0-(/3-n-
14222. gluco-4-enepyranosyluronic acid)-D-glucose.

12199

This is an Open Access article under the CC BY license.

 Hyaluronic Acid Biosynthesis

followed by counting in a Packard Tri-Carb liquid scintillation spec-

trometer.
Isolation and Characterization of Hyaluronic Acid-Following
incubation of a large scale mixture, containing 10 times the amounts
of components of routine assays, 2 mg of umbilical cord hyaluronic :
0 40
acid were added and the material was treated for 48 h with 0.5 N KOH ;i
at 4°C neutralized with glacial acetic acid, and protein was precipi-
E
tated with trichloroacetic acid at a final concentration of 5%. Follow- ’ 20
ing centrifugation at 10,000 x g for 10 min. the supernatant solution E
was dialyzed against water, made 0.03 N with respect to NaCl, and 0.1 0”
-4
ml of 10% cetylpyridinium chloride was added. The resulting precip- ,A---
ii/
itate, collected by centrifugation, was dissolved in 2 M LiCl and the 60 120 240
glycosaminoglycans were precipitated by ethanol at 80% concentra- Min
tion. Ethanol precipitates were washed with ethanol/ether and air-
dried.
The glycosaminoglycans were dissolved in 0.1 M pyridine acetate
buffer, pH 5.5, and chromatographed on a column (0.8 x 100 cm) of
Sepharose 4B eluted with the same buffer. The void volume fractions
were pooled, lyophilized,
gests were passed through
and digested with leech hyaluronidase.
Sepharose 4B to determine degradation
Di-
by
1
shift of radioactivity from void volume to included volume fractions.

RESULTS

Sedimentation Fractionation of Hyaluronic Acid Synthe-

sizing Activity-Table I shows that all recoverable hyaluronic
acid synthesizing activity could be sedimented at 100,000 x g
for 1 h. The bulk of activity recovered was associated with
material precipitating between 600 x g and 10,000 x g, which
is expected to contain large membrane fragments. In prelimi- I:
nary experiments comparing sonication and homogenization
by Teflon-glass Potter-Elvehjem homogenizer, sonication pro-
duced higher yields of enzyme. Less than 2% of the total FIG. 1. Effects of time of incubation and enzyme protein
activity was associated with the 600 x g precipitate. No concentration on hyaluronate synthetase activity. Incubation
activity was detectable in the 100,000 x g supernatant solu- mixtures contained 4.8 pmol of UDP-[‘4C]GlcUA, 10 nmol of UDP-
GlcNAc, 0.84 pmol of MgC12, 0.24 pmol of dithiothereitol, 0.5 amol of
tion. For all fractions, the total synthetic activity and specific ATP, 2.4 pmol of sodium phosphate, pH 6.7, and 100,000 x g enzyme
activity were significantly greater for Marfan preparations protein in a final volume of 60 al. Incubations were for 2 h at 37°C or
than for normal preparations. for the times indicated in the inset. The results for enzyme from
Properties of the Hyaluronic Acid Synthesizing System- normal fibroblasts (0- - -O), and enzyme from Marfan fibroblasts
Fig. 1 shows that synthesis of hyaluronic acid, as measured in (U) are shown.
incorporation of UDP-[“‘C]GlcUA increased with increasing
amount of protein. The synthesis of hyaluronic acid was also fragments (Fig. 2). These results indicate that the [‘4C]GlcUA-
linear with time (Fig. 1). The difference in synthetic capacity labeled high molecular weight product produced by normal
between normal and Marfan preparations was observed at all and Marfan preparations is hyaluronic acid.
levels of enzyme protein and at all times of incubation. If MgCl* was omitted from the standard incubation, no
When large scale incubations of normal and Mar-fan partic- hyaluronic acid synthesis was observed. Fig. 3 shows maximal
ulate enzyme were subjected to procedures for isolating hy- stimulation of hyaluronic acid synthesis at a concentration of
aluronic acid, followed by gel filtration chromatography, all of 13.3 mM MgC12; at the same concentration, other divalent
the radioactivity appeared in the void volume of Sepharose cations tested failed to stimulate synthesis to the same degree
4B (Fig. 2). When this material was treated with leech hya- as Mg2+ (Table II). Monovalent cations did not substitute for
luronidase and rechromatographed on Sepharose 4B, all of Mg’+.
the radioactivity was recovered as small molecular weight The omission of ATP from reaction mixtures resulted in an
absence of hyaluronic acid synthesis. Fig. 4 shows maximal
TABLE I stimulation of hyaluronic acid synthesis at a final concentra-
Fractionation of hyaluronic acid synthetic activity from normal tion of 10 mM ATP. At concentrations above 10 mu, there
and Marfan fibroblasts was considerable inhibition of synthesis. ATP had little effect
Fibroblasts were harvested as described under “Experimental Pro- on the extent of breakdown of UDP-[‘4C]GlcUA, but mark-
cedures” and the indicated fractions were used as enzyme sources in
edly inhibited breakdown of UDP-[3H]GlcNAc (Table III).
the assay. Synthetic activity was determined as described under
“Experimental Procedures” using IJDP-[‘4C]GlcUA in the standard The stimulatory effect of ATP may be due to its “sparing” of
incubation mixture. the UDP-GlcNAc by competition for hydrolysis by phospha-
Total activity Specific activity tases in the membrane preparation.
Preparation Maximal stimulation of hyaluronic acid synthesis was ob-
NOUd Marfan Normal MWfan
tained at a concentration of 40 mM phosphate buffer. When
pm&/h pmol/h/mg
the pH of incubations was varied between 6 and 7, maximal
Sonicate 118 763 4.2 12.2
synthesis was observed in a pH range of 6.4 to 6.8 for both
600 x g precipitate 7 17 3.2 4.1
600 X g supernatant 87 715 1.1 14.3 normal and Marfan preparations (Fig. 5).
10,000 X g precipitate 33 265 2.7 23.8 The rate of synthesis of hyaluronic acid by the particulate
10,000 X g supernatant 15 67 0.5 6.2 cell-free system was determined as a function of nucleotide
100,000 X g precipitate 10 79 1.8 23.1 sugar concentration in order to detect possible changes in
100,000 X g superna- 0 0 0 0 Marfan-derived enzyme which could account for an increased
tant rate of hyaluronic acid synthesis. Marfan and normal enzymes
 Hyaluronic Acid Biosynthesis 12201

