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Vitamin A and Mucopolysaccharide Biosynthesis
Vitamin A and Mucopolysaccharide Biosynthesis GEORGE WOLF AND B. CONNOR JOHNSON Radiocarbon Laboratory and Department of Animal Science, University of Illinois, Urbann, Illinois
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... 11. In Viuo Observations.. ........................ .................... 1. Vitamin A Deficiency ................................................. 2. Hypervitaminosis A . . . . . .................
Page 439 440 440
IV. In Vitro Observations. . . . . . . . . .
1. Chemistry and Biochemistry of Mucopolysaccharides. . . . . . . . . . . . . . . . . . 443
4. Vitamin A and Net Synthesis of Mucopolysaccharides in Vitro. . . . . . . 452 5. Vitamin A and Keratinization V. Conclusions. .................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. INTRODUCTION In a search for a biochemical function of vitamin A in metabolism, when looking over the existing literature on the effects of vitamin A deficiency or excess, one is immediately struck by the importance of the influence of vitamin A on mucus formation and on mucosal tissue. Moore (1957a), in his monumental work on vitamin -4,states: “If we consider the role of vitamin A on the widest possible basis, therefore, we may say that it is necessary for the formation of large molecules containing glucosamine.” Such large molecules are the mucopolysaccharides which, in addition, contain galactosamine, glucuronic acid, sulfate, and in certain cases also sialic acid, mannose, and fucose. Any hypothesis of a systemic function of vitamin A, therefore, has to take into account its effect on the biosynthesis of the mucopolysaccharides. These occur in various forms, and with various functions, in almost all tissues of the mammalian organism, but principally, and in largest quantities, in two locations: in the mucus secreted by mucous epithelium; and in the extracellular matrix of cartilage, mainly as chondroitin sulfate. Attention will be focused in this review on the influence of vitamin A on these two tissues. 439
440
GEOltGE: WOLF A N D €3.
CONNOR JOHNSON
11. I n Vivo OBSERVATIONS 1 . Vitamin A Deficiency
Vitamin A deficiency shows its first manifestation in a disruption and atrophy of all those epithelial tissues that function in mucus secretion: in the respiratory, intestinal, and urinary tracts and in the genital system (Wolbach and Howe, 1925). A decrease in the number and activity of mucus-secreting cells of the gastrointestinal tract occurs (Manville, 1937). Ultimately, a new type of epithelium replaces the mucus-secreting tissue. It becomes keratinized and similar to epidermis. The effect of the deficiency on the other important mucopolysaccharide of the mammal, the chondroitin sulfate of cartilage, is quite different : increased activity of osteoblasts, periosteal overgrowth with the formation of larger, soft bones (Mellanby, 1950). The effect of the deficiency on mucus formation in the genital system, especially vaginal epithelium, is particularly instructive, as here there are cells that at one time of the estrous cycle become mucussecreting, at another time keratinized. The whole picture is well summarized by Moore (1957b) : the vaginal epithelium, even in the absence of estrogen, becomes keratinized in vitamin A deficiency. In the absence of estrogen, but with excess vitamin A, it is transformed into mucus-secreting epithelium; a similar change being caused by progesterone, but only in the presence of normal levels of vitamin A. Estrogen, even in the presence of vitamin A, stimulates keratinization. The conclusion from these observations is that the normal state of this epithelium is to be keratinized; vitamin A stimulates the transformation to mucous epithelium. One could then further speculate that estrogen inhibits this action of vitamin A, whereas progesterone overcomes the inhibition. Since esterified sulfate represents an important part of the mucopolysaccharide molecule, a number of investigations have considered the effect of vitamin A deficiency on sulfate incorporation into cartilage. Dziewiatkowsky (1954) isolated chondroitin sulfate, the principal substance, apart from collagen, of cartilage matrix, after injection of labeled sulfate into vitamin A-deficient, normal, and vitamin A-treated deficient rats. The level of labeled sulfate was higher in the normal and vitamin A-treated deficient rats than in the deficient. This author also found increased breakdown as well as synthesis of chondroitin sulfate in the vitamin A-treated animals. Somewhat different results were obtained by Frape et al. (1959) working with vitamin A-deficient pigs. Here, vitamin A treatment of the deficient animals caused decreased sulfate uptake into cartilage as the primary response (Table I).
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
44 1
2. Hypervitaminosis A
Vitarniii A is distiiwt from other vitamins in that, it, acts when administered in excess, the effect being generally the reverse of that obtained in deficiency. Maturat ion of cartilage cells is accelerated; cartilage, as well TABLE I Sa* ACTIVITY I N TISSUESFROM PIOS ON VARIOUS DIETARYVITAMINA LEVELS~
A added
per pound of feed
5th Rib costochondrial junctionb
Ear cartilageb
0 55 208 790 1540 3000 11393
87581 42622 48601 32554 46789 54014 53221
21213 26764 5902 10811 12812 17082 22238
I.U.
Lunge
counts/min./gm. of fresh tissue"
7053 3442 4033 1731 1346 1761
2087
From Rape et 02. (1969). b Values are average of two samples.
Values are average of three samples. Counting rates are corrected for background, decay, and extrapolatedto zero thickness of precipitate.
TABLE I1 EFFECTOF HYPERVITAMINOSIS A ON SERUMCONCENTRATIONS OF MU COP ROTE IN^ Group*
Number
Vitamin A dose
Age at killing (days)
An Ah Cn Ch
2 2 3 4
0 0 lo00 lo00
34-40 39-40 39-40 39-46
Serum mucoproteinc (mg. tyrosine %)
6.8(2) 6.3(2) 10.3(2) 16.3(4)
f 0.05 0.70 f 0.05 f 3.74 &
~~
From Cohen et ol. (1955).
* n Refers to the group of animals on the diet normal in calcium; h refers to thaae on a diet high in osl-
cium content. The figures in parenthesis refer to the number of animals or cheinical determinations in the group.
as bone, is rapidly absorbed, resulting in spontaneous fractures (Wolbach, 1947). Normal vaginal and uterine epithelium is replaced by noncornifying epithelium (Hedenberg, 1954). Mucoprotein levels in blood are greatly increased (Cohen et al., 1955) (Table 11). Recently, Thomas el al. (1960) showed that, in the intact animal, hypervitaminosis A resulted in a depletion of cartilage matrix, due to loss of cartilage chondromucoprotein, and a consequent rise in serum chondroitin
4.42
GEORGE WOLF AND B. CONNOR JOHNSON
sulfate. It should be noted that this is the reverse of the effect of vitamin A excess on mucous tissue, where the hypervitaminosis results in a stimulation of mucus secretion, reflecting, no doubt, a different mode of action of vitamin A on mucopolysaccharide of connective tissue and of mucous tissue.
111. OBSERVATIONS ON TISSUE CULTURE All the work reported here on the effect of vitamin A on tissue culture has been carried out by H. B. Fell and her collaborators (reviewed by Fell, 1953). The objective was to determine whether the effects of vitamin A deficiency and hypervitaminosis A described in the preceding sections were indirect and mediated through an endocrine organ, or directly on the tissue. First, in their work on explants of undifferentiated chick embryo bones, cultured in a medium with excess vitamin A, Fell and Mellanby (1952) found the bone to turn soft and gelatinous, growth to diminish, and fractures to occur. The cartilage matrix ceased t o stain with basophile dyes; metachromasia, that is, the response of the stain toluidine blue to the acid groups of chondroitin sulfate, disappeared. It was thought that, whereas there occurred dissolution of the chondroitin sulfate, the collagen part of the matrix remained intact. With fully developed fetal mouse bones, the situation was similar-cartilage matrix and bone were absorbed and disappeared. The vitamin was present in a concentration similar to that found in the blood. There was then no doubt that the action of vitamin A was direct, though only on growing cells. In their second set of experiments, Fell and Mellanby (1953) cultured undifferentiated embryonic chick ectoderm. This normally develops a keratinizing layer over squamous epithelium. Cultured in excess vitamin A, it formed instead ciliated columnar cells with secretory vacuoles, actively secreting mucus, closely similar to nasal epithelium. When returned to normal medium, mucus secretion continued, but no new mucous cells differentiated and ultimately a new layer of squamous cells was formed which eventually keratinized. The changes described are all basically those seen in the whole animal, though greatly exaggerated. Similar experiments with skin from mammals or birds showed less pronounced transformations than with embryonic skin. These important results demonstrate that the action of vitamin A is twofold: (1) it causes dissolution of chondroitin sulfate of cartilage matrix; (2) it affects epithelial basal cells in such a manner as to make them differentiate into mucus-secreting cells, when they would normally form keratinizing cells. The conclusion is inescapable that vitamin A has an action akin to that of a hormone in determining the course of differentiation of the basal cells of epithelium.
