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NAD-dependent l -fucose dehydrogenase from sheep liver
NAD-Dependent
L-Fucose
Iteceived
Febrll:rry
Dehydrogenase
25, 1970;
accepted
from
.4pril
Sheep
Liver’
21, 1970
L-Frmxe dehydrogenase has been purified :ibo\lt &fold from the solrrble fraction of sheep liver. The enzyme accepts cithcr r,-fucose or wurabinosr as srlbstrntes but is specific for NAI) xs coenzyrne. IXsc-gel elcctrophoresis failed to separate the ,zctivit,irs obtained with r,-fucose and warwbinose substrates. The pr1 maximum is 10.4; at this ])H, the K, va111es are 1.5, 7.2, and 0.19 mM for I,-fucose, i)~ar:tbinose, and XAI), respectively. The product obtained with the L-fucose s~~bstr:~te under these conditions was r,-fuconic acid; with n wxrnbinose substrate, o-xxbonic acid was formed. II(~~I~IcosP, u-g:tl:mtose, o-xylose, and r,-ar:~binose were not sltl)~tr:lirs for I his enzyme.
I,-l~ucow
[(j-deox--L-galactosc] is 3 sugu of blood group substances, plxsmn gl~xqroteins, and cell membranes (l-3). Elex-nted protein-bound fucose levels Il:r\-e brrn wported to occur in the plasma of clixbtstics (4). C.hrrently, little is l;no\vr~ of th: pt,ll\v:~ys of cat:zbolism of this sugar. Ho\\-wer, ScIlacllter et al. (5) hvc recently ITlwtvtl :I study of pig liver L-fucose delrydrog’Il:l~~~, nntl thy postulnte tliatl hlris c~rizyme Inay phy :L role in mxnmahrt fucose catabolism. Other dellydrogtwwes Iraring :lrt,ivitJ. with structurnlly similar aldose subs;tr:ltcs h:~v(~ been found in mnmm:Aan livrkrs by Scllim:w:~ et al. (0, 7) and b)llctzger and Wick (8). In t’llis I):~pcr, n-e report the parti:d purific:ltion and clltrr:tcterizut,ioll of :I SLID-del)endcnt L-fucose dellydrogrnaw from sheep liver. 13oth I,-fucose and u-arabinow serve :I’ substrxtrs for the enzyme, but the I<,,( is lower for the former.
adjusted to pII i.5 Iwforc 1~. I)-Ar:ll)ollo-y-l:ictone was obtained from IX anal K I,nbnratorics and was twice rccryst:Illizcd before IIY~. 1,.Fuconic acid was prepared ))y the niethotl of Clark (!,), All other chemicals rmployed were commercial products arid were usctl without, fllrther purific:Ltion. Ens!/mc assrcy. The fornation of ShI)H w:ts followed at 340 111~ Iwing :t ISeckrn:tn I)B spectrophotornclcr. Incl~bation niist~wes of 1.0 ml I ot :rl vollm~c: collt:lincd the following: 0.05 11 buffer, 0.89 rii>l XAl I or NhL)P, 0.05 hl substrate, plus an aliquot of I he cnz~me prep:uxt,ioll. Control incubatioii mixtllres lncketl slibstral(~ except. in iests at high pIT, in which case the cnzyrnc preparation w:Ls omitted. All assays wcrc ruin at 25”. Protein W‘:LS tlctcrminetl either by t hc Ijiuret met,hod or that of Idowry cl al., borh ai; cksc*ribetl bl, I,:ryne (loj f)i.rc-ye1 c,l~~~l,upko,~c.sis. hnalytiwl gels were run on a ~::11131cY) :inalytical disc-gel clectrophorrsis :rppnr:ttlls. The gels were 7“; in polyacq-lxnitle alid wcrc run aI 3 rtiA per tubule. Protein I)antls \vercl stained with :unitlo black while wtivilirs \verc detected :IS tledchydrogemw scribed by 8chiww:i ci nl. (6).
