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Synthesis of uridine diphosphate l -rhamnose by enzymes of Chlorella pyrenoidosa
ARCHIVES
OF
BIOCHEMISl~l~Y
Synthesis
AND
BIOPHYSICS
of Uridine
118,
Diphosphate Chlorella
G. A. BARBER Deparintent
of Biochemistry
Lij<~-(i(ii~
and
(196i)
I-Rhamnose
by Enzymes
of
Pyrenoidosa ASD
Biophysics,
RI-.
T. ‘I-. CHAS-G
1 7niz~er.~il,r/
oj Hawaii,
Honululu,
Hawaii
968%
An enzyme system obtained from light-grown p~tenoic~osa, is shown ttt cat.alyae l.he reduction
cultures of the green alga, Chlorel2a and epimerization of rlridine diphosphate u-glllcose t,o a compolund characterized as uridille diphosphate L-rhamnose; TPNH is required in the reaction. IJridine diphosphate I)-galactose and thymidine diphosphate o-glucose participate to 75 and 2572, the extent of uridine diphosphate D-glUCOSe. Evidence is presented that the expected intermediate, uridine diphosphate 4-keto-G-deoxy-D-glucose, is produced in the interconversion of the two sugar nncleotides.
The B-deoxyhexose, L-rhamnose, is reported to be one of the sugars present in a cell-wall heteropolysaccharide of the green alga, Chlorella pyenoidosa (1). Evidence is presented in this paper t,hat indicates t’hat C. pyrmoidosa brings about the formation of that, monosaccharide by way of the sugar nucleotide, uridine diphosphate L-rhamnose. The synthesis of UDP-L-rhamnose has previously been shown to be catalyzed by enzymes extracted from the leaves of various higher plarlts (Z), and the compound itself was isolated from a speciesof golden brown algae, Ochromonas malhamensis (3). In the bacteria, on the other hand, the synthesis of L-rhamnose is usually mediated by the thymidine diphosphate derivative (4), although recently UDP-L-rhamnose ma.sisolated from a strain of Salmonella t&imwium (5). Thus, it might have been predicted from the evolutionary relationship of algae to vascular plants that L-rhamnose in Chlorella would be formed as the uridine diphosphate compound. AS far as in ~~3.0 reactions can be correlated wit,h in N&J processes, this does indeed seem to be t,he case. MATERIALS Reagents. labeled with
AND
METHODS
a-u-Glucose l-phosphate uniformly I% (62 &/pmole) ~tvas obta.ined from 659
llC-labeled sucrose by use of the enzyme sucrose phosphorylase from Pseudomonas sacchnwphila (6). UDP-u-Glucose aud dTDP-n-glllcose labeled with ‘“C in the o-glucosyl moieties were synthesized enzymically from that radioactive LY-I-glucase l-phosphate with the appropriate pyrophosphorylase preparation from mung beaus (Phaseolus auretts) (7, 8). IJDF-u-(;alactose-1-“C (35 &/wmole) was also produced ellzymically from D-galactose-l-‘% with o-galactose kinase (9) prepared from galactose adapt’ed yeast (Sigma Chemical Co.) and UDP-u-galactose p~ropho~phorylase (7). QaercetitGgl\lcoside (isoqr[ercitrin) and tideox,v-u-glucose were gifts of Dr. T. A. (ieissman and 1)r. Winifred Watkins, respect,ively, md rutinose was prepared by the enzymic hydrolysis of rutin with rhamnodiastase (10). All ot#her reagents were obtained from commercial sources. A culture of C. pvrenoidosa of unknown straili was supplied by Dr. H. Yamamot,o. Paper chromatoyraphy and electrophoresis. The following solvent systems were used for chromatography OIL Whatman No. 1 or Schleicher and Schuell No. 589 (Blue Label) paper: (I) n-propanol-ethyl acetat.e-water, 7: 1:2; (II) n-buta~~olacetic acid-n-at,er, 52:13:35; (III) butanolleacetic acid-water, 8:l:l; (IV) 957; ethanol-l 3, ammonium acetate, pH, 7 7:3. Electrophoresis was carried out ill an apparatus sl~ch as that described by Crestfield and Allen (11) on washed paper (S and S, 589) at 25530 V/cm. Radioactive compomlds were located on paper
660
BARBER
AND
by exposure to X-ray film or with a rat,e-meter and mica-window Geiger tube. When eluted from the paper, they were counted with a thin-window counter and conventional scaler 011 metal planchets. Unlabeled reducing sugars were locat,ed by spraying papers with the p-anisidine phosphate reagent (10). Chemical analysis. For the chemical analysis of the sugar nucleotide, L-rhamnose was determined by the cysteine-sulfuric acid method of Dische and Shettles (12), reducing sugar by the met,hod of Park and Johnson (13), and total phosphat,e by the method of Ames and Dubin (14). The optical absorbancy of the compound was measured at 262 rnp (at pH 7), and its concentration was estimated from the molar absorbancy given for UDP-uglucose, 10 X lo3 (15). Growth of algae. Subcultures of C. pyrenoidosa were grown on sugar slants in test tubes on the window sill for l-2 weeks at 25”. The nutrient medium consisted of the salt solution given by Davis and Dedrick (16) plus 2y0 agar and 1% D-glucose. Algae from one of these slants were harvested in sterile water and used to inoculate 800 ml of the salts medium in a flat bottle. Carbon dioxide (5%) in air was bubbled through the culture which was incubated for 6-7 days in a water tank at 24-26” under constant, illumination (indirect sunlight during the day and the light of four 40-W fluorescent lamps at night). The yield of cells from 800 ml of such a culture was from 4 t,o 5 gm fresh weight. Preparation of algul enzyme. Algae were collected by centrifugation