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Glycogen synthesis in the insect fat body
BIOCHIMICA ET BIOPHYSICAACTA
505
BBA 12229
GLYCOGEN SYNTHESIS IN T H E INSECT FAT BODY* A. VARDANIS Agricultural Research Institute, Canada Department of Agriculture, University Sub Post O~ce, London, Ontario (Canada)
(Received January 4th, 1962)
SUMMARY A tissue extract prepared from the fat body of the american cockroach (Periplaneta american¢, L.) has been shown to contain UDPG-pyrophosphorylase (EC 2.7.7.9) and UDPG-glycogen transglucosylase (EC 2.4.1.11) activity. The latter enzyme is not stimulated by Glc-6-P. Addition of Glc-I-P to the system results in a substantial enhancement of [14C~glucose incorporation from UDPG. The present evidence suggests that Glc-:c-P is a true activator of UDPG-glycogen transglucosylase of the cockroach fat body. INTRODUCTION The enzyme UDPG-glycogen transglucosylase (UDP glucose: a-l,4-glucan a- 4glucosyl transferase, EC 2.4.1.11 ) is generally thought to be responsible for the synthesis of a-l, 4 bonds in glycogen. Since its discovery by LELOIR AND GARDINI1, it has been found in a variety of tissues 2-s and in microorganismsS, 1°. Glc-6-P is reported 1to stimulate the mammalian enzyme to a greater or lesser extent depending upon the tissue of origin and subsequent treatment of the preparation6,S, 11. The possible importance of Glc-6-P stimulation has recently been underscored by the findings of LARNER and his group 12 who have been able to distinguish between two species of the enzyme, one dependent and the other independent of Glc-6-P for activity, and have shown their interconversion through an ATP-mediated phosphorylation reaction 18. So far, a short note has appeared in the literature on the existence of UDPGglycogen transghicosylase in tissue extracts of the locust 14, and a histochemical demonstration of UDPG-mediated glycogen synthesis in muscle from the same insect is has been reported. The purpose of this paper is to present the results of a study of UDPG-glycogen transglucosylase in extracts of the fat body of the american cockroach (Periplaneta americana L.). MATERIALS AND METHODS Materials
The following substances used in this investigation were commercial prepa* Contribution No. 245. Biochim. Biophys. Acta, 73 (1963) 565-573
566
A. VARDANIS
rations: NADP, UDPG, UTP, Glc-6-P and muscle phosphorylase-a (EC2.4.I.I) (Sigma Chemical Co.); maltose (Nutritional Biochemicals Corp.); Glc-I-P (C. F. B5hringer p n d Srhne) ; glycogen (shellfish, Mann Research Laboratories) ; E14CJstarch (Atomic E n e r g y of Canada Ltd.); and sweet potato fl-amylase (EC3.2.I.2), (uamylase free (EC 3.2.1.1)) (California Corporation for Biochemical Research).
Preparation of extracts One month old male cockroaches bred in our laboratory were used. Fat bodies were dissected out, immersed in ice cold water (0.3 ml per fat body) and homogenized at o ° for approx. I min in a Potter-Elvehjem unit fitted with a Teflon pestle. The homogenate was spun at 2000 × g for IO min at 00-4 ° in the Servall refrigerated centrifuge, and the sediment was discarded. The supernate was quick-frozen in a mixture of acetone and solid COz and stored at --25 °. Under these conditions there was no loss of the UDPG-glycogen transglucosylase activity of the preparation, for periods of up to twenty days. Before use, the extract was allowed to thaw, it was centrifuged at 12 ooo x g for IO min and the supernate was removed and dialyzed against distilled water for 2 h in the cold. The dialysis step was omitted in the determination ofGlc-6-P dehydrogenase (EC I.I.I.49), phosphoglucomutase (EC 2.7.5.1 ) and U D P G pyrophosphorylase (EC 2.7.7.9) activities.
Preparation of UDP[X~C]glucose* E14C]starch was initially incubated with phosphorylase a. Subsequently U T P was added in excess, as well as a cell free extract of Xanthomonas phaseoli (utilized as a source of UDPG-pyrophosphorylase). The synthesized UDP[X~C]glucose was isolated b y chromatography (ethanol : i M ammonium acetate (7:3) and elution. The product was not contaminated with unreacted [14C]starch or [14C]-Glc-I-P.
