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Galactose metabolism of the red cell
E:cp. Eye Res. (1971) 11, 402--t14 Galactose
Metabolism
of the Red
Cell*
Wo,x- O. No
Depa rtment.~ oj" B iochemi.,'try and Pcdiatric.s', University of Souther~ Californi~, ,School of llietlicitw. Los ,4 ,~geles, Calif. 90033, U.S.A. (~7l(l
Children's Hospit¢H of Los Angele.% Los Angelica, Calif. 90027. U.S...I. The human erTthrocyt¢ offers an opportunity for investigation of galactos¢ metxLbohsm because the complete normal pathway exists in thin accessible tissue. The erythrocyte lu~. proven to be u.~eful not only in identifit.ation of the hetevozygous and homoT.ygous states in galactosemia and in the galactokinasa defect, but. a].~o irt the .~ttt(t)" Of variant.s. 1b is recognized, however, that, the erythrocyto is a special tyl~3 of cell aI~(l that findings from its study should not be extend~l to other tissues without reservation. With rc.'~pczct to the ervthrocyte itself, there still is much to be learned concerning intnxcellular kinetics under di~'(~re[~t conditions and the nature of the enzymes involved. Introduction O u r i n t e r e s t in g a l a c t o s e m e t a b o l i s m has e x t e n d e d o v e r a n u m b e r of years, a n d t h e h u m a n e r y t h r o c y t e h a s b e e n used in m u c h of this wo rk . T h e c o m p l e t e p a t h w a y s of g a l a c t o s e m e t a b o l i s m is p r e s e n t in red cells f r o m n o r m a l i n d i v i d u a l s , p r o v i d i n g t h e o p p o r t u n i t y for e x a m i n a t i o n of b i o c h e m i c a l e v e n t s in d i s o r d e r s of this p a t h w a y . T h e Principal Pathway T h e p r i n c i p a l p a t h w a y for t h e m e t a b o l i s m of g a l a c t o s e in the h u m a n e r y t h r o c y t e w a s s h o w n ( I s s e l b a c h e r , A n d e r s o n , K u r a h a s h i a n d K a l c k a r , 1956) to follow t h e s a m e s e q u e n c e of r e a c t i o n s w o r k e d o u t o r i g i n a l l y by a n u m b e r of i n v e s t i g a t o r s ibr t h e m e t a b o l i s m of g a l a c t o s e in y e a s t ( K o s t e r l i t z , 1943; C a p u t t o , Leloir, Card in i a n d P a l a d i n i , 1949; C a p u t t o , Leloir, Cardirri a n d P a l a d i n i , 1950; ~Leloir, 1951; K a l c k a r , : B ra ga nc a a n d M u n c h - P e t e r s o n , 1953): A T P + Galactose -~-Gal-I-P U D P - G l c + GaI-1-P ~,-~-UDP-Gal + G l c - I - P UDP-Ga] ,~-UDP-Glc
(I) (2) (3)
S u m : A T P + Galactose-->Glc-1-P + A D P T h i s p a t h w a y is k n o w n as t h e L e l o i r p a t h w a y ( F i s c h e r a n d W e i n l a n d , 1965). T h e r e a c t i o n s a r e c a t a l y z e d b y t h e e n z y m e s : (1) g a l a c t o k i n a s e , (2) g a l a c t o s e - l - p h o s p h a t e u r i d y l t r a n s f e r a s e ( G a l - I - P u r i d y l t r a n s f e r a s e ) a n d (3) u r i d i n e d i p h o s p h a t e - g a l a c t o s e - 4 e p i m e r a s e ( L r D P - G a l - 4 - e p i m e r a s e ) . T h e o v e r a l l r e s u l t is t h e c o n v e r s i o n of g a l a c t o s c a n d t h e f o r m a t i o n of g l u c o s e - l - p h o s p h a t e ( G l c - I - P ) , w h i c h t h e n is c o n v e r t e d to g l u c o s e - 6 - p h o s p h a t e (Glc-6-P) a n d f u r t h e r m e t a b o l i z e d t h r o u g h t h e g l y c o l y t i c p a t h w a y a n d t h e h e x o s e m o n o p h o s p h a t e s h u n t . This is also t h e m a i n p a t l ~ w a y for t h e m e t a b o l i s m of g a l a c t o s e i n t h e m a m m a l i a n l i v e r ( S t r o m i n g e r , 1960). * This investigation wins supporte.d by grants (A~f-04135, A~L04837, I:ID-00800 and RR-0086) from the National Institutes of Health, U.S. Public Health Service. 402
(;AI,A(~'I'OSI';
METABOLISM
OF
'FtIE
ICED
CELL
403
The s t u d y of e r y t b r o c y t e galaetose mctabolisln in m a n was greatly s t i m u l a t e d b y the loealizatio,~ of the enzymic defect in galactosemia a n d the d e m o n s t r a t i o n t h a t the erythroeyt,e is a suitable diagnostic tissue. Galactosemia is an inborn error of inetal,olism characterized by failure to thrive, susceptibility to infecbion, jaundice, hel)atomegaly, amino aciduria, ca[erects and m e n t a l r e t a r d a t i o n . The disease results fro,,: abse,we of (;el-1-P u r i d y l t r a n s f e r a s e (transferase) act, ivity in tissues (Reaction 2) (Kalclmr, Andcr.~on and lsselbacher, 1956; Anderson, K a l c k a r and Isselbacher, 1957a). The enzymic deficiency leads to a c c u m u l a t i o n of Gal-I-P in tissues when lactose or other galactose-containing foods are ingested (Schwarz, Goldberg, Komrow(,r and Itolzel, 1956). A nothl,:" met.al)olie disorder involving the galactose p a t h w a y has been described. The defect has t.~et.n demonstrat.ed to be in galaetokinase act.ivity (Gitzelmalm, 1965). The I)rinmry clinical m a n i f e s t a t i o n is the presence of cataracts. No case of U-D P-Gal4-epimerase (cpimer~sc) defect, has been reported.
Enzymes of the Principal Pathway Existing if'formation concerning the individual enzymes of the principal p a t h w a y of galactosc in the e r y t h r o c y t e has been derived prim'arily from studies with hemolysates. "Fhc rate c o n s t a n t s of the galactokinase, transferase and epimerase reactions, however, have been measured in i n t a c t red blood cells as well as in h e m o l y s a t e s (Hobinso,~, 1963; I'uck and Ilill, 1967; Hill and Puck, 1970). The relative a c t i v i t y values arc similar in both systems. B o t h gulactokinase and transferase of e r y t h r o c y t e s have been p a r t i a l l y purified (Mathai and Beutler, I967; ]liabov, Inouye, P a r k e r and I-Isle, 1965; 13cutler a n d Baluda, 19(;t;c) aml their kinetic properties have been examined. I s o l a t i o n of e r y t h r o c y t e transferase and of epinmrase has been in progress in our l a b o r a t o r y . Transfcrase preparat.io~s of greater purit, y t h a n those reported h a v e been o b t a i n e d . K i n e t i c st.udies ha:'e yielded x'alues concordant, with those o b t a i n e d by others. :Epimerase fractions, free of ~ransferase activity, have resulted as a b y - p r o d u c t of t h e t r a n s f e r a s e isolation. A consistent problem in isolation is increasing i n s t a b i l i t y of b o t h enzymes with c o n t i n u i n g purification. T.t~*LS 1
Relationship between galactosc oxidatio~ a.nd enzyme activity
(4rou p
Newborn infants Adults
14C02 f r o m [ l - ~ C ] G al
3,t,000 11,500
]Relative activity* of galactose enzymes Kinoze T. Epi~nerase
0-83 0"26
8-1 7-3
2-84 1 "39
* Aet.ix~ity is e x p r e s s e d a s m i e r o m o l e s o f s u b s t r a t e c o u x - e r t e d p e r h o u r p e r m i l l i l i t e r p a c k e d r e d b l o o d ceils.
The c r y t h r o c y t c s of n e w b o r n infants, in c o n t r a s t w i t h those of adults, h a v e a greater c a p a b i l i t y of utilizing galactose, as i n d i c a t e d b y t h e finding of increased galactose c o n s u m p t i o n , oxygen u p t a k e a n d m e t h e m o g l o b i n r e d u c t i o n (Betke, Baltz ~4
404
~V. G. N G
and Mass, 1960; Lachhein. Grube, J o h n i g k and Matt.hies, 1961). ~Ve have found t,h a t the r a t e of IaCO,, production from [1-1aC]Gal by intact e r v t h r o c v t e s of newborn infants is a b o u t three times that. of adulL~. Also, in hemolysates of newborn infanLs the act;ivity of all e]lzymes of the galaetose p a t h w a y were noted to be incroased (Table I). Transferase activity is itw.reased slightly, but galacrokinase and epimerase acl~ivities arc two to three times thost~ found for adults (Ng, Donnell, I [ o d g m a n and ]3ergren, 1967c; Ng, Bergren, Donnell and l-[odgman, 1967b; l.-)onne[I, Ng. l [ o d g m a n and ]3ergren, 1967b). Galactokinase and transferase activities are also elevated in e r y t h r o c y t e s of children wit, h D o w n ' s s y n d r o m e ( B r a n d t , Froland, Mikkelsen, Nielsen and Tolstrup, 1963; Krone. Wolf, Goedde and Bait.sch. 1965; Ng, Bergren and I)onnell, 1964a, b; Donne[l, Ng, Bergren, Melnyk and Koch, 1965; Rosner. Ong, Paine and M a h a n a n d , 1965). In h e m o l y s a t e s of adults, epimerase act.ivit.v is d e p e n d e n t on addctl nicot, inatnide adeifine dinucleotide (N,LD) (I~selbacher et al.. !956) because of the destruction of endogenous N:LD by stromal nicot, inamide adeniae dinucleotide nueleosidmse (NADase) liberated in the hemolvsates ( K i r k m a n and Maxwell, 1960). In contrast, we have found in h e n m l y s a t e s of newborn infants t h a t NADase v-ctivitv is ext, re1~mlv low and t h a t epimerase a c t i v i t y can be d e m o n s t r a t e d wit, hour addition of NA1) (Bergren, Ng and Donnell, 1967). H e m o l y s a t e s from certain adult.n resemble those of newborn infants in this respect. Such individuals have been found to have a geneticallyd e t e r m i n e d e r y t h r o c y t e N:kDase deficiency (Ng, Donnell and Bergren, 1968). The presence of N A D not only aft'eeLs epimerase activity but also changes the molecular form of the enzyme, as d e m o n s t r a t e d by starch gel electrophoresis (Fig. 1) O
÷ NADOSe
NAD
I
II
FIO. 1. S t a r c h gel clectrophoresis of U 1 ) P . G a l - , t - e p i m e r a s e from h e m o l y a a t e ~ . FluorcacenL b a n d s corre~Jponding to c p i m e r a s c a c t i v i t y are i n d i c a t e d b y solid b l a c k r e c t a n g l e s : the h a t c h e d r e c t a n g l e s repr,esent hemoglobirm.