at 20009 for 10 minutes and washed two times with cold water. The packed cells could then be stored at -10” for at least several mont)hs with no loss of activity in the subsequent enzyme extracts. In a typical preparation, 5 gm of the fresh or thawed frozen cells were suspended in 20 ml of cold buffer, pH 7.0, containing 0.1 M sodiumpotassium phosphate, 0.05 M mercaptoethanol, and 5% polyvinylpyrrolidone. A protective effect on the enzyme was not obtained consistently by the inclusion of the latter two substances, but they were rolltinely added to the buffer. All t,he operations following were carried out in the cold. The cell suspension was passed twice through a French cell at a pressure of about 15,000 pounds per square inch. Microscopic examination of the suspension indicated that this procedure disrupted more than 757, of the cells. Whole cells and particulate material were removed from the homogenate by centrifugation at 10,OOOg for 10 minutes. The supernatant solution was treated with a neutral saturated solution of ammonium sulfate. The protein fraction which precipitated bet,ween
CHANG 35 and 55y0 ammonium sulfate saturation carried the bulk of the enzyme activity. This fract.ic)ll was dialyzed overnight against two l-liter volumes of 0.025 hr sodium-potassium phosphate:0.05 JI mercapto-ethanol buffer, pH 7.0. If it was not to be used immediately after dialysis, the enzyme preparation was lyophilized and stored at -10”. A solution of the dialyzed enzyme lost activity in a day or two at this temperature, but the lyophilized preparation remained active for at least several weeks. Assay of enzyme uclivity. The ext,ent of formation of UDP-L-rhamnose was measured essentially as it had been done earlier (2). RESULTS
Table I gives t’he substrate requirement for production of L-rhamnose by the enzyme extract of C. pyrenoidosa. The reactivity of dTDP-n-glucose suggests that the enzyme is not absolutely specific for the pyrimidine portion of the nucleotide triphosphate. It is of course possible that there may be two enzyme systems, one reacting with UDP-Dglucose and one with dTDP-n-glucose, but this seems unlikely in view of the results of studies made of 6-deoxy-sugar formation in other organisms (4, 5). The participation of UDP-n-galactose in the reaction was not unexpected since it had been observed incidentally in this laboratory that ChZoreZZa extracts will catalyze the epimerization of UDP-u-galactose to UDPn-glucose. However, until the epimerase is removed from the preparation, it cannot be stated unequivocally that the rhamnoseforming enzyme does not react with UDP-Dgalactose. The conversion of UDP-n-glucose to UDP-L-rhamnose was proportional to time and to enzyme concentration. There was no activity in the absence of TPNH (5 X 1O-3 M) while DPNH in the same concentration was without effect. Neither DPN (3 X 1OF -3 X 10e4~), EDTA (1.6 X 10e3 M), cysteine (3.2 X 1O-3 M), glutathione (3.2 X 10e3 M), MgClz (3 X 1O-3 M), nor MnCL (3 X 1O-3 M) caused any consistent stimulation of the reaction rate. Characterization of UDP-L-rhamnose. In order to obtain a quantity of the rhamnosyl nucleotide large enough for microchemical analysis, the following mixture was prepared
SY’NTHESIS TABLE sl-BSTHA.~E
~I’ECIFICI’I’B
SYNTHESIS
OF
OF
URIl)TTI;:
DIPHOSPHATR
I IN
THE
&zVMIC
L-RHBMNOSE
The react,ion mixtlue contained 20 ~1 of the algal enzyme (0.5 mg protein in 0.025 M sodiumpotassilun phosphate brlffer, pH 7.0), 0.15 rmole TI’TH, 0.01 rmole I>PN, and 0.01 /*mole (about 0.05 WC) of the ‘YXabeled sugar nucleotide. The mixture was incubated in a capillary for GO minr~ies at 37”. The extent of formation of L-rhamnose was estimated as dewrihed in the t.ext. Substrate’”
‘i ‘4C incorporated into the L-rhamnose derivative
38 20 0 7
U1)I’-D-Glucosc
lTDl’-u-Galactose dTDP-1).Glwose Q WI-‘-o-Glucose, glucose l-phosphate enzyme system.
GDP-wglrrcose, were not acted
alld Q-Dupon by the
and incubated at] 37” for 60 minutes: 0.3 /.mole UDP-I,-glucose-‘4C (0.2 PC), 4.5 pmoles TI’SH, 0.3 bmole DPN, 0.75 pmole sodium EDTA, and 0.6 ml of the lyophilized enzyme fraction (about 15 mg of protein) in 0.025 M sodium-pot,assium phosphate buffer, pH 7, in a t)otal volume of 0.75 ml. UDP-Hexoses were isolated from t,he mixt,ure by elect~rophoresis on paper in 0.1 Y ammonium format,e, pH 3.7. Aft,er elution from the paper, the material was chromatographed on washed paper in solvent IV. To separate UDP-n-glucose from UDP-L-rhamnose, the paper was developed and dried three successive times with the solvent (a), and to remove residual ammonium acetate, it was developed one additional kc with 80 % etjhanol (2). Aft,er this treatment, CDP-n-rhamnose is found slightly ahead of UDP-I)-glucose. The radioac*tive nucleotides were lwat,ed by exposure of the paper to X-ray film and by t,heir absorption of ultraviolet8 light,, and material in the position of GDP-n-rhamnose was clutcd from the paper with water. Another skip of paper was developed in the same way, :md a (*orresponding area was clutcd with water and used as a blank in all the subsequent,analyses. The yield of UDl’-L-rharllriose, as estimated by its nbsorhanc~y at 262 111~ against, the paper blank, was 0.0.58 qnolc.