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
443
It is of interest to note that the effect of excess vitamin A in tissue culture on the chondroitin sulfate of cartilage leads to dissolution and dis2ppearance, whereas on the related mucoid substances of mucous epithelium, it causes sulfate uptake and synthesis (Fell et al., 1954). Though the properties and identity of the mucopolysaccharidesof mucus are not completely established (see Section IV, l), they are certainly different from chondroitin sulfate of cartilage. It is possible therefore to conclude that excess vitamin A increases the mucus type and decreases the connective tissue t,ype of mucopolysaccharide. The reverse, a decrease in mucus and increase in connective tissue mucopolysaccharide, along with keratinization, occurs in deficiency. These generalizations help to interpret the i n vivo data discussed in Section I1: decrease in mucus secretion and periosteal overgrowth in vitamin A deficiency; increased mucus secretion, increased breakdown of chondroitin sulfate from cartilage, less sulfate uptake into cartilage, when vitamin A is administered. IV. In Vitro OBSERVATIONS 1 . Chemistry and Biochemistry of Mucopolysacchorides
Many excellent reviews exist of recent work in the chemistry of the mucopolysaccharides (American Society of Biological Chemists Symposium, 1958; Springer, 1958). However, for a clearer understanding of the subsequent discussion, a brief summary of the chemistry and biochemistry of those mucopolysaccharides on which vitamin A has an influence, is relevant. Three different chondroitin sulfates have been isolated (Meyer, 1958). Chondroitin sulfate A, found in cartilage, bone, cornea, and aorta, is a polymer of D-glucuronic acid and sulfated N-acetylgalactosamine, with N-acetylchondrosine sulfate (I) as the repeating unit. The two subunits are linked in a ,f3-1,3-glucuronidic link and a 8-1,Chexosaminidic link, wit,h sulfate esterified on position 4 of the acetylgalactosamine. Chondroitin sulfate B (repeating unit, 11), found in skin, tendon, and aorta, is identical with A, except for epimerization at C-5 of the glucuronic acid, leading to L-iduronic acid. In chondroitin sulfate C (repeating unit, 111), obtained from cartilage, umbilical cord, tendon, and sclera, the sulfate group is shifted to position 6 of the acetylgalactosamine moiety. Heparin is defined by Jeanloz (1958) as “dextrorotatory sulfated polysaccharides composed of D-glucosaniine and D-glucuronic acid” of various degrees of sulfation. The sulfate in these compounds is linked to the amino group in a sulfamic link, although compounds related to heparin, containing glucosamine and glucuronic acid with N-acetyl and O-sulfate-glucosamine have been found (heparitin sulfate).
444
,rT:rp\
GEORGE WOLF AND B. CONNOR JOHNSON
z
6CHr OH
CH~OH
\oo:!” OH
0
NHCOCHI
OH
NHCOCHj
OH
(1)
FfjQ\
(11)
HzCOSOjH
’0
NHCOCHI OH (111)
The mucopolysaccharides of the mucous epithelium, and of secreted mucus, have not been well characterized. Werner (1953) extracted three main types from pig colon mucosa, the prevalent one having L-fucose (IV) as its most characteristic carbohydrate constituent and therefore being called fucomucin ; it also contains N-acetylglucosamine, N-acetylgalactosamine, and D-galactose, the hexosamines constituting about one-half of the sugar content. The second type of epithelial mucopolysaccharide was found to contain sialic acid (V), and was therefore called sialomucin. It has an equivalent amount of N-acetylgalactosamine. The third group are the sulfated mucopolysaccharides. The identity of these is still in question. Smith and Gallop (1953) isolated two chondroitin sulfates from gastric mucosa, which appear to be chondroit,in sulfates A or C, and B (Meyer, 1958), as well as heparin. Meyer et al. (1937) isolated heparitin sulfate from gastric mucosa. CH, OH CHOH
(W
(V)
In t,he tissues examined, t,he choiidroitin sulfates exist as protein complexes, called chondromucoprotein (Schubert, 1958), a water-soluble compound of chondroitin sulfate and a noncollagenous protein, with a link
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
445
that is not saltlike. It is probable that in mucus as well, at least some of the mucopolysaccharides are linked to protein. The pathway of biosynt,hesis of acid mucopolysaccharides has recently become clear (reviewed by Dorfman et al., 1958). Markovitz e2 al. (1959), for instance, uses a bacterial enzyme system which synthesizes hyaluronic acid as a model for mucopolysaccharide. A brief summary of the enzymatic reaction sequence involved is given here (compare Fig. 1) : glucose 6-phosGlycogen
Glucose 1-P
1 Glucose 1-P
1
I
Glucose 6-P
I
glutamine
Uridine triphasphate
Glucosamine 6 P CH1COSCoA
I U r i d i n e diphosphoglucose
N-Acetyl glucosamine 6 P
1
N-Acetyl glucosamine 1-P
1
Uridine diphosphoglucuronic acid
Uridine triphasphate
Uridine diphospho-N-acetylglucosamine
I-
I
PAPS
ATP ATP FIG.1. Possible pathway of mucopolysaccharide biosynthesis (P, phosphate; CHaCO-SCoA, acetyl coenzyme A; IIPN, diphosphopyridine nucleotide; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; APS, adenosine 5'-phosphosulfate). Heparitin sulfuric acid
phate reacts with glutamine to form glucosamine 6-phosphate. The latter is acetylated by acetyl coenzyme A to produce N-acetylglucosamine 6-phosphate, which is isomerized to the 1-phosphate and reacts with uridine triphosphate to form uridine diphosphoacetylglucosamine. Similarly, glucose 1-phosphate forms uridine diphosphoglucose with uridine triphosphate, which is oxidized by diphosphopyridine nucleotide to uridine diphosphoglucuronic acid. This, with the above uridine diphosphoacetylglucosamine, is polymerized to hyaluronic acid. The introduction of the sulfate group into chondroitin sulfate presents a problem, since it may enter at the level of the uridine nucleotides or the
446
GEORGE WOLF AND B . CONNOR JOHNSON
fully formed polymer. Recent evidence (Suzuki and Strominger, 19GO) would favor the second possibility. Sulfate, before entering this reaction, is activated by reaction with adenosine triphosphate to give adenosine 5’-phosphosulfate, which is further phosphorylated to 3’-phosphoadenosine 5’-phosphosulfatc (“active sulfate”) (Rohhins and Lipmaan, 1957). 2 . Studies with Colon Homogenatm and Vitamin A
From in vivo observatioiis and tissue culture data, indications were strongly suggestive of an involvement of vitamin A in a controlling step in mucopolysaccharide biosynthesis, in particular in stimulation of such synthesis in a mucus-forming system. For this purpose, Wolf and Varandani (1960) developed an in vitro system, consisting of rat colon segments INCORPORATION
OF
TABLE I11 GLUCO~E-U-C“ INTO HEXOSAMINES O F MUCOPOLYSACCHARIDES BY COLON HOMOGENATES~ SYNTHESIZED c.p.m.
Total (314-mucopolysaccharidesisolated Total C14-hexosamine obtained therefrom Specific activity of hexosamine After addition of carrier (54 pmoles) After 2nd crystallization of osazone After 3rd crystallization of osazone
15,200 6,232 c.p.m./mmole 5,000 6,800 6,750
4 The complete system consisted of 10 pmoles ATP, 6 pmoles of MgCli , 1 pniole glutamhe, 3 pmoles of DPN, 2Opmoles NadO,, and 1 X Id c.p.m. of glucose-U-CII.
or homogenates, which could incorporate S36-labeledsulfate or C14-glucose into mucopolysaccharide on incubation. Since the level of incorporation was small, the identity of the radioactive mucopolysaccharides produced had to be rigorously established. This was done by paper chromatography and paper electrophoresis, with coincidence of the CI4- and S35-radioactive spots of synthesized mucopolysaccharide with those of known chondroitin sulfate, and with response to the specific toluidine blue color spray; hydrolysis of the C14-mucopolysaccharidesand isolation and identification of resulting C14-hexosaminesby ion exchange and paper chromatography and crystallization of derivatives with carrier glucosamine (Table 111) ; precipitation of the C14-mucopolysaccharide along with carrier chondroitin sulfate by cetylpyridinium bromide, a specific precipitation reagent, and nondialyzability of the precipitated C14-mucopolysacccharide;hydrolysis of the S~~-mucopolysaccharide t o give labeled sulfate under conditions that lead to hydrolysis of the sulfate group of chondroitin sulfate.
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
447
Colon segments of deficient and normal rats were incubated with Sa5sulfate in Krebs-Ringer-phosphate buffer, pH 7.4. The mucopolysaccharides synthesized and secreted into the medium were isolated by precipitation and purified by paper chromatography, and their radioactivity was determined. Incorporation of S35into the mucopolysaccharides by deficient colon segments was found to be about one-half compared to that of normal colons. When a suspension containing 10 pg. of vitamin A was added to the incubation medium of the deficient colons, incorporation was raised to the level of the normal (Table IV). Vitamin A aldehyde was also effective. The conditions under which incorporation of S35-sulfateinto mucopolysaccharides of rat colon homogenates would take place were first determined. Incorporation was highest when the medium contained diphosTABLE IV EFFECTOF ADDEDVITAMINA ON INCORPORATION OF Sa60i-MUCOPOLYSACCHARIDES BY COLONSEGMENTSO Vitamin A status
Vitamin A added
-
-
-
+
10 pg. (alcohol) 10 pg. (aldehyde) -
INTO
Mucopolysaccharide (c.p.m.) 1279 3515 3001 2685
(4) (4) (4)
(4)
The values are averages of the number of incubations ahown in parentheam. Incubation period, 3 s hours; activity added, 19 X 106 c.p.m.; = normal rat; = deficient rat.
+
-
phopyridine nucleotide, glucose, glutamine, and adenosine triphosphate (“complete system”). No incorporation was obtained if the two last-named constituent,s were omitted. Homogenates were then prepared from vitamin A-deficient and normal rat colons tind incubated with the complete system. The results are summarized in Table V. The S35-sulfateincorporated into mucopolysaccharides by homogenates from deficient colons was once again about one-half that of normal colons. The addition of vitamin A suspension to the deficient homogenates restored incorporation t o normal. The suspending medium alone (serum albumin-ethanol) had no effect. In Table VI can be seen the specificity tests with other fat-soluble vitamins. Vitamin A only is capable of completely reversing decreased activity. The next step in this investigation was to localize the function of vitamin A in a specific step in mucopolysaccharide biosynthesis. It was found that the vitamin is not involved in the formation of hexosamines, because glucosamine or galactosamine, when substituted for glucose and glutamine in the incubation medium of deficient homogenates, could not restore the decrease in incorporation.