comlwntM
83
84
MOBLEY,
METZGER,
sucrose and centrifuged at 25,000g for 20 min.2 The resulting supernatant solution was poured through cheese cloth and solid protamine sulfate was added, with constant stirring, in an amount equivalent to one-fifth the total milligrams of protein present. The precipitate from this treatment was removed by centrifugation and discarded. To the protamine-treated supernatant, solut.ion was added 176 g of ammonium sulfate per liter of preparation. The mixture was stirred constantly for 20 min and then centrifuged. The pellet was discarded and the supernatant solution treated with an additional 198 g of ammonium sulfate per liter of preparation. After stirring for 20 min, (he material was cent.rifuged and the pellet suspended in a minimal amount of buffer. The enzyme solution was then dialyzed overnight against 10 times its volume of buffer; the buffer solution was changed once during the course of the dialysis. After dialysis, the enzyme solution was applied to a 4.5 X 70.cm DEAE-cellulose column. The column was washed with buffer and eluted with a linear gradient ranging from 0 to 0.3 >\I 9aCl in a total of 2 1. of buffer. 9 large peak of activity eluted from the column at a concentration of about 0.1 M NaCl. The tubes containing activity were pooled and the volume of the enzyme preparation reduced by use of an ilmicon ultjrafiltration cell fitted with the UM-10 membrane, which retains molecules with a molecular weight greater than 10,000. The concentrated solution was applied to a 3 X 35.cm Sephadex (;-lo0 colu~rm and eluted with bulfer. A typical elution pattern from the Sephadex G-100 colu~nn is shown in Fig. 1. The tubes containing the enzymatic activity were pooled; this was the preparation employed in the experiments to be described. The purification procedure is summarized in Table I. RESULTS
Throughout the purification procedure, a high n-arabinose dehydrogenase activity coincided with the L-fucose dehydrogenase activity as seen, for example, in Fig. 1. Analytical disc-gel electrophoresis of the purified enzyme solution showed four distinct protein bands when stained with amido black reagent. Staining for dehydrogenase activity with n-arabinose and L-fucose substrates gave one band. Further evidence that a single enzyme possessed both activities was 2 Centrifugation of sheep liver homogenates at 88,OOOg for 1 hr gave a supernatant that had activity comparable to the 25,000g supernatant.
ANLI
WICK
FIG. 1. Ii:lution pattern of t-fucosr dehydrogenase and n-arabinose dehydrogenase activit,ies from a Sephadex G-100 column. Protein levels (0) were estimated by the absorbance of the solut,ions at 280 mp. Dehydrogenase activities were estimated by following the increase in absorbance at 340 mp in the presence of I.-fucose (0) and I,-arabinose (a) substrates.
Procedure 6’ ..-
Crude supernatant 30-(iO$b ammonium sulfat,e 11EAE (;-loo a Units
are pmoles
(__
1030.13 540.19 NADH
~~~~
/ 10 12.3 i 2.5l47.5 formed
11.8 44.X
min.
obtained when gels which were cut longitudinally and incubated separately with either n-arabinose or L-fucose gave a single continuous band when the gels were r+ joined. The rate of reaction was constant \vith time of incubation and proportional to the enzyme concentration. The optimum pH fol I,-fucose dehydrogenase activity is approsimat,ely 10.4, as shown in Fig. 2. At this pH, the I<, is 1.5 rnl\r for L-fucose, 7.2 rn.\r for rj-nrabinose, and 0.19 rnlr for SAD. At pH 8.5, the substrate K, values were lower: 0.074 m\z for L-fucose and 0.40 ml1 for IF ;ybinosn, as shown in Fig. 3. The relativr lllaX values for n-arabinose and L-fucose A pH 10.4 are approximat’ely equal; houever,
i
PH
14’1~;. 2. \Yarintion ill L-fucose dehydrogcrlxsr activity with pH. The purified enzyme was :I+ snycd :IS described in the text, using 0.05 M Trisrrralc:rte (A). 0.05 hf g1ycylylycine (Cl), or 0.05 tir glycincl (0) bluffer at the appropriate pH. The CIII’VC for the mar;~biuose dehydrogenast: act ivit 1 (*oillcidm1 wit.h that shown :rbovr.
