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peroxidase method for glucose assay leads to overestimation of the inhibition of gluconeogenesis by aminopyrine
152
BBA Report
BBA 2O229
Use of the glucose oxidase/peroxidase method for glucose assay leads to overestimation of the inhibition of gluconeogenesis
by aminopyrine P a w e I L a w s a n d A n n a B. W o j t c z a k
Key ~ d s : G~o~e ass~. A ~ J ~
4-A~no~.
G ] . ~ ;
(~p~-~);
(~=~,~)
4 - A m l m m m ~ r ~ e ~ m a ~ Zahi~its g ~ m s e ~ ~ m e gJacose o x l d a s e / p e r o z i d a s e / ~ a s s ~ bat does at~ iatedete /a the a~ay ~ ~pl~pl~te d e l ~ j d m g e ~ hexel~ase a~l ATP. A s a ~ file ~ of gbumaeob~esis by ambmp,jriae nepoded to be . f ~ - ~ ( B i a b e g ~ G . M m ~ , J . Ante~, F. msd Garz6, T. (19~0 Bh~hlm. BioldO~ Acta 92~, 406-41Q is s~magly ov~g:~thtmlzd aml ~ to oaly I0-30~.
The effect of aminop3aine on gluco~ex~enesLs from lactate and other precmsors was studied in perfesed rat liver [i] and isohted mouse hepatocytes [2,3] in phenobarbital-induced and untreated animals. These studies indicated that ghconeogenesls was inhibited by concomitant oxidation (demethylalion) of aminopyrine. This obserwafion was interpreted as showing that gluconeogenesis and aminopyrine oxida~on compete for malate which is used as a percursor for glucose synthesis and, via malate dehydrogeaase (decarboxylating) (EC 1.L1.38), can also pwduce N A D P H utilized for aminopyrin¢ oxidation by the microsomal moaooxygenase system. Thunuan and Scholz [1] showed that, in perfused liveT of slarved rats, ~ e n e s / s from lactate was suppressed by about 20~ in the presence of aminopyrine. This inhibition increased up to 50% in livers of phenobarbital-treated rats. On the other hand, B~mhegyi et aL [2,3] reported about g0~ inhibition of glucon¢ogenesis by aminop3a'ine in isolated mouse hepatocytes. In our experiments with hepatocytes isolated from fed rats we have measured a substantially lower level of glucose if aminopyrine was present in the incubation medium. Under these conditions gluco~ was derived from glycogen, since gluconeogenes/s is very low in Abbreviation: EGTA,ethylene gly¢~ bls(~-anfiaoethylFN, N.N'.N "tetraacetie acid. Correspondence: A.B, Wojtczak. Department of Cc~ular Biochemistry. Nencki Institute of E x ~ t a l B/ology. Pasteura 3, 02-093. Wmsaw,Poland.
hcpatocyces from fed rats. As there is no/nd~athm flint am/nopyrine inhibits glycogenoI3~ we assumed that the product of aminopyrin¢ oxidation may /nteffere with glucose determination. In fact, ~ : found that 4aminoamlipyrlne strongly interfered with the assay for glucose based on glucose oxidase ~ 1.1-3.4), peroxidase (EC 1.11.1.7) and d;anicidine [4]. Fig. 1 shows that glucose determination by this assay was in~'bited to the same extent by authentic 4-aminoantipyrine and by the product of aminopyrine oxidation by ,n/crosomes added as the n e u ~ perchloric a ~ d extract from an incubation mixture of rat fiver mk:rosonM~ aml an~nopyrine~ The inhibition increased with decreasing glucose/4-aminoaufipyrine ratio in the assay med~ung In this assay system glucose is oxidized by. glucose oxidase to gluconic acid with a stoich/omeU~ fo~aalion of H202, the latter being determ/ned due t~ the f o r t ~ tion of an o r a ~ ¢ peroxid~tion product of d/anis/dine in the presence