448
GEORGE WOLF AND B. CONNOR JOHNSON
Next, experiments were undertaken to determine whether vitamin A is required in a step before or after the involvement of the uridine nucleotides in mucopolysaccharide synthesis (see Fig. 1). First, a system requiring the uridine nucleotides, and capable of incorporating Sa6-sulfateinto mucopolysaccharideby rat colon homogenates, was developed. This system TABLE V
INCORPORATION O F
S"'O4-
- I N T O MUCOPOLYSACCHARIDES BY COLON HOMOQENATES"
Vitamin A status
Vitamin A added (10r g . )
+-
+
Mucopolysaccharide (c.p.m.)
-
Serum albumin-ethanol
346 (3) 805 (2) 936 (4) 268 (2)
0 The values are averagcm of the number of incubations shown in parentheses. The complete system contained 10pmole.qof ATP, Bpmoles of M p , 1pmole of glubmine, apmoles of DPN, and IOpmolea of gluwefinal volume 1 ml. Each incubation contained 10.8 X 108 c.p.m. of radioactive sulfate and 14 mg. of protein; normal rat; - = defiaient rat.
+
-
TABLE VI MUCOPOLYSACCHARIDES BY COLON HOMOOENATES. SPECIFICITYOF VITAMINAm
INCORPORATION OF
Status of animal Normal Deficient Deficient Deficient Deficient Deficient Deficient
S"'O4-
-
INTO
Addition (10rg.1 -
-
Vitamin A alcohol Vitamin D Vitamin E Vitamin E vitamin A Vitamin KI
+
Mucopolysaccharide (c .p.m.) 896 483 841 232
(2) (2) (2) (1) 608 (2) 752 (2) 435 (2)
O The values are averages of the number of incubations, shown in parentheses. The complete system WBB that shown in Table V. Each incubation contained 9.78 X 101 c.p.m. of activity and 14 mg.of protein.
showed an absolute requirement for glutamine, uridine diphosphoacetylglucosamine, ane uridine diphosphoglucuronic acid, and a partial requirement for uridine diphosphoglucose and acetylglucosamine. When incubated with deficient colon homogenates, it once again showed lowered incorporation of SS6-sulfateinto mucopolysaccharide, restorable with added vitamin A (Table VII). Therefore, vitamin A functions at some step beyond the synthesis of the uridine nucleotides. In an attempt to achieve independence from the use of vitamin A-deficient animals, and to find a means of destroying vitamin A in vitro, the
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
449
colon homogenates were preincubated with lipoxidase, an enzyme known to destroy vit,amin A. This procedure, as shown in Table VIII, lowered or abolished S36-sulfatcincorporation. Addition of vitamin A restored incorporatio11 only at low concentrations of lipoxidase, presumably because t,he TABLE VII A ON THE INCORPORATION OF S W - - INTO EFFECTOF VITAMIN MUCOPOLYSACCHARIDES BY COLON HOMOQENATEB CONTAININQ UDP-DERIVATIVEB~ Vitamin A status
Addition of vita- Mucopolysaccharide min, A 1Opg. (c.p.m.)
The values are averages of tbe number of incubations, shown in parentheme. The complete iystem conaisted of 1 pmole of UDPGA, 1 pmole of UDPAG, 1 pmole of UDPG, 1 pmole of glutamine, 8pmolen of AQ (all obtained from Sigma Chemical Corporation), 1pmole of ATP, and 10pmoles of Mg*. Each incubation contained 8.6 X 106 c.p.m. of activity and 14 mg. of protein; normal rat; - = deficient rat.
+
-
TABLE VIII EFFECTOF LIPOXIDASE ON Sss04-- INCORPORATION INTO MUCOPOLYSACCHARIDEB BY COLON HOMOQENATESO Preincubation for 1 hour with lipoxidase (3 mg./ml.) 0.01 ml. 0.01 ml. 0.04 ml. 0.04 ml. 0.07 ml. 0.07 ml.
Addition of vita- Mucopolyaaccharide min A, pg. (c.p.m.)
-
+ ++
306 538 268 0 0 0
Complete ayatem waa that ahown in Table V. Each incubation contained 10.6 X 106 0.p.m. of activity and 14 mg. of protein. Crystalline soybean lipoxitlase was obtained from Nutritional Biochemical Corporation.
added vitamin was destroyed by the excess lipoxidase present in the incubation medium. The assay used for mucopolysaccharide synthesis in the above experiments was the uptake of labeled sulfate from the medium into the final product of a many-step synthesis. Hence, the requirement of vitamin A for any particular step can only be derived by inference. One can infer from the results described that the vitamin is not required for the following steps: the conversion of glucose to hexosamines; the acetylation of hexosamines; the formation of the uridine diphosphoacetylhexoamines;
450
GEORGE WOLF AND B. CONNOR JOHNSON
the oxidation of uridine diphosphoglucose to uridine diphosphoglucuronic acid. By a process of elimination, therefore, one could conclude that vitamin A functions either in the polymerization of the uridine nurleotides, or the activation or transfer of sulfate t,o the polymer. 3. Studies with Pig Colon l h x y m e Fractions and Vitamin A
Since rat colon mucosa were difficult to obtain free from muscle and in sufficient quantity, a new approach was sought by the use of pig colon mucosa (Wolf et al. 19GO). In homogenates of that tissue, radioactivity vas incorporated into mucopolysaccharide from S3s-labeled sulfate or C14-labeled glucose. Mucopolysaccharide was identified in a manner similar to TABLE IX MUCOPOLYSACCHARIDE B Y SUBCELLULAR PH 5 ENZYMES OF P I Q COLONMUCOSA~
INCORPORATION OF s a 6 - s U L F A T E INTO
FRACTIONS AND
Subcellular fraction Supernatant free from mitochondria and nuclei Supernatant free from mitochondria, nuclei, and microsomes Supernatant free from mitochondria, nuclei, microsomes, and pH 5 enzymes Microsomes pH 5 Enzymes pH 5 Enzymes and microsomes
Mucopolysaccharide (c.p.m./mg. protein) 1293 (4) 15’21 (4) 326 516 1728 1770
(1) (4) (4) (3)
4 The values are averages of the number of incubations, shown in parenthesee. The complete system consistedof lopmoles ATP, 6pmoles MgClr ,3pmolea DPN, lrmoleglutamine, and lOpmoles glucoee. The radioactivity added per incubation, 9 X 108 c.p.m. made to final volume of I ml. with phosphate buffer, pH 7.4.
that described for rat colon mucopolysaccharide. The synthesizing activity was located in the supernatant solution, after removal of nuclei, mitochondria, and microsomes from the homogenate (Table IX) and could be precipitated a t pH 5.2. This “pH 5 enzyme” fraction contained 48.6% of the total vitamin A content of the mucosa (3.7 pg. per 100 gm. mucosa). It required uridine triphosphate, adenosine triphosphate, glucose, and glutamine for activity (Table X) and showed a pH maximum between 6.2 and 7.2. This enzyme fraction when obtained from vitamin A-deficient pigs was less active (Table XI) ; lipoxidase also lowered its activity, which in both cases could be restored by added vitamin A (Table XII). To exclude the involvement of vitamin A in sulfate transfer from 3’phosphoadenosine 5’-phosphosulfat,e (“active sulfate”) to the polymer, pH 5 enzyme fraction was incubated with active sulfate and model sulfate acceptors such as chondroitin and nitrophenol. No differences were found
451
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
TABLE X REQUIREMENTS OF COFACTORS nY PH 5 ENZYMES FOR INCORPORATION OF Sas0,- - INTO MUCOPOLYSACCHARIDESO
ATP, 10 pmoles
DPN, 3 pmoles
Glutamine, 1 Cole
Mucopolysaccharide (c.p.m./mg. protein)
Glucose, UTP, 10 @moles 4 pmoles
++ + +-
+ + + ++
-
2234 2349 1713 798 560 5723
+-
(2) (2) (2) (2) (2) (2)
The values are averages of the number of incubations, shown ie the parentheses. Radioactivityadded per incubation, 9 X 106 c.p.m. made to final volume of 1 ml. with phosphate buffer, p H 7.4.
TABLE X I EFFECT OF VITAMINA DEFIENCY O N THE INCORPORATION OF Sa601-INTO MUCOPOLYSACCHARIDE A N D SPECIFICITY OF VITAMINA IN PH 5 ENZYMES' Vitamin A status
Addition of vitamin A,
+-
10 ag.
-
Vitamin A Vitamin E Vitamin D
Mucopolysaccharide (c.p.m./mg. protein) 2349 1407 2556 1564 1530
(2) (2) (2) (2) (2)
a The values are average8 of the number of incubations, shown in the parentheses. The complete system consisted of lOrinoles glucoae, 1 prnole glutamine, 3 rinoles DPN, and 8Wa-- 19 X loa c.p.m., made up to I Inl. with phosphate buffer, pH 6.8.
TABLE X I 1 EFFECT OF PREINCUBATION WITH LIPOXIDASE ON THE INCORPORATION OF S J 6 0 r - - INTO MUCOPOLYSACCHARIDE SYNTHESIZED BY PH 5 ENZYMESO Lipoxidttse, 0.3
Added vitamin A, 10 rg.
0.1 ml. 0 . 1 ml.
-
mg./ml.