Enzyme assays Incubation temperature was 22 ° throughout. UDPG-pyrophosphorylase activity was assayed spectrophotometrically. N A D P reduction was followed at 34o m/~, using U D P G as substrate, in the presence of endogenous phosphoglucomutase and Glc-6-P dehydrogenase. UDPG-glycogen transglucosylase activity was measured as follows: Reactions with UDPE14C]glucose were stopped b y immersing the tubes in a boiling water b a t h for I rain. The precipitated protein was spun down and an aliquot of the supernatant solution was spotted on W h a t m a n No. i paper. The papers were developed overnight in ethanol I 1 : I M ammonium acetate (7:3) in a descending system. Under these conditions glycogen did not move from the origin and it was thus possible to measure the extent of [l~C]glucose incorporation by counting the origin before and after chromatographic development 10. Radioactivity was estimated with a mica windowed Geiger-Miiller probe. Experiments showed that absorption of x4C b y W h a t m a n No. I paper was a constant percentage of the total, over a wide range of activities spotted. * The author is indebted to Dr. N. B. MADSEN, Department of Biochemistry, University of Alberta, for providing the particulars of this technique.
Biochim. Biophys. Aaa, 73 (I963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
507
Since all calculations were on the basis of radioactivity on paper, self absorption corrections; were unnecessary. Chromatographic sprays Glc-I-P was locatec~ by the technique of HANES-ISHERWOODre. Maltose was detected ~dth a benzidine spray as recommended by HORROCKSxT. Analytical methods Inorganic phosphate was determined by the method of FISKE AND SOBBAROW18. Protein concentration was estimated by the procedure of LOWRY et al. 19.
EXPERIMENTAL RESULTS
U D PG-pyrop hosphorylase This enzyme, required for the synthesis of the substrate of UDPG-glycogen transglucosylase, catalyzes the reaction: G l c - I - P + U T P ~- U D P G + P P t
Providing our tissue extract contained phosphoglucomutase and Glc-6-P dehydrogenase activities, we should be able to demonstrate Glc-I-P formation from UDPG and PPt through NADP reduction in the Glc-6-P dehydrogenase reaction. Preliminary experiments (Fig. I) showed that the two enzyme activities, required for the indirect assay of UDPG-pyrophosphorylase, were present. Subsequently (Fig. 2) NADPH 2 formation, dependent on the addition of both UDPG and PPt was shown. Extracts .of the cockroach fat body were thus found to contain the first enzyme involved in the incorporation of glucose from Glc-I-P to glycogen through UDPG.
0.~
.......'"'"" ...*" /" / :: /s - / .:/ /"
0.4
0.3
o.a
//
¢o .io
I. 0.1
.Q
<
!
!
5
10
Time (rain)
Fig. I. Glc-6-P d e h y d r o g e n a s e a n d p h o s p h o g l u c o m u t a s e a c t i v i t y o f f a t b o d y e x t r a c t . C u v e t t e c o n t e n t s (in 3 m l ) : g l y c y l - g l y c i n e buffer ( p H 7.8), 3 oo # m o l e s ; MgC1t, 3 o # m o l e s ; N A D P (added a t zero t i m e ) , o. 3 # m o l e ; e x t r a c t , o. 4 m l (i.2 m g protein) a n d s u b s t r a t e as s h o w n , 3 o # m o l e s (added in o.o 5 m l a t t h e t i m e i n d i c a t e d b y a r r o w ) . . . . . , Glc-6-P; . ., G l c - I - P ; , no substrate.