of peroxidas~L As ah'eady point~l out in the original description of this procedure [4], compounds which compet~ for the hydrogen peroxide formed will thus result in an underesfin~ti~n of glucose. One of such compmmds is 4-aminematipyrine which, in the presence of peroxidas~ reacts with H202 forming a hydraTo- and subsequently an a z o - ~ [5]. However, glucose does not inhibit the assay for 4-aminoanfipyrine, which is carried out in the presence of an express of H202, and the peroxidation prodtu::t is condensed with phenol forming red quinoaimlne [6.7]. On the other hand, it has been ascez'ufined 4-amino~nfipyrine and aminopyfine at I m M concentrations do not interfere in glucose delermie~fion
03044165,/89/503,50 © 1989 ElsewierS c i e ~ PabllshexsB.V.(Bionzedi~d ~ o a )
153
m
~oOA A st"
0
A
"6" toc
0 A --
I
A
r,,,.,e(,~) Wtf~2. Ttme-com-~ of $1uco~ and 4-arninoanttpydae formati~ ia I~eamc~.es ftem 12 h stam:d rats. Hepatet'y~ (6 n~ d ~ weight/n~ ~cd ~ m Ben~ a~l Fr~ul 191v,ere incubated at 37°C mt~a~r~ *~,~th~ m~m"9~g O.z+ $% CO.z in the IOebs-El~se~t bwad~omm~ ~edmm |10] ~u~dnln~ I mM CaCIz, 1~ defatted and dia~zgd bo~ne tmum a~bum~ l0 mM laclate and. where i ~ 1 mM a~mp~jdme. I ~ b a t k m was s ~ p e d by additima of pe~hlm~ add m a f'm~ ~ t r a t ~ a ¢d $~. G l ~ t e a~d dl.amiaeantipy¢i~ wet~ ~ in mmtral~d pe~ldor~ acid ~xtga~ts.Ciluc~ was ,ka~d w~ e~me-6~uae dehydrogena~ he~,~.inase and ATP m de~crt~bed~ ~ad. g .~1 ~ t i p y l i n e as in Figr I. ~ O. 4 - ~ (remJ~ from two ex~petimeats). Glumse alter i ~ mbat~m witl~et 01) and with ( ~ amia~pyriue.
.,x
.
.
.
.
.
.
.
.
.
O
~
by t ~ I#mm* m i d ~ at~'y. ~ a , co~ ' r ~ ~ as demnt'~*l R ~ 4 w~b a ~ p n ~ from Boe]ui~er ~ F.R~G.~. of f~l ~ W'ma~ ra~ ~u-e paf~a~l ba ~m ~ l h 0.9~e gca~ tbtm
~'fc~s~,~ ~ aud fux,~mys ~ p ~ d in 1® ~ K ~ / 2 0 t i m T ~ I I C I f~H 7.4k ~ (1-2 m~ l ~ m ~ / m [ ) ~ mc u b a ~ l ~m ~0 mM Tt~s.l.1.,Cl (pH 75)/LS0 mM KCI/10 mm m 3~0 C l a m ~ i ~ ~ ~
s t e ~ l by ~
using glucose-6-phesphate dehydrogenase ( E C 1.1.1.49), he~okinase ( E C 2.7.L1) a n d A T P [8] (not shown). Therefm-e, in ~ experiment depicted in Fig. 2 this assay system for g l u c ~ e determination was applied. It was slmwn t h a t 81tr.ose production from l a c t a t e by hepato~jtes isolated from starved rats was linear for a t least 3 0 rain, although it was lower b y a b o u t 2 5 ~ in the preseuce o f I m M aminopyrlne, which is in agreement with Ref. I but much less t h a n in Refs. 2 a n d 3- T h e
~ pet~d~ a~l
pe:g&~k ;gid c a t n ~ by ~ e me~0d c~ Tfmd~ ~] az
c ~ e # ~ ngu~-~2~ e m r a ~ from ~ a~aopyri~_ G~uome ~ d ~
~ e d ~ith ce~mU~o~ ~
TABLE l ~ ~:re ~olaled frma rms ~ r ~ e d f ~ 12 h. They w~z~~ a d m ~ l at the c~enu-~ion of 4~6 mg dry weight/~ m ~ go F-zg. 2 h~ ~ e Kn~s-Hcmsdeil I~-bou,~e medima , - ~ i mM Ca~2. 1% bot~e se~m ~ ~ 10 mM | ~ Amiaop,~iae (1 re.M}aad ~ (I WM)wev~added wI~m'ei m t ~ z ~ Im:ubafima w~m~opped af¢or ~ ~ a ~ t ~ ~ i ~ of ~ m the ~
~ 1 m ¢1~:h~emis to F~gs~1 a~d 2_The value~age m ~ u ~ : S.D. f ~ 4-7 ~
l~-odtgtiem( p n ~ / h per g dry ~-,~ht ) 8]uco~ ~ ~ ~
Lactate Lactate+~.z~m Py*uvate
t
e
wi~ ~ f ~ t
dehy~
~l ~
method
con~el
~-amlmap~iae
%id~ b~iee
comrol
+amLnopyrme
~ inhlbitiea
149~ 16 235±32 143::k32
17ili 94+20 50± 5
8g 60 65
128~ 13 229~a0 135~32
87+14 1"/2-t-4~. 121±29
32 2~. 16
.