(Bergren, 1967). ~Iemolysates from the newborn show two well-defined fluorescent bands, in cont=rast wibh the si~gle band of different mobiJity for hemoiysatcs from adults. W h e n a newborn h e m o l y s a t e is t r e a t e d wi~h N.&Dase, the two-banded epimerase is converted to the one-banded form. Couversely, when an a d u l t hemolysate is p r e i n e u b a t e d with N A D : two b a n d s c o m p a r a b l e to those for a newborn irdanb are found. W h e n NA_D is added to the starch gel itself, all hemolysates show two bands of ephnerase activity. Two e]ectrophoretic forms of epimerase also h a v e been reported to be present in bovine m a m m a r y gland preparations (Tsai, ]~[olmberg a n d Ebr/er, 1970). Other ]Pathways of Galactose Metabolism
I n addition to the principal p a t h w a y of galact~se metabolism, other p a t h w a y s h a v e been described or postulated as possible routes for the disposition of galactose (:Fig. 2).
GAI.AC'I'OSE
MI';TAIIOLISM
OF TIIE
B, E D C E L L
405
The conversion of galactose to galactitol, catalyzed b y aldose reductasc, w i t h reduced nieotinamide ade~line dinucleotide p h o s p h a t e (NADPI-I) as cofactor, has been described in aniJnal tissues (van l-leyningen, 1959; Kinoshita, Merola, Satoh and l)ikmark, 19(32). Substantial amount,s of galactitol have been found in autopsy samples of lens, brain, kidney and other tissues of galactosemie patients (Quan-Ma, Wells, Wells, S}~crman and Egan, 1966). A high concentration of galactitol is present in tim urine of patients with the transferase defect and also in those with galactokinase deficiency (~,Vells, Pitotmftn and Egan, 196¢; Gitzehnann, Curtius and :M/iUer, 1966). In rats fed galactose, galactitol has been shown to be present in red blood cells and in other t i ~ u e s (Quan-Ma and \Vells, 1965). UTP
\
G,QIQ¢Iilol
Gol
PP
u~Dp~ El
K J - GaI-I-P.-~,...-,~' (K)
/
C~1!6_P
{T) '~.._,...Glu-I-P GI!-6-P
Loctic ocid
Fro. 2. P a t h w a y s of gM~ctose metabolism. The principal p a t h w a y is indicated b y h e a v y aryows, K, '[" and E designate gab~ctoki,mse, Gal-1-1' uridyl t ransferase, tuld UDP-Gal-4-cpimcritse, respectively.
On the basis of studies in rat liver, it, was suggested t h a t with increasing age galaetosemics m i g h t a d a p t to ga lact.ose ~hrough an increase in the activity of liver U D P - G a l 1)yrophosphorylase (Isselbacher, 1957). This e n z y m e catalyzes the conversion of Gal-I-I' to UDP-Gal, with uridine triphosphate (UTP) as the co-substrate, instead. of UDP-glucose (UDP-Glc) ( [sselbacher, 1958). However, when this e n z y m e was studied in h u m a n liver homogenates, it was found that it,s a c t i v i t y did not.change from the newborn period to old age (Abraham and 1-Iowell, 1969) and t h a t the UDP-G-al pyrophosphorylase p a t h w a y does not contribute significantly to the disposition of Gal-I-P. In call" liver, both U1)P-Glc pyrophosphorylase and I f D P - G a ! pyro.phosphorylase activities appear to be properties of the same protein, but, wiCh a m a r k e d l y lower activity with Gal-I-P as the substrate (Ting and tIansen, 1968). U D P - G a l pyrophosphorylmse has not been d e m o n s t r a t e d in hemolysates (tsselbacher, 1960), while the a c t i v i t y of UDP-GIo pyrophosphorylase ca~t be m e a s u r e d read.ily in such preparations. A purified U D P - G l c p3,u-ophosphorylase from h u m a n red blood ceils had negligible a c t i v i t y toward Gal-I-P (Tsuboi, F u k u n a g a a n d :Petricciani,
1969). An oxidative l?r~thway has been postulated to explain the small in vivo 14COz produetion from [l-14C]Ga.1 in Caucasian galactosemic patients (Segal a n d Cuatreoasas, 1968). This involves the conversion of galactose t~ galactonic acid on t h e w a y to ('.O~ and D-xyluIose. However, ]3cutler (1967), a n d S r i v a s t a v a and :Beutler (1969) could not confirm the presence of such a n e n z y m e in m a m m a l i a n liver which could oxidize galactose to galactonic acid. i t has been reported t h a t G al-6-P can be formed in erythrocy~es of galactosemic patient~ (Inouye, T a n n e n b a u m and l:Isia, 196-2). This has not been confirmed.
406
~,V. G . N G
Influence of Glucose on Galactose Utilization
Galactose a n d glucose share a c o m m o n i n t e r m e d i a t e in G1c-6-1). and the question arises concerning the effect in the i n t a c t e r y t h r o c y t e of glucose oa galactose metabolism. I t has been shown t h a t t h e utilization of galactose by the red cell is slower t h a n t h a t of glucose (Lachhein ct al., 1961; Va|cntine ct el., 1967). In the absence of glucose in t h e i n c u b a t i o n medium, b o t h decreased o x y g e n c o n s u m p t i o n and u~ethcmoglobin r e d u c t i o n h a v e been d e m o n s t r a t e d , even in the presence of mct.hylcnc bluc (Betke et el., 1960; BeutlEr and Collins, 1965). I t has been c o n v e n i e n t to e m p l o y a m e t h o d involving the production of lat)elcd CO~ from [1-1aC]GaI as an index o f g a l a c t o s e utilization by the i n t a c t e r y t h r o c y t e (\Veiubcrg, 1961). The measurements, while useful in relation to the hexose m o n o p h o s p h a t e shunt, are n o t to be i n t e r p r e t e d in terms of ovcrall metabolism of galactosc b)" the cell. E x a m i n a t i o n of labeled i n t e r m e d i a t e s under various conditions of iacul)~tion can provide more extensive i n f o r m a t i o n (Bartlett, 1959). I n our own l a b o r a t o r y , it has been observed t h a t in the presence of glucose at a c o n c e n t r a t i o n of 1 mg/ml, there is a s u b s t a n t i a l p r o d u c t i o n of labeled COe by n o r m a l red blood cells from [1-14C]Gal. This is s t i m u l a t e d a p p r o x i m a t e l y 13-fold by m c t h ) ' l c a e blue (Table II). The effects of lower concel~trations of glucose also have been studied. TABLE I [
Effect of methylene blue (M.B.) on 14C02 ?)rr.~luctionfrom [1-1~C]Ga1 Galactose added (mg/ml)
0.075 0-125 0-175
0-225
M.B.
T o t a l 1ICOn (et/min)*
Stimulation b y M. B . t
-~--}--
139,000 9740 161,000 12,200
14x
~ --
15¢),800 11,420
13~
-i-
148,100
13 x
--
1.I , 8 3 0
13x
* C o r r e c t e d to c o n s t a n t specific a c t i v i t y o f g a [ a ~ t o s e . ~f ~ ' I e t h y l e n e b l u e c o n c e n t r a t i o n i n t h e i n c u b a t i o n m e d i u m is 0-061 m g / m l .
ll~ a t y p i c a l e x p e r i m e n t it wa~ f o u n d t h a t a t lower c o n c e n t r a t i o n of glucose, the a m o u n t of C02 formed from galactose in a given time period was inversely proport i o n a l to t h e glucose c o n c e n t r a t i o n . The r a t e of p r o d u c t i o n of C02 from galactose was c o n s t a n t a t glucose c o n c e n t r a t i o n s higher t h a n 0-05 m g / m l (Fig. 3). One e x p l a n a t i o n for this effect of glucose is isotope d i l u t i o n of i n t e r m e d i a t e s (Sturman, 1969). I n similar experiments, t h e effect was e x a m i n e d of v a r y i n g t h e galactose c o n c e n t r a tion (with [1-14C]Gal a t c o n s t a n t specific a c t i v i t y a n d with a c o n s t a n t c o n c e n t r a t i o n of u n l a b e l e d glucose a t 1 mg/mI). I n o t h e r studies, t h e p r o d u c t i o n of labeled c a r b o n dioxide from [1-14C]Glc in t h e absence of galactose was determined. I t was f o u n d
(IALAC'I'OSE
ME'I.'AIIOLISM
OF
THE
ICED
CELl,
407
(Fig. 4) t h a t the c o n c e n t r a t i o n a t s a t u r a t i o n level for CO 2 p r o d u c t i o n is surprisingly low for both sugqrs. For glucose i~ is a b o u t 0-l mg/ml, and for galactose a b o u t 0-0,5 rag/ ml. At sat.uration levels, and with specific activities expressed o n the same basis, the a p p a r e n t rate of CO., t)roduction from glucose is four to five times t h a t from galaetose under the particular e x p e r i m e n t a l conditions. w o
8O
7O I
A i
o
50 40 30
E
-
20I0-
0
0
l
I
0-05
0"I0
Glucose
Fh~. 3. T h e cll,'ct o f glueo.~e c o n c e n t r a t i o n
/
~r¢'9
x
0 0 05
Hexose
F I o . 4. C o m p a r i s o n centrations.
of
ttCOs
production
'
0-15 0.10
I
0-20
,,
,,|
,
0-25
0~30
o n t h e p r o d u c t i o n o f ItCO~ f r o m [1-taO]Oal.
..
~
,
(mg/ml)
r
I !
O
1
0"15
14
I
0-25 0 20
.
//
--
0 40 0 60 0-:30 0 50
(mglml)
f r o m [ I - I ' C ] G a l a n d [1-14C]CIc a t (tifforenC s u b s t r a t e
con-
Intermediates of Galactose Metabolism
Pulse-labeling studies of galactose m e t a b o l i s m in i n t a c t e r y t h r o c y t e s have been carried o u t in our l a b o r a t o r y . The products of i n c u b a t i o n were e x a m i n e d b y D o w e x - I formate columl:, c h r o m a t o g r a p h y . The results of a typiea[ e~rly e x p e r i m e n t are shown in ]rigs 5 and 6. Two inert c o n t a m i n a n t s present in the labeled galactose used a t t h a t time appeared in the column fractions. -Wighin the first 5 rain, the carbon label from [i-14C]Gal a p p e a r e d in Gal-I-P a n d in UDP-hexose. After 15 rain, Gal-I-P c o n t i n u e d to increase in a m o u n t , b u t there was o n l y a slighL rise in UDP-hexose. A t this time, labeled d i p h o s p h o g l y c e r a t e was detected. P e a k n u m b e r 5 represented several compomnds, including triose p h o s p h a t e s a n d hexose d i p h o s p h a t e s . After 30 rain of i n c u b a t i o n (:Fig. 6), there was f u r t h e r a c c u m u l a t i o n of GM-1-P,
408
W.G.