Biochlm. Biophys. Acta, 73 (I963) 565-573
568
A. VARDANIS
//// 1/
0.3
0~ 0 . 2 ~1'
~o o.1 .a
NADP
o
o
I
5
I
I
10 15 Time (rain)
I
20
/
25
Fig. 2. U D P G - p y r o p h o s p h o r y l a s e a c t i v i t y of f a t b o d y extract. C u v e t t e c o n t e n t s (in 3 ml) : g l y c y l glycine buffer (pH 7.8), 3 o o / , m o l e s ; MgCll, 30 p m o l e s ; U D P G , 3o/*moles; s o d i u m p y r o p h o s p h a t e , 3 ° / , m o l e s ; e x t r a c t , o. 4 m l (1.2 m g protein) a n d N A D P , o. 3 / * m o l e (added a t zero time). C u r v e I, all c o m p o n e n t s p r e i n c u b a t e d for io m i n before t h e a d d i t i o n o f N A D P , C u r v e s 2 a n d 3, as a b o v e w i t h either U D P G ( ) (Curve 2) or s o d i u m p y r o p h o s p h a t e ( . . . . ) (Curve 3) o m i t t e d u n t i l t h e t i m e i n d i c a t e d b y arrow.
UDPG-glycogen transglucosylase Evidence for the presence of this enzyme was obtained by incubating the extract with UDP[14C]glucose and measuring the extent of incorporation of [14C]glucose into glycogen (see MATERIALSAND METHODS). Table I gives the results. It can be seen that Ex4C]glucose incorporation was dependent upon the addition of primer glycogen and that the activity of the extract was virtually destroyed by heating. Addition of Glc6-P, a known stimulator of the mammalian enzymed,s, resulted in decreased radioTABLE I UDPG-GLYCOGEN TRANSGLUCOSYLASE ACTIVITY AND THE E F F E C T O F GLC-6-P T h e c o m p l e t e i n c u b a t i o n m i x t u r e c o n t a i n e d (in 0.2 ml) : Tris buffer (pH 7.5), 5 / , m o l e s ; E D T A , o.i /,mole; glycogen, I.O m g ; UDP[llC]glucose, o. 5 pmole, a d d e d l a s t a t zero t i m e ( c o n t a i n i n g a p p r o x . 2o ooo c o u n t s / m i n on paper) a n d e x t r a c t (15o/~g protein). A f t e r 60 m i n t h e t u b e s were placed in a boiling w a t e r b a t h for I m i n , cooled, c e n t r i f u g e d a n d o.o5 ml of t h e s u p e r n a t a n t solution s u b j e c t e d to p a p e r c h r o m a t o g r a p h y . I n c o r p o r a t i o n o f [xIC]glucose into p o l y s a c c h a r i d e w a s d e t e r m i n e d as e x p l a i n e d in t h e t e x t . [xICJglu.Cos¢
i 2 3 4 5 6 7
Incubation mixture
incorporated (m/~moles/mg t~,omn)
Complete O m i t glycogen Omit extract As in 3 + h e a t e d e x t r a c t * C o m p l e t e + o . 5 / , m o l e Glc-6-P C o m p l e t e + i . o / * m o l e Glc-6-P C o m p l e t e + 2 . o / , m o l e s Glc-6-P
2i i 13 o 8 2oo lO 3 lO2
* E x t r a c t t r e a t e d a t i o o ° for I min.