0
100
200
300
400
500 u
600
100
800
900
9
171~;. 3. Iktcrminatiou of K,,, aud I-,,,;,, values for I.-fllcose dchydrogemse (0) mtl u-arsbinose tlchydrogeume (A) nctivitics at pII 8.5. VelociI its are given in terms of the change in absorbnnr~ at S-10 mp per 5 miu; substrate concentkations arc itt units of ruolarity. The assay was performed :IS ~leacribctl in the test, iming 0.05 II Tris-Cl buffer.
;It pH $5, tlw ratio of the ly,,,,, for w:wbi110w compared to that of L-fucow is 1.1. Thus, at, lower pH, warnbinose is oxidized at :t gwat,er rate than I,-fucose. The substrate specificity of the enzyme Inyp:w:ltion wit,11 compounds other than Lfr~ose and n-nrabinose is also altered by pH. gives about, ‘It pH 10.4, Zdeoxy-u-ribose 10 f A of the activity found w&h the L-fucose wxylose, I)+ubst’rattl, while D-galactose, ribose, I)-glucose, and L-:wabinosr yielded ltw than 5 “; activity at this pH. IZt pH ,\..j, L’-d~,oxv-r)-l.ibose ga\:o 42 5’; and wribose g;:rw 10’ P of the activity found \vith :I I)-
while the other comambinose substlatr, pounds liskd above yielded ncgligiblr act,ivity. L-Gaktct~ose, :I compound which may :~lso br a substrate for L-fncose dehydrogen:w (S), was un:~v:&,blc for testing. At botSh pH values, the activitv with iYADP was 5 5 of the value found using SAID. The purified c’nzyme 1~:~s sitible at 4” for periods up to 1 month in the presence of KAD. The moltakr weight of the enzyme \\-:IS determined to be approximately !%,OOO by Srphndex G-100 chrom:ltogr:lpll~, as described b). Whit:tl;cr (11). Tllc product of e:Ai react ion WIS identified by paper chromatogr:~phy and t lrin-layel cllroma.tof;rnpll;v. using either etliyl :tcet:~te: formic acid: wai er (3: 1 : 1) or tr~butanol: :LCP~ic acid: \vater (4: 2: I) as solvrnt systems. l)d ection of the products 1~:~s ~w2omplishrd using cithcr :I periodat ebenzidine spr:l>’ (12) or hydroxylnmineferric chloride spray for l:~ctonrs (12). Thr> product obtained using L-fucose as subst rat c migrated idtwi ically If-it h tire r,-fuconic acid standard. Similarly, the product using :I r)-wabinose substrai e migrnt rd identically to tllc I)-alnborlol:rctoIle st:md:ud. Tlrc stnndunder conditions :irds \vcw equilibrated similar to the enzyme incubation systtm, so tliat tlic standards rcprcsent :L mlxutru.11 nf t hr approprial P frer acid and Iact 0110. Purifird mamm:Lli:~n chnzymcs having I,fucose and n-arabinose dehydrogenase wtivities have only recently been reported (.5--S). The structural requirements of the substrates for tile slipup liver I,-fncose dnhydrogenasc art similar to thrw enzyme systems. The sheep liver I,-fucosr dehydrogcnase differs from the rat livrr enzyme (S) in its cocnzyme specificit\(KAD for shtq, K&IDE’ for rat), and from both pork liver (5) and rat, liver prep:w:~tions in its higher pH maximum. d SAD-dcpcndcnt pentose dehydrogcnase from pork liver (6) has ;I, knilar pH maxkmml, but, this cnzl-me \V:LSnot, extensively purificbd nor WIS L-fucosct reported as :I substratjr. The substrate specificity of the sliecp liver I,-fucosr delrydrogrnnse dist~inguislies it from &her :ddose deh~~drogen:w;rs which act, on wgy1acto.w
(13, 14), I,-glucose (1.5), and n-sylose (16, 17). The catabolic f:LtC’ of L-fuco;ie and I)arabinose in sheep is not l~nown, but n role for L-fucose dehydrogenase in the metnbolism of these sugars is a possibility. wArabon& is known to be metabolized by rat liver preparations (IS). A4n enzyme acting upon a L-fuconnte substrate, r.-fuconat’e dehydratase, occurs in pork liver (19). Thus, L-fucose dehydrogenase n-a? initiate a path IV:L~ of L-fucosc and u-arabmose catabolism six&r to the patliwng which exists in wrtain bacteria (20).
t.
ti('~In-.\~.\,
G.,
W.,
nical
Rlrs.