+
Mucopolysaccharide (c.p .m ./mg. protein) 2706 (4) 1213 (4) 2188 (4)
The values are average8 of the number of incubations, shown in the parentheses. The complete system consiated of 10 rnioles glucose, 1 pmole glutamine, 3 pnioles DPN, and S"Oa--, 12 X 10' c.p.m., made up to 1 nil. with phosphate buffer pH, 8.8. (1
452
GEORGE WOLF AND B. CONNOR JOHNSON
in sulfate transferase activity between normal and lipoxidase-treated fractions, thus excluding vitamin A function in sulfate transfer. Such a conclusion, however, ‘has to be treated with caution, since none of the natural acceptors occurring in colon have so far been tried as acceptors in this reaction. On the other hand, evidence has now been obtained (Varandani et al. 1960) which shows that colon homogenates of deficient rats form phosphoadenosine phosphosulfate less readily than normal homogenates (Table XIII), a lesion which could be corrected by addition of vitamin A. One could therefore conclude that vitamin A is involved in mucopolysaccharide biosynthesis at the sulfate activation step. TABLE XIII EFFECTOF VITAMIN A DEFICIENCY ON PAPSa6SYNTHESIS I N RATCOLON HOMOQENATE’ PAPW Vitamin A status of rats Adequate Deficient Deficient Deficient
Addition
-
Vitamin A, u)pg. in propylene glycol, 5 pl. propylene glycol, 5 pl.
Expt. I1 Expt. I (c.p.m. /mg. protein) 19,200 8,440
59,000 28,700
24,300 -
54,100 31,400
NOTE:Aotivity added: Expt. I. 8.4 X 1Osc.p.m.; Expt. 11, 16.8 X 10So.p.m. a
Abbraviation: PAF@”, 3’-phmphardenosine 6’-phmphosulfata labeled with 8”.
4. Vitamin A
and Net Synthesis of Mucopolysaccharides in Vitro
Since up to this point only minute amounts of mucopolysaccharide, detectable only by radioactivity, have been synthesized, it was thought necessary to investigate net synthesis of colon mucopolysaccharide in vitro, and the effect of vitamin A thereon. Total hexosamine content of rat colon was determined by Moretti and Wolf (unpublished observations), by acid hydrolysis of colon homogenates and isolation and assay of hexosamines. Normal colons contained an average of 11.58 f I .2 pmoles, colons from vitamin A-deficient rat,s an average of 7.38 f 1.0 pnioles per 100 mg. of protein. Actually, “total hexosamine” may include not only that in mucopolysaccharide, but, also in the various hexosaminc-cont,airiiirg intermediates (cf. Fig. 1). Therefore, it was necessary to separate mucopolysaccharide from low-molecular weight material by precipitation and dialysis. Again, the deficient colons showed lowered mucopolysaccharide hexosamine to
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
453
the extent of about GO% (average of 5.00 f 0.4 pmoles hexosamines per 100 mg. of protein), compared to the normal colon (average of 8.04 f 0.9 pmoles per 100 mg. of protein). The ratio glucosamine/galactosamine of 0.5 in samples of normal colon was increased to 0.85 in those from deficient animals; hence vitamin A deficiency caused a decrease mainly in the content of galactosamine containing mucopolysaccharides in colon. To study net synthesis of mucopolysaccharide, and the effect of vitamin A thereon, a system was developed using rat colon homogenates incubated with glutamine, glucose G-phosphate, diphosphopyridine nucleotide, uridine triphosphate, and adenosine triphosphate. The samples were assayed for total hexosamine by acid hydrolysis before and after incubation. The increase of total hexosamine content after incubation of deficient colon homogenates was about one half compared to that of normal colons. The synthetic capacity of the deficient colon homogenates could be restored almost to normal by addition to the incubation mixture of vitamin A. The minimum quantity which showed this stimulation was 1.25 X pmoles (11.5 I.U.) added per incubation (per 11-13 mg. of protein). This action appeared to be not graded, but all-or-none. Net synthesis was not restored to normal in deficient homogenates by addition to the incubation mixture of vitamin A acid, vitamins E, K 1 , and Ds . Again, it was necessary to confirm that, since the total hexosamine assayed included the intermediates in mucopolysaccharide biosynthesis, the effect of t,he vitamin was truly on mucopolysaccharide-bound hexosamine. Therefore, it was first shown that vitamin A had no effect on the formation of glucosamine G-phosphate. Secondly, mucopolysaccharide was separated from low-molecular weight material by precipitation and dialysis. The net formation of mucopolysaccharide-bound hexosamine (precipit,able and nondialyzable) of normal colon homogenates was between 7 and 12 times greater than that of deficient homogenates. The net synthesis of the latter was raised about four-fold by addition of vitamin A to the incubation. These results, t,herefore, definitely establish an effect of vitamiii A on the content, and net synthesis of mucopolysaccharide in colon. 5. Vitamin A and Keratinizatim
Though not, st rict,ly coming under the heading of mucopolysaccharide biosynthesis, the theories of vitamin A function in the inhibition of keratinizatioii should here be ment,ioiied. Balakhowskii and Drozdova (1957) consider keratinizstioii in vitamin A deficiency to be due to a disturbance of oxidative processes. In particular, the catalytic activity of copper in the oxidation of cysteine to cystine, which the authors regard as the rate-
454
GEORGE WOLF AND B. CONNOR JOHNSON
limiting step in keratinization, was found to be inhibited by carotenoid compounds in model systems. The same authors (1956) also found the catalytic action of copper in ascorbinase to be lowered in presence of carotenoids. Vitamin A-deficient rats excreted more free labeled sulfate than normal rats after injection of Sas-methionine (Balakhowskii and Drozdova, 1958), which the authors again assume to be due t o a release from the inhibitory action of vitamin A on the copper-catalyzed oxidation of SHgroups. Another interpretation of this result would be to consider it due t o increased excretion of sulfate caused by lowered mucopolysaccharide biosynthesis. V. CONCLUSIONS From the data presented, especially the tissue culture studies of H. B. Fell, t,he conclusion is inescapable that vitamin A has a controlling action on the basal cells of epithelium. The presence of vitamin A stimulates them to produce mucus, and its absence permits them to form keratin. The vitamin therefore has an action similar to that of a hormone and, in that sense, we know as little about the mode of its biochemical function as about that of other hormones. On the other hand, taking its stimulation of mucus formation as a starting point, results have been presented which show that vitamin A has a direct, possibly coenzymatic, function in the polymerization reaction of the uridine nucleotides to form mucopolysaccharide, or in the sulfation of the latter. Perhaps the vitamin could control differentiation of basal cells by being the essential factor in a rate-limiting step of a reaction sequence leading to mucus formation. REFERENCES American Society of Biological Chemists Symposium. 1958. Federation Proc. 17,10741105. Balakhowskii, S.D.,and Drozdova, N . N. 1956. Doklady Akad. Nauk S.S.S.R. 109, 355-357. Balakhowskii, S.D.,and Drozdova, N . N . 1957. Biokhimiya 22,33&335. Balakhowskii, S. D., and Drozdova, N . N. 1958. Doklady Akad. Nauk S.S.S.R. 118, 331-333. Cohen, J., Maddock, C. L., and Wolbach, S. B . 1955. A . M . A . ilrch. Pathol. 69, 723726. Dorfman, A., Markovitz, A., and Cifonelli, J . A. 1958. Federation Proc. 17, 1093-1099. Uziewiatkowsky, 13. D . , 1954. J . Exptl. Med. 100, 10-24. Fell, H.B. 1953. In “Connective Tissues,” Transaction of the Fourth Conference (C. Ragan, ed.), 142 pp. Josiah Macy, Jr. Foundation, New York. Fell, H . B., and Mellanby, E. 1952. J . Physiol. (London) 116,320-349. Fell, H.B.,and Mellanby, E.1953. J . Physiol. (London) 119,47&488. Fell, H.B., Mellanby, E . , and Pelc, S. R . 1954. Brit. Med. J . 11, 611.
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHEBIS
455
Frape, U. L., Allen, R. S., Spear, V. C., Hays, W. V., and Catron, D.V. 1959.J . Nutrition 68, 189-201. Hedenberg, I. 1954.Acta Clin. Scand. Suppl. 192,87. Jeanloz, R.W. 1958.Federation Proc. 17, 1082-1086. Manville, I. A. 1937.Science 86,44. Markovits, A., Cifonelli, J. A., and Dorfman, A. 1959.J . B i d . Chem. 2S4.2343-2350. Mellanby, E. 1950. “A Story of Nutritional Research; The Effect of Some Dietary Factors on Bones and the Nervous System.” Williams & Wilkins, Baltimore, Maryland. Meyer, K. 1958.In “Polysaccharides in Biology,” Transactions of the Fourth Conference, (G. F. Springer, ed.), pp. 11-56. Josiah Macy, Jr. Foundation, New York. Meyer, K., Smyth, E. M., and Palmer, J. W. 1937. J . Biol. Chem. 119,73-84. Moore, T.1957a. “Vitamin A,” p. 535.Elsevier, New York. Moore, T. 1957b. “Vitamin A,” p. 518. Elsevier, New York. Robbins, P. W., and Lipmann, F. 1957. J . Biol. Chem. 229,837-851. Schubert, M.1958.Federation Proc. 17, 1099-1105. Smith, H.,and Gallop, R. C. 1953. Biochem. J . 63,66&671. Springer, G. F., ed. 1958. “Polysaccharides in Biology,” Transactions of the Fourth Conference. Josiah Macy, Jr. Foundation, New York. Susuki, S., and Strominger, J. L. 1960.J . B i d . Chem. 236,257-276. Thomas, L., McCluskey, R. T,. Potter, J. L., and Weissmann, G. 1960.J . Ezptl. Med. 111, 704-718. Varandani, P. T., Wolf, G. and Johnson, B. C. 1960. Biochem. Biophys. Research Communs. 3, 97-100. Werner, I. R . 1953.Acta SOC.Med. Upsaliensis [N.S.] 68, 1-55. Wolbach, S. B. 1947.J . Bone and Joint Surg. 29, 171. Wolbach, S.B., and Howe, P. R . 1925.J . Ezptl. Med. 42,753-777. Wolf, G., and Varandani, P. T. 1960.Biochim. et Biophys. Acta 45, 601-512. Wolf, G., and Varandani, P. T., and Johnson, B.C.1960.Biochim. et Biophys. Acta, in press.