66 1
L-IIHAXISOSE
The rat,ios of the absorbnncies at various wavelengths are compared with the published tigurrs for UDl’-n-glucwc in Table II (15). These results indicut,e that the base asswiated with the c~ompou~ld is uracil. The results of the c*heniicnl tleteriiiiJiations are given in Table III. These arc also wrksterlt with the proposed structure. The sugar moiety of cnzymically prodwed I;I)l’-L~rhamrlose-‘4(: was further characterized c~hrontat,ographically. UDf’-L-Rlmnr~ose-‘~C was synthesized as dcwribed in the ~Iet~hotls section and was isolated by c,lIromatogrxpl,y in solvent I\‘. Xfter hydrolysis at pFI 2 (0.01 s HCl, 10 minutes at 100’) :mtl admixture with authentic L-rhamnosc, the hydrolyzxtr was c~hromatographed in solvcnts I and III and rlect,rophoresed in 0.05 31sodium trtrabornte buffer, pH 9. In eac~hsystcw the ratlionctivc COII~pourd and authentic2 I)-rhamnosc wirwided csact ly . Additional cviderwc for the proposed struc%ure of the w~~~pountl was provided by an oxpcriment, in which radionc~tivc rutin [:3-clucrc~etirl-0-Lu-L-rh:lnlnosyl (1 -+ 6) P-J:glucoside] was formed eiizyniic*nlly from UDl’-L-rli~~iiiJiosC-14(‘ ‘ 5I :mtl the :rppropriatje acceptor. The details of this enzymica TC:IVtion w-erc published wrlier (10). An xmmonium sulfate fraction was prep:tretl from TG%F: RATIOS
OF
THE
STNTHESIZEI)
UDP-
r,-rbamnose
mp III@
E;NZPMICALLY
Published value for UDP-~-glucose (15)
0.7G 0.43
TABLE CHEMICAL
OF
l:DP-1:1~HAYSOHE Enzymic
Ratio
250/2ao 280/260
II
~4BSORB.iNCIES
ANALYSIS
OF
0.74 0.38
III ENZYMICALLY
E’oR~IED
UI)P-I.-RHAMNOSE Analysis .~
Uridine L-Rhamnose Reducing sugar (after hydrolysis at pH 2, 15 minrltt=s at, 100”) Total phosphate
Total hmole) -~______---.--
Ratio
0.058 0.058 0.061
1.00 1.00 1.05
0.123
2.12
662
BARBER
mung bean leaves and was incubated with algal UDP-L-rhamnose-i4C which had been synthesized and purified as described in the preceding paragraph. The reaction mixture conbained 20 ~1 mung bean leaf enzyme (about 1.0 mg protein in 0.025 M Tris.HCI0.01 M mercaptoethanol buffer, pH 7.5), 30 pg quercet-in-3-glucoside, 0.3 pmole ATP, and 1.6 X 10F4 bmole UDP-L-rhamnose-14C (0.01 PC) in a total volume of 25 ~1. The mixture was incubated for 45 minutes at 37”. It was added to 0.75 ml of methanol, and t,he precipitate was removed by centrifugation. Authentic rutin was added to the met’hanolic supernatant solution which was then evaporat,ed to a small volume and chromatographed on washed paper with solvent’ II. Rutin was located by its characteristic yellow fluorescence under ultraviolet light when the paper was exposed to ammonia vapor. The radioactive compound and rutin coincided exactly. Further evidence that t,he compound formed in this way was i4C-labeled rutin was obtained from the demonstration that radioactive rutinose (0-cu-L-rhamnosyl (1 -+ 6) n-glucose) could be released from the compound by mild acid hydrolysis. 14C-Labeled rutin was eluted from the paper with methanol and evaporated to dryness in a tube. It was treated with 10% acebic acid at 100” for 60 minutes (17). The hydrolyzate was evaporat)ed to dryness in vacua and chromatographed in solvent I. Two radioactive areas were detect,ed on the chromatogram, one in the position of rutin and the ot)her in the position of authentic rutinose. Formation of an intermediate. An intermediate which has been found in the enzymic formation of most of the 6-deoxyhexoses and 3,6-dideoxyhexoses investigated so far (4) is the 4-keto-6-deoxysugar nucleotide derivative. The algal system responsible for the synthesis of UDP-n-rhamnose seems to follow this same general pattern. Thus, when a mixture was prepared and incubated as described in the Methods section, except that TPNH was omitted, a radioactive compound could be isolated from the mixture which showed the properties to be expected of UDP-4-keto-6-deoxy-n-glucose (2). This compound migrated upon electrophoresis at pH 3.7 like a UDP-hexose. Upon hydrolysis
AND
CHANG