NG
w h i l e t h e a m o u n t of U D l ) - h e x o s e r e m a i n e d essentially u n c h a n g e d . T h e r e was a n i n c r e a s e in triose p h o s p h a t e s , h e x o s e d i p h o s p h a t e s and d i p h o s l ) h o g l y c e r a t e . Labeled lactic acid first w a s e v i d e n t a t G0 rain of i n c u b a t i o n . G a l - l - P was still accumulat.ing, as was d i p h o s p h o g l y c e r a t e . 800
-
2 I00 .~_ E
0
15 m m
800]-
2
too6
5O
I00
Tube number :FIG. 5. Labeled interme
2
2000 ~000':
30
__
4 .a
6
I00 ~ E t,3,
A
0 2000
"-
I 000""
m,n
60 rain
=
6
4
gi
'°I /t 0~
50
I00
Tube n u m b e r
Fro. 6. L a b e l e d i n t e r m e d i a t ~ f r o m e r y t h r o c y t e i n c u b a t i o n :Fig. 5 for e x p l a n a t i o n o f labeled peaks.
with []-x~CJGal
(normal adult).
Sea
T h e s e e x p e r i m e n t s s u g g e s t e d t h a t t h e m e t a b o l i s m of g a l a c t o s e in t h e i n t a c t red cell is a r e l a t i v e l y slow process a n d t h a t t h e r e m o v a l of G a l - I - P m a y be a r a t e - l i m i t i n g factor. I n t e r m s of t h e t r a n s f e r a s e r e a c t i o n , t h e a c c u m u l a t i o n of G a l - I - P c a n be influenced b y t h e c o n c e n t r a t i o n of G l c - I - P , U I ) P - G l c a n d U D P - G a l . T h e r a t i o of t h e l a t t e r t w o c o m p o u n d s is r e g u l a t e d in t u r n b y e p i m e r a s e a c t i v i t y .
(;-kI,ACTOSE
METABOLISM
OF THE
I'~ED C E L L
409
Results of columll chrom~ltogral)hy of l)roducts forl~led from [lJ4C]Gal in a 90-rain il~cubati~ with ('rvthro('vtcs of nor~ml individuals and of galactosemic patients arc compared in l"iv,. 7 (Ng et el., 1967a, b). For these studies, [lJ~C]Gal had been i)uritb(I. With gala(-toscmie patienu% tile cxl)ecte(1 large peal: of G a l - l - P was found. 2000,
!
^
4
,
(a
0 8000
"~
(b)
E
200 ~ 0 8000,
(c)
A
-j
• i
0
.
.
.
.
.
50
lO0
Tube number
I,'.;. 7. ('o.[i)~u'ison o{" labeled galactose intermcdiat~s from e r y t h r o c y t e ineubaC~ions. ,4, Patt~l'n from l~.ram; B awt C from g a l a e t o s e m i c patients. T h e peak n u m b e r s r e p r e s e n t e o m p o i m n t s as f¢,ll,~u'~: I. lactic a c i d : 2. (;~d. I - l ' ; 3, L : D P - h c x o s c : 4, triosc i)hosphate~ a n d hexo~c d i p h o s p h a t e s ; trod 5, liphouphoglyeero.t ~,.
tlormal
In addition, a sl~lal] peak corresponding to U D P - h e x o s e also was noted. F o r three ~tmo~g thc galactoscmic itldividuals studied, the U D P - h e x o s e p e a k was increased a n d labeled diphost)hoglycerate was detected. I t is possible that, some galactosemic patients m a y h a v e a slight residual transferase a c t i v i t y (Schwarz, ~Vells, Holzel a n d Komrower, 196l; IIsia, Tannenbatm~, Schneider, H u a n g a n d Simpson. 1960), b u t tlle possibility of participation of a subsidiary p a t h w a y c a n n o t y e t be, eliminated. Ingestiou of galactose l)y galactosemic patients results iI1 the accunuflation of Gel-1-P in erythL'oeytcs. The conceutration m a y become as high as 0-2 m g / m l a f t e r an oral galactose tolerance test (Donncll, Bcrgren, P e r r y and Koch, ~.963). t-Iowever, 2,t hr later the G a l - I - P level has r e t u r n e d to the baseline level. During in vitro experiments to simul~tte the in vivo conditions, accumulation of labeled Gal-I-:P was produced b y preincubation of e r y t h r o c y t e s with radioactive galactose (Donnell, Bergrcn a n d Ng, 1967a). I t was found during tile s u b s e q u e n t 24 hr t h a t the l a t e cellular concentration of G a l - l - P decreased substantially and t h a t a corresponding a m o t m t of r a d i o a c t i v i t y a p p e a r e d as free g a l a c b s e in the medium. I t was p o s t u l a t e d t h a t the decrease in G a l - l - P could result from nonspecii~c p h o s p h a t a s e activity. Similar ob~erx'ations have been reported recently (Gitzelman, 1969). ~ ' t e r an extended period of incubation of galaetose a n d red blood cells from galactosemic patients, in t h e absence of glucose, a drop in adenosine t r i p h o s p h a t e (ATP) level was observed (I)enington a n d P r a n k e r d , 1958). The inability to m a i n t a i n A T P c o n c e n t r a t i o n could a c c o u n t for a n increase in p o t a s s i u m flux in galaetosemic
410
\V. G. N O
er).%hrocytes when galactose is the only c a r b o h y d r a t e source (13ear and Gordon, 1964). However, i t has been shown when glucose is present, a c c u m u l a t e d O a l - l - P in e r y t h r o c y t e s of galactosemics does n o t affect the level of :VI'P or of gl.vcolytic i n t e r m e d i a t e s (Zipursky, l t o w l a n d . Ford. 1-Iaworth and Israels, 1965). Screening for Galactosemia
A l t h o u g h t h e frequency of occurrcnce of t,he transferase defect and of the galactokinase defect is low (Schwarz et al., 1961" Beutler, Baluda. Sturgeon and l)av, 8 it. is i m p o r t a n t to be aMe to detect, these genetic 1966 ; Mayes and Guthrie, I96,:), disorders by relatively simple procedures. In our laboratory, in the past ten ~nonths alone, we have formal one transferase defect in our own hospital and have confirmed six new cases referred from o t h e r hospitals on tim basis of screening 1)roeedures or of clinical suspicion. I n addition, an example of the galactokinasc detk'ct was found (galactokinase defec"c samples referred by :Dr I). Frazicr of Los Angeles Count) ..... U.S.C. 5iedical Center). A n u m b e r of c o n v e n i e n t tests now are available for the detection and confirmation of these defects (Table I I I ) . TA!)L)" I I I
Methods f o r detccHon o f !talac~osemia Screeni~lg proceclure~ :
I. ])apcr c h r o m a t o g r a p h y _'2. ~iicrobiologieal 3. M e t h y l e n e blue r e d u c t i o n 4. ~ A D I ) H fluore.~ceneo 5. I~CO~ from [1-1'C]Gal
Specific ussays : I. 2. 3. 4.
U D P - G k : cons-eruption ~Ianometric tl~dio~ctive K i n e t i c coupling
Some screening m e t h o d s are based on the presence of exce~i ve a m o u n t s of galactosc in uxine or blood, as identified by paper c h r o m a t o g r a p h y ( H a w o r t h and B a r c b u k , 1967; D a h l q v i s t , J a g e n b u r g a n d Mark, 1969) or .by a microbiological i n h i b i t i o n assay (Guthrie, 1968). O t h e r procedures utilize coupling to the hexose m o n o p h o s p h a t c s h u n t . . F o r example, t h e presence or absence of transferase a c t i v i t y in e r y t h r o e y t e s can be d e t e c t e d readily b y m e t h y l e n e blue reduction or by the a p p e a r a n c e of fluorescence due to g e n e r a t i o n of NA]:)P]-I (Brewer and Tarlov, 1963; Beutler, Balud~ a n d :Donnell, 1964; B e u t l e r a n d Baluda, 1966b; I:lochella and Hill, 1969). z ~ m t h e r a p p r o a c h involves the f o r m a t i o n of labeled C02 from [1-1aC]Gal b y intact, red blood cells (London, M a r y ~ m n t a n d Fuld, 1964). Specific e n z y m e assays are essential for confirmation. Transferase assays can be p e r f o r m e d b y t h e U-D:P-Glc c o n s u m p t i o n procedure (Anderson, Kalckar, K u r a h a s h i a n d Isselbacher, 1957b; B r e t t h a u e r , :Hansen, ]-)onnell and :Bergren, 1959; Melhn~n a n d Tedesco, 1965; :Beutler a n d Baluda, 1966a), by oxygen c o n s u m p t i o n ( K i r k m a n and B y n u m , 1959), b y t h e i n c o r p o r a t i o n of labeled GaI-1-P into 17DP-Gal (:Ng, Bergren a n d Donnell, 1967a ; I n o u y e , N a d l e r and Hsia, 1968 ; Sawicka and Chojnack, 1959; T r o n a n d Milhaud, 1969), or by s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of coupling
l +l +.,, "+"i.; I. iilii+l
it
('q~ii~l~+'lJ'i.,.++++i
lll'~'+lilil~',l
<~I','i",'ilit'+,(.,,'t~
+'1+,>.~ +\ll~t'h'+'"
+ tt'iili,
ll+l:ll,'t~.~'.<_'
|i+11tt'i'ii.,.t
'.l'h~'+ iril+lt.-lliu+,l
~.+II +'+t+tt'<'lt
I)txttttl'll
<~t'l t'(+t'( '+>t llt>','+~++
t>t.tx+'(.,,tL
t'~,lli'ost++lit+ "'I+.++\. +. iilili,,'lthl+ll.
iltJl'lltili
GALACTOSE
M E'I'AIiOL1SM OF THE
REI') CELL
411
to t.be hexose monophosphate s h u n t (Colombo, Moser, :Rosin, Richterich and :Rossi, 1965; Beuth;r and Mitchell, 1968; Copeahaver, Bausch and Fitzgibbons, 1969). Galactokinase has been assayed by the incorporation of labeled galactose into Gal-I-P. in the presence of A T P (Ng, Donncl[ and Bcrgren, 1965; de Verdier, 1966). The details, a d v a n t a g e s and disadvant~ges of some of these methods h a v e been presented in a symposium on gnlaetosemia. (Hsia, 1969). Genetic Variants of GaI-I-P Uridyltransferase
In genetic counseling, the capability of identifying heterozygotes is i m p o r t a n t . \Vhile ~'.arriers of galactosen~ia have halfknormal e r y t h r o c y t e transferase activity, the occm'rence of transforase variants in the population presents a problem in identification of the gala,-t.oscmic hcterozygotes. The a s y m p t o m a t i c D u a r t e v a r i a n t homozygotc also Ires half-nor,~ml e r y t h r o e y t c transferase activity (Beutler et el., 1966). I n starch gel elect rophorcsis, using a phosphate buffer, the D u a r t e v a r i a n t enzyme migrates as a single band of higher mobility than t h a t for the normal enzyme, b a t the ~wo forms t)resent in hcmolysates from ] ) u a r t e - n o r m a l heterozygotes c a n n o t be se[)arat(,',I clearly in this system (Me thai and Beutler, 1966). This problem has been resolved (Ng. Bcr.m'cn, Fields and DonnelJ, 1969), however, by use of Tris-g!ycine buIlbr at, p | I 8-8. In d~c moditicd eiectrophoretic procedure, the D u a r t e v a r i a n t enzyme exhibits triple bands while only one is sce~} with the normal enzyme (Fig. 8). W i t h the D u a r t e Hemoglobin Ocm ~- I
.......