TABLE II
Effect of cations on hyaluronic acid synthesis
Enzyme preparations were 100,000 x g precipitates. Assays for
synthesis of hyaluronic acid by incorporation of UDP-[‘“C]GlcUA
were carried out as described under “Experimental Procedures” with
the addition of the indicated salts at a concentration of 13.3 mM.
Specific activity
Addition
NOlTd Marfan
pmol/h/mgprotein
None 0 0
MgClz 2.7 10.5
MnCls 1.7 5.0
CaC12 1.2 3.2

VOLUME (ML)
FIG. 2. Characterization of hyaluronic acid produced by the mM
cell-free synthetic system. Enzyme incubations contained 10 times FIG. 4. Effect of ATP concentration on cell-free hyaluronic
the amounts of reactants indicated in Fig. 1 and were carried out for acid synthesis. Incubation mixtures were as described in the legend
2 h. The hyaluronic acid was isolated as indicated under “Experimen- to Fig. 1 with ATP at the indicated concentrations. Enzyme from
tal Procedures.” Sepharose 4B chromatography was carried out with normal cells (0- - -0) and enzyme from Marfan cells (O---+) are
0.1 M pyridinium acetate, pH 5.5, on columns (0.8 x 100 cm). A, indicated.
chromatography of product of incubation with enzyme from normal
fibroblasts; B, chromatography of incubation products of Marfan-
TABLE III
derived enzyme. The patterns for intact polysaccharide isolated as
described under “Methods,” (O-----O) and for polysaccharide after Effect of ATP on stability of UDP sugars
digestion (0- - -0) with leech hyaluronidase are indicated. The cell-free synthesis of hyaluronic acid was carried out using
either UDP-[‘%]GlcUA (84,500 cpm) or UDP-[“HlGlcNAc (375,000
cpm) in the presence of increasing concentrations of ATP. UDP-
[‘%]GlcUA and UDP-[3H]GlcNA c were localized by paper chroma-
tography as described under “Experimental Procedures.”
UDP-[“C]GlcUA re- UDP-[:‘H]GlcNAc
ATP added maining remaining
nmf %
0 48 3
4.2 61 5
8.3 72 82
16.7 76 79
41.7 86 77