Jlldith
(:uilmette
for
her
tech-
1<. G,,L~.
engl.
11. 12.
2. SPIRO,
IL. C,., A:. Engl. H. B., AXD 190 (19G9).
164, 4.
XICMILL.\N,
I).
5. SCHACHTI:R, AND
(1969).
If., HOSEK~~,
.\XD
s..
J.
/jj0/.
SI:~,\L,
S.,
.I.
Ij’iol.
241,
5910
C~~~TRIX.IS.~S,
?~II,:TzGI:R,
1:.
I’.,
(1969).
18.
S.~X.I:Y, S., J.
J., Biol.
129
M&EIRE, Chem.
(19GS).
19.
IX. J., 244, 4785
~1l:TZGER, L)iego 1967. YUEN, II.,
(1906). W~I,cos, C%em. Las~~:rz,
R., Fed.
11..
NV~OKORO, Fed.
pp.
S. S.,
313,
.LNI)
7!)1.
Wrc~i,
240, 2767 11065). L., Riochen/. Netl. Biochwr.
1%. P., Doctoral State and Univ.
I’roc. 29, 675 (1970).
20.
Press,
~?hmrn:r-
SI:C+\I.,
1043
16,
K.,
5904 (1966). P., .mn
C~J;\TI~IICAS~\S,
281,
A?es.
~IIACEK,
241,
l-ork
J. Med.
I<:., Clin.
/jl'r,~,hc,,/.
Academic (1963). P., AND
(1969).
S. S., Science
I.,
“I%per
Chem. 14.
-4.
H.ws. I. M., tography,” New
13.
ARTD WICK,
3, -248 (Mii) 35, 1950 (1963).
991
MARTIN,
349,
LIYNI:, 15., Jlethods Enzynzol. W~IMIT.\I
281,
3. BOSZVL~NN,
\(:I;,
9. 10.
P.,
J. Med.
REFERENCES 1. SPlRO,
I)OV (‘hcttt.
Biophys. 1l’c.s. Commun. 26, 742 (1~7). Cr,~ne, E, P., J. Biob. Chem. 54, 65 (1922’).
A. N., J. Biol. T,;., .\sI) IG. Wmm, 53 (1968). VAN HEYSINGEN, li. (1958).
assistance.
ANI)
s.
15. thank
W.,
%. l’hy.siol.
1582 (19G8). &.II:TZGl~:R, R
(‘hem. We
DO&L~CHKE,
Hoppe-Seyler’s
I.
IJissert Cxlif. N.,
Amer.
ASI)
2,
69,
-181
alion, 5an Sntt I)icgo, STH.L(~TKK,
Sac.
~'.\I,LI.:RosI, N. J., :\ND ~)OUl,OROPI~, Rd. Clwm. 223, 199 (1956).
h’xp.
Iliol. hl.,
./.
L-Fucose
Iteceived
Febrll:rry
Dehydrogenase
25, 1970;
accepted
from
.4pril
Sheep
Liver’
21, 1970
L-Frmxe dehydrogenase has been purified :ibo\lt &fold from the solrrble fraction of sheep liver. The enzyme accepts cithcr r,-fucose or wurabinosr as srlbstrntes but is specific for NAI) xs coenzyrne. IXsc-gel elcctrophoresis failed to separate the ,zctivit,irs obtained with r,-fucose and warwbinose substrates. The pr1 maximum is 10.4; at this ])H, the K, va111es are 1.5, 7.2, and 0.19 mM for I,-fucose, i)~ar:tbinose, and XAI), respectively. The product obtained with the L-fucose s~~bstr:~te under these conditions was r,-fuconic acid; with n wxrnbinose substrate, o-xxbonic acid was formed. II(~~I~IcosP, u-g:tl:mtose, o-xylose, and r,-ar:~binose were not sltl)~tr:lirs for I his enzyme.