I
Fluorescent !
i
I
I
l
I
I
bands tO ~
..
i
g
0
oo
I
$
0 O0
I-i-]
+
Fro. 8. Migration pt~ttern of c r y t h r o c y t a Gal-1-]? uridyltransferase in starch gel electrophoresis in Tris-glycinc buffer a t p K 8.8. ~NN. uormal homozygote; ND, n o r m a l / D u a r t e heterozygotd; ]319, :Duarte homozygotc; and (ID, gnlactoscmic/Duarte hctcrozygotc.
homozygote ~here are three bands, the two faster b a n d s p r e d o m i n a n t . Three b a n d s also are seen with the ] ) u a r t e - n o r m a l heterezygote, b u t the slowest band, corresponding in position to Che single b a n d of a. normal control, is outstanding. The D u a r t e galactosemic hef, erozygote shows three faint bands. A n o t h e r e r y t h r o e y t e transfera.se v a r i a n t t e n t a t i v e l y n a m d d the " L o s Angeles" variant, has been ictentified in our laboratory. The iudixdduals p r e s u m e d to be homozygous have elevated transferase activity in c o n t r a s t with the reduced activity for the Y)uarte variant. The elec~rophoretic p a t t e r n s are similar to i:hose for the D u a r t e v a r i a n t (Plate 1). A n u m b e r of " L o s Angeles" v a r i a n t families h a v e been studied, and the Kndings are eonsisten~ with a u t o s o m a l inheritance. The distribution of e r y t h r o c y t e transfe.rase activity a m o n g know-n genotypes is summarized in Table IV. I n t e r m s of m e a n value, t h e gal~ctosemic-normal heterozygore has o n e - h a ~ n o r m a l activity, while t h a t for the galactoscmic homozygote is
412
~V, G. ~NTG
negligible. I n r e l a t i o n to n o r m a l e r y t h r o c y t e t r a n s f e r a s e v a l u e s , t h e D u a r t e - n o r m a l h e t e r o z y g o t e e x h i b i t s t h r e e - f o u r t h s , t h e D u a r t e h o m o z y g o t e one-half, t h e D u a r t e galactosemic h e t e r o z y g o t e one-fourth, the Los Angeles-normal hcterozygote somew h a t e l e v a t e d a c t i v i t y , a n d t h e p r e s u m e d Los A n g e l e s v a r i a n t h o m o z y g o t c significantly increased activity. One p r e s u m e d Los-Angeles-galactosemic heterozygote h a s b e c n f o u n d to h a v e o n e - h a l f n o r m a l a c t i v i t y . I t is e v i d e n c t h a t w i t h o u t e l e c t r o p h o r e s i s it is n o t possible to d i s t i n g u i s h a m o n g all of th ese g e n o t y p e s . 3?Ar~rm IV
Erythrocyte transferase activity distributioqt among differ¢mt genotypes Prc.sumed genotype
No.
N-~NG-N G-G D-~N D-D D-G I.~Y-N LA-LA LA-G
123 69 9 40 2 3 36 5 I
Transfcra~ act iv ity* Mean Range 24 "8 10.9 {)-2 17-9 10-8 5-7 26-7 32-0 12-7
18"/-33-3 5.6-16.5 0-0-8 12-6-24-3 9-9-11.7 5.6-5-8 18-8-35-5 2 L-0--40.5 --
* Activity is expressed as nfieromoles of Gal-l-PV'~neorporated into UDP-Gal per h per g hemoglobin. I n a s t u d y in o u r l a b o r a t o r y , w h i c h so f a r has i n c l u d e d 400 blood s a m p l e s f r o m C a u c a s i a n s , t h e f r e q u e n c y of D u a r t e - n o r m a l h e t e r o z y g o t e o c c u r r e n c e has b e e n f o u n d to be a b o u t 12°/o, c or t f i r m i ng earlier" r e p o r t s ( B e u t l e r et al., 1966; M e l l m a n , T e d e s c o a n d Feigl, 1968), a n d t h a t of t h e L o s A n g e l e s - n o r m a l h e t e r o z y g o t e a b o u t 4°//o. The. o c c u r r e n c e of t h e s e v a r i a n t s in 110 A m e r i c a n N e g r o e s zo far s t u d i e d is a b o u t t h e s a m e as t h a t in Caucasians, w h e r e a s o n l y t w o D u a r t e - n o r m a l h e t e r o z y g o t e s w e r e f o u n d a m o n g 105 u n r e l a t e d O r i e n t a l a d u l t s . T w o o t h e r e r y t h r o e y t e t r a n s f e r a s e v a r i a n t s of g a l a c t o s e m i a h a v e b e e n r e p o r t e d ( S c h a p i r a a n d K a p l a n , 1969; C h a c k o , C h r i s t i a n a n d N a d l e r , 1970). A C K N Ox,V L E D G 3 I E N T S I wish to express m y appreciatiml to Drs William 1%. ]3ezgren a u d George N. D o n n e l | for t h e o p p o r t u n i t y to p a r t i c i p a t e ill collaborative efforts over a period of years, a n d for their suggestions a n d criticism in th e p r e s e n t work. R k~'~l~ E N C E S Abraham, ~,Ynderson, Anderson, 2~led.
H. E. E. 50,
D. and Howell, i%. l-l. (1960). J. 13ioi. Ohem. 244, 545. P., ]~alckar, l:[. ~I. and Isselbacher, K. J. (1957a). Scienve, N.Y. 125, 113. I~., Kalckar, 11. hl., Kurahashi, K. and Isselbacher, K. J. (1957b). J. Lab. Clin. 469.
:Baar, H. S. and Gordon, ~[. (.1964). zVature (London) 201, 1223. :Bartlett, G. I~. (1959). J..Biol. Chem. 234, 459. ~Bergren, \V. lZ. (1967). In I:Iereditary Disorders of Erythrocyte l-tIetabolia'm, p. 83. (Ed. by :Beutler, E.) Grune sad Stratton, New York.
(:ALACTOSE
METAI¢OLISM OF THE
RED CELL
413
Bergren, \V. 1~., Ng, \V. G. and ])onnell, G. N. (1967). Pediatrics 40, 136. Betkc, b:., Baltz. A. a n d Maa8, U. (19(i0). Z. Ki?ulerheil. 84, 226. Beutler. l':. (1967). Science, N . Y . 156, 1516. Beu 1ler, 1'3. and l htluda, 31. C. (1966a). Clin. Chir~. A c ~ 13, 369. .Beu! ler, 1",. a n d Baluda, M. C. (1966b). J . Lab. Olin. ,lied. 68, 137. l;eutler, E. a n d l¢aluda, M. C. (1966c). J . l.~b. Olin. Aled. 6 7 , 9 4 7 . 13eutler, 1~., ]~aluda, M. a n d Dommll, G. N. (19t)4). J . l_,e~b. Cli~. M e d . 67, 694. E.. I3ah~d~, 31. C., Sturgeon, P. a n d Day, R. ~,V. (1966). J . . L a b . Olin. M e d . 68, 646. Beut.ler, E. a n d (?ol)ins, Z. (1965). Sc~,nd. ,]. ]lae,uatol. 2, 377. Beutler. E. a~(! Mitcb-l]. ),1. (1968). ,1. [.~b. Clin. Aled. 72, 527. B r a m l l , N. J . , Fro]and. A., Mikkelse,~, M., ~Nielsen. A. and Tolstrup, N. (1963). Lancet ii, 700. Brctth~u(.r, R. K., }t:Lnse.n. 14. G., Doanc|l, G. N. a n d ~¢ergron, \V. I4. (1959). Pro~. N a t . Ac,ad. Sci. L'.S'..-i. 45, 328. 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A., l-Iuang, I. c~nd Simpson, K . (1960). J . I_x~b. Olin. .~lal. 5 6 , 3 6 8 . I n o u y e , T., ~Nadler, H . L. a n d ]tsia, D.Y.-Y. (1968). Clin. Chin*.. A e t a 19, 169. J n o u y e , "]7, T a n n e n b a u m , ~1. a n d Hsia. D . Y . - Y . (1962). N a t u r e (Lo~don) 15)3, 67. lsse|bacher, 1,~. J. (195"/). Science, N . Y . 126, 652. ]s,~elbacher, K. J. (1958). J . l~iol. Chem. 2 3 2 , 429. lssolbacher. K. J. (1960). I n T h e :ffetabolic, ]3a.sia o f I n h , rited/)/.seaae, p. 208. ]~d. J. J3. S t . a n b u r y , J . B. \ V y n g a r d e n a n d 1). S. Fred.rickson. McGraw-.~[iil, New Y o r k . ]sselbacher, K. ,)'., A n d e r s o n , ~ . P., K u r a h n s h i , K . a n d ~Kalckar. ]:I. (1956). Science, . N . Y . 123, 635. K a l c k a r , ]]. M., :knderson, E. P. a n d ][s,~olbachcr, ]~. J. (1956). Proc. N a t . A c a d . Sci., U . S . A . 42, 49. K a l c k a r , ]-{. M., ]~r~ganca, ]3. and M u n c h - P e t e r s o n , A. (1953). W a t u r e (London,) I72, 1038. 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414
Ng, Ng, Ng, Ng, Ng,
W.G.
