The specificity of UDP-glucose 4-epimerase from the yeast Saccharomyces fragilis

484

BIOCHIMICAET BIOPHYSICAACTA

BBA 65687 T H E S P E C I F I C I T Y OF UDP-OLU(LOSE 4 - E P I M E R A S E FROM T H E YEAST SACCHAROMYCES FRAGILIS*

W. L. S A L e , J. H. N O R I ) I N ~*, D. R. PI';TI';RSON, R. D. 13EVIIA,"*" AND S. K I R K W O O D

l)epart~ent of BiochemistJ y, ('olh,ge of Biological Scie~ces, Ul~iz,ersity of 3Ibzm~s~ta, St. Paid, 3[i~J~.

(~7.s..4.)

(Received August -'sth, 19o7)

SUMMARY

I. The reactivity of UDP-glucose 4-epimerase (EC 5.I.3.2) from the yeast Saccharontyces fragilis toward I I analogs of UDPG has been examined. The compounds tested involve stereochemical modifications of the hexosyl and ribosyl moieties of UDPG. 2. Of the i i compounds only UDP-fi-L-arabinose, UDP-D-fucose and UDP-Dxylose are epimerized. However, evidence is presented indicating that these epimerizations are due to contaminating enzymes and are not due to UDPG 4-epimerase. 3. The substances UDP-1)-mannose, UDP-N-acetyl-D-galactosamine, UDP-Dallose and UDP-3-O-methyl-D-glucose were not epimerized. These observations are in agreement with the hypothesis of BUDOWSK¥ et aZ.9 since the postulated hexose: uracil hydrogen bond, which is necessary for enzyme action in their mechanism, would be hindered in these substances. 4- dUDPG is not acted upon by the yeast epimerase. It has been reported to serve as a substrate for the epimerase from calf liver. 5. Other substrates which were not epimerized are UDP-fi-~-glucose, UDP-Dglucuronic acid, UDP-4-0-methyl-D-glucose, ADPG, CDPG, GDPG, IDPG and dTDPG.

INTROI) UCTION

The biological interconversion of glucose and gatactose has been the object of considerable interest for many years. The reaction shown in Eqn. ~ was discovered A b b r e v i a t i o n s : UDPA, UI)P-fl-L-arabinose; U D I ' X , ~!DP-D-XVlOSe; UDPI;, UDP-Dfucose; UDPQ, 1 DP-D-quinovose. * Paper No. 033i Scientific J o u r m d Series, 1Tniversity of Minnesota \gricultural Experinlent Station. ** Present address: D e p a r t m e n t of Biochemistry, University of Massachusetts, Amherst, l~Iass., U.S.A. *** Present address: D e p a r t m e n t of Molecular Biology, ..klbert Einstein College of Medicine, i3oo Morris Park Ave., Bronx, N.Y. [o,t6[, U.S.A.

l~iochim. Bioph.vs. ,qctc~, I5~ ([9(i8) 4,g4 492

SPECIFICITY OF U D P G

4-EPIMERASE

485

by LELOIR1 and is catalyzed by UDP-glucose 4-epimerase (EC 5.1.3.2) hereafter called epimerase. UDPG ~- UDPGal

(i)

The epimerase occurs widely in nature and has been studied from a variety of sources 2-4. Although the enzymes differ according to source they all require NAD + for activity. In earlier studies in this laboratory 5 it was observed that epimerase preparations from yeast (Saccharomyces fragilis) were capable of epimerizing UDP-fl-L-arabinose (UDPA) and it had been reported by NEUFELD et al. ~ that yeast epimerase failed to act upon UDPA. It has also been reported that mung bean extracts contain UDPA4-epimerase activity although it has not been separated from UDPG-4-epimerase activity or distinguished from it in any other wayS, 2. It is evident that, if the epimerization of UDPGal and UDPA are catalyzed by different enzymes, these enzymes should exhibit a strong specificity toward the groups at C-5 of the pyranose ring of the substrate, since UDPGal and UDPA differ only in substitution at C-5. Thus it was felt profitable to extend the work on the specificity pattern of the enzyme toward the pyranose moiety of the substrate and to determine whether or not the previous specificity results reported from this laboratory were influenced by the presence of more than one enzyme activity in the preparation used. In addition, such work would allow a further evaluation of the hypothesisS, 9 that UDPG 4-epimerase requires a specific secondary structure in its substrates. METHODS AND MATERIALS