I,-l~ucow
[(j-deox--L-galactosc] is 3 sugu of blood group substances, plxsmn gl~xqroteins, and cell membranes (l-3). Elex-nted protein-bound fucose levels Il:r\-e brrn wported to occur in the plasma of clixbtstics (4). C.hrrently, little is l;no\vr~ of th: pt,ll\v:~ys of cat:zbolism of this sugar. Ho\\-wer, ScIlacllter et al. (5) hvc recently ITlwtvtl :I study of pig liver L-fucose delrydrog’Il:l~~~, nntl thy postulnte tliatl hlris c~rizyme Inay phy :L role in mxnmahrt fucose catabolism. Other dellydrogtwwes Iraring :lrt,ivitJ. with structurnlly similar aldose subs;tr:ltcs h:~v(~ been found in mnmm:Aan livrkrs by Scllim:w:~ et al. (0, 7) and b)llctzger and Wick (8). In t’llis I):~pcr, n-e report the parti:d purific:ltion and clltrr:tcterizut,ioll of :I SLID-del)endcnt L-fucose dellydrogrnaw from sheep liver. 13oth I,-fucose and u-arabinow serve :I’ substrxtrs for the enzyme, but the I<,,( is lower for the former.
adjusted to pII i.5 Iwforc 1~. I)-Ar:ll)ollo-y-l:ictone was obtained from IX anal K I,nbnratorics and was twice rccryst:Illizcd before IIY~. 1,.Fuconic acid was prepared ))y the niethotl of Clark (!,), All other chemicals rmployed were commercial products arid were usctl without, fllrther purific:Ltion. Ens!/mc assrcy. The fornation of ShI)H w:ts followed at 340 111~ Iwing :t ISeckrn:tn I)B spectrophotornclcr. Incl~bation niist~wes of 1.0 ml I ot :rl vollm~c: collt:lincd the following: 0.05 11 buffer, 0.89 rii>l XAl I or NhL)P, 0.05 hl substrate, plus an aliquot of I he cnz~me prep:uxt,ioll. Control incubatioii mixtllres lncketl slibstral(~ except. in iests at high pIT, in which case the cnzyrnc preparation w:Ls omitted. All assays wcrc ruin at 25”. Protein W‘:LS tlctcrminetl either by t hc Ijiuret met,hod or that of Idowry cl al., borh ai; cksc*ribetl bl, I,:ryne (loj f)i.rc-ye1 c,l~~~l,upko,~c.sis. hnalytiwl gels were run on a ~::11131cY) :inalytical disc-gel clectrophorrsis :rppnr:ttlls. The gels were 7“; in polyacq-lxnitle alid wcrc run aI 3 rtiA per tubule. Protein I)antls \vercl stained with :unitlo black while wtivilirs \verc detected :IS tledchydrogemw scribed by 8chiww:i ci nl. (6).