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... 11. In Viuo Observations.. ........................ .................... 1. Vitamin A Deficiency ................................................. 2. Hypervitaminosis A . . . . . .................
Page 439 440 440
IV. In Vitro Observations. . . . . . . . . .
1. Chemistry and Biochemistry of Mucopolysaccharides. . . . . . . . . . . . . . . . . . 443
4. Vitamin A and Net Synthesis of Mucopolysaccharides in Vitro. . . . . . . 452 5. Vitamin A and Keratinization V. Conclusions. .................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. INTRODUCTION In a search for a biochemical function of vitamin A in metabolism, when looking over the existing literature on the effects of vitamin A deficiency or excess, one is immediately struck by the importance of the influence of vitamin A on mucus formation and on mucosal tissue. Moore (1957a), in his monumental work on vitamin -4,states: “If we consider the role of vitamin A on the widest possible basis, therefore, we may say that it is necessary for the formation of large molecules containing glucosamine.” Such large molecules are the mucopolysaccharides which, in addition, contain galactosamine, glucuronic acid, sulfate, and in certain cases also sialic acid, mannose, and fucose. Any hypothesis of a systemic function of vitamin A, therefore, has to take into account its effect on the biosynthesis of the mucopolysaccharides. These occur in various forms, and with various functions, in almost all tissues of the mammalian organism, but principally, and in largest quantities, in two locations: in the mucus secreted by mucous epithelium; and in the extracellular matrix of cartilage, mainly as chondroitin sulfate. Attention will be focused in this review on the influence of vitamin A on these two tissues. 439
440
GEOltGE: WOLF A N D €3.
CONNOR JOHNSON
11. I n Vivo OBSERVATIONS 1 . Vitamin A Deficiency
Vitamin A deficiency shows its first manifestation in a disruption and atrophy of all those epithelial tissues that function in mucus secretion: in the respiratory, intestinal, and urinary tracts and in the genital system (Wolbach and Howe, 1925). A decrease in the number and activity of mucus-secreting cells of the gastrointestinal tract occurs (Manville, 1937). Ultimately, a new type of epithelium replaces the mucus-secreting tissue. It becomes keratinized and similar to epidermis. The effect of the deficiency on the other important mucopolysaccharide of the mammal, the chondroitin sulfate of cartilage, is quite different : increased activity of osteoblasts, periosteal overgrowth with the formation of larger, soft bones (Mellanby, 1950). The effect of the deficiency on mucus formation in the genital system, especially vaginal epithelium, is particularly instructive, as here there are cells that at one time of the estrous cycle become mucussecreting, at another time keratinized. The whole picture is well summarized by Moore (1957b) : the vaginal epithelium, even in the absence of estrogen, becomes keratinized in vitamin A deficiency. In the absence of estrogen, but with excess vitamin A, it is transformed into mucus-secreting epithelium; a similar change being caused by progesterone, but only in the presence of normal levels of vitamin A. Estrogen, even in the presence of vitamin A, stimulates keratinization. The conclusion from these observations is that the normal state of this epithelium is to be keratinized; vitamin A stimulates the transformation to mucous epithelium. One could then further speculate that estrogen inhibits this action of vitamin A, whereas progesterone overcomes the inhibition. Since esterified sulfate represents an important part of the mucopolysaccharide molecule, a number of investigations have considered the effect of vitamin A deficiency on sulfate incorporation into cartilage. Dziewiatkowsky (1954) isolated chondroitin sulfate, the principal substance, apart from collagen, of cartilage matrix, after injection of labeled sulfate into vitamin A-deficient, normal, and vitamin A-treated deficient rats. The level of labeled sulfate was higher in the normal and vitamin A-treated deficient rats than in the deficient. This author also found increased breakdown as well as synthesis of chondroitin sulfate in the vitamin A-treated animals. Somewhat different results were obtained by Frape et al. (1959) working with vitamin A-deficient pigs. Here, vitamin A treatment of the deficient animals caused decreased sulfate uptake into cartilage as the primary response (Table I).
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
44 1
2. Hypervitaminosis A
Vitarniii A is distiiwt from other vitamins in that, it, acts when administered in excess, the effect being generally the reverse of that obtained in deficiency. Maturat ion of cartilage cells is accelerated; cartilage, as well TABLE I Sa* ACTIVITY I N TISSUESFROM PIOS ON VARIOUS DIETARYVITAMINA LEVELS~
A added
per pound of feed
5th Rib costochondrial junctionb
Ear cartilageb
0 55 208 790 1540 3000 11393
87581 42622 48601 32554 46789 54014 53221
21213 26764 5902 10811 12812 17082 22238
I.U.
Lunge
counts/min./gm. of fresh tissue"
7053 3442 4033 1731 1346 1761
2087
From Rape et 02. (1969). b Values are average of two samples.
Values are average of three samples. Counting rates are corrected for background, decay, and extrapolatedto zero thickness of precipitate.
TABLE I1 EFFECTOF HYPERVITAMINOSIS A ON SERUMCONCENTRATIONS OF MU COP ROTE IN^ Group*
Number
Vitamin A dose
Age at killing (days)
An Ah Cn Ch
2 2 3 4
0 0 lo00 lo00
34-40 39-40 39-40 39-46
Serum mucoproteinc (mg. tyrosine %)
6.8(2) 6.3(2) 10.3(2) 16.3(4)
f 0.05 0.70 f 0.05 f 3.74 &
~~
From Cohen et ol. (1955).
* n Refers to the group of animals on the diet normal in calcium; h refers to thaae on a diet high in osl-
cium content. The figures in parenthesis refer to the number of animals or cheinical determinations in the group.
as bone, is rapidly absorbed, resulting in spontaneous fractures (Wolbach, 1947). Normal vaginal and uterine epithelium is replaced by noncornifying epithelium (Hedenberg, 1954). Mucoprotein levels in blood are greatly increased (Cohen et al., 1955) (Table 11). Recently, Thomas el al. (1960) showed that, in the intact animal, hypervitaminosis A resulted in a depletion of cartilage matrix, due to loss of cartilage chondromucoprotein, and a consequent rise in serum chondroitin
4.42
GEORGE WOLF AND B. CONNOR JOHNSON
sulfate. It should be noted that this is the reverse of the effect of vitamin A excess on mucous tissue, where the hypervitaminosis results in a stimulation of mucus secretion, reflecting, no doubt, a different mode of action of vitamin A on mucopolysaccharide of connective tissue and of mucous tissue.
111. OBSERVATIONS ON TISSUE CULTURE All the work reported here on the effect of vitamin A on tissue culture has been carried out by H. B. Fell and her collaborators (reviewed by Fell, 1953). The objective was to determine whether the effects of vitamin A deficiency and hypervitaminosis A described in the preceding sections were indirect and mediated through an endocrine organ, or directly on the tissue. First, in their work on explants of undifferentiated chick embryo bones, cultured in a medium with excess vitamin A, Fell and Mellanby (1952) found the bone to turn soft and gelatinous, growth to diminish, and fractures to occur. The cartilage matrix ceased t o stain with basophile dyes; metachromasia, that is, the response of the stain toluidine blue to the acid groups of chondroitin sulfate, disappeared. It was thought that, whereas there occurred dissolution of the chondroitin sulfate, the collagen part of the matrix remained intact. With fully developed fetal mouse bones, the situation was similar-cartilage matrix and bone were absorbed and disappeared. The vitamin was present in a concentration similar to that found in the blood. There was then no doubt that the action of vitamin A was direct, though only on growing cells. In their second set of experiments, Fell and Mellanby (1953) cultured undifferentiated embryonic chick ectoderm. This normally develops a keratinizing layer over squamous epithelium. Cultured in excess vitamin A, it formed instead ciliated columnar cells with secretory vacuoles, actively secreting mucus, closely similar to nasal epithelium. When returned to normal medium, mucus secretion continued, but no new mucous cells differentiated and ultimately a new layer of squamous cells was formed which eventually keratinized. The changes described are all basically those seen in the whole animal, though greatly exaggerated. Similar experiments with skin from mammals or birds showed less pronounced transformations than with embryonic skin. These important results demonstrate that the action of vitamin A is twofold: (1) it causes dissolution of chondroitin sulfate of cartilage matrix; (2) it affects epithelial basal cells in such a manner as to make them differentiate into mucus-secreting cells, when they would normally form keratinizing cells. The conclusion is inescapable that vitamin A has an action akin to that of a hormone in determining the course of differentiation of the basal cells of epithelium.