Enzymes Yeast UDPG 4-epimerase was employed at three levels of purification. Prep. I and 2, purified by the procedure of MAXWELL AND DE ROBICHON-SZULMAJSTER 2, had specific activities of 262 and 620 mU/mg* of protein, respectively. Prep. 3, generously donated by Dr. H. KALCKAR, had a specific activity of 14 830 mU/mg of protein and migrated as a single component upon acrylamide gel electrophoresis. UDPG dehydrogenase (EC 1.1.1.22) had a specific activity of 202 mU/mg of protein after purification through Step 6 and assay according to STROMINGERet ai. 1°. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49 ) and phosphoglucomutase (EC 2.7.5.1 ) were purchased from California Biochemicals Incorp.

Determination of specific activity of epimerase Reactions were run in quartz cuvettes containing suitable enzyme concentrations, o.I/,mole UDPGal, I/,mole NAD+, IO mU of UDPG dehydrogenase and 15o/,moles of glycine buffer (pH 8.7) in a final volume of 0.5 ml. Reaction was followed by measuring NADH production at 24 ° in a Beckman DU spectrophotometer equipped with pinhole filter and photomultiplier. The rate of change of absorbance was kept between O.Ol-O.O4 per rain and the change in absorbance was converted to /,moles of UDPG oxidized per rain using an E3a0 mu for NADH of 6.2 • lO3 M -1. cm -1. " The units are those r e c o m m e n d e d b y the I n t e r n a t i o n a l Union of Biochemistry and are expressed in k~moles/mir/.

Biochim. Biophys. Acta, ISI (1968) 484-492

486

w . t . . SALO ct al.

Preparation of carbohydrates D-Allose was synthesized from a commercial preparation of D-ribose by cyanohydrin synthesis and subsequent reduction of D-a-allonolactone n. The yield of crystalline fl-D-allose ([a i]~ + 13 ° (c I in H20)) was 63% based on D-allonolactone and it had a melting point of 127--129 °. The 3-0-methyl-D-glucose was prepared from 1,2:5,6-di-0-isopyropylidene-a-D-glucofuranose in 51% yield (m.p. 165.5-168°) 12. The 4-0-methyl-D-glucose was obtained as an oil in 54% yield from 2,3:5,6-di-0-isopropylidene-D-glucose dimethyl acetal and characterized as its phenylosazone (m.p. I56-I57°)12,13. The D-fucose obtained as a syrup was prepared in 80% yield by LiA1H 4 reduction of 1,2:3,4-di-0-isopropylidine-6-0-tosyl-n-galactosO 4. It was characterized by paper chromatography in Solvent A and found to contain only a trace of galactose. The D-p4Qglucose was purchased from New England Nuclear Corp., Boston, Mass. D-Glucose, D-galactose, D-Xylose, L-arabinose and D-mannose were all commercial preparations from Pfanstiehl Laboratories. Reference samples of 6-deoxy-D-glucose (quinovose), D-talose, 3-0-methyl-D-galactose, and 4-0methyl-D-galaetose, were supplied by Dr. B. A. LEwis of this department.

Preparation of sugar acetates and sugar phosphates All of the carbohydrate acetates were prepared by common laboratory procedures 15 and isolated in crystalline form where possible. The sugar phosphates were all prepared from their respective acetates by the MACDONALD method ~n except for the fi-anomer of D-glucose I-phosphate. It was prepared by previously described procedures 1v-19, crystallized as its bis cyclohexylammonium salt, and recrystallized TABLE PHYSICAL

1 DATA

ON SUGAR

PHOSPHATES

Compound

Sugar z-phosphates

m.p.