UDP-GlcNAc determined from the Lineweaver-Burk plot

(Fig. 6) was 26 PM for enzyme preparations from Marfan cells
and 21 pM for normal preparations.
The rate of hyaluronic acid synthesis at various concentra-
tions of UDP-GlcUA is shown in Fig. 7 (inset). Again, satu-
ration kinetics are displayed by both normal and Marfan-
FIG. 3. Effect of MgCl, concentration on hyaluronic acid
synthesis. Incubations were for 2 h as indicated in Fig. 1 with MgClz derived enzyme preparations. The apparent K,,, values for
concentrations as indicated. Enzyme from Marfan cells (M) and UDP-GlcUA calculated from the data in Fig. 7 are 3.2 PM for
enzyme from normal cells (0- - -0) are recorded. the normal enzyme and 2.2 pM for enzyme preparations from
Marfan cells. Similar Km determinations were done on enzyme
demonstrated similar kinetics with respect to UDP-GlcNAc preparations from a number of different cultures. The appar-
concentration. Fig. 6 (inset) indicates that both preparations ent Km for UDP-GlcUA for enzymes from normal cell cultures
demonstrate similar saturation kinetics for UDP-GlcNAc. Re- was 2.9 f 1.0 (SD.) pM while the enzyme from Marfan cultures
action velocity for Marfan enzymes remained more than 3 gave a value of 3.2 + 1.1 PM. Due to the particulate nature of
times normal at most concentrations. The apparent Km for the system, the K,,, values must be considered approximations.
12202 Hyaluronic Acid Biosynthesis

30-
e
=z 20-
E
Q IO-

6.0 6.5 7.0

PH
FIG. 5. Effect of pH gf incubation mixture on the cell-free
synthesis of hyaluronic acid by extracts of normal and Marfan /”
fibroblasts. The enzyme source was the 100,000 x g particulate /
enzyme. Incubation mixtures were as described under “Methods,” but
the pH of the sodium phosphate was varied to give the indicated pH.
dP l
k N- I- I- I I
The pH is the final pH after addition of all components of the reaction
mixtures. Indicated are activities for enzyme prepared from normal I 2 3 4
(0- - -0) and Marfan syndrome (M) fibroblasts. ,uM-’ X IO2
FIG. 7. Effect of UDP-GlcUA concentration on rate of cell-

1
free synthesis of hyaluronic acid. Incubation mixtures contained
particulate enzyme, UDP-GlcNAc, cofactors, and buffers as described
under “Experimental Procedures.” The concentration of UDP-
lOOk [‘%]GlcUA was varied as indicated. Values indicated are for enzyme
preparations from Marfan (O-----O) and normal (0- - -0) fibroblasts.

each mixture was approximately that expected from the sum

of activities assayed separately. Thus, there were no soluble
stimulatory or inhibitory factors in either preparation which
could explain the difference in hyaluronic acid synthetic ac-
tivity. The rate of heat inactivation at 55°C was similar for
the extracts of both normal and Mar-fan fibroblasts.