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
443
It is of interest to note that the effect of excess vitamin A in tissue culture on the chondroitin sulfate of cartilage leads to dissolution and dis2ppearance, whereas on the related mucoid substances of mucous epithelium, it causes sulfate uptake and synthesis (Fell et al., 1954). Though the properties and identity of the mucopolysaccharidesof mucus are not completely established (see Section IV, l), they are certainly different from chondroitin sulfate of cartilage. It is possible therefore to conclude that excess vitamin A increases the mucus type and decreases the connective tissue t,ype of mucopolysaccharide. The reverse, a decrease in mucus and increase in connective tissue mucopolysaccharide, along with keratinization, occurs in deficiency. These generalizations help to interpret the i n vivo data discussed in Section I1: decrease in mucus secretion and periosteal overgrowth in vitamin A deficiency; increased mucus secretion, increased breakdown of chondroitin sulfate from cartilage, less sulfate uptake into cartilage, when vitamin A is administered. IV. In Vitro OBSERVATIONS 1 . Chemistry and Biochemistry of Mucopolysacchorides
Many excellent reviews exist of recent work in the chemistry of the mucopolysaccharides (American Society of Biological Chemists Symposium, 1958; Springer, 1958). However, for a clearer understanding of the subsequent discussion, a brief summary of the chemistry and biochemistry of those mucopolysaccharides on which vitamin A has an influence, is relevant. Three different chondroitin sulfates have been isolated (Meyer, 1958). Chondroitin sulfate A, found in cartilage, bone, cornea, and aorta, is a polymer of D-glucuronic acid and sulfated N-acetylgalactosamine, with N-acetylchondrosine sulfate (I) as the repeating unit. The two subunits are linked in a ,f3-1,3-glucuronidic link and a 8-1,Chexosaminidic link, wit,h sulfate esterified on position 4 of the acetylgalactosamine. Chondroitin sulfate B (repeating unit, 11), found in skin, tendon, and aorta, is identical with A, except for epimerization at C-5 of the glucuronic acid, leading to L-iduronic acid. In chondroitin sulfate C (repeating unit, 111), obtained from cartilage, umbilical cord, tendon, and sclera, the sulfate group is shifted to position 6 of the acetylgalactosamine moiety. Heparin is defined by Jeanloz (1958) as “dextrorotatory sulfated polysaccharides composed of D-glucosaniine and D-glucuronic acid” of various degrees of sulfation. The sulfate in these compounds is linked to the amino group in a sulfamic link, although compounds related to heparin, containing glucosamine and glucuronic acid with N-acetyl and O-sulfate-glucosamine have been found (heparitin sulfate).
444
,rT:rp\
GEORGE WOLF AND B. CONNOR JOHNSON
z
6CHr OH
CH~OH
\oo:!” OH
0
NHCOCHI
OH
NHCOCHj
OH
(1)
FfjQ\
(11)
HzCOSOjH
’0
NHCOCHI OH (111)
The mucopolysaccharides of the mucous epithelium, and of secreted mucus, have not been well characterized. Werner (1953) extracted three main types from pig colon mucosa, the prevalent one having L-fucose (IV) as its most characteristic carbohydrate constituent and therefore being called fucomucin ; it also contains N-acetylglucosamine, N-acetylgalactosamine, and D-galactose, the hexosamines constituting about one-half of the sugar content. The second type of epithelial mucopolysaccharide was found to contain sialic acid (V), and was therefore called sialomucin. It has an equivalent amount of N-acetylgalactosamine. The third group are the sulfated mucopolysaccharides. The identity of these is still in question. Smith and Gallop (1953) isolated two chondroitin sulfates from gastric mucosa, which appear to be chondroit,in sulfates A or C, and B (Meyer, 1958), as well as heparin. Meyer et al. (1937) isolated heparitin sulfate from gastric mucosa. CH, OH CHOH
(W
(V)
In t,he tissues examined, t,he choiidroitin sulfates exist as protein complexes, called chondromucoprotein (Schubert, 1958), a water-soluble compound of chondroitin sulfate and a noncollagenous protein, with a link
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
445
that is not saltlike. It is probable that in mucus as well, at least some of the mucopolysaccharides are linked to protein. The pathway of biosynt,hesis of acid mucopolysaccharides has recently become clear (reviewed by Dorfman et al., 1958). Markovitz e2 al. (1959), for instance, uses a bacterial enzyme system which synthesizes hyaluronic acid as a model for mucopolysaccharide. A brief summary of the enzymatic reaction sequence involved is given here (compare Fig. 1) : glucose 6-phosGlycogen
Glucose 1-P
1 Glucose 1-P
1
I
Glucose 6-P
I
glutamine
Uridine triphasphate
Glucosamine 6 P CH1COSCoA
I U r i d i n e diphosphoglucose
N-Acetyl glucosamine 6 P
1
N-Acetyl glucosamine 1-P
1
Uridine diphosphoglucuronic acid
Uridine triphasphate
Uridine diphospho-N-acetylglucosamine
I-
I
PAPS
ATP ATP FIG.1. Possible pathway of mucopolysaccharide biosynthesis (P, phosphate; CHaCO-SCoA, acetyl coenzyme A; IIPN, diphosphopyridine nucleotide; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; APS, adenosine 5'-phosphosulfate). Heparitin sulfuric acid
phate reacts with glutamine to form glucosamine 6-phosphate. The latter is acetylated by acetyl coenzyme A to produce N-acetylglucosamine 6-phosphate, which is isomerized to the 1-phosphate and reacts with uridine triphosphate to form uridine diphosphoacetylglucosamine. Similarly, glucose 1-phosphate forms uridine diphosphoglucose with uridine triphosphate, which is oxidized by diphosphopyridine nucleotide to uridine diphosphoglucuronic acid. This, with the above uridine diphosphoacetylglucosamine, is polymerized to hyaluronic acid. The introduction of the sulfate group into chondroitin sulfate presents a problem, since it may enter at the level of the uridine nucleotides or the
446
GEORGE WOLF AND B . CONNOR JOHNSON
fully formed polymer. Recent evidence (Suzuki and Strominger, 19GO) would favor the second possibility. Sulfate, before entering this reaction, is activated by reaction with adenosine triphosphate to give adenosine 5’-phosphosulfate, which is further phosphorylated to 3’-phosphoadenosine 5’-phosphosulfatc (“active sulfate”) (Rohhins and Lipmaan, 1957). 2 . Studies with Colon Homogenatm and Vitamin A
From in vivo observatioiis and tissue culture data, indications were strongly suggestive of an involvement of vitamin A in a controlling step in mucopolysaccharide biosynthesis, in particular in stimulation of such synthesis in a mucus-forming system. For this purpose, Wolf and Varandani (1960) developed an in vitro system, consisting of rat colon segments INCORPORATION
OF
TABLE I11 GLUCO~E-U-C“ INTO HEXOSAMINES O F MUCOPOLYSACCHARIDES BY COLON HOMOGENATES~ SYNTHESIZED c.p.m.
Total (314-mucopolysaccharidesisolated Total C14-hexosamine obtained therefrom Specific activity of hexosamine After addition of carrier (54 pmoles) After 2nd crystallization of osazone After 3rd crystallization of osazone
15,200 6,232 c.p.m./mmole 5,000 6,800 6,750
4 The complete system consisted of 10 pmoles ATP, 6 pmoles of MgCli , 1 pniole glutamhe, 3 pmoles of DPN, 2Opmoles NadO,, and 1 X Id c.p.m. of glucose-U-CII.
or homogenates, which could incorporate S36-labeledsulfate or C14-glucose into mucopolysaccharide on incubation. Since the level of incorporation was small, the identity of the radioactive mucopolysaccharides produced had to be rigorously established. This was done by paper chromatography and paper electrophoresis, with coincidence of the CI4- and S35-radioactive spots of synthesized mucopolysaccharide with those of known chondroitin sulfate, and with response to the specific toluidine blue color spray; hydrolysis of the C14-mucopolysaccharidesand isolation and identification of resulting C14-hexosaminesby ion exchange and paper chromatography and crystallization of derivatives with carrier glucosamine (Table 111) ; precipitation of the C14-mucopolysaccharide along with carrier chondroitin sulfate by cetylpyridinium bromide, a specific precipitation reagent, and nondialyzability of the precipitated C14-mucopolysacccharide;hydrolysis of the S~~-mucopolysaccharide t o give labeled sulfate under conditions that lead to hydrolysis of the sulfate group of chondroitin sulfate.
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
447
Colon segments of deficient and normal rats were incubated with Sa5sulfate in Krebs-Ringer-phosphate buffer, pH 7.4. The mucopolysaccharides synthesized and secreted into the medium were isolated by precipitation and purified by paper chromatography, and their radioactivity was determined. Incorporation of S35into the mucopolysaccharides by deficient colon segments was found to be about one-half compared to that of normal colons. When a suspension containing 10 pg. of vitamin A was added to the incubation medium of the deficient colons, incorporation was raised to the level of the normal (Table IV). Vitamin A aldehyde was also effective. The conditions under which incorporation of S35-sulfateinto mucopolysaccharides of rat colon homogenates would take place were first determined. Incorporation was highest when the medium contained diphosTABLE IV EFFECTOF ADDEDVITAMINA ON INCORPORATION OF Sa60i-MUCOPOLYSACCHARIDES BY COLONSEGMENTSO Vitamin A status
Vitamin A added
-
-
-
+
10 pg. (alcohol) 10 pg. (aldehyde) -
INTO
Mucopolysaccharide (c.p.m.) 1279 3515 3001 2685
(4) (4) (4)
(4)
The values are averages of the number of incubations ahown in parentheam. Incubation period, 3 s hours; activity added, 19 X 106 c.p.m.; = normal rat; = deficient rat.
+
-
phopyridine nucleotide, glucose, glutamine, and adenosine triphosphate (“complete system”). No incorporation was obtained if the two last-named constituent,s were omitted. Homogenates were then prepared from vitamin A-deficient and normal rat colons tind incubated with the complete system. The results are summarized in Table V. The S35-sulfateincorporated into mucopolysaccharides by homogenates from deficient colons was once again about one-half that of normal colons. The addition of vitamin A suspension to the deficient homogenates restored incorporation t o normal. The suspending medium alone (serum albumin-ethanol) had no effect. In Table VI can be seen the specificity tests with other fat-soluble vitamins. Vitamin A only is capable of completely reversing decreased activity. The next step in this investigation was to localize the function of vitamin A in a specific step in mucopolysaccharide biosynthesis. It was found that the vitamin is not involved in the formation of hexosamines, because glucosamine or galactosamine, when substituted for glucose and glutamine in the incubation medium of deficient homogenates, could not restore the decrease in incorporation.