' (t,J?2a l)

!

salt

a-D- Allopyranose i-phosphate fi-L-Arabinopyranose i-phosphate a-D-Galactopyranose 1-phosphate (~-D- rl4C]Glucose I-phosphate fl-D-Glucopyranose 1-phosphate I~-D-3-O-M ethyl-glucopyranose 1-phosphate

a-D-4-O-Methyl-glucopyranosc I-phosphate a-D-Fucopyranose 1-phosphate a-D-Xylopyranosc I-phosphate Cz-D-Mannopyranose 1-phosphate

Bis cyclohexylammonium salt Bis cyclohexylammonium salt l'otassium Bis cyclohexylammonium salt* 13is cyclohexylammonium salt Brucine Brucine Bis cyclohexylammonium salt Bis cyclohexylammonium salt Bis cyclohexylammonium salt

i 7 9 - i 8 o '~ 187-188.5 ° -

(¢ 2 in HzO )

726 t-9o.o' @91.5

°

(c 2 in H 2 0 ) (c 0. 5 in H=O)

i66-17o°d

~ 6i °

i65'd

4. io' (c ~ in H 2 0 ) - 7 8 . 5 .... (c 2 in H20)

i59-164°d

{ 6.3 °

(c 5,3 in H=O)

i84.5-I86 °

t 67.5 '~

(c z in H 2 0 )

~52-I58~)

~56.8 J

(c 2 in H 2 0 )

183-185 °

+3o/)

(c t, 5 in H 2 0 )

(c z in H 2 0 )

* Recrystallized to a constant specific activity of 83 ooo disint./niin per/*mole. ** Rotation taken with potassium salt since brucine salt has a negative rotation. B i o c h i m . B i o p h y s . A c t a , [51 (I968) 484 4 9 2

SPECIFICITY OF UDPG

487

4-EPIMERASE

until free of a-D-glucose 1-phosphate as determined spectrophotometricaUy with phosphoglucomutase, glucose-6-phosphate dehydrogenase and NAD+ (ref. 20). All of the sugar phosphates were recrystallized 3 times to rid them of any possible contamination with the undesired anomer. This number of recrystallizations was found to be sufficient to obtain constant specific activity with a-D-[14Clglucose 1-phosphate. They all had a ratio of hexose or pentose to orthophosphate of I : i after hydrolysis in o.I M HC1 at IOO° for 15 min. Physical data on the sugar phosphates are presented in Table I.

Preparation of nucleotides and nucleotide sugars Nucleotide morpholidates were synthesized and analyzed as described by MOFFATT AND KHORANAzl. dUMP was prepared from dCMP by oxidative deamination with NaNO, (ref. 22). The product was obtained as its calcium salt in 79°/0 yield. The product was pure dUMP as determined by spectral analysis at pH's 2 and 12 and paper chromatography in Solvent D which clearly separates dCMP and dUMP. Nucleoside monophosphates, NAD+, NADP+, dCMP and UDPG were purchased from P.L. Biochemicals Inc., Milwaukee, Wisc. All chemically synthesized nucleotide sugars were prepared on a microscale by a modification of the procedure of ROSEMAN et al. ~s, using a glass vacuum manifold system. Details of this procedure have been published elsewhere .4. UDP-D-fucose (UDPF), UDPA and UDPGal were also synthesized on a large scale .3. Analytical data for some nucleotide sugars is presented in Table II. UDPGal was assayed TABLE

II

ANALYTICAL DATA ON NUCLEOTIDE SUGARS

Compound

UDP-D-fUcose UDP-/~-L-arabinose UDV-D-mannose UDP-D-xylose UDP-D-allose UDP-/~-D-glucose UDP-3-O-methyl-D-glucose UDP-4-O-methyl-D-glucose

Molar ratio Base

Reducing Total sugar phosphate

i.oo I.OO i .oo i.oo I.OO I.OO i.oo I.OO

0.96 0.99 i .oi 1.o8 1.o2 0.99 0.98 I.O2

1.97 1.96 2.02 2.oo 2.02 2.o2 2.04 2.08

enzymatically with epimerase and found to be 95% pure. UDP-D-L14Clglucuronic acid was prepared in 97~/o yield by enzymic oxidation of UDP-D-[14Clglucose with UDPG dehydrogenase 7. UDP-D-[14C3galactosamine (7.5 #C//zmole) was a gift from Dr. D. M. CARLSON, Western Reserve University.

Analytical methods Reducing sugar was determined by either the P A R K - J O H N S O N ~5 o r NELSON-SOMOGYI~e procedures. Protein was determined by a modified Folin-Lowry method 27 or a modified biuret procedure 2s, and phosphorous by the method of FISKE AND Biochim. Biophys. Acta, 151 (1968) 4 8 4 - 4 9 2

488

w.I.. s.\~.o el al.