DISCUSSION

This paper presents the fist report of cell-free synthesis of

hyaluronic acid by extracts of cultured human skin fibroblasts.
In all previously studied systems for the cell-free synthesis
of hyaluronic acid, the activity has been found to be particu-
late (4, 8, 10, 13-15). Because methods of cell disruption and
centrifugation differed in studies on preparations from various
tissues and cells, it is not possible to make an extensive
comparison of activity among particulate fractions such as
FIG. 6. Effect of UDP-GlcNAc concentration on rate of cell- that of cell membranes. Using homogenates of rat fibrosar-
free synthesis of hyaluronic acid. Incubation mixtures contained coma, Hopwood and Dorfman’ found 5 times as much total
particulate enzyme which had sedimented at 100,000 x g, UDP- synthetic activity associated with a fraction sedimenting at
[“C]GlcUA and cofactors as described under “Experimental Proce- 10,000 x g than with the fraction sedimenting between 10,000
dures.” The concentration of UDP-GlcNAc was varied as indicated. X g and 100,000 X g. Ishimoto et al. (13), also using homoge-
Enzyme activity is expressed as picomoles of disaccharide units nization, reported that 90% of the synthetic activity of the
formed and isolated as described in the text. Values indicated are for
enzyme preparation from Marfan (O-----O) and normal (0- - -0) crude homogenate of chick embryo fibroblasts could be re-
fibroblasts. covered in the 10,000 x g precipitate fraction. Although this
fraction is heterogeneous, it is expected that large membrane
The differences in K, values between Marfan and normal fragments, especially particles derived from the cell mem-
preparations were not significant. brane, would be present.
There was no effect on hyaluronic acid synthesis as a result Addition of ATP has been reported to be required for cell-
of addition of any of the following compounds to the standard free synthesis of hyaluronic acid in preparations derived from
incubation mixture: GlcNAc, GlcUA, p-nitrophenyl-GlcNAc, fibroblasts, from rat fibrosarcoma (16), and hyalocytes (11).
hyaluronic acid, or heparan sulfate. The effect of several However, it is not needed in cell-free systems derived from
detergents was studied. At 0.1% (v/v) Triton X-100 and NP- streptococcus (8-10) and was not added to systems derived
40, there was complete inhibition of hyaluronic acid synthesis. from other eukaryotic cells (12-15). The principal role of ATP
Tween 80 at the same concentration inhibited synthesis by in the fibroblast cell-free system appears to be prevention of
about 50%. When the polyisoprenoids, dolichol and dolichol hydrolysis of UDP-GlcNAc.
phosphate, were added in the presence of 0.1% Tween 80, In addition to characterizing the hyaluronic acid synthesiz-
activity was less than that observed with no additions.
When enzyme preparations from normal and Marfan fibro- * J. J. Hopwood and A. Dorfman (University of Chicago), unpub-
blasts were mixed, the hyaluronic acid synthetic capacity in lished observations.
 Hyaluronic Acid Biosynthesis 12203