comlwntM
83
84
MOBLEY,
METZGER,
sucrose and centrifuged at 25,000g for 20 min.2 The resulting supernatant solution was poured through cheese cloth and solid protamine sulfate was added, with constant stirring, in an amount equivalent to one-fifth the total milligrams of protein present. The precipitate from this treatment was removed by centrifugation and discarded. To the protamine-treated supernatant, solut.ion was added 176 g of ammonium sulfate per liter of preparation. The mixture was stirred constantly for 20 min and then centrifuged. The pellet was discarded and the supernatant solution treated with an additional 198 g of ammonium sulfate per liter of preparation. After stirring for 20 min, (he material was cent.rifuged and the pellet suspended in a minimal amount of buffer. The enzyme solution was then dialyzed overnight against 10 times its volume of buffer; the buffer solution was changed once during the course of the dialysis. After dialysis, the enzyme solution was applied to a 4.5 X 70.cm DEAE-cellulose column. The column was washed with buffer and eluted with a linear gradient ranging from 0 to 0.3 >\I 9aCl in a total of 2 1. of buffer. 9 large peak of activity eluted from the column at a concentration of about 0.1 M NaCl. The tubes containing activity were pooled and the volume of the enzyme preparation reduced by use of an ilmicon ultjrafiltration cell fitted with the UM-10 membrane, which retains molecules with a molecular weight greater than 10,000. The concentrated solution was applied to a 3 X 35.cm Sephadex (;-lo0 colu~rm and eluted with bulfer. A typical elution pattern from the Sephadex G-100 colu~nn is shown in Fig. 1. The tubes containing the enzymatic activity were pooled; this was the preparation employed in the experiments to be described. The purification procedure is summarized in Table I. RESULTS
Throughout the purification procedure, a high n-arabinose dehydrogenase activity coincided with the L-fucose dehydrogenase activity as seen, for example, in Fig. 1. Analytical disc-gel electrophoresis of the purified enzyme solution showed four distinct protein bands when stained with amido black reagent. Staining for dehydrogenase activity with n-arabinose and L-fucose substrates gave one band. Further evidence that a single enzyme possessed both activities was 2 Centrifugation of sheep liver homogenates at 88,OOOg for 1 hr gave a supernatant that had activity comparable to the 25,000g supernatant.
ANLI
WICK
FIG. 1. Ii:lution pattern of t-fucosr dehydrogenase and n-arabinose dehydrogenase activit,ies from a Sephadex G-100 column. Protein levels (0) were estimated by the absorbance of the solut,ions at 280 mp. Dehydrogenase activities were estimated by following the increase in absorbance at 340 mp in the presence of I.-fucose (0) and I,-arabinose (a) substrates.
Procedure 6’ ..-
Crude supernatant 30-(iO$b ammonium sulfat,e 11EAE (;-loo a Units
are pmoles
(__
1030.13 540.19 NADH
~~~~
/ 10 12.3 i 2.5l47.5 formed
11.8 44.X
min.
obtained when gels which were cut longitudinally and incubated separately with either n-arabinose or L-fucose gave a single continuous band when the gels were r+ joined. The rate of reaction was constant \vith time of incubation and proportional to the enzyme concentration. The optimum pH fol I,-fucose dehydrogenase activity is approsimat,ely 10.4, as shown in Fig. 2. At this pH, the I<, is 1.5 rnl\r for L-fucose, 7.2 rn.\r for rj-nrabinose, and 0.19 rnlr for SAD. At pH 8.5, the substrate K, values were lower: 0.074 m\z for L-fucose and 0.40 ml1 for IF ;ybinosn, as shown in Fig. 3. The relativr lllaX values for n-arabinose and L-fucose A pH 10.4 are approximat’ely equal; houever,
i
PH
14’1~;. 2. \Yarintion ill L-fucose dehydrogcrlxsr activity with pH. The purified enzyme was :I+ snycd :IS described in the text, using 0.05 M Trisrrralc:rte (A). 0.05 hf g1ycylylycine (Cl), or 0.05 tir glycincl (0) bluffer at the appropriate pH. The CIII’VC for the mar;~biuose dehydrogenast: act ivit 1 (*oillcidm1 wit.h that shown :rbovr.
0
100
200
300
400
500 u
600
100
800
900
9
171~;. 3. Iktcrminatiou of K,,, aud I-,,,;,, values for I.-fllcose dchydrogemse (0) mtl u-arsbinose tlchydrogeume (A) nctivitics at pII 8.5. VelociI its are given in terms of the change in absorbnnr~ at S-10 mp per 5 miu; substrate concentkations arc itt units of ruolarity. The assay was performed :IS ~leacribctl in the test, iming 0.05 II Tris-Cl buffer.