448
GEORGE WOLF AND B. CONNOR JOHNSON
Next, experiments were undertaken to determine whether vitamin A is required in a step before or after the involvement of the uridine nucleotides in mucopolysaccharide synthesis (see Fig. 1). First, a system requiring the uridine nucleotides, and capable of incorporating Sa6-sulfateinto mucopolysaccharideby rat colon homogenates, was developed. This system TABLE V
INCORPORATION O F
S"'O4-
- I N T O MUCOPOLYSACCHARIDES BY COLON HOMOQENATES"
Vitamin A status
Vitamin A added (10r g . )
+-
+
Mucopolysaccharide (c.p.m.)
-
Serum albumin-ethanol
346 (3) 805 (2) 936 (4) 268 (2)
0 The values are averagcm of the number of incubations shown in parentheses. The complete system contained 10pmole.qof ATP, Bpmoles of M p , 1pmole of glubmine, apmoles of DPN, and IOpmolea of gluwefinal volume 1 ml. Each incubation contained 10.8 X 108 c.p.m. of radioactive sulfate and 14 mg. of protein; normal rat; - = defiaient rat.
+
-
TABLE VI MUCOPOLYSACCHARIDES BY COLON HOMOOENATES. SPECIFICITYOF VITAMINAm
INCORPORATION OF
Status of animal Normal Deficient Deficient Deficient Deficient Deficient Deficient
S"'O4-
-
INTO
Addition (10rg.1 -
-
Vitamin A alcohol Vitamin D Vitamin E Vitamin E vitamin A Vitamin KI
+
Mucopolysaccharide (c .p.m.) 896 483 841 232
(2) (2) (2) (1) 608 (2) 752 (2) 435 (2)
O The values are averages of the number of incubations, shown in parentheses. The complete system WBB that shown in Table V. Each incubation contained 9.78 X 101 c.p.m. of activity and 14 mg.of protein.
showed an absolute requirement for glutamine, uridine diphosphoacetylglucosamine, ane uridine diphosphoglucuronic acid, and a partial requirement for uridine diphosphoglucose and acetylglucosamine. When incubated with deficient colon homogenates, it once again showed lowered incorporation of SS6-sulfateinto mucopolysaccharide, restorable with added vitamin A (Table VII). Therefore, vitamin A functions at some step beyond the synthesis of the uridine nucleotides. In an attempt to achieve independence from the use of vitamin A-deficient animals, and to find a means of destroying vitamin A in vitro, the
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
449
colon homogenates were preincubated with lipoxidase, an enzyme known to destroy vit,amin A. This procedure, as shown in Table VIII, lowered or abolished S36-sulfatcincorporation. Addition of vitamin A restored incorporatio11 only at low concentrations of lipoxidase, presumably because t,he TABLE VII A ON THE INCORPORATION OF S W - - INTO EFFECTOF VITAMIN MUCOPOLYSACCHARIDES BY COLON HOMOQENATEB CONTAININQ UDP-DERIVATIVEB~ Vitamin A status
Addition of vita- Mucopolysaccharide min, A 1Opg. (c.p.m.)
The values are averages of tbe number of incubations, shown in parentheme. The complete iystem conaisted of 1 pmole of UDPGA, 1 pmole of UDPAG, 1 pmole of UDPG, 1 pmole of glutamine, 8pmolen of AQ (all obtained from Sigma Chemical Corporation), 1pmole of ATP, and 10pmoles of Mg*. Each incubation contained 8.6 X 106 c.p.m. of activity and 14 mg. of protein; normal rat; - = deficient rat.
+
-
TABLE VIII EFFECTOF LIPOXIDASE ON Sss04-- INCORPORATION INTO MUCOPOLYSACCHARIDEB BY COLON HOMOQENATESO Preincubation for 1 hour with lipoxidase (3 mg./ml.) 0.01 ml. 0.01 ml. 0.04 ml. 0.04 ml. 0.07 ml. 0.07 ml.
Addition of vita- Mucopolyaaccharide min A, pg. (c.p.m.)
-
+ ++
306 538 268 0 0 0
Complete ayatem waa that ahown in Table V. Each incubation contained 10.6 X 106 0.p.m. of activity and 14 mg. of protein. Crystalline soybean lipoxitlase was obtained from Nutritional Biochemical Corporation.
added vitamin was destroyed by the excess lipoxidase present in the incubation medium. The assay used for mucopolysaccharide synthesis in the above experiments was the uptake of labeled sulfate from the medium into the final product of a many-step synthesis. Hence, the requirement of vitamin A for any particular step can only be derived by inference. One can infer from the results described that the vitamin is not required for the following steps: the conversion of glucose to hexosamines; the acetylation of hexosamines; the formation of the uridine diphosphoacetylhexoamines;
450
GEORGE WOLF AND B. CONNOR JOHNSON
the oxidation of uridine diphosphoglucose to uridine diphosphoglucuronic acid. By a process of elimination, therefore, one could conclude that vitamin A functions either in the polymerization of the uridine nurleotides, or the activation or transfer of sulfate t,o the polymer. 3. Studies with Pig Colon l h x y m e Fractions and Vitamin A
Since rat colon mucosa were difficult to obtain free from muscle and in sufficient quantity, a new approach was sought by the use of pig colon mucosa (Wolf et al. 19GO). In homogenates of that tissue, radioactivity vas incorporated into mucopolysaccharide from S3s-labeled sulfate or C14-labeled glucose. Mucopolysaccharide was identified in a manner similar to TABLE IX MUCOPOLYSACCHARIDE B Y SUBCELLULAR PH 5 ENZYMES OF P I Q COLONMUCOSA~
INCORPORATION OF s a 6 - s U L F A T E INTO
FRACTIONS AND
Subcellular fraction Supernatant free from mitochondria and nuclei Supernatant free from mitochondria, nuclei, and microsomes Supernatant free from mitochondria, nuclei, microsomes, and pH 5 enzymes Microsomes pH 5 Enzymes pH 5 Enzymes and microsomes
Mucopolysaccharide (c.p.m./mg. protein) 1293 (4) 15’21 (4) 326 516 1728 1770
(1) (4) (4) (3)
4 The values are averages of the number of incubations, shown in parenthesee. The complete system consistedof lopmoles ATP, 6pmoles MgClr ,3pmolea DPN, lrmoleglutamine, and lOpmoles glucoee. The radioactivity added per incubation, 9 X 108 c.p.m. made to final volume of I ml. with phosphate buffer, pH 7.4.
that described for rat colon mucopolysaccharide. The synthesizing activity was located in the supernatant solution, after removal of nuclei, mitochondria, and microsomes from the homogenate (Table IX) and could be precipitated a t pH 5.2. This “pH 5 enzyme” fraction contained 48.6% of the total vitamin A content of the mucosa (3.7 pg. per 100 gm. mucosa). It required uridine triphosphate, adenosine triphosphate, glucose, and glutamine for activity (Table X) and showed a pH maximum between 6.2 and 7.2. This enzyme fraction when obtained from vitamin A-deficient pigs was less active (Table XI) ; lipoxidase also lowered its activity, which in both cases could be restored by added vitamin A (Table XII). To exclude the involvement of vitamin A in sulfate transfer from 3’phosphoadenosine 5’-phosphosulfat,e (“active sulfate”) to the polymer, pH 5 enzyme fraction was incubated with active sulfate and model sulfate acceptors such as chondroitin and nitrophenol. No differences were found
451
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
TABLE X REQUIREMENTS OF COFACTORS nY PH 5 ENZYMES FOR INCORPORATION OF Sas0,- - INTO MUCOPOLYSACCHARIDESO
ATP, 10 pmoles
DPN, 3 pmoles
Glutamine, 1 Cole
Mucopolysaccharide (c.p.m./mg. protein)
Glucose, UTP, 10 @moles 4 pmoles
++ + +-
+ + + ++
-
2234 2349 1713 798 560 5723
+-
(2) (2) (2) (2) (2) (2)
The values are averages of the number of incubations, shown ie the parentheses. Radioactivityadded per incubation, 9 X 106 c.p.m. made to final volume of 1 ml. with phosphate buffer, p H 7.4.
TABLE X I EFFECT OF VITAMINA DEFIENCY O N THE INCORPORATION OF Sa601-INTO MUCOPOLYSACCHARIDE A N D SPECIFICITY OF VITAMINA IN PH 5 ENZYMES' Vitamin A status
Addition of vitamin A,
+-
10 ag.
-
Vitamin A Vitamin E Vitamin D
Mucopolysaccharide (c.p.m./mg. protein) 2349 1407 2556 1564 1530
(2) (2) (2) (2) (2)
a The values are average8 of the number of incubations, shown in the parentheses. The complete system consisted of lOrinoles glucoae, 1 prnole glutamine, 3 rinoles DPN, and 8Wa-- 19 X loa c.p.m., made up to I Inl. with phosphate buffer, pH 6.8.
TABLE X I 1 EFFECT OF PREINCUBATION WITH LIPOXIDASE ON THE INCORPORATION OF S J 6 0 r - - INTO MUCOPOLYSACCHARIDE SYNTHESIZED BY PH 5 ENZYMESO Lipoxidttse, 0.3
Added vitamin A, 10 rg.
0.1 ml. 0 . 1 ml.
-
mg./ml.
+
Mucopolysaccharide (c.p .m ./mg. protein) 2706 (4) 1213 (4) 2188 (4)
The values are average8 of the number of incubations, shown in the parentheses. The complete system consiated of 10 rnioles glucose, 1 pmole glutamine, 3 pnioles DPN, and S"Oa--, 12 X 10' c.p.m., made up to 1 nil. with phosphate buffer pH, 8.8. (1
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GEORGE WOLF AND B. CONNOR JOHNSON
in sulfate transferase activity between normal and lipoxidase-treated fractions, thus excluding vitamin A function in sulfate transfer. Such a conclusion, however, ‘has to be treated with caution, since none of the natural acceptors occurring in colon have so far been tried as acceptors in this reaction. On the other hand, evidence has now been obtained (Varandani et al. 1960) which shows that colon homogenates of deficient rats form phosphoadenosine phosphosulfate less readily than normal homogenates (Table XIII), a lesion which could be corrected by addition of vitamin A. One could therefore conclude that vitamin A is involved in mucopolysaccharide biosynthesis at the sulfate activation step. TABLE XIII EFFECTOF VITAMIN A DEFICIENCY ON PAPSa6SYNTHESIS I N RATCOLON HOMOQENATE’ PAPW Vitamin A status of rats Adequate Deficient Deficient Deficient
Addition
-
Vitamin A, u)pg. in propylene glycol, 5 pl. propylene glycol, 5 pl.