ing system in cultured fibroblasts, we have utilized the cell- Recently, Lamberg (20) has reported that fibroblasts from
free assay in an attempt to elucidate the basis for the increase patients with Marfan disease have an increased stimulation of
in hyaluronic acid synthesis in fibroblasts from Mar-fan pa- hyaluronic acid synthesis as a response to added hyaluronic
tients. In the cell-free assay described here, extracts derived acid. In addition to a change in glycosyltransferase properties,
from fibroblasts of Mar-fan patients synthesized at least 3 there are a number of other factors which could influence the
times more hyaluronic acid than did comparable preparations activity of the hyaluronate synthesis system. Since the defect
from control fibroblasts. This finding is consistent with pre- of Marfan disease is apparent in a cell-free system with added
vious reports from this laboratory showing that the accumu- nucleotide sugars, the excessive hyaluronic acid production is
lation of hyaluronic acid in Marfan fibroblast cultures is due unlikely to be due to changes in nucleotide sugar pools or
to excess synthesis (2,3) and with the finding that degradation precursor metabolites. Within the limits of assay in the system
of hyaluronic acid proceeds at the same rate in control and described here, normal and Marfan preparations failed to
Marfan cultures (3). We have found that fibroblast extracts show any significant differences in properties other than rate
from a large number of Mar-fan patients show a close corre- of synthesis. The nature of the genetic defect that results in
lation between the amount of hyaluronic acid in culture and the increase in hyaluronic acid synthesis remains unknown.
the level of hyaluronic acid synthetic activity.
With respect to cellular distribution of synthetic activity, REFERENCES
size of material synthesized, cofactor, pH optimum, heat sta-
bility, and failure to be stimulated with exogenous agents, no 1. McKusick, V. A. (1973) in Heritable Disorders of Connective
Tissue (McKusick, V. A., ed) 4th Ed, pp. 61-224, C. V. Mosby,
differences between normal and Mar-fan preparations were
St. Louis
observed. The slight differences between normal and Marfan 2. Matalon, R., and Dorfman, A. (1968) Biochem. Biophys. Res.
preparations observed in apparent K,,, values for UDP- Commun. 32,150-154
GlcNAc and UDP-GlcUA would not appear, in themselves, to 3. Lamberg, S. I., and Dorfman, A. (1973) J. Clin. Znuest. 52,2428-
be responsible for the increased hyaluronic acid synthesis in 2433
the Marfan preparation. The approximate nature of these K,,, 4. Hopwood, J. J., and Dorfman, A. (1977) J. Biol. Chem. 252,4777-
4785
values must be emphasized since the enzyme complex remains 5. Pessac, B., and Defendi, V. (1972) Science 175,898-900
membrane-bound. It is impossible to know the actual concen- 6. Wasteson, A., Westermark, B., Lindahl, U., and Ponten, J. (1973)
tration of UDP-sugar substrates in contact with the enzyme. Znt. J. Cancer 12, 169-178
Many details of the biosynthesis of hyaluronic acid remain 7. Toole, B., Jackson, G., and Gross, J. (1972) Proc. Natl. Acad. Sci.
to be resolved. For example, the role of lipid intermediates in U. S. A. 69, 13841386
the synthesis of this polysaccharide has been suggested (19). 8. Markovitz, A., Cifonelli, J. A., and Dorfman, A. (1959) J. Biol.
Chem. 234,2343-2350
Failure of tunicamycin to inhibit synthesis of hyaluronic acid 9. Stoolmiller, A. C., and Dorfman, A. (1969) J. Biol. Chem. 244,
in fibrosarcoma tissues and in cell-free extracts of fibrosar- 236-246
coma3 or in the streptococcal system (10) suggests that a 10. Sugahara, K., Schwartz, N. B., and Dorfman, A. (1979) J. Biol.
dolichol pyrophosporyl intermediate is not involved. It has Chem. 254,6252-6261
also been suggested that there is a cooperative effect of the 11. Gsterlin, S. E., and Jacobson, B. (1968) Exp. Eye Res. 7,497-510
two membrane-bound glycosyltransferases with a growing 12. Glaser, L., and Brown, D. H. (1955) Proc. N&Z. Acad. Sci.
U. S. A. 41, 253-260
hyaluronate chain which promotes polysaccharide synthesis 13. Ishimoto, N., Temin, H. M., and Strominger, J. L. (1966) J. Biol.
(8, 9). Changes in activity of such intermediates and mem- Chem. 241,2052-2057
brane changes could increase the rate of hyaluronic acid 14. Tomida, M., Koyama, H., and Ono, T. (1974) Biochim. Biophys.
synthesis in Marfan syndrome without increasing the number Acta 338,352-363
of enzymic biosynthetic units. 15. Tomida, M., Koyama, H., and Ono, T. (1977) J. Cell. Physiol. 91,
A number of parameters of culture conditions have been 323-328
16. Hopwood, J. J., Fitch, F., and Dorfman, A. (1974) Biochem.
described to influence hyaluronic acid production in cultured
Biophys. Res. Commun. 61,583-5X)
cells. Disturbance in the control mechanisms influenced by 17. Matalon, R., and Dorfman, A. (1966) Proc. Natl. Acad. Sci.
these culture conditions could lead to the excess hyaluronic U. S. A. 56, 1310-1316
acid production of Marfan disease. Among these conditions 18. Lowrv. 0. H.. Rosebrouah. N. J.. Farr. A. L.. and Randall. R. J.
may be cell culture density and viral transformation (4). (l&l) J. Biol. Chem. 193, 2651275
19. Hopwood, J. J., and Dorfman, A. (1977) Biochem. Biophys. Res.
’ A. Appel, A. L. Horwitz, and A. Dorfman, unpublished observa- Commun. 75,472-479
tions. 20. Lamberg, S. I. (1978) J. Znuest. Dermatol. 71, 391-395