in acid (0.1 N HCl, 10 minutes at 100”) a radioactive subst’ance was produced witjh n mobility in solvent I slightly less than that of L-rhamnose. When the UDP-hexose was first reduced chemically with YaBH4 (1 5 pmoles phosphate buffer, pmole NaBH4, pH 7.0, in a total of 20 ~1; allowed to react 15 minutes at 25’), elecbrophoresed again to remove salts, and t,hen hydrolyzed as before, t’wo new radioactive compounds were produced. Upon chromatography in solvent I and solvent III, ‘kc-labeled compounds with the mobilities of authentic nglucose, L-fucose, and 6-deoxy-n-glucose were observed. The epimers to be expected after the chemical reduction of the 4-keto group of such an intermediate would be n-fucose and 6-deoxy-n-glucose. (Solvent 111 readily separates the three 6-deoxyhexoses, fucose, rhamnose, and 6-deoxyglucose.) DISCUSSION
The reproductive process in Chlorella is somewhat more elaborate than it is in many other unicellular organisms. In brief, at a certain stage in its life cycle a Chlorella cell enlarges and produces two, four, eight, or more autospores which are released into the surrounding medium by rupture of the wall of the mother cell (18). Since only a very small proportion of cells in the total population is in a particular stage of this process at any given time, it is undoubtedly important to know if a compound of enzymological interest is produced continuously or only at one short, period in the life of the cell. It appears, for example, that the format,ion of cell walls in Chlorella occurs during autospore generation and over a relatively short period of time (18). It is thus quite possible that the synthesis of what are presumably cell wall precursors, such as UDP-n-rhamnose and other sugar nucleotides, also occurs during only a portion of the cell’s life (19). It is unfortunate t,hat this possibility was not considered during the course of the research reportsed here. It is hoped that in future studies of sugar nucleotide metabolism and cell-wall formation in this group of organisms synchronous cultures at some given stage will provide a richer source of enzyme. Despit’e the heterogeneous population of
algacl usctl for the extraction of the rhamnose-synthesizing enzyme, there seems, lit& doubt1 kat extrarts of C. pyrenoidosa do form lJl)P-r,-rharnnose. It, remains now to show the function of that) wmpound in the nwtabolism of the algal c*ell.
AhI
This work 10(%2-01
was supported PC.
in part
by NIIL
grant
10. B.IRISEI~, G. A., Hiwhemislry 11.
Chem. 12. I)~scxE, Chem.
13. I?\ItK,
1). II., (:OULDING, K. J., .\ND R,. W., B&hem. J. 70, 391 (1958). BARBER, G. A., ‘lrch. Kiochem. Biophgs. 103, 276 (19(i3). K.zr-SS, H., Biochem. Biophys. Res. Commun. 18, Ii0 (1965). GINYBCRG, V., d duan. Enzymol. 26, 35 (19fi4). GINSBURG, V., J. Biol. Chem. 241, 3750 (19%).
4. 5.
6. H.\SSID, w. z., Enzymol. 7. XECFELD,
10,123 F.,
E.
.YND
M., ;~daan.
~)O~DOROFF, V.,
PUTNAM,
15. iY.\~r~Ios.~I,
16.
FANSHIER,
17. 18.
E.
W. Z., .lwh. Bioch,enz. Biophys. 69, 602 (1957). 8. NEUFELD, fi:. F., Biochem. Biophys. Res. conlm~rn. 7, 461 (1962). 9. Tnucco, R. E., CAPUTTO, II., LELOIR, L. F., AND MITTEI.M.~N, pr'., Llrch. Biochem. 18, 137 (1948). W.,
I).,
J.
Chem.
(1950). GIKSBURG, AND
.\SD
T.,
AND
1, 463 (1962). F. W.,
ALLE?;,
~OHSSUN,
181, l-19 (1949). 14. AMES, B. h’., AND I)YRIN,
HOHNE,
3.
&I.,
27, 422 (3955). Z., .INI) SHIGTTLES, 175, 595 (I9G).
,!nd.
I,.
B.,
J.
Ijiol.
51.
J.,
J.
f{iOl.
T.,
J.
/jiol.
I~ASSII~,
19.
11.
235, 769 (1960). AC.WEJIY-
OF
SCIENCES-SATIONAL
COINCIDE, “Specificat,ions and Criteria for Biochemical Compounds,” 1’111~ liratiw il9. Washingt>on, 11.C. (1960). I>.\vIs, 141. A., .\sI) I)EIMICK, K. C:., in “Algal Cldt,lu-e from Laboratory to Pilot Plant” (J. S. Blulew, ed.), l’ubliratiou (iO0, p. 119. Carnegie Institute, Washington, D.C. (1953). 41t.1~.1n..1~ II., Nippon k’nyaku Zasshi 77, 1314 (19%) [Chem. A&s/r. 53, 20316d, (1959)j. MVR.UL~MI, S., MONIM~-ELI, Y., ANI) TIK.\wY.1, A., in “Studies 01~ Microalgae and Photosynthetic Bacteria“ (Japallese Sot. I’l. Physiol., ed.), p. 65. Univ. of Tokyo Press (1963). IV-.~uIL~, T., KANAZ.LWI, T., .\ND K~N.Iz.Y\v.L, K., in “Studies on Rlicroalgae and Photosyllthet.ic Bacteria” (Japanese Sot. 1’1. Physiol, cd.) 11. 58i. Univ. Tokyo Press (1903). RESEAIWH
1. xORTHCOTE,
A.