NG
W. G., .Bergren, W. R. and Dommll. C. N. (1964a). Cl6~. Chim. Acta 10, 337. W. G., Bergren, W. R. and Donnell, C. N. (1964b). Nature (Lo~ulon) 203, 845. 'W. G. Bergren, W. R. and l)om~tell, G. N. (1967a). Clin. Chim. Acta 15, 489. W. G., Bergren, W. R., Donnell, G. N. and Hodgman, J. E. (1967b). P~liatri,'.~ 39, 293. W. G., Bergren, W. 1R..,Fields, M. and Donnell, G. N. (1969). Biochem. Biophys. Res. Commu~t. 37, 354. Ng, W. G., Dotumll, G. N. and Bergren. W. R. (1965). J. Lab. Clin. Med. 66, i 15. Ng, W. G., Don.uell, G. N. and Borgren, W'. R. (1968). Nature (Londo~) 217, 64, Ng. \V. G., Dormell, G. N., Hodgtaan,]J. E. and Bergrea, W. I:L (19{~7c). A'ature (London)214, 283. Penington, J. S. and Praakerd, T. A. J. (1958). Clin. Sci. 17, 385. Puck. T. T. and Hill, H. Z. (1967). Pro(:. Nat. Ace~d. Sci. U.S.A. 57, 167(i. Qu:~rl.Ma, ~R. and Wells, W. \V. (1965). Biochem. B&ph.qs. lic,~. Commun. 20, 4~(;. Qu0,n-3Ia, R., Wells, H. J., Wells, W. W., Sherman, F. E. and Egan, T. J. (1966). Amer. J. Dis. Child. 112~ 477. Riabo~', S., Inouye. T.. Parker, D. ,~n(t Hsia. D.Y.-Y. (1965). Biochim. Biophys. :Ic~ 99, 17:~. Robinson, A. (1963). J. Exp. 3Ied. ll8, 359. Rosner, F., Ong. B. H., Paine, R. S. and Mahanaad, D. (1965). New E~ufl. J. Med. 273, 135(;. Sawicka, T. and Chojnack, T. (1969). Clin. CMm. Acta 23, 463. Schapira, F. and Kaplan, J. C. (1969). Biochem. Biophys. Reds. Commun. 35° 451. Schwarz, V., Goldberg, L.. Komrowcr, G. M. and tlolzel. A. (1956). Biochem. J. 62, :14. Schwarz, V., Wells, A. R., Holzcl. -k. and Komrower, (:~ M. (1961). Ann. llum. ~/eaetic.~. 25, 179. Segal. S. and Cuatrecmsas, P. (1968). Amer. J. Mezl. 44, 340. Srivastava, S. 14. and Beutler, E. (1969). J. Biol. Chem. 244, 6377. Strominger, J. L. (1960). Physiol. Rev. 40, 55. Sturman, J. A. (1969). Clin. Chim. Acla 26, 135. Ting, W. K. and Hansen, 1~. G. (1968). P r ~ . Soc. Exp. Biol. Med. 127~ 960. Tron, P. and Milhaud, G. (1969). Rev. fr. Etud. Clin. Biol. 14, 12. Tsai, C. M., Holmberg, N. and Ebner, K. E. (1970). Arch. Biochem. Biophys. 136, 2:33. Tsuboi, K. K., Fukunag,~, K. and Petricciani, J. C. (1969). J. Biol. Chem. 244~ 1008. Valentine, W. N., Oski, F. A., Paglia, D. E., Baughan, M. A., Schneider, A. S. and Naiman, J. L. (1967). ~n Hereditary Di,~orders of E','ythrocyte ~]lelaboli~m, p. 288. Ecl. by Bcutlcr, E., (;rune and Stratton. ,.New York. de Verdier, C. H. (1966). Scarul. J. Clin. Lab. Invest. 8-92, 156. Weinberg, A. N. (1961). Metabolism 10, 728. Wells, W. W., Pittman, T. A. and Egan, T. J. (1964). J. Biol. Chem,. 239, 3192. Zipursky, A., :Rowland, M., :Ford, J. D., Haworth, J. C. and lsracls, L. G. (1965). Pediatric~ 35,126.
Metabolism
of the Red
Cell*
Wo,x- O. No
Depa rtment.~ oj" B iochemi.,'try and Pcdiatric.s', University of Souther~ Californi~, ,School of llietlicitw. Los ,4 ,~geles, Calif. 90033, U.S.A. (~7l(l
Children's Hospit¢H of Los Angele.% Los Angelica, Calif. 90027. U.S...I. The human erTthrocyt¢ offers an opportunity for investigation of galactos¢ metxLbohsm because the complete normal pathway exists in thin accessible tissue. The erythrocyte lu~. proven to be u.~eful not only in identifit.ation of the hetevozygous and homoT.ygous states in galactosemia and in the galactokinasa defect, but. a].~o irt the .~ttt(t)" Of variant.s. 1b is recognized, however, that, the erythrocyto is a special tyl~3 of cell aI~(l that findings from its study should not be extend~l to other tissues without reservation. With rc.'~pczct to the ervthrocyte itself, there still is much to be learned concerning intnxcellular kinetics under di~'(~re[~t conditions and the nature of the enzymes involved. Introduction O u r i n t e r e s t in g a l a c t o s e m e t a b o l i s m has e x t e n d e d o v e r a n u m b e r of years, a n d t h e h u m a n e r y t h r o c y t e h a s b e e n used in m u c h of this wo rk . T h e c o m p l e t e p a t h w a y s of g a l a c t o s e m e t a b o l i s m is p r e s e n t in red cells f r o m n o r m a l i n d i v i d u a l s , p r o v i d i n g t h e o p p o r t u n i t y for e x a m i n a t i o n of b i o c h e m i c a l e v e n t s in d i s o r d e r s of this p a t h w a y . T h e Principal Pathway T h e p r i n c i p a l p a t h w a y for t h e m e t a b o l i s m of g a l a c t o s e in the h u m a n e r y t h r o c y t e w a s s h o w n ( I s s e l b a c h e r , A n d e r s o n , K u r a h a s h i a n d K a l c k a r , 1956) to follow t h e s a m e s e q u e n c e of r e a c t i o n s w o r k e d o u t o r i g i n a l l y by a n u m b e r of i n v e s t i g a t o r s ibr t h e m e t a b o l i s m of g a l a c t o s e in y e a s t ( K o s t e r l i t z , 1943; C a p u t t o , Leloir, Card in i a n d P a l a d i n i , 1949; C a p u t t o , Leloir, Cardirri a n d P a l a d i n i , 1950; ~Leloir, 1951; K a l c k a r , : B ra ga nc a a n d M u n c h - P e t e r s o n , 1953): A T P + Galactose -~-Gal-I-P U D P - G l c + GaI-1-P ~,-~-UDP-Gal + G l c - I - P UDP-Ga] ,~-UDP-Glc
(I) (2) (3)
S u m : A T P + Galactose-->Glc-1-P + A D P T h i s p a t h w a y is k n o w n as t h e L e l o i r p a t h w a y ( F i s c h e r a n d W e i n l a n d , 1965). T h e r e a c t i o n s a r e c a t a l y z e d b y t h e e n z y m e s : (1) g a l a c t o k i n a s e , (2) g a l a c t o s e - l - p h o s p h a t e u r i d y l t r a n s f e r a s e ( G a l - I - P u r i d y l t r a n s f e r a s e ) a n d (3) u r i d i n e d i p h o s p h a t e - g a l a c t o s e - 4 e p i m e r a s e ( L r D P - G a l - 4 - e p i m e r a s e ) . T h e o v e r a l l r e s u l t is t h e c o n v e r s i o n of g a l a c t o s c a n d t h e f o r m a t i o n of g l u c o s e - l - p h o s p h a t e ( G l c - I - P ) , w h i c h t h e n is c o n v e r t e d to g l u c o s e - 6 - p h o s p h a t e (Glc-6-P) a n d f u r t h e r m e t a b o l i z e d t h r o u g h t h e g l y c o l y t i c p a t h w a y a n d t h e h e x o s e m o n o p h o s p h a t e s h u n t . This is also t h e m a i n p a t l ~ w a y for t h e m e t a b o l i s m of g a l a c t o s e i n t h e m a m m a l i a n l i v e r ( S t r o m i n g e r , 1960). * This investigation wins supporte.d by grants (A~f-04135, A~L04837, I:ID-00800 and RR-0086) from the National Institutes of Health, U.S. Public Health Service. 402
(;AI,A(~'I'OSI';
METABOLISM
OF
'FtIE
ICED
CELL
403
The s t u d y of e r y t b r o c y t e galaetose mctabolisln in m a n was greatly s t i m u l a t e d b y the loealizatio,~ of the enzymic defect in galactosemia a n d the d e m o n s t r a t i o n t h a t the erythroeyt,e is a suitable diagnostic tissue. Galactosemia is an inborn error of inetal,olism characterized by failure to thrive, susceptibility to infecbion, jaundice, hel)atomegaly, amino aciduria, ca[erects and m e n t a l r e t a r d a t i o n . The disease results fro,,: abse,we of (;el-1-P u r i d y l t r a n s f e r a s e (transferase) act, ivity in tissues (Reaction 2) (Kalclmr, Andcr.~on and lsselbacher, 1956; Anderson, K a l c k a r and Isselbacher, 1957a). The enzymic deficiency leads to a c c u m u l a t i o n of Gal-I-P in tissues when lactose or other galactose-containing foods are ingested (Schwarz, Goldberg, Komrow(,r and Itolzel, 1956). A nothl,:" met.al)olie disorder involving the galactose p a t h w a y has been described. The defect has t.~et.n demonstrat.ed to be in galaetokinase act.ivity (Gitzelmalm, 1965). The I)rinmry clinical m a n i f e s t a t i o n is the presence of cataracts. No case of U-D P-Gal4-epimerase (cpimer~sc) defect, has been reported.
Enzymes of the Principal Pathway Existing if'formation concerning the individual enzymes of the principal p a t h w a y of galactosc in the e r y t h r o c y t e has been derived prim'arily from studies with hemolysates. "Fhc rate c o n s t a n t s of the galactokinase, transferase and epimerase reactions, however, have been measured in i n t a c t red blood cells as well as in h e m o l y s a t e s (Hobinso,~, 1963; I'uck and Ilill, 1967; Hill and Puck, 1970). The relative a c t i v i t y values arc similar in both systems. B o t h gulactokinase and transferase of e r y t h r o c y t e s have been p a r t i a l l y purified (Mathai and Beutler, I967; ]liabov, Inouye, P a r k e r and I-Isle, 1965; 13cutler a n d Baluda, 19(;t;c) aml their kinetic properties have been examined. I s o l a t i o n of e r y t h r o c y t e transferase and of epinmrase has been in progress in our l a b o r a t o r y . Transfcrase preparat.io~s of greater purit, y t h a n those reported h a v e been o b t a i n e d . K i n e t i c st.udies ha:'e yielded x'alues concordant, with those o b t a i n e d by others. :Epimerase fractions, free of ~ransferase activity, have resulted as a b y - p r o d u c t of t h e t r a n s f e r a s e isolation. A consistent problem in isolation is increasing i n s t a b i l i t y of b o t h enzymes with c o n t i n u i n g purification. T.t~*LS 1
Relationship between galactosc oxidatio~ a.nd enzyme activity
(4rou p
Newborn infants Adults
14C02 f r o m [ l - ~ C ] G al
3,t,000 11,500
]Relative activity* of galactose enzymes Kinoze T. Epi~nerase
0-83 0"26
8-1 7-3
2-84 1 "39
* Aet.ix~ity is e x p r e s s e d a s m i e r o m o l e s o f s u b s t r a t e c o u x - e r t e d p e r h o u r p e r m i l l i l i t e r p a c k e d r e d b l o o d ceils.