;It pH $5, tlw ratio of the ly,,,,, for w:wbi110w compared to that of L-fucow is 1.1. Thus, at, lower pH, warnbinose is oxidized at :t gwat,er rate than I,-fucose. The substrate specificity of the enzyme Inyp:w:ltion wit,11 compounds other than Lfr~ose and n-nrabinose is also altered by pH. gives about, ‘It pH 10.4, Zdeoxy-u-ribose 10 f A of the activity found w&h the L-fucose wxylose, I)+ubst’rattl, while D-galactose, ribose, I)-glucose, and L-:wabinosr yielded ltw than 5 “; activity at this pH. IZt pH ,\..j, L’-d~,oxv-r)-l.ibose ga\:o 42 5’; and wribose g;:rw 10’ P of the activity found \vith :I I)-
while the other comambinose substlatr, pounds liskd above yielded ncgligiblr act,ivity. L-Gaktct~ose, :I compound which may :~lso br a substrate for L-fncose dehydrogen:w (S), was un:~v:&,blc for testing. At botSh pH values, the activitv with iYADP was 5 5 of the value found using SAID. The purified c’nzyme 1~:~s sitible at 4” for periods up to 1 month in the presence of KAD. The moltakr weight of the enzyme \\-:IS determined to be approximately !%,OOO by Srphndex G-100 chrom:ltogr:lpll~, as described b). Whit:tl;cr (11). Tllc product of e:Ai react ion WIS identified by paper chromatogr:~phy and t lrin-layel cllroma.tof;rnpll;v. using either etliyl :tcet:~te: formic acid: wai er (3: 1 : 1) or tr~butanol: :LCP~ic acid: \vater (4: 2: I) as solvrnt systems. l)d ection of the products 1~:~s ~w2omplishrd using cithcr :I periodat ebenzidine spr:l>’ (12) or hydroxylnmineferric chloride spray for l:~ctonrs (12). Thr> product obtained using L-fucose as subst rat c migrated idtwi ically If-it h tire r,-fuconic acid standard. Similarly, the product using :I r)-wabinose substrai e migrnt rd identically to tllc I)-alnborlol:rctoIle st:md:ud. Tlrc stnndunder conditions :irds \vcw equilibrated similar to the enzyme incubation systtm, so tliat tlic standards rcprcsent :L mlxutru.11 nf t hr approprial P frer acid and Iact 0110. Purifird mamm:Lli:~n chnzymcs having I,fucose and n-arabinose dehydrogenase wtivities have only recently been reported (.5--S). The structural requirements of the substrates for tile slipup liver I,-fncose dnhydrogenasc art similar to thrw enzyme systems. The sheep liver I,-fucosr dehydrogcnase differs from the rat livrr enzyme (S) in its cocnzyme specificit\(KAD for shtq, K&IDE’ for rat), and from both pork liver (5) and rat, liver prep:w:~tions in its higher pH maximum. d SAD-dcpcndcnt pentose dehydrogcnase from pork liver (6) has ;I, knilar pH maxkmml, but, this cnzl-me \V:LSnot, extensively purificbd nor WIS L-fucosct reported as :I substratjr. The substrate specificity of the sliecp liver I,-fucosr delrydrogrnnse dist~inguislies it from &her :ddose deh~~drogen:w;rs which act, on wgy1acto.w
(13, 14), I,-glucose (1.5), and n-sylose (16, 17). The catabolic f:LtC’ of L-fuco;ie and I)arabinose in sheep is not l~nown, but n role for L-fucose dehydrogenase in the metnbolism of these sugars is a possibility. wArabon& is known to be metabolized by rat liver preparations (IS). A4n enzyme acting upon a L-fuconnte substrate, r.-fuconat’e dehydratase, occurs in pork liver (19). Thus, L-fucose dehydrogenase n-a? initiate a path IV:L~ of L-fucosc and u-arabmose catabolism six&r to the patliwng which exists in wrtain bacteria (20).
t.
ti('~In-.\~.\,
G.,
W.,
nical
Rlrs.
Jlldith
(:uilmette
for
her
tech-
1<. G,,L~.
engl.
11. 12.
2. SPIRO,
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