C’hem.
REFEREXCES
2.
CI~ESTFIELD,
OF
BIOCHEMISl~l~Y
Synthesis
AND
BIOPHYSICS
of Uridine
118,
Diphosphate Chlorella
G. A. BARBER Deparintent
of Biochemistry
Lij<~-(i(ii~
and
(196i)
I-Rhamnose
by Enzymes
of
Pyrenoidosa ASD
Biophysics,
RI-.
T. ‘I-. CHAS-G
1 7niz~er.~il,r/
oj Hawaii,
Honululu,
Hawaii
968%
An enzyme system obtained from light-grown p~tenoic~osa, is shown ttt cat.alyae l.he reduction
cultures of the green alga, Chlorel2a and epimerization of rlridine diphosphate u-glllcose t,o a compolund characterized as uridille diphosphate L-rhamnose; TPNH is required in the reaction. IJridine diphosphate I)-galactose and thymidine diphosphate o-glucose participate to 75 and 2572, the extent of uridine diphosphate D-glUCOSe. Evidence is presented that the expected intermediate, uridine diphosphate 4-keto-G-deoxy-D-glucose, is produced in the interconversion of the two sugar nncleotides.
The B-deoxyhexose, L-rhamnose, is reported to be one of the sugars present in a cell-wall heteropolysaccharide of the green alga, Chlorella pyenoidosa (1). Evidence is presented in this paper t,hat indicates t’hat C. pyrmoidosa brings about the formation of that, monosaccharide by way of the sugar nucleotide, uridine diphosphate L-rhamnose. The synthesis of UDP-L-rhamnose has previously been shown to be catalyzed by enzymes extracted from the leaves of various higher plarlts (Z), and the compound itself was isolated from a speciesof golden brown algae, Ochromonas malhamensis (3). In the bacteria, on the other hand, the synthesis of L-rhamnose is usually mediated by the thymidine diphosphate derivative (4), although recently UDP-L-rhamnose ma.sisolated from a strain of Salmonella t&imwium (5). Thus, it might have been predicted from the evolutionary relationship of algae to vascular plants that L-rhamnose in Chlorella would be formed as the uridine diphosphate compound. AS far as in ~~3.0 reactions can be correlated wit,h in N&J processes, this does indeed seem to be t,he case. MATERIALS Reagents. labeled with
AND
METHODS
a-u-Glucose l-phosphate uniformly I% (62 &/pmole) ~tvas obta.ined from 659
llC-labeled sucrose by use of the enzyme sucrose phosphorylase from Pseudomonas sacchnwphila (6). UDP-u-Glucose aud dTDP-n-glllcose labeled with ‘“C in the o-glucosyl moieties were synthesized enzymically from that radioactive LY-I-glucase l-phosphate with the appropriate pyrophosphorylase preparation from mung beaus (Phaseolus auretts) (7, 8). IJDF-u-(;alactose-1-“C (35 &/wmole) was also produced ellzymically from D-galactose-l-‘% with o-galactose kinase (9) prepared from galactose adapt’ed yeast (Sigma Chemical Co.) and UDP-u-galactose p~ropho~phorylase (7). QaercetitGgl\lcoside (isoqr[ercitrin) and tideox,v-u-glucose were gifts of Dr. T. A. (ieissman and 1)r. Winifred Watkins, respect,ively, md rutinose was prepared by the enzymic hydrolysis of rutin with rhamnodiastase (10). All ot#her reagents were obtained from commercial sources. A culture of C. pvrenoidosa of unknown straili was supplied by Dr. H. Yamamot,o. Paper chromatoyraphy and electrophoresis. The following solvent systems were used for chromatography OIL Whatman No. 1 or Schleicher and Schuell No. 589 (Blue Label) paper: (I) n-propanol-ethyl acetat.e-water, 7: 1:2; (II) n-buta~~olacetic acid-n-at,er, 52:13:35; (III) butanolleacetic acid-water, 8:l:l; (IV) 957; ethanol-l 3, ammonium acetate, pH, 7 7:3. Electrophoresis was carried out ill an apparatus sl~ch as that described by Crestfield and Allen (11) on washed paper (S and S, 589) at 25530 V/cm. Radioactive compomlds were located on paper
660
BARBER
AND
by exposure to X-ray film or with a rat,e-meter and mica-window Geiger tube. When eluted from the paper, they were counted with a thin-window counter and conventional scaler 011 metal planchets. Unlabeled reducing sugars were locat,ed by spraying papers with the p-anisidine phosphate reagent (10). Chemical analysis. For the chemical analysis of the sugar nucleotide, L-rhamnose was determined by the cysteine-sulfuric acid method of Dische and Shettles (12), reducing sugar by the met,hod of Park and Johnson (13), and total phosphat,e by the method of Ames and Dubin (14). The optical absorbancy of the compound was measured at 262 rnp (at pH 7), and its concentration was estimated from the molar absorbancy given for UDP-uglucose, 10 X lo3 (15). Growth of algae. Subcultures of C. pyrenoidosa were grown on sugar slants in test tubes on the window sill for l-2 weeks at 25”. The nutrient medium consisted of the salt solution given by Davis and Dedrick (16) plus 2y0 agar and 1% D-glucose. Algae from one of these slants were harvested in sterile water and used to inoculate 800 ml of the salts medium in a flat bottle. Carbon dioxide (5%) in air was bubbled through the culture which was incubated for 6-7 days in a water tank at 24-26” under constant, illumination (indirect sunlight during the day and the light of four 40-W fluorescent lamps at night). The yield of cells from 800 ml of such a culture was from 4 t,o 5 gm fresh weight. Preparation of algul enzyme. Algae were collected by centrifugation at 20009 for 10 minutes and washed two times with cold