SUBBAROw29. 14C deternfinations were made on liquid samples using a Nuclear Chicago scintillation spectrometer and on paper chromatograms b y the use of a Nuclear Chicago 4 n radiochromatogram scanner. Paper c h r o m a t o g r a p h y of sugars, sugar phosphates, and nucleotide sugars was performed on W h a t m a n No. I paper using the following solvents (all concentrations v/v)" Solvent A, pyridine ethyl acetate-water 2 : 5 : 7 (upper phase)(ref. 3o); Solvent B, I-butanol pyridine water 6 : 4 : 3 (ref. 31); Solvent C, I-butanol ethanol water 4o:11:19 (ref. 31); Solvent D, absolute ethanol I M a m m o n i u m acetate (pH 7-5) 75:25 (ref. 32); Solvent E, absolute ethanol nlethylethylket(me o.5 M morpholiniuin tetraborate (pH 8.0) 7:2:13 (ref. 33): and Solvent F, I - b u t a n o l acetic acid-water 2 : I : I (ref. 34). Ultraviolet-absorbing compounds were detected on paper with a Mineralight h a n d lamp, Model 3I- 2537. Free sugars were detected by the method of TREVELYAN, PROCTER AN I) HARRISONaa. Sugar phosphates were detected using the procedure of HANES AX'> [SHERWOODa6 using an ultraviolet light to develop the coloraL Incubation of nucleotide sugar analogs with epimerase The nucleotide sugars were incubated for 8 h at 37 ° with 2~2 lll~_i of enzyme and 240/~moles of glvcine buffer (pH 8.7) in a total volmne of Lo ml. The a m o u n t of substrate incubated with the enzyme varied from 0. 5/zmole of the non-radioactive analogs to I()ooo counts/nfin of the radioactive analogs. A boiled enzyme control was run with every incubation. After incubation, Lo ml of I M HCl was added to the reaction mixture which was then hydrolyzed for io rain at IOO°. After cooling, the solution was deionized by passage through columns of IR-I2O (H ~) and I R-45 (OH-). It was then streaked on W h a t m a n No. I paper and chronmtographed with a solvent chosen for its ability to separate the particular reactant sugar from its 4-epimer. An alternate method was used to determine the extent of reaction with solne of the analogs. The reaction was stopped by heating at Ioo c for 45 sec and the reaction mixture then streaked on washed W h a t m a n 3-ram paper and chromatographed using Solvent D. After c h r o m a t o g r a p h y the nucleotide sugar band was cut out and eluted with water. The eluant was concentrated, streaked on W h a t m a n No. i paper, and c h r o m a t o g r a p h e d in Solvent E. This system will separate U D P F from UDP-D-quinow)se (UI)PQ), U D P ( ; from UDPGal, and UDP-D-xvlose (UDPX) from UDPA. The U D P - s u g a r bands were eluted and their concentration determined by reading the absorbance of the eluant, against water, at 262 lll tl. :\ correction for contaminating ultraviolet-absorbing material from the paper and the solvent can be applied b y subtracting the absorbance obtained at 3 t8 m,u. This is possible since the absorbance of the contaminants at 318 m# is the same as their absorbance at 2()2 lU/t while uridine has no absorbance at 318 int~. I~ESUI.TS

Reactivity of nudcotide sztgars with cpimerase A m o n g the substrate analogs tested with epimerase preparations only U I ) P N , U D P A and U D P F were acted upon. 1)-Fucose, 1)-fucose I-phosphate, I~ arabinose, /3-L-arabinose I-phosphate and xy]ose were not epimerized. D-Xvh,se i phosphate showed slight reaction with ephnerase Prep. 2, but no reaction with epimerase Prep. 3. Biochim. Biophys. .I c/a, 151 (19()8)484 4o2

489

SPECIFICITY OF U D P G 4-EPIMERASE TABLE III R E A C T I V I T Y OF S U B S T R A T E S W I T H E P I M E R A S E

See METHODS for incubation of nucleotide sugar analogs with epimerase; the alternate method was used.

Substrate

Molar ratio after incubation with epimerase

Equilibrium ratio

UDPG

UDPG UDPGal

2.82~.2o

3.0 (ref. I)

UDPX

UDPX UDPA

I.O9~.o8

I.O (ref. 7)

UDPA

UDPA UDPX

1.26~-.o9

I.O (ref. 7)

UDPF

UDPF UDPQ

-- 1.62~ .I 4

*

--*

The equilibrium ratio of the U D P F UDPQ interconversion has not been determined.