m ~ 1~ m ~mv~. ~d ~
To ~ n
~ ~
4-Amino.qntipyrine formation (p.mol/h per g dt-y weigh0 43± 2 M':i: I0 46~: 13
154
of 4-amino~tipyr~e production shows (Fig, 2) that its rate was h i g h ~ during the lust 10 nfin than during the subseqoe~t 10-30 rain p~iod. lower/rig of 4-an~hu~utipyrine formation may be caused by product inhibition as observed by Thutman and
~1 ~ t s to 10-30~, depe~llag c~ the g l ~ e u m genh: precursor. We also want to da~w attention to a ~ i t e d appl~abi~ty of the gh~ese ox~ase/petoxidase assay for glucose [4]; namely, caution ~ a s t be takea that no mher n:actio~ utgt~ng H20~_ are i m a m s . T l ~
Tabie I shows the effect o f amlnopyrine on gluconeogenesis from lactate and p ~ a v a t c as measured by the two assay systems for g l ~ . In agreement with Ref. 2, g l u ~ production from p3~'uvate was inhibited to a lower extent than from lactate. ~ , the degree of inhibition by aminowjrine d~ffered very much whea measored with the t~o methods, being mueh lower wRh the ghw.osc-6-phosphate dchydrogen~e procedure. Glucagon, as ~ s ~ d y increased g l u ~ e production from lactate, bet decreased the formation of 4-aminoantipyrine. The ~atte~ observ~tioa is new and in line with a recent report by B~u~hegyi et al. [11] who found a n inhibitory effect of dibutyryI-~.MP on the oxidation of aminopyrine in isolated mouse liver hepatocy[~ and m ~ o m e ~ N e s ~ ' t h e l ~ also in tbis publicafio~ glucose p r o d ~ n was probably uaderestimated as ~t was measured ~ith the glucose oxidase procedure. In conclu~on, the pres~at paper shows that the lm,g¢ apparent inbi'bition of gluc~3neoo~sis by aminopyrh~ as described by B ~ e s ~ i ct aL [2,3,11], is an artifact due to the interferen~ of the aminopyrine oxidation product. 4-amlnoantlpy~n~ in the glucose assay used by thes~ authors. The real inhibition is much smaller
shown in Table 1, van be aleut'bed in ~
detail
This work was supported by G r a m CI'BP O4.01 o f t ~ PoI~h Academy of Sciences.
1 ~ 2 m~si,
R.G. aed ,f,&e~ R. (I~r/3) E.~r.]. B i C ~ L 38. 64--7~_ G.~LAa~.F.a~Gar~T.il~l)B~m A~a 927,4 ~ * 1 6 .
Bie~em. 20,101-104. 4 Be~me)er. H.U. a ~ Beret. E {1974) ~,, M ~ c l s d ~ & ~ y ~ IBez~te,ser. ELU. e~L),v e t 3. pp. 12~-i2|5. ~ew YeA.
$ Karts, P. (1950)Orglm~C ~ , Ne~ yor~
C~
3L p. 45L E~,-der.
6 T t ~ r . PJ. (1969) Am~. CEm. Biodte~L 6, .7A--36_ 7 GemmL EL f1977} J. C~L Chem. C't~ Biodae~ gS. ( ~ 9 - ~ . S B u r A m ~ , BLU. a e r ~ E~ S , : I u ~ . F . ~ Srm~-kit. (1974) i= Mee~ds d F . ~ z ~ l ~ Aeal)~s ( ~ . I L U . e~.). V~L 3, Ep.
11~6-120L ~ Press, ~ York. 9 Ber~, M.N. a~l Frk~d, D~q.(1~9) $. Cell BioL430 5~6-5~. 10 ~ HA. Ed ~ l . IC (1932) ~ s 7-Che~. 210, 3~66. 1| B ~ ' i , G. Garzb, T. Meszagt~G~ Far,go. A_ ,q~f~e~ F. aetd M.~di. J. f l g ~ ) B i ~ h ~ L P ~ a ~ a c ~ 37, 8~-~64.