water. The packed cells could then be stored at -10” for at least several mont)hs with no loss of activity in the subsequent enzyme extracts. In a typical preparation, 5 gm of the fresh or thawed frozen cells were suspended in 20 ml of cold buffer, pH 7.0, containing 0.1 M sodiumpotassium phosphate, 0.05 M mercaptoethanol, and 5% polyvinylpyrrolidone. A protective effect on the enzyme was not obtained consistently by the inclusion of the latter two substances, but they were rolltinely added to the buffer. All t,he operations following were carried out in the cold. The cell suspension was passed twice through a French cell at a pressure of about 15,000 pounds per square inch. Microscopic examination of the suspension indicated that this procedure disrupted more than 757, of the cells. Whole cells and particulate material were removed from the homogenate by centrifugation at 10,OOOg for 10 minutes. The supernatant solution was treated with a neutral saturated solution of ammonium sulfate. The protein fraction which precipitated bet,ween
CHANG 35 and 55y0 ammonium sulfate saturation carried the bulk of the enzyme activity. This fract.ic)ll was dialyzed overnight against two l-liter volumes of 0.025 hr sodium-potassium phosphate:0.05 JI mercapto-ethanol buffer, pH 7.0. If it was not to be used immediately after dialysis, the enzyme preparation was lyophilized and stored at -10”. A solution of the dialyzed enzyme lost activity in a day or two at this temperature, but the lyophilized preparation remained active for at least several weeks. Assay of enzyme uclivity. The ext,ent of formation of UDP-L-rhamnose was measured essentially as it had been done earlier (2). RESULTS
Table I gives t’he substrate requirement for production of L-rhamnose by the enzyme extract of C. pyrenoidosa. The reactivity of dTDP-n-glucose suggests that the enzyme is not absolutely specific for the pyrimidine portion of the nucleotide triphosphate. It is of course possible that there may be two enzyme systems, one reacting with UDP-Dglucose and one with dTDP-n-glucose, but this seems unlikely in view of the results of studies made of 6-deoxy-sugar formation in other organisms (4, 5). The participation of UDP-n-galactose in the reaction was not unexpected since it had been observed incidentally in this laboratory that ChZoreZZa extracts will catalyze the epimerization of UDP-u-galactose to UDPn-glucose. However, until the epimerase is removed from the preparation, it cannot be stated unequivocally that the rhamnoseforming enzyme does not react with UDP-Dgalactose. The conversion of UDP-n-glucose to UDP-L-rhamnose was proportional to time and to enzyme concentration. There was no activity in the absence of TPNH (5 X 1O-3 M) while DPNH in the same concentration was without effect. Neither DPN (3 X 1OF -3 X 10e4~), EDTA (1.6 X 10e3 M), cysteine (3.2 X 1O-3 M), glutathione (3.2 X 10e3 M), MgClz (3 X 1O-3 M), nor MnCL (3 X 1O-3 M) caused any consistent stimulation of the reaction rate. Characterization of UDP-L-rhamnose. In order to obtain a quantity of the rhamnosyl nucleotide large enough for microchemical analysis, the following mixture was prepared
SY’NTHESIS TABLE sl-BSTHA.~E
~I’ECIFICI’I’B
SYNTHESIS
OF
OF
URIl)TTI;:
DIPHOSPHATR
I IN
THE
&zVMIC
L-RHBMNOSE
The react,ion mixtlue contained 20 ~1 of the algal enzyme (0.5 mg protein in 0.025 M sodiumpotassilun phosphate brlffer, pH 7.0), 0.15 rmole TI’TH, 0.01 rmole I>PN, and 0.01 /*mole (about 0.05 WC) of the ‘YXabeled sugar nucleotide. The mixture was incubated in a capillary for GO minr~ies at 37”. The extent of formation of L-rhamnose was estimated as dewrihed in the t.ext. Substrate’”
‘i ‘4C incorporated into the L-rhamnose derivative
38 20 0 7
U1)I’-D-Glucosc
lTDl’-u-Galactose dTDP-1).Glwose Q WI-‘-o-Glucose, glucose l-phosphate enzyme system.
GDP-wglrrcose, were not acted
alld Q-Dupon by the
and incubated at] 37” for 60 minutes: 0.3 /.mole UDP-I,-glucose-‘4C (0.2 PC), 4.5 pmoles TI’SH, 0.3 bmole DPN, 0.75 pmole sodium EDTA, and 0.6 ml of the lyophilized enzyme fraction (about 15 mg of protein) in 0.025 M sodium-pot,assium phosphate buffer, pH 7, in a t)otal volume of 0.75 ml. UDP-Hexoses were isolated from t,he mixt,ure by elect~rophoresis on paper in 0.1 Y ammonium format,e, pH 3.7. Aft,er elution from the paper, the material was chromatographed on washed paper in solvent IV. To separate UDP-n-glucose from UDP-L-rhamnose, the paper was developed and dried three successive times with the solvent (a), and to remove residual ammonium acetate, it was developed one additional kc with 80 % etjhanol (2). Aft,er this treatment, CDP-n-rhamnose is found slightly ahead of UDP-I)-glucose. The radioac*tive nucleotides were lwat,ed by exposure of the paper to X-ray film and by t,heir absorption of ultraviolet8 light,, and material in the position of GDP-n-rhamnose was clutcd from the paper with water. Another skip of paper was developed in the same way, :md a (*orresponding area was clutcd with water and used as a blank in all the subsequent,analyses. The yield of UDl’-L-rharllriose, as estimated by its nbsorhanc~y at 262 111~ against, the paper blank, was 0.0.58 qnolc.