Expt. I1 Expt. I (c.p.m. /mg. protein) 19,200 8,440
59,000 28,700
24,300 -
54,100 31,400
NOTE:Aotivity added: Expt. I. 8.4 X 1Osc.p.m.; Expt. 11, 16.8 X 10So.p.m. a
Abbraviation: PAF@”, 3’-phmphardenosine 6’-phmphosulfata labeled with 8”.
4. Vitamin A
and Net Synthesis of Mucopolysaccharides in Vitro
Since up to this point only minute amounts of mucopolysaccharide, detectable only by radioactivity, have been synthesized, it was thought necessary to investigate net synthesis of colon mucopolysaccharide in vitro, and the effect of vitamin A thereon. Total hexosamine content of rat colon was determined by Moretti and Wolf (unpublished observations), by acid hydrolysis of colon homogenates and isolation and assay of hexosamines. Normal colons contained an average of 11.58 f I .2 pmoles, colons from vitamin A-deficient rat,s an average of 7.38 f 1.0 pnioles per 100 mg. of protein. Actually, “total hexosamine” may include not only that in mucopolysaccharide, but, also in the various hexosaminc-cont,airiiirg intermediates (cf. Fig. 1). Therefore, it was necessary to separate mucopolysaccharide from low-molecular weight material by precipitation and dialysis. Again, the deficient colons showed lowered mucopolysaccharide hexosamine to
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHESIS
453
the extent of about GO% (average of 5.00 f 0.4 pmoles hexosamines per 100 mg. of protein), compared to the normal colon (average of 8.04 f 0.9 pmoles per 100 mg. of protein). The ratio glucosamine/galactosamine of 0.5 in samples of normal colon was increased to 0.85 in those from deficient animals; hence vitamin A deficiency caused a decrease mainly in the content of galactosamine containing mucopolysaccharides in colon. To study net synthesis of mucopolysaccharide, and the effect of vitamin A thereon, a system was developed using rat colon homogenates incubated with glutamine, glucose G-phosphate, diphosphopyridine nucleotide, uridine triphosphate, and adenosine triphosphate. The samples were assayed for total hexosamine by acid hydrolysis before and after incubation. The increase of total hexosamine content after incubation of deficient colon homogenates was about one half compared to that of normal colons. The synthetic capacity of the deficient colon homogenates could be restored almost to normal by addition to the incubation mixture of vitamin A. The minimum quantity which showed this stimulation was 1.25 X pmoles (11.5 I.U.) added per incubation (per 11-13 mg. of protein). This action appeared to be not graded, but all-or-none. Net synthesis was not restored to normal in deficient homogenates by addition to the incubation mixture of vitamin A acid, vitamins E, K 1 , and Ds . Again, it was necessary to confirm that, since the total hexosamine assayed included the intermediates in mucopolysaccharide biosynthesis, the effect of t,he vitamin was truly on mucopolysaccharide-bound hexosamine. Therefore, it was first shown that vitamin A had no effect on the formation of glucosamine G-phosphate. Secondly, mucopolysaccharide was separated from low-molecular weight material by precipitation and dialysis. The net formation of mucopolysaccharide-bound hexosamine (precipit,able and nondialyzable) of normal colon homogenates was between 7 and 12 times greater than that of deficient homogenates. The net synthesis of the latter was raised about four-fold by addition of vitamin A to the incubation. These results, t,herefore, definitely establish an effect of vitamiii A on the content, and net synthesis of mucopolysaccharide in colon. 5. Vitamin A and Keratinizatim
Though not, st rict,ly coming under the heading of mucopolysaccharide biosynthesis, the theories of vitamin A function in the inhibition of keratinizatioii should here be ment,ioiied. Balakhowskii and Drozdova (1957) consider keratinizstioii in vitamin A deficiency to be due to a disturbance of oxidative processes. In particular, the catalytic activity of copper in the oxidation of cysteine to cystine, which the authors regard as the rate-
454
GEORGE WOLF AND B. CONNOR JOHNSON
limiting step in keratinization, was found to be inhibited by carotenoid compounds in model systems. The same authors (1956) also found the catalytic action of copper in ascorbinase to be lowered in presence of carotenoids. Vitamin A-deficient rats excreted more free labeled sulfate than normal rats after injection of Sas-methionine (Balakhowskii and Drozdova, 1958), which the authors again assume to be due t o a release from the inhibitory action of vitamin A on the copper-catalyzed oxidation of SHgroups. Another interpretation of this result would be to consider it due t o increased excretion of sulfate caused by lowered mucopolysaccharide biosynthesis. V. CONCLUSIONS From the data presented, especially the tissue culture studies of H. B. Fell, t,he conclusion is inescapable that vitamin A has a controlling action on the basal cells of epithelium. The presence of vitamin A stimulates them to produce mucus, and its absence permits them to form keratin. The vitamin therefore has an action similar to that of a hormone and, in that sense, we know as little about the mode of its biochemical function as about that of other hormones. On the other hand, taking its stimulation of mucus formation as a starting point, results have been presented which show that vitamin A has a direct, possibly coenzymatic, function in the polymerization reaction of the uridine nucleotides to form mucopolysaccharide, or in the sulfation of the latter. Perhaps the vitamin could control differentiation of basal cells by being the essential factor in a rate-limiting step of a reaction sequence leading to mucus formation. REFERENCES American Society of Biological Chemists Symposium. 1958. Federation Proc. 17,10741105. Balakhowskii, S.D.,and Drozdova, N . N. 1956. Doklady Akad. Nauk S.S.S.R. 109, 355-357. Balakhowskii, S.D.,and Drozdova, N . N . 1957. Biokhimiya 22,33&335. Balakhowskii, S. D., and Drozdova, N . N. 1958. Doklady Akad. Nauk S.S.S.R. 118, 331-333. Cohen, J., Maddock, C. L., and Wolbach, S. B . 1955. A . M . A . ilrch. Pathol. 69, 723726. Dorfman, A., Markovitz, A., and Cifonelli, J . A. 1958. Federation Proc. 17, 1093-1099. Uziewiatkowsky, 13. D . , 1954. J . Exptl. Med. 100, 10-24. Fell, H.B. 1953. In “Connective Tissues,” Transaction of the Fourth Conference (C. Ragan, ed.), 142 pp. Josiah Macy, Jr. Foundation, New York. Fell, H . B., and Mellanby, E. 1952. J . Physiol. (London) 116,320-349. Fell, H.B.,and Mellanby, E.1953. J . Physiol. (London) 119,47&488. Fell, H.B., Mellanby, E . , and Pelc, S. R . 1954. Brit. Med. J . 11, 611.
VITAMIN A AND MUCOPOLYSACCHARIDE BIOSYNTHEBIS
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Frape, U. L., Allen, R. S., Spear, V. C., Hays, W. V., and Catron, D.V. 1959.J . Nutrition 68, 189-201. Hedenberg, I. 1954.Acta Clin. Scand. Suppl. 192,87. Jeanloz, R.W. 1958.Federation Proc. 17, 1082-1086. Manville, I. A. 1937.Science 86,44. Markovits, A., Cifonelli, J. A., and Dorfman, A. 1959.J . B i d . Chem. 2S4.2343-2350. Mellanby, E. 1950. “A Story of Nutritional Research; The Effect of Some Dietary Factors on Bones and the Nervous System.” Williams & Wilkins, Baltimore, Maryland. Meyer, K. 1958.In “Polysaccharides in Biology,” Transactions of the Fourth Conference, (G. F. Springer, ed.), pp. 11-56. Josiah Macy, Jr. Foundation, New York. Meyer, K., Smyth, E. M., and Palmer, J. W. 1937. J . Biol. Chem. 119,73-84. Moore, T.1957a. “Vitamin A,” p. 535.Elsevier, New York. Moore, T. 1957b. “Vitamin A,” p. 518. Elsevier, New York. Robbins, P. W., and Lipmann, F. 1957. J . Biol. Chem. 229,837-851. Schubert, M.1958.Federation Proc. 17, 1099-1105. Smith, H.,and Gallop, R. C. 1953. Biochem. J . 63,66&671. Springer, G. F., ed. 1958. “Polysaccharides in Biology,” Transactions of the Fourth Conference. Josiah Macy, Jr. Foundation, New York. Susuki, S., and Strominger, J. L. 1960.J . B i d . Chem. 236,257-276. Thomas, L., McCluskey, R. T,. Potter, J. L., and Weissmann, G. 1960.J . Ezptl. Med. 111, 704-718. Varandani, P. T., Wolf, G. and Johnson, B. C. 1960. Biochem. Biophys. Research Communs. 3, 97-100. Werner, I. R . 1953.Acta SOC.Med. Upsaliensis [N.S.] 68, 1-55. Wolbach, S. B. 1947.J . Bone and Joint Surg. 29, 171. Wolbach, S.B., and Howe, P. R . 1925.J . Ezptl. Med. 42,753-777. Wolf, G., and Varandani, P. T. 1960.Biochim. et Biophys. Acta 45, 601-512. Wolf, G., and Varandani, P. T., and Johnson, B.C.1960.Biochim. et Biophys. Acta, in press.