BBA Report
BBA 2O229
Use of the glucose oxidase/peroxidase method for glucose assay leads to overestimation of the inhibition of gluconeogenesis
by aminopyrine P a w e I L a w s a n d A n n a B. W o j t c z a k
Key ~ d s : G~o~e ass~. A ~ J ~
4-A~no~.
G ] . ~ ;
(~p~-~);
(~=~,~)
4 - A m l m m m ~ r ~ e ~ m a ~ Zahi~its g ~ m s e ~ ~ m e gJacose o x l d a s e / p e r o z i d a s e / ~ a s s ~ bat does at~ iatedete /a the a~ay ~ ~pl~pl~te d e l ~ j d m g e ~ hexel~ase a~l ATP. A s a ~ file ~ of gbumaeob~esis by ambmp,jriae nepoded to be . f ~ - ~ ( B i a b e g ~ G . M m ~ , J . Ante~, F. msd Garz6, T. (19~0 Bh~hlm. BioldO~ Acta 92~, 406-41Q is s~magly ov~g:~thtmlzd aml ~ to oaly I0-30~.
The effect of aminop3aine on gluco~ex~enesLs from lactate and other precmsors was studied in perfesed rat liver [i] and isohted mouse hepatocytes [2,3] in phenobarbital-induced and untreated animals. These studies indicated that ghconeogenesls was inhibited by concomitant oxidation (demethylalion) of aminopyrine. This obserwafion was interpreted as showing that gluconeogenesis and aminopyrine oxida~on compete for malate which is used as a percursor for glucose synthesis and, via malate dehydrogeaase (decarboxylating) (EC 1.L1.38), can also pwduce N A D P H utilized for aminopyrin¢ oxidation by the microsomal moaooxygenase system. Thunuan and Scholz [1] showed that, in perfused liveT of slarved rats, ~ e n e s / s from lactate was suppressed by about 20~ in the presence of aminopyrine. This inhibition increased up to 50% in livers of phenobarbital-treated rats. On the other hand, B~mhegyi et aL [2,3] reported about g0~ inhibition of glucon¢ogenesis by aminop3a'ine in isolated mouse hepatocytes. In our experiments with hepatocytes isolated from fed rats we have measured a substantially lower level of glucose if aminopyrine was present in the incubation medium. Under these conditions gluco~ was derived from glycogen, since gluconeogenes/s is very low in Abbreviation: EGTA,ethylene gly¢~ bls(~-anfiaoethylFN, N.N'.N "tetraacetie acid. Correspondence: A.B, Wojtczak. Department of Cc~ular Biochemistry. Nencki Institute of E x ~ t a l B/ology. Pasteura 3, 02-093. Wmsaw,Poland.
hcpatocyces from fed rats. As there is no/nd~athm flint am/nopyrine inhibits glycogenoI3~ we assumed that the product of aminopyrin¢ oxidation may /nteffere with glucose determination. In fact, ~ : found that 4aminoamlipyrlne strongly interfered with the assay for glucose based on glucose oxidase ~ 1.1-3.4), peroxidase (EC 1.11.1.7) and d;anicidine [4]. Fig. 1 shows that glucose determination by this assay was in~'bited to the same extent by authentic 4-aminoantipyrine and by the product of aminopyrine oxidation by ,n/crosomes added as the n e u ~ perchloric a ~ d extract from an incubation mixture of rat fiver mk:rosonM~ aml an~nopyrine~ The inhibition increased with decreasing glucose/4-aminoaufipyrine ratio in the assay med~ung In this assay system glucose is oxidized by. glucose oxidase to gluconic acid with a stoich/omeU~ fo~aalion of H202, the latter being determ/ned due t~ the f o r t ~ tion of an o r a ~ ¢ peroxid~tion product of d/anis/dine in the presence of peroxidas~L As ah'eady point~l out in the original description of this procedure [4], compounds which compet~ for the hydrogen peroxide formed will thus result in an underesfin~ti~n of glucose. One of such compmmds is 4-aminematipyrine which, in the presence of peroxidas~ reacts with H202 forming a hydraTo- and subsequently an a z o - ~ [5]. However, glucose does not inhibit the assay for 4-aminoanfipyrine, which is carried out in the presence of an express of H202, and the peroxidation prodtu::t is condensed with phenol forming red quinoaimlne [6.7]. On the other hand, it has been ascez'ufined 4-amino~nfipyrine and aminopyfine at I m M concentrations do not interfere in glucose delermie~fion
03044165,/89/503,50 © 1989 ElsewierS c i e ~ PabllshexsB.V.(Bionzedi~d ~ o a )
153
m
~oOA A st"
0
A
"6" toc
0 A --
I
A
r,,,.,e(,~) Wtf~2. Ttme-com-~ of $1uco~ and 4-arninoanttpydae formati~ ia I~eamc~.es ftem 12 h stam:d rats. Hepatet'y~ (6 n~ d ~ weight/n~ ~cd ~ m Ben~ a~l Fr~ul 191v,ere incubated at 37°C mt~a~r~ *~,~th~ m~m"9~g O.z+ $% CO.z in the IOebs-El~se~t bwad~omm~ ~edmm |10] ~u~dnln~ I mM CaCIz, 1~ defatted and dia~zgd bo~ne tmum a~bum~ l0 mM laclate and. where i ~ 1 mM a~mp~jdme. I ~ b a t k m was s ~ p e d by additima of pe~hlm~ add m a f'm~ ~ t r a t ~ a ¢d $~. G l ~ t e a~d dl.amiaeantipy¢i~ wet~ ~ in mmtral~d pe~ldor~ acid ~xtga~ts.Ciluc~ was ,ka~d w~ e~me-6~uae dehydrogena~ he~,~.inase and ATP m de~crt~bed~ ~ad. g .~1 ~ t i p y l i n e as in Figr I. ~ O. 4 - ~ (remJ~ from two ex~petimeats). Glumse alter i ~ mbat~m witl~et 01) and with ( ~ amia~pyriue.
.,x
.
.
.
.
.
.
.
.
.
O
~
by t ~ I#mm* m i d ~ at~'y. ~ a , co~ ' r ~ ~ as demnt'~*l R ~ 4 w~b a ~ p n ~ from Boe]ui~er ~ F.R~G.~. of f~l ~ W'ma~ ra~ ~u-e paf~a~l ba ~m ~ l h 0.9~e gca~ tbtm
~'fc~s~,~ ~ aud fux,~mys ~ p ~ d in 1® ~ K ~ / 2 0 t i m T ~ I I C I f~H 7.4k ~ (1-2 m~ l ~ m ~ / m [ ) ~ mc u b a ~ l ~m ~0 mM Tt~s.l.1.,Cl (pH 75)/LS0 mM KCI/10 mm m 3~0 C l a m ~ i ~ ~ ~
s t e ~ l by ~
using glucose-6-phesphate dehydrogenase ( E C 1.1.1.49), he~okinase ( E C 2.7.L1) a n d A T P [8] (not shown). Therefm-e, in ~ experiment depicted in Fig. 2 this assay system for g l u c ~ e determination was applied. It was slmwn t h a t 81tr.ose production from l a c t a t e by hepato~jtes isolated from starved rats was linear for a t least 3 0 rain, although it was lower b y a b o u t 2 5 ~ in the preseuce o f I m M aminopyrlne, which is in agreement with Ref. I but much less t h a n in Refs. 2 a n d 3- T h e
~ pet~d~ a~l
pe:g&~k ;gid c a t n ~ by ~ e me~0d c~ Tfmd~ ~] az
c ~ e # ~ ngu~-~2~ e m r a ~ from ~ a~aopyri~_ G~uome ~ d ~
~ e d ~ith ce~mU~o~ ~
TABLE l ~ ~:re ~olaled frma rms ~ r ~ e d f ~ 12 h. They w~z~~ a d m ~ l at the c~enu-~ion of 4~6 mg dry weight/~ m ~ go F-zg. 2 h~ ~ e Kn~s-Hcmsdeil I~-bou,~e medima , - ~ i mM Ca~2. 1% bot~e se~m ~ ~ 10 mM | ~ Amiaop,~iae (1 re.M}aad ~ (I WM)wev~added wI~m'ei m t ~ z ~ Im:ubafima w~m~opped af¢or ~ ~ a ~ t ~ ~ i ~ of ~ m the ~
~ 1 m ¢1~:h~emis to F~gs~1 a~d 2_The value~age m ~ u ~ : S.D. f ~ 4-7 ~
l~-odtgtiem( p n ~ / h per g dry ~-,~ht ) 8]uco~ ~ ~ ~
Lactate Lactate+~.z~m Py*uvate
t
e
wi~ ~ f ~ t
dehy~
~l ~
method
con~el
~-amlmap~iae
%id~ b~iee
comrol
+amLnopyrme
~ inhlbitiea
149~ 16 235±32 143::k32
17ili 94+20 50± 5
8g 60 65
128~ 13 229~a0 135~32
87+14 1"/2-t-4~. 121±29
32 2~. 16
.