66 1
L-IIHAXISOSE
The rat,ios of the absorbnncies at various wavelengths are compared with the published tigurrs for UDl’-n-glucwc in Table II (15). These results indicut,e that the base asswiated with the c~ompou~ld is uracil. The results of the c*heniicnl tleteriiiiJiations are given in Table III. These arc also wrksterlt with the proposed structure. The sugar moiety of cnzymically prodwed I;I)l’-L~rhamrlose-‘4(: was further characterized c~hrontat,ographically. UDf’-L-Rlmnr~ose-‘~C was synthesized as dcwribed in the ~Iet~hotls section and was isolated by c,lIromatogrxpl,y in solvent I\‘. Xfter hydrolysis at pFI 2 (0.01 s HCl, 10 minutes at 100’) :mtl admixture with authentic L-rhamnosc, the hydrolyzxtr was c~hromatographed in solvcnts I and III and rlect,rophoresed in 0.05 31sodium trtrabornte buffer, pH 9. In eac~hsystcw the ratlionctivc COII~pourd and authentic2 I)-rhamnosc wirwided csact ly . Additional cviderwc for the proposed struc%ure of the w~~~pountl was provided by an oxpcriment, in which radionc~tivc rutin [:3-clucrc~etirl-0-Lu-L-rh:lnlnosyl (1 -+ 6) P-J:glucoside] was formed eiizyniic*nlly from UDl’-L-rli~~iiiJiosC-14(‘ ‘ 5I :mtl the :rppropriatje acceptor. The details of this enzymica TC:IVtion w-erc published wrlier (10). An xmmonium sulfate fraction was prep:tretl from TG%F: RATIOS
OF
THE
STNTHESIZEI)
UDP-
r,-rbamnose
mp III@
E;NZPMICALLY
Published value for UDP-~-glucose (15)
0.7G 0.43
TABLE CHEMICAL
OF
l:DP-1:1~HAYSOHE Enzymic
Ratio
250/2ao 280/260
II
~4BSORB.iNCIES
ANALYSIS
OF
0.74 0.38
III ENZYMICALLY
E’oR~IED
UI)P-I.-RHAMNOSE Analysis .~
Uridine L-Rhamnose Reducing sugar (after hydrolysis at pH 2, 15 minrltt=s at, 100”) Total phosphate
Total hmole) -~______---.--
Ratio
0.058 0.058 0.061
1.00 1.00 1.05
0.123
2.12
662
BARBER
mung bean leaves and was incubated with algal UDP-L-rhamnose-i4C which had been synthesized and purified as described in the preceding paragraph. The reaction mixture conbained 20 ~1 mung bean leaf enzyme (about 1.0 mg protein in 0.025 M Tris.HCI0.01 M mercaptoethanol buffer, pH 7.5), 30 pg quercet-in-3-glucoside, 0.3 pmole ATP, and 1.6 X 10F4 bmole UDP-L-rhamnose-14C (0.01 PC) in a total volume of 25 ~1. The mixture was incubated for 45 minutes at 37”. It was added to 0.75 ml of methanol, and t,he precipitate was removed by centrifugation. Authentic rutin was added to the met’hanolic supernatant solution which was then evaporat,ed to a small volume and chromatographed on washed paper with solvent’ II. Rutin was located by its characteristic yellow fluorescence under ultraviolet light when the paper was exposed to ammonia vapor. The radioactive compound and rutin coincided exactly. Further evidence that t,he compound formed in this way was i4C-labeled rutin was obtained from the demonstration that radioactive rutinose (0-cu-L-rhamnosyl (1 -+ 6) n-glucose) could be released from the compound by mild acid hydrolysis. 14C-Labeled rutin was eluted from the paper with methanol and evaporated to dryness in a tube. It was treated with 10% acebic acid at 100” for 60 minutes (17). The hydrolyzate was evaporat)ed to dryness in vacua and chromatographed in solvent I. Two radioactive areas were detect,ed on the chromatogram, one in the position of rutin and the ot)her in the position of authentic rutinose. Formation of an intermediate. An intermediate which has been found in the enzymic formation of most of the 6-deoxyhexoses and 3,6-dideoxyhexoses investigated so far (4) is the 4-keto-6-deoxysugar nucleotide derivative. The algal system responsible for the synthesis of UDP-n-rhamnose seems to follow this same general pattern. Thus, when a mixture was prepared and incubated as described in the Methods section, except that TPNH was omitted, a radioactive compound could be isolated from the mixture which showed the properties to be expected of UDP-4-keto-6-deoxy-n-glucose (2). This compound migrated upon electrophoresis at pH 3.7 like a UDP-hexose. Upon hydrolysis
AND
CHANG