The c r y t h r o c y t c s of n e w b o r n infants, in c o n t r a s t w i t h those of adults, h a v e a greater c a p a b i l i t y of utilizing galactose, as i n d i c a t e d b y t h e finding of increased galactose c o n s u m p t i o n , oxygen u p t a k e a n d m e t h e m o g l o b i n r e d u c t i o n (Betke, Baltz ~4
404
~V. G. N G
and Mass, 1960; Lachhein. Grube, J o h n i g k and Matt.hies, 1961). ~Ve have found t,h a t the r a t e of IaCO,, production from [1-1aC]Gal by intact e r v t h r o c v t e s of newborn infants is a b o u t three times that. of adulL~. Also, in hemolysates of newborn infanLs the act;ivity of all e]lzymes of the galaetose p a t h w a y were noted to be incroased (Table I). Transferase activity is itw.reased slightly, but galacrokinase and epimerase acl~ivities arc two to three times thost~ found for adults (Ng, Donnell, I [ o d g m a n and ]3ergren, 1967c; Ng, Bergren, Donnell and l-[odgman, 1967b; l.-)onne[I, Ng. l [ o d g m a n and ]3ergren, 1967b). Galactokinase and transferase activities are also elevated in e r y t h r o c y t e s of children wit, h D o w n ' s s y n d r o m e ( B r a n d t , Froland, Mikkelsen, Nielsen and Tolstrup, 1963; Krone. Wolf, Goedde and Bait.sch. 1965; Ng, Bergren and I)onnell, 1964a, b; Donne[l, Ng, Bergren, Melnyk and Koch, 1965; Rosner. Ong, Paine and M a h a n a n d , 1965). In h e m o l y s a t e s of adults, epimerase act.ivit.v is d e p e n d e n t on addctl nicot, inatnide adeifine dinucleotide (N,LD) (I~selbacher et al.. !956) because of the destruction of endogenous N:LD by stromal nicot, inamide adeniae dinucleotide nueleosidmse (NADase) liberated in the hemolvsates ( K i r k m a n and Maxwell, 1960). In contrast, we have found in h e n m l y s a t e s of newborn infants t h a t NADase v-ctivitv is ext, re1~mlv low and t h a t epimerase a c t i v i t y can be d e m o n s t r a t e d wit, hour addition of NA1) (Bergren, Ng and Donnell, 1967). H e m o l y s a t e s from certain adult.n resemble those of newborn infants in this respect. Such individuals have been found to have a geneticallyd e t e r m i n e d e r y t h r o c y t e N:kDase deficiency (Ng, Donnell and Bergren, 1968). The presence of N A D not only aft'eeLs epimerase activity but also changes the molecular form of the enzyme, as d e m o n s t r a t e d by starch gel electrophoresis (Fig. 1) O
÷ NADOSe
NAD
I
II
FIO. 1. S t a r c h gel clectrophoresis of U 1 ) P . G a l - , t - e p i m e r a s e from h e m o l y a a t e ~ . FluorcacenL b a n d s corre~Jponding to c p i m e r a s c a c t i v i t y are i n d i c a t e d b y solid b l a c k r e c t a n g l e s : the h a t c h e d r e c t a n g l e s repr,esent hemoglobirm.
(Bergren, 1967). ~Iemolysates from the newborn show two well-defined fluorescent bands, in cont=rast wibh the si~gle band of different mobiJity for hemoiysatcs from adults. W h e n a newborn h e m o l y s a t e is t r e a t e d wi~h N.&Dase, the two-banded epimerase is converted to the one-banded form. Couversely, when an a d u l t hemolysate is p r e i n e u b a t e d with N A D : two b a n d s c o m p a r a b l e to those for a newborn irdanb are found. W h e n NA_D is added to the starch gel itself, all hemolysates show two bands of ephnerase activity. Two e]ectrophoretic forms of epimerase also h a v e been reported to be present in bovine m a m m a r y gland preparations (Tsai, ]~[olmberg a n d Ebr/er, 1970). Other ]Pathways of Galactose Metabolism
I n addition to the principal p a t h w a y of galact~se metabolism, other p a t h w a y s h a v e been described or postulated as possible routes for the disposition of galactose (:Fig. 2).
GAI.AC'I'OSE
MI';TAIIOLISM
OF TIIE
B, E D C E L L
405
The conversion of galactose to galactitol, catalyzed b y aldose reductasc, w i t h reduced nieotinamide ade~line dinucleotide p h o s p h a t e (NADPI-I) as cofactor, has been described in aniJnal tissues (van l-leyningen, 1959; Kinoshita, Merola, Satoh and l)ikmark, 19(32). Substantial amount,s of galactitol have been found in autopsy samples of lens, brain, kidney and other tissues of galactosemie patients (Quan-Ma, Wells, Wells, S}~crman and Egan, 1966). A high concentration of galactitol is present in tim urine of patients with the transferase defect and also in those with galactokinase deficiency (~,Vells, Pitotmftn and Egan, 196¢; Gitzehnann, Curtius and :M/iUer, 1966). In rats fed galactose, galactitol has been shown to be present in red blood cells and in other t i ~ u e s (Quan-Ma and \Vells, 1965). UTP
\
G,QIQ¢Iilol
Gol
PP
u~Dp~ El
K J - GaI-I-P.-~,...-,~' (K)
/
C~1!6_P
{T) '~.._,...Glu-I-P GI!-6-P
Loctic ocid
Fro. 2. P a t h w a y s of gM~ctose metabolism. The principal p a t h w a y is indicated b y h e a v y aryows, K, '[" and E designate gab~ctoki,mse, Gal-1-1' uridyl t ransferase, tuld UDP-Gal-4-cpimcritse, respectively.
On the basis of studies in rat liver, it, was suggested t h a t with increasing age galaetosemics m i g h t a d a p t to ga lact.ose ~hrough an increase in the activity of liver U D P - G a l 1)yrophosphorylase (Isselbacher, 1957). This e n z y m e catalyzes the conversion of Gal-I-I' to UDP-Gal, with uridine triphosphate (UTP) as the co-substrate, instead. of UDP-glucose (UDP-Glc) ( [sselbacher, 1958). However, when this e n z y m e was studied in h u m a n liver homogenates, it was found that it,s a c t i v i t y did not.change from the newborn period to old age (Abraham and 1-Iowell, 1969) and t h a t the UDP-G-al pyrophosphorylase p a t h w a y does not contribute significantly to the disposition of Gal-I-P. In call" liver, both U1)P-Glc pyrophosphorylase and I f D P - G a ! pyro.phosphorylase activities appear to be properties of the same protein, but, wiCh a m a r k e d l y lower activity with Gal-I-P as the substrate (Ting and tIansen, 1968). U D P - G a l pyrophosphorylmse has not been d e m o n s t r a t e d in hemolysates (tsselbacher, 1960), while the a c t i v i t y of UDP-GIo pyrophosphorylase ca~t be m e a s u r e d read.ily in such preparations. A purified U D P - G l c p3,u-ophosphorylase from h u m a n red blood ceils had negligible a c t i v i t y toward Gal-I-P (Tsuboi, F u k u n a g a a n d :Petricciani,
1969). An oxidative l?r~thway has been postulated to explain the small in vivo 14COz produetion from [l-14C]Ga.1 in Caucasian galactosemic patients (Segal a n d Cuatreoasas, 1968). This involves the conversion of galactose t~ galactonic acid on t h e w a y to ('.O~ and D-xyluIose. However, ]3cutler (1967), a n d S r i v a s t a v a and :Beutler (1969) could not confirm the presence of such a n e n z y m e in m a m m a l i a n liver which could oxidize galactose to galactonic acid. i t has been reported t h a t G al-6-P can be formed in erythrocy~es of galactosemic patient~ (Inouye, T a n n e n b a u m and l:Isia, 196-2). This has not been confirmed.
406
~,V. G . N G
Influence of Glucose on Galactose Utilization
Galactose a n d glucose share a c o m m o n i n t e r m e d i a t e in G1c-6-1). and the question arises concerning the effect in the i n t a c t e r y t h r o c y t e of glucose oa galactose metabolism. I t has been shown t h a t t h e utilization of galactose by the red cell is slower t h a n t h a t of glucose (Lachhein ct al., 1961; Va|cntine ct el., 1967). In the absence of glucose in t h e i n c u b a t i o n medium, b o t h decreased o x y g e n c o n s u m p t i o n and u~ethcmoglobin r e d u c t i o n h a v e been d e m o n s t r a t e d , even in the presence of mct.hylcnc bluc (Betke et el., 1960; BeutlEr and Collins, 1965). I t has been c o n v e n i e n t to e m p l o y a m e t h o d involving the production of lat)elcd CO~ from [1-1aC]GaI as an index o f g a l a c t o s e utilization by the i n t a c t e r y t h r o c y t e (\Veiubcrg, 1961). The measurements, while useful in relation to the hexose m o n o p h o s p h a t e shunt, are n o t to be i n t e r p r e t e d in terms of ovcrall metabolism of galactosc b)" the cell. E x a m i n a t i o n of labeled i n t e r m e d i a t e s under various conditions of iacul)~tion can provide more extensive i n f o r m a t i o n (Bartlett, 1959). I n our own l a b o r a t o r y , it has been observed t h a t in the presence of glucose at a c o n c e n t r a t i o n of 1 mg/ml, there is a s u b s t a n t i a l p r o d u c t i o n of labeled COe by n o r m a l red blood cells from [1-14C]Gal. This is s t i m u l a t e d a p p r o x i m a t e l y 13-fold by m c t h ) ' l c a e blue (Table II). The effects of lower concel~trations of glucose also have been studied. TABLE I [
Effect of methylene blue (M.B.) on 14C02 ?)rr.~luctionfrom [1-1~C]Ga1 Galactose added (mg/ml)
0.075 0-125 0-175
0-225
M.B.
T o t a l 1ICOn (et/min)*
Stimulation b y M. B . t
-~--}--
139,000 9740 161,000 12,200
14x
~ --
15¢),800 11,420
13~
-i-
148,100
13 x
--
1.I , 8 3 0
13x
* C o r r e c t e d to c o n s t a n t specific a c t i v i t y o f g a [ a ~ t o s e . ~f ~ ' I e t h y l e n e b l u e c o n c e n t r a t i o n i n t h e i n c u b a t i o n m e d i u m is 0-061 m g / m l .
ll~ a t y p i c a l e x p e r i m e n t it wa~ f o u n d t h a t a t lower c o n c e n t r a t i o n of glucose, the a m o u n t of C02 formed from galactose in a given time period was inversely proport i o n a l to t h e glucose c o n c e n t r a t i o n . The r a t e of p r o d u c t i o n of C02 from galactose was c o n s t a n t a t glucose c o n c e n t r a t i o n s higher t h a n 0-05 m g / m l (Fig. 3). One e x p l a n a t i o n for this effect of glucose is isotope d i l u t i o n of i n t e r m e d i a t e s (Sturman, 1969). I n similar experiments, t h e effect was e x a m i n e d of v a r y i n g t h e galactose c o n c e n t r a tion (with [1-14C]Gal a t c o n s t a n t specific a c t i v i t y a n d with a c o n s t a n t c o n c e n t r a t i o n of u n l a b e l e d glucose a t 1 mg/mI). I n o t h e r studies, t h e p r o d u c t i o n of labeled c a r b o n dioxide from [1-14C]Glc in t h e absence of galactose was determined. I t was f o u n d
(IALAC'I'OSE
ME'I.'AIIOLISM
OF
THE
ICED
CELl,
407
(Fig. 4) t h a t the c o n c e n t r a t i o n a t s a t u r a t i o n level for CO 2 p r o d u c t i o n is surprisingly low for both sugqrs. For glucose i~ is a b o u t 0-l mg/ml, and for galactose a b o u t 0-0,5 rag/ ml. At sat.uration levels, and with specific activities expressed o n the same basis, the a p p a r e n t rate of CO., t)roduction from glucose is four to five times t h a t from galaetose under the particular e x p e r i m e n t a l conditions. w o
8O
7O I
A i
o
50 40 30
E
-
20I0-
0
0
l
I
0-05
0"I0
Glucose
Fh~. 3. T h e cll,'ct o f glueo.~e c o n c e n t r a t i o n
/
~r¢'9
x
0 0 05
Hexose
F I o . 4. C o m p a r i s o n centrations.
of
ttCOs
production
'
0-15 0.10
I
0-20
,,
,,|
,
0-25
0~30
o n t h e p r o d u c t i o n o f ItCO~ f r o m [1-taO]Oal.