U D P F was epimerized to a new nucleotide sugar upon incubation with all three epimerase preparations. This new ultraviolet-absorbing compound was separated from U D P F by paper chromatography in Solvent E, RUDPF 1.2. It had a ufidine spectrum and when subjected to mild acid hydrolysis released a sugar with a mobility identical to 6-deoxy-D-glucose (quinovose) in Solvents A, B, and C. Similar experi=

TABLE IV NUCLEOTIDE SUGARSTESTED WITH EPIMERASE Modification of UDPG or UDPGal: H, hexosyl moiety; C-number, position of modification iu the sugar ring; C, chemical modification; S, stereochemical modification; R, ribosyl moiety.

Compound

Modification

UDP-D-glucose UDP-D-galactose UDP-D-xylose" UDP-/~-L-arabinose" UDP-D-fucose* UDP-D-El*C~glucuronic acid UDP-4-O-methyl-D-glucose UDP-3-O-methyl-D-glucose UDP-D-allose UDP-D-nlannose UDP-N-acetyl-D-[14C]galactosamine UDP-/J-D-glucose dUDP-D-E14C]glucose ADP [l*C~glucose CDP [14C]glucose GDP [14Clglucose I D P [14CJglucose dTDP [14C]glucose

Natural substrate Natural substrate H, C-5, C H, C-5, C H, C-5, C H, C-5, C H, C-4, C H, C-3, C H, C-3, S H, C-2, S H, C-2, C H, C-I, S R, C-2, C Base Base Base Base Base

These substrates were epimerized, but by contaminating enzymes. None of the other substances, with the exception of UDPG and UDPGal, were acted upon.

Biochim. Biophys. ,4cta, 151 (1968) 484-492

490

w. L, SALO g¢ al.

ments using Solvents A, B, and F demonstrated arabinose in the hydrolyzate of the product from the incubation of U D P X with epimerase and xylose in the hydrolyzate of the product from the incubation of UDPA. U D P F , UDPX, and UDPA failed to inhibit the conversion of UDPGal to U D P G at concentrations up to 2oo times that of UDPGal. Inhibition would certainly be expected if these substances were epimerized by the same enzyme that acts upon UDPGal. In order to investigate this matter further, the Km for U D P F was determined and found to be 2.5" IO-4 M. The magnitude of this Michaelis constant, in comparison with that for UDPGal (1.86.1o -~ M) leaves no doubt whatever that inhibition should have been observed at the 2oo-fold concentration of UDPF. In view of this it was assumed that the epimerization of the three substances was catalyzed b y enzymes other than U D P G 4-epimerase. More evidence in support of this idea was gained by investigating the activity of U D P G 4-epimerase preparations at different stages of purification. A crude preparation (Prep. I) readily catalyzed the epimerization of UDI)G, UDPX, UDPA and U D P F in the standard 8-h incubation period ('Fable III). When U D P X and

Fig. I. The effect of degree of purification on the ability of a U D P G - 4 - e p i m e r a s e p r e p a r a t i o n t catalyze the epimerization of U D P F . The time of incubation was 8 h.

F3iochim. Biophys. Acta, 151 (t968) 484-492

SPECIFICITY OF

UDPG 4-EPIMERASE

491

UDPA were incubated with Prep. 2 or 3, only traces of the epimerized product could be detected b y paper chromatography of the free sugars after hydrolysis. The epimerization of U D P F was also very much reduced upon purification of the enzyme as seen in Fig. I. It is therefore concluded that the epimerization of UDPX, UDPA, and U D P F is the result of contaminating epimerase activities in even the purest available preparations of UDPG 4-epimerase. A list of the substrate analogs, together with the nature of the modification of their structures from that of the natural substrates, is presented in Table IV. DISCUSSION