in acid (0.1 N HCl, 10 minutes at 100”) a radioactive subst’ance was produced witjh n mobility in solvent I slightly less than that of L-rhamnose. When the UDP-hexose was first reduced chemically with YaBH4 (1 5 pmoles phosphate buffer, pmole NaBH4, pH 7.0, in a total of 20 ~1; allowed to react 15 minutes at 25’), elecbrophoresed again to remove salts, and t,hen hydrolyzed as before, t’wo new radioactive compounds were produced. Upon chromatography in solvent I and solvent III, ‘kc-labeled compounds with the mobilities of authentic nglucose, L-fucose, and 6-deoxy-n-glucose were observed. The epimers to be expected after the chemical reduction of the 4-keto group of such an intermediate would be n-fucose and 6-deoxy-n-glucose. (Solvent 111 readily separates the three 6-deoxyhexoses, fucose, rhamnose, and 6-deoxyglucose.) DISCUSSION
The reproductive process in Chlorella is somewhat more elaborate than it is in many other unicellular organisms. In brief, at a certain stage in its life cycle a Chlorella cell enlarges and produces two, four, eight, or more autospores which are released into the surrounding medium by rupture of the wall of the mother cell (18). Since only a very small proportion of cells in the total population is in a particular stage of this process at any given time, it is undoubtedly important to know if a compound of enzymological interest is produced continuously or only at one short, period in the life of the cell. It appears, for example, that the format,ion of cell walls in Chlorella occurs during autospore generation and over a relatively short period of time (18). It is thus quite possible that the synthesis of what are presumably cell wall precursors, such as UDP-n-rhamnose and other sugar nucleotides, also occurs during only a portion of the cell’s life (19). It is unfortunate t,hat this possibility was not considered during the course of the research reportsed here. It is hoped that in future studies of sugar nucleotide metabolism and cell-wall formation in this group of organisms synchronous cultures at some given stage will provide a richer source of enzyme. Despit’e the heterogeneous population of
algacl usctl for the extraction of the rhamnose-synthesizing enzyme, there seems, lit& doubt1 kat extrarts of C. pyrenoidosa do form lJl)P-r,-rharnnose. It, remains now to show the function of that) wmpound in the nwtabolism of the algal c*ell.
AhI
This work 10(%2-01
was supported PC.
in part
by NIIL
grant
10. B.IRISEI~, G. A., Hiwhemislry 11.
Chem. 12. I)~scxE, Chem.
13. I?\ItK,
1). II., (:OULDING, K. J., .\ND R,. W., B&hem. J. 70, 391 (1958). BARBER, G. A., ‘lrch. Kiochem. Biophgs. 103, 276 (19(i3). K.zr-SS, H., Biochem. Biophys. Res. Commun. 18, Ii0 (1965). GINYBCRG, V., d duan. Enzymol. 26, 35 (19fi4). GINSBURG, V., J. Biol. Chem. 241, 3750 (19%).
4. 5.
6. H.\SSID, w. z., Enzymol. 7. XECFELD,
10,123 F.,
E.
.YND
M., ;~daan.
~)O~DOROFF, V.,
PUTNAM,
15. iY.\~r~Ios.~I,
16.
FANSHIER,
17. 18.
E.
W. Z., .lwh. Bioch,enz. Biophys. 69, 602 (1957). 8. NEUFELD, fi:. F., Biochem. Biophys. Res. conlm~rn. 7, 461 (1962). 9. Tnucco, R. E., CAPUTTO, II., LELOIR, L. F., AND MITTEI.M.~N, pr'., Llrch. Biochem. 18, 137 (1948). W.,
I).,
J.
Chem.
(1950). GIKSBURG, AND
.\SD
T.,
AND
1, 463 (1962). F. W.,
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~OHSSUN,
181, l-19 (1949). 14. AMES, B. h’., AND I)YRIN,
HOHNE,
3.
&I.,
27, 422 (3955). Z., .INI) SHIGTTLES, 175, 595 (I9G).
,!nd.
I,.
B.,
J.
Ijiol.
51.
J.,
J.
f{iOl.
T.,
J.
/jiol.
I~ASSII~,
19.
11.
235, 769 (1960). AC.WEJIY-
OF
SCIENCES-SATIONAL
COINCIDE, “Specificat,ions and Criteria for Biochemical Compounds,” 1’111~ liratiw il9. Washingt>on, 11.C. (1960). I>.\vIs, 141. A., .\sI) I)EIMICK, K. C:., in “Algal Cldt,lu-e from Laboratory to Pilot Plant” (J. S. Blulew, ed.), l’ubliratiou (iO0, p. 119. Carnegie Institute, Washington, D.C. (1953). 41t.1~.1n..1~ II., Nippon k’nyaku Zasshi 77, 1314 (19%) [Chem. A&s/r. 53, 20316d, (1959)j. MVR.UL~MI, S., MONIM~-ELI, Y., ANI) TIK.\wY.1, A., in “Studies 01~ Microalgae and Photosynthetic Bacteria“ (Japallese Sot. I’l. Physiol., ed.), p. 65. Univ. of Tokyo Press (1963). IV-.~uIL~, T., KANAZ.LWI, T., .\ND K~N.Iz.Y\v.L, K., in “Studies on Rlicroalgae and Photosyllthet.ic Bacteria” (Japanese Sot. 1’1. Physiol, cd.) 11. 58i. Univ. Tokyo Press (1903). RESEAIWH
1. xORTHCOTE,
A.
C’hem.
REFEREXCES
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