..
~
,
(mg/ml)
r
I !
O
1
0"15
14
I
0-25 0 20
.
//
--
0 40 0 60 0-:30 0 50
(mglml)
f r o m [ I - I ' C ] G a l a n d [1-14C]CIc a t (tifforenC s u b s t r a t e
con-
Intermediates of Galactose Metabolism
Pulse-labeling studies of galactose m e t a b o l i s m in i n t a c t e r y t h r o c y t e s have been carried o u t in our l a b o r a t o r y . The products of i n c u b a t i o n were e x a m i n e d b y D o w e x - I formate columl:, c h r o m a t o g r a p h y . The results of a typiea[ e~rly e x p e r i m e n t are shown in ]rigs 5 and 6. Two inert c o n t a m i n a n t s present in the labeled galactose used a t t h a t time appeared in the column fractions. -Wighin the first 5 rain, the carbon label from [i-14C]Gal a p p e a r e d in Gal-I-P a n d in UDP-hexose. After 15 rain, Gal-I-P c o n t i n u e d to increase in a m o u n t , b u t there was o n l y a slighL rise in UDP-hexose. A t this time, labeled d i p h o s p h o g l y c e r a t e was detected. P e a k n u m b e r 5 represented several compomnds, including triose p h o s p h a t e s a n d hexose d i p h o s p h a t e s . After 30 rain of i n c u b a t i o n (:Fig. 6), there was f u r t h e r a c c u m u l a t i o n of GM-1-P,
408
W.G.
NG
w h i l e t h e a m o u n t of U D l ) - h e x o s e r e m a i n e d essentially u n c h a n g e d . T h e r e was a n i n c r e a s e in triose p h o s p h a t e s , h e x o s e d i p h o s p h a t e s and d i p h o s l ) h o g l y c e r a t e . Labeled lactic acid first w a s e v i d e n t a t G0 rain of i n c u b a t i o n . G a l - l - P was still accumulat.ing, as was d i p h o s p h o g l y c e r a t e . 800
-
2 I00 .~_ E
0
15 m m
800]-
2
too6
5O
I00
Tube number :FIG. 5. Labeled interme
2
2000 ~000':
30
__
4 .a
6
I00 ~ E t,3,
A
0 2000
"-
I 000""
m,n
60 rain
=
6
4
gi
'°I /t 0~
50
I00
Tube n u m b e r
Fro. 6. L a b e l e d i n t e r m e d i a t ~ f r o m e r y t h r o c y t e i n c u b a t i o n :Fig. 5 for e x p l a n a t i o n o f labeled peaks.
with []-x~CJGal
(normal adult).
Sea
T h e s e e x p e r i m e n t s s u g g e s t e d t h a t t h e m e t a b o l i s m of g a l a c t o s e in t h e i n t a c t red cell is a r e l a t i v e l y slow process a n d t h a t t h e r e m o v a l of G a l - I - P m a y be a r a t e - l i m i t i n g factor. I n t e r m s of t h e t r a n s f e r a s e r e a c t i o n , t h e a c c u m u l a t i o n of G a l - I - P c a n be influenced b y t h e c o n c e n t r a t i o n of G l c - I - P , U I ) P - G l c a n d U D P - G a l . T h e r a t i o of t h e l a t t e r t w o c o m p o u n d s is r e g u l a t e d in t u r n b y e p i m e r a s e a c t i v i t y .
(;-kI,ACTOSE
METABOLISM
OF THE
I'~ED C E L L
409
Results of columll chrom~ltogral)hy of l)roducts forl~led from [lJ4C]Gal in a 90-rain il~cubati~ with ('rvthro('vtcs of nor~ml individuals and of galactosemic patients arc compared in l"iv,. 7 (Ng et el., 1967a, b). For these studies, [lJ~C]Gal had been i)uritb(I. With gala(-toscmie patienu% tile cxl)ecte(1 large peal: of G a l - l - P was found. 2000,
!
^
4
,
(a
0 8000
"~
(b)
E
200 ~ 0 8000,
(c)
A
-j
• i
0
.
.
.
.
.
50
lO0
Tube number
I,'.;. 7. ('o.[i)~u'ison o{" labeled galactose intermcdiat~s from e r y t h r o c y t e ineubaC~ions. ,4, Patt~l'n from l~.ram; B awt C from g a l a e t o s e m i c patients. T h e peak n u m b e r s r e p r e s e n t e o m p o i m n t s as f¢,ll,~u'~: I. lactic a c i d : 2. (;~d. I - l ' ; 3, L : D P - h c x o s c : 4, triosc i)hosphate~ a n d hexo~c d i p h o s p h a t e s ; trod 5, liphouphoglyeero.t ~,.
tlormal
In addition, a sl~lal] peak corresponding to U D P - h e x o s e also was noted. F o r three ~tmo~g thc galactoscmic itldividuals studied, the U D P - h e x o s e p e a k was increased a n d labeled diphost)hoglycerate was detected. I t is possible that, some galactosemic patients m a y h a v e a slight residual transferase a c t i v i t y (Schwarz, ~Vells, Holzel a n d Komrower, 196l; IIsia, Tannenbatm~, Schneider, H u a n g a n d Simpson. 1960), b u t tlle possibility of participation of a subsidiary p a t h w a y c a n n o t y e t be, eliminated. Ingestiou of galactose l)y galactosemic patients results iI1 the accunuflation of Gel-1-P in erythL'oeytcs. The conceutration m a y become as high as 0-2 m g / m l a f t e r an oral galactose tolerance test (Donncll, Bcrgren, P e r r y and Koch, ~.963). t-Iowever, 2,t hr later the G a l - I - P level has r e t u r n e d to the baseline level. During in vitro experiments to simul~tte the in vivo conditions, accumulation of labeled Gal-I-:P was produced b y preincubation of e r y t h r o c y t e s with radioactive galactose (Donnell, Bergrcn a n d Ng, 1967a). I t was found during tile s u b s e q u e n t 24 hr t h a t the l a t e cellular concentration of G a l - l - P decreased substantially and t h a t a corresponding a m o t m t of r a d i o a c t i v i t y a p p e a r e d as free g a l a c b s e in the medium. I t was p o s t u l a t e d t h a t the decrease in G a l - l - P could result from nonspecii~c p h o s p h a t a s e activity. Similar ob~erx'ations have been reported recently (Gitzelman, 1969). ~ ' t e r an extended period of incubation of galaetose a n d red blood cells from galactosemic patients, in t h e absence of glucose, a drop in adenosine t r i p h o s p h a t e (ATP) level was observed (I)enington a n d P r a n k e r d , 1958). The inability to m a i n t a i n A T P c o n c e n t r a t i o n could a c c o u n t for a n increase in p o t a s s i u m flux in galaetosemic
410
\V. G. N O
er).%hrocytes when galactose is the only c a r b o h y d r a t e source (13ear and Gordon, 1964). However, i t has been shown when glucose is present, a c c u m u l a t e d O a l - l - P in e r y t h r o c y t e s of galactosemics does n o t affect the level of :VI'P or of gl.vcolytic i n t e r m e d i a t e s (Zipursky, l t o w l a n d . Ford. 1-Iaworth and Israels, 1965). Screening for Galactosemia
A l t h o u g h t h e frequency of occurrcnce of t,he transferase defect and of the galactokinase defect is low (Schwarz et al., 1961" Beutler, Baluda. Sturgeon and l)av, 8 it. is i m p o r t a n t to be aMe to detect, these genetic 1966 ; Mayes and Guthrie, I96,:), disorders by relatively simple procedures. In our laboratory, in the past ten ~nonths alone, we have formal one transferase defect in our own hospital and have confirmed six new cases referred from o t h e r hospitals on tim basis of screening 1)roeedures or of clinical suspicion. I n addition, an example of the galactokinasc detk'ct was found (galactokinase defec"c samples referred by :Dr I). Frazicr of Los Angeles Count) ..... U.S.C. 5iedical Center). A n u m b e r of c o n v e n i e n t tests now are available for the detection and confirmation of these defects (Table I I I ) . TA!)L)" I I I
Methods f o r detccHon o f !talac~osemia Screeni~lg proceclure~ :
I. ])apcr c h r o m a t o g r a p h y _'2. ~iicrobiologieal 3. M e t h y l e n e blue r e d u c t i o n 4. ~ A D I ) H fluore.~ceneo 5. I~CO~ from [1-1'C]Gal
Specific ussays : I. 2. 3. 4.