m ~ 1~ m ~mv~. ~d ~
To ~ n
~ ~
4-Amino.qntipyrine formation (p.mol/h per g dt-y weigh0 43± 2 M':i: I0 46~: 13
154
of 4-amino~tipyr~e production shows (Fig, 2) that its rate was h i g h ~ during the lust 10 nfin than during the subseqoe~t 10-30 rain p~iod. lower/rig of 4-an~hu~utipyrine formation may be caused by product inhibition as observed by Thutman and
~1 ~ t s to 10-30~, depe~llag c~ the g l ~ e u m genh: precursor. We also want to da~w attention to a ~ i t e d appl~abi~ty of the gh~ese ox~ase/petoxidase assay for glucose [4]; namely, caution ~ a s t be takea that no mher n:actio~ utgt~ng H20~_ are i m a m s . T l ~
Tabie I shows the effect o f amlnopyrine on gluconeogenesis from lactate and p ~ a v a t c as measured by the two assay systems for g l ~ . In agreement with Ref. 2, g l u ~ production from p3~'uvate was inhibited to a lower extent than from lactate. ~ , the degree of inhibition by aminowjrine d~ffered very much whea measored with the t~o methods, being mueh lower wRh the ghw.osc-6-phosphate dchydrogen~e procedure. Glucagon, as ~ s ~ d y increased g l u ~ e production from lactate, bet decreased the formation of 4-aminoantipyrine. The ~atte~ observ~tioa is new and in line with a recent report by B~u~hegyi et al. [11] who found a n inhibitory effect of dibutyryI-~.MP on the oxidation of aminopyrine in isolated mouse liver hepatocy[~ and m ~ o m e ~ N e s ~ ' t h e l ~ also in tbis publicafio~ glucose p r o d ~ n was probably uaderestimated as ~t was measured ~ith the glucose oxidase procedure. In conclu~on, the pres~at paper shows that the lm,g¢ apparent inbi'bition of gluc~3neoo~sis by aminopyrh~ as described by B ~ e s ~ i ct aL [2,3,11], is an artifact due to the interferen~ of the aminopyrine oxidation product. 4-amlnoantlpy~n~ in the glucose assay used by thes~ authors. The real inhibition is much smaller
shown in Table 1, van be aleut'bed in ~
detail
This work was supported by G r a m CI'BP O4.01 o f t ~ PoI~h Academy of Sciences.
1 ~ 2 m~si,
R.G. aed ,f,&e~ R. (I~r/3) E.~r.]. B i C ~ L 38. 64--7~_ G.~LAa~.F.a~Gar~T.il~l)B~m A~a 927,4 ~ * 1 6 .
Bie~em. 20,101-104. 4 Be~me)er. H.U. a ~ Beret. E {1974) ~,, M ~ c l s d ~ & ~ y ~ IBez~te,ser. ELU. e~L),v e t 3. pp. 12~-i2|5. ~ew YeA.
$ Karts, P. (1950)Orglm~C ~ , Ne~ yor~
C~
3L p. 45L E~,-der.
6 T t ~ r . PJ. (1969) Am~. CEm. Biodte~L 6, .7A--36_ 7 GemmL EL f1977} J. C~L Chem. C't~ Biodae~ gS. ( ~ 9 - ~ . S B u r A m ~ , BLU. a e r ~ E~ S , : I u ~ . F . ~ Srm~-kit. (1974) i= Mee~ds d F . ~ z ~ l ~ Aeal)~s ( ~ . I L U . e~.). V~L 3, Ep.
11~6-120L ~ Press, ~ York. 9 Ber~, M.N. a~l Frk~d, D~q.(1~9) $. Cell BioL430 5~6-5~. 10 ~ HA. Ed ~ l . IC (1932) ~ s 7-Che~. 210, 3~66. 1| B ~ ' i , G. Garzb, T. Meszagt~G~ Far,go. A_ ,q~f~e~ F. aetd M.~di. J. f l g ~ ) B i ~ h ~ L P ~ a ~ a c ~ 37, 8~-~64.