UDPG 4-epimerase from yeast appears to have a strict specificity for the hexosyl moiety of its substrate. Three substrate analogs were epimerized; however, the evidence suggests that these three analogs, UDPF, UDPX and UDPA, were epimerized by enzymes other than UDPG 4-epimerase. This being so, the epimerase has a strict specificity for the hydroxymethyl group at C-5 of the pyranose ring. It is also sensitive to structural or stereochemical modifications at C-I, C-2, and C-3, and to structural modification at C-4. dUDPG was not epimerized by the yeast enzyme although it has been reportedS, 9 that calf-liver epimerase will epimerize this molecule. Thus, the specificity of yeast epimerase is slightly different from that of calf-liver epimerase. This difference may be because yeast epimerase has either a more stringent requirement for intramolecular hydrogen bonding in the substrate, or it requires a binding of the 2' hydroxyl group to some specific site on the enzyme. dTDPG was not epimerized but the lack of reactivity could be due entirely to the lack of the 2' hydroxyl group. In order to determine if thymine will substitute for uracil, T D P G must be made. None of the base substitutions for uracil resulted in molecules which were reactive with the yeast epimerase. It has been suggested that the base moiety is important for enzyme specificity in reactions involving nucleotide sugars as, i.e. a sort of handle. However, from the work reported here it is obvious that the sugar moiety is also very important. Calf-liver epimerase can accommodate several modifications of the base moiety without complete loss of reactivityS, 9. These studies led to a proposed secondary structure for the substrate. The secondary structure hypothesis has the merit that it offers an explanation for specificities involving the entire substrate molecule and not just isolated non-interacting moieties. Four substrates which would have hindered hexose:uracil hydrogen bonding of the type described by BUDOWSKY et al. 9 failed to react with epimerase. These were UDP-D-allose, UDP-3-0-methyl-D-glucose ' UDP-D-mannose, and UDP-Nacetyl-D-galactosamine. Models of UDP-fl-I)-glucose would not readily form the proposed secondary structure. These observations tend to support the secondary structure hypothesis and also explain why the hexosyl moiety is very important in determining specificity. A secondary structure similar to that of BUDOWSKY et al. was proposed earlier 39. It was pointed out that a knowledge of the three dimensional structure of UDPG and UDPGal would be of great value in interpreting the mechanism of action of the epimerase. l~iochim, lgiophys. ,4cta, 151 (1968) 484-492

W . L . SAI.O C! a/.

492 ACKNOWLEDGEMENTS

This work was supported by a grant from the National Institutes of Health. The authors are indebted to Dr. H. G. FLETChEr< Jr. and to Dr. N. K. R~CHTMYER for a generous sample of .,>allose. RF, F E R E N C E S 1

2 3 4 5 0 7 8 9 1o

LI,;LOIR, /D'C]I. lJiochem. BiotShYs., 33 (195L) I80. MAXWl'LL ANl) It. D E ROBICHON-SZULMAJSTER, .[. Hi(t/. (Them., 235 1191}o) 3 o& MAXWELL, J. l:;iol. (;hem., 229 119571 139. WILSON AND D. S. IIOGNV:SS, J. Biol. (;hem., -'39 119041 2409. NORDTX, ll. 11. BEVILL, W . L..%ALO, P. T. WlCKLUND AND .%. NIRKWOOD. l"~defali,)~z Proc., 24 (1905) 411. E. F. ~NEUFELD, V. GINSBURG, E . W . PUTMAN, D. IrANSHJER AND \ ¥ . Z. H-aSSlD, ~lJch. B i o chem. Biophys., 09 11957) 6o2. D. S. I:EINGOLD, 17.. F. NEUFELD AND ~,V. Z. HASStD, J. Biol. Chem., 235 1190o) 9~o. T. N. DRUSttININ-a, ~/. A. NOVIKOVA AND G. g . ZItDANOV, Dokl. Alead. N a u k U S S R , 164 1t9651 1175. E. 1. ]~UDOWSKY, T..N..I)RUStlININA, (;. 1. HLISEI';VA, N. D. GABRIb;LYAN, N. b2. NOCHETKOV, M. A. NOVIKOVA, V. N. SHIBAI';V AND G-. k . ZHDANOV, Biochim. Biophys. Acta, 12-' (I966) 213. J. L. ~TROMINGER, E. S. ~{AXWELL, J. AXELROD AND H. M[. KALCKAR, ol. Biol. Chem., "-'4 I,. F. E. S. E. S. D. B. J" H.

(~957) 79-

I 1 t ?. L . HUMOLLI';R, i n 1{. L. \ ¥ H I S T L E R AND ~1. g . \VOLFROM, Methods i*z Carbohydr**te Cltemislry,

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