U D P - G k : cons-eruption ~Ianometric tl~dio~ctive K i n e t i c coupling
Some screening m e t h o d s are based on the presence of exce~i ve a m o u n t s of galactosc in uxine or blood, as identified by paper c h r o m a t o g r a p h y ( H a w o r t h and B a r c b u k , 1967; D a h l q v i s t , J a g e n b u r g a n d Mark, 1969) or .by a microbiological i n h i b i t i o n assay (Guthrie, 1968). O t h e r procedures utilize coupling to the hexose m o n o p h o s p h a t c s h u n t . . F o r example, t h e presence or absence of transferase a c t i v i t y in e r y t h r o e y t e s can be d e t e c t e d readily b y m e t h y l e n e blue reduction or by the a p p e a r a n c e of fluorescence due to g e n e r a t i o n of NA]:)P]-I (Brewer and Tarlov, 1963; Beutler, Balud~ a n d :Donnell, 1964; B e u t l e r a n d Baluda, 1966b; I:lochella and Hill, 1969). z ~ m t h e r a p p r o a c h involves the f o r m a t i o n of labeled C02 from [1-1aC]Gal b y intact, red blood cells (London, M a r y ~ m n t a n d Fuld, 1964). Specific e n z y m e assays are essential for confirmation. Transferase assays can be p e r f o r m e d b y t h e U-D:P-Glc c o n s u m p t i o n procedure (Anderson, Kalckar, K u r a h a s h i a n d Isselbacher, 1957b; B r e t t h a u e r , :Hansen, ]-)onnell and :Bergren, 1959; Melhn~n a n d Tedesco, 1965; :Beutler a n d Baluda, 1966a), by oxygen c o n s u m p t i o n ( K i r k m a n and B y n u m , 1959), b y t h e i n c o r p o r a t i o n of labeled GaI-1-P into 17DP-Gal (:Ng, Bergren a n d Donnell, 1967a ; I n o u y e , N a d l e r and Hsia, 1968 ; Sawicka and Chojnack, 1959; T r o n a n d Milhaud, 1969), or by s p e c t r o p h o t o m e t r i c m e a s u r e m e n t of coupling
l +l +.,, "+"i.; I. iilii+l
it
('q~ii~l~+'lJ'i.,.++++i
lll'~'+lilil~',l
<~I','i",'ilit'+,(.,,'t~
+'1+,>.~ +\ll~t'h'+'"
+ tt'iili,
ll+l:ll,'t~.~'.<_'
|i+11tt'i'ii.,.t
'.l'h~'+ iril+lt.-lliu+,l
~.+II +'+t+tt'<'lt
I)txttttl'll
<~t'l t'(+t'( '+>t llt>','+~++
t>t.tx+'(.,,tL
t'~,lli'ost++lit+ "'I+.++\. +. iilili,,'lthl+ll.
iltJl'lltili
GALACTOSE
M E'I'AIiOL1SM OF THE
REI') CELL
411
to t.be hexose monophosphate s h u n t (Colombo, Moser, :Rosin, Richterich and :Rossi, 1965; Beuth;r and Mitchell, 1968; Copeahaver, Bausch and Fitzgibbons, 1969). Galactokinase has been assayed by the incorporation of labeled galactose into Gal-I-P. in the presence of A T P (Ng, Donncl[ and Bcrgren, 1965; de Verdier, 1966). The details, a d v a n t a g e s and disadvant~ges of some of these methods h a v e been presented in a symposium on gnlaetosemia. (Hsia, 1969). Genetic Variants of GaI-I-P Uridyltransferase
In genetic counseling, the capability of identifying heterozygotes is i m p o r t a n t . \Vhile ~'.arriers of galactosen~ia have halfknormal e r y t h r o c y t e transferase activity, the occm'rence of transforase variants in the population presents a problem in identification of the gala,-t.oscmic hcterozygotes. The a s y m p t o m a t i c D u a r t e v a r i a n t homozygotc also Ires half-nor,~ml e r y t h r o e y t c transferase activity (Beutler et el., 1966). I n starch gel elect rophorcsis, using a phosphate buffer, the D u a r t e v a r i a n t enzyme migrates as a single band of higher mobility than t h a t for the normal enzyme, b a t the ~wo forms t)resent in hcmolysates from ] ) u a r t e - n o r m a l heterozygotes c a n n o t be se[)arat(,',I clearly in this system (Me thai and Beutler, 1966). This problem has been resolved (Ng. Bcr.m'cn, Fields and DonnelJ, 1969), however, by use of Tris-g!ycine buIlbr at, p | I 8-8. In d~c moditicd eiectrophoretic procedure, the D u a r t e v a r i a n t enzyme exhibits triple bands while only one is sce~} with the normal enzyme (Fig. 8). W i t h the D u a r t e Hemoglobin Ocm ~- I
.......
I
Fluorescent !
i
I
I
l
I
I
bands tO ~
..
i
g
0
oo
I
$
0 O0
I-i-]
+
Fro. 8. Migration pt~ttern of c r y t h r o c y t a Gal-1-]? uridyltransferase in starch gel electrophoresis in Tris-glycinc buffer a t p K 8.8. ~NN. uormal homozygote; ND, n o r m a l / D u a r t e heterozygotd; ]319, :Duarte homozygotc; and (ID, gnlactoscmic/Duarte hctcrozygotc.
homozygote ~here are three bands, the two faster b a n d s p r e d o m i n a n t . Three b a n d s also are seen with the ] ) u a r t e - n o r m a l heterezygote, b u t the slowest band, corresponding in position to Che single b a n d of a. normal control, is outstanding. The D u a r t e galactosemic hef, erozygote shows three faint bands. A n o t h e r e r y t h r o e y t e transfera.se v a r i a n t t e n t a t i v e l y n a m d d the " L o s Angeles" variant, has been ictentified in our laboratory. The iudixdduals p r e s u m e d to be homozygous have elevated transferase activity in c o n t r a s t with the reduced activity for the Y)uarte variant. The elec~rophoretic p a t t e r n s are similar to i:hose for the D u a r t e v a r i a n t (Plate 1). A n u m b e r of " L o s Angeles" v a r i a n t families h a v e been studied, and the Kndings are eonsisten~ with a u t o s o m a l inheritance. The distribution of e r y t h r o c y t e transfe.rase activity a m o n g know-n genotypes is summarized in Table IV. I n t e r m s of m e a n value, t h e gal~ctosemic-normal heterozygore has o n e - h a ~ n o r m a l activity, while t h a t for the galactoscmic homozygote is
412
~V, G. ~NTG
negligible. I n r e l a t i o n to n o r m a l e r y t h r o c y t e t r a n s f e r a s e v a l u e s , t h e D u a r t e - n o r m a l h e t e r o z y g o t e e x h i b i t s t h r e e - f o u r t h s , t h e D u a r t e h o m o z y g o t e one-half, t h e D u a r t e galactosemic h e t e r o z y g o t e one-fourth, the Los Angeles-normal hcterozygote somew h a t e l e v a t e d a c t i v i t y , a n d t h e p r e s u m e d Los A n g e l e s v a r i a n t h o m o z y g o t c significantly increased activity. One p r e s u m e d Los-Angeles-galactosemic heterozygote h a s b e c n f o u n d to h a v e o n e - h a l f n o r m a l a c t i v i t y . I t is e v i d e n c t h a t w i t h o u t e l e c t r o p h o r e s i s it is n o t possible to d i s t i n g u i s h a m o n g all of th ese g e n o t y p e s . 3?Ar~rm IV
Erythrocyte transferase activity distributioqt among differ¢mt genotypes Prc.sumed genotype
No.
N-~NG-N G-G D-~N D-D D-G I.~Y-N LA-LA LA-G
123 69 9 40 2 3 36 5 I
Transfcra~ act iv ity* Mean Range 24 "8 10.9 {)-2 17-9 10-8 5-7 26-7 32-0 12-7
18"/-33-3 5.6-16.5 0-0-8 12-6-24-3 9-9-11.7 5.6-5-8 18-8-35-5 2 L-0--40.5 --
* Activity is expressed as nfieromoles of Gal-l-PV'~neorporated into UDP-Gal per h per g hemoglobin. I n a s t u d y in o u r l a b o r a t o r y , w h i c h so f a r has i n c l u d e d 400 blood s a m p l e s f r o m C a u c a s i a n s , t h e f r e q u e n c y of D u a r t e - n o r m a l h e t e r o z y g o t e o c c u r r e n c e has b e e n f o u n d to be a b o u t 12°/o, c or t f i r m i ng earlier" r e p o r t s ( B e u t l e r et al., 1966; M e l l m a n , T e d e s c o a n d Feigl, 1968), a n d t h a t of t h e L o s A n g e l e s - n o r m a l h e t e r o z y g o t e a b o u t 4°//o. The. o c c u r r e n c e of t h e s e v a r i a n t s in 110 A m e r i c a n N e g r o e s zo far s t u d i e d is a b o u t t h e s a m e as t h a t in Caucasians, w h e r e a s o n l y t w o D u a r t e - n o r m a l h e t e r o z y g o t e s w e r e f o u n d a m o n g 105 u n r e l a t e d O r i e n t a l a d u l t s . T w o o t h e r e r y t h r o e y t e t r a n s f e r a s e v a r i a n t s of g a l a c t o s e m i a h a v e b e e n r e p o r t e d ( S c h a p i r a a n d K a p l a n , 1969; C h a c k o , C h r i s t i a n a n d N a d l e r , 1970). A C K N Ox,V L E D G 3 I E N T S I wish to express m y appreciatiml to Drs William 1%. ]3ezgren a u d George N. D o n n e l | for t h e o p p o r t u n i t y to p a r t i c i p a t e ill collaborative efforts over a period of years, a n d for their suggestions a n d criticism in th e p r e s e n t work. R k~'~l~ E N C E S Abraham, ~,Ynderson, Anderson, 2~led.
H. E. E. 50,
D. and Howell, i%. l-l. (1960). J. 13ioi. Ohem. 244, 545. P., ]~alckar, l:[. ~I. and Isselbacher, K. J. (1957a). Scienve, N.Y. 125, 113. I~., Kalckar, 11. hl., Kurahashi, K. and Isselbacher, K. J. (1957b). J. Lab. Clin. 469.
:Baar, H. S. and Gordon, ~[. (.1964). zVature (London) 201, 1223. :Bartlett, G. I~. (1959). J..Biol. Chem. 234, 459. ~Bergren, \V. lZ. (1967). In I:Iereditary Disorders of Erythrocyte l-tIetabolia'm, p. 83. (Ed. by :Beutler, E.) Grune sad Stratton, New York.
(:ALACTOSE
METAI¢OLISM OF THE
RED CELL
413
Bergren, \V. 1~., Ng, \V. G. and ])onnell, G. N. (1967). Pediatrics 40, 136. Betkc, b:., Baltz. A. a n d Maa8, U. (19(i0). Z. Ki?ulerheil. 84, 226. Beutler. l':. (1967). Science, N . Y . 156, 1516. Beu 1ler, 1'3. and l htluda, 31. C. (1966a). Clin. Chir~. A c ~ 13, 369. .Beu! ler, 1",. a n d Baluda, M. C. (1966b). J . Lab. Olin. ,lied. 68, 137. l;eutler, E. a n d l¢aluda, M. C. (1966c). J . l.~b. Olin. Aled. 6 7 , 9 4 7 . 13eutler, 1~., ]~aluda, M. a n d Dommll, G. N. (19t)4). J . l_,e~b. Cli~. M e d . 67, 694. E.. I3ah~d~, 31. C., Sturgeon, P. a n d Day, R. ~,V. (1966). J . . L a b . Olin. M e d . 68, 646. Beut.ler, E. a n d (?ol)ins, Z. (1965). Sc~,nd. ,]. ]lae,uatol. 2, 377. Beutler. E. a~(! Mitcb-l]. ),1. (1968). ,1. [.~b. Clin. Aled. 72, 527. B r a m l l , N. J . , Fro]and. A., Mikkelse,~, M., ~Nielsen. A. and Tolstrup, N. (1963). Lancet ii, 700. Brctth~u(.r, R. K., }t:Lnse.n. 14. G., Doanc|l, G. N. a n d ~¢ergron, \V. I4. (1959). Pro~. N a t . Ac,ad. Sci. L'.S'..-i. 45, 328. 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