Biochim. Biophys. Aaa, 73 (1963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
509
activity in the synthesized glycogen. This effect is probably due to competition of two enzymes for a common substrate, since UDPG and Glc-6-P are known to undergo the reaction: UDPG + Glc-6-P -+ Trehalose phosphate + UDP in the insect fat body ~°. The results agree well with those of TRIVELLON114who has stated that under the same conditions [nC]trehalose formation was detected by paper chromatography. It appears that Glc-6-P at the level of I #mole saturates trehalose phosphate synthetase (EC 2.4.1.15) as far as competition for available UDPG is concerned. The absence of additional effects at higher levels of Glc-6-P indicates that UDPG-glycogen transglucosylase as such, is insensitive to Glc-6-P. The effect of Glc-z-P on UDPG-glycogen transglucosylase
UDP[:t4C]glucose could theoretically give rise to [14C]glycogen through the UDPGpyrophosp]aorylase, glycogen phosphorylase series of reactions. This was not probable in our incubation mixtures since it was already shown (Fig. 2) that the presence of a high concentration of pyrophosphate was necessary for the formation of Glc-I-P from UDPG, Nevertheless, as an additional safeguard against the above possibility, incubations wffh added, non-radioactive Glc-I-P were carried out. It was reasoned that if phosphorylase was responsible for the observed glycogen synthesis, addition of Glc-I-P should reduce glycogen radioactivity by isotopic dilution. Table I I gives the results obtained in these experiments. Unexpectedly, the TABLE II EFFECTOF GLC-I-P ON UDPG-GLYCOGENTRANSGLUCOSYLACEACTIVITY Complete system, incubation, chromatography and radioactivity determination as in Table I. [~'C]glueose
Incubationmixture
I 2 3 4 5 6 7
Complete Omit glycogen Complete + i/*mole Glc-I-P Complete + 2/*moles Glc-I-P As in 2 + 2/*moles Glc-I-P As in 4 + 2o/,moles NaF Complete + I mg glycogen
incor1~orated (mr, moles/rag tn'otein)
16i 3 527 690 171 7o8 18o
addition of Glc-I-P effected a large stimulation of UDPG conversion. Although a stimulation was observed in the absence of added glycogen the bulk of the radioactivity recovered was dependent on the addition of primer. The degree of stimulation was not influenced b y high concentrations of NaF which is known to inhibit phosphoglucomutase in fat-body homogenates 21. This finding together with the ineffectiveness of Glc-6-P (Table I) indicates that stimulation is due to Glc-I-P, rather than any of its catabolic metabolites. It was at first essential to show that the radioactivity on the basis of which Biochim. Biophys. Acta. 7:t (io6a~ ~6~-v,.,
570
A. VARDANIS
[14C]glucose incorporation was measured, consisted solely of [14C]glycogen. This was done in the following manner:Radioactivity at the origin from experiments with and without Glc-i-P, was eluted with a small volume of water. The eluates were incubated with either muscle phosphorylase a (at pH 7.0 and 30 °) or r-amylase (at pH 4.8 and 22°). Reactions were stopped by heating and the supernates were spotted on Whatman No. r paper and developed in ethanol: i M ammonium acetate (7:3), containing o.I °/o EDTA, for 20 h. In all cases 95% of the iadioactivity applied travelled away from the origin as a single spot, coinciding with either GIc-I-P (Re o.4 I) or maltose (Re 0.70 ) in the phosphorylase and amylase incubations respectively. In an effort to understand the mechanism of stimulation by Glc-I-P we considered the possibility that Glc-I-P can independently synthesize glycogen through phosphorylase and thus provide more primer (i.e. additional outer branches assuming the "brancher" enzyme is present) for UDPG-glycogen transglucosylase. On the basis of results of Expt. 7, Table II and those reported in Table IV this explanation does not seem likely. Alternatively, a more efficient primer could be produced from Glcx-P through extension of primer outer branches. KORNFELDzz working with muscle transglucosylase has shown that the optimal outer chain length of the primer ranges from about xo-24 glucose units; with longer chains the reaction is inhibited. Table III
TABLE III PHOSPHORYLASE
ACTIVITY
OF THE FAT BODY EXTRACT
The complete incubation mixture contained (in 0.4 ml) : Tris buffer (pH 7.5), i0/tmoles; MgCI,, i /~mole; EDTA, o.z/tmoles; glycogen, 2 mg; Glc-I-P, 4/tmoles; NaF, 4 °/~moles and extract (260/tg protein). After the appropriate incubation time the tubes were placed in a boiling water bath for I min, cooled, centrifuged and the supernatant solution analyzed for orthophosphate. Incubation time (rain) Incubation mixture
30 60 Ne~ total P~ .found
(~moles)
I 2 3 4
Complete Omit glycogen Omit glycogen and NaF Omit NaF
I.O3 0.35 o.3z I.I6
1.8I o.7I -
-
--
shows that in our tissue extract, and, under the conditions of the transglucosylase assay, there was considerable phosphorylase activity. Unfortunately, activity was not dependent on the presence of NaF, indicating that hydrolysis of Glc-I-P by phosphatase and conversion of phosphorylase a to phosphorylase b did not take place to any great extent under these conditions. Otherwise, at this stage, we might have been able to dissociate independent glycogen synthesis from Glc-r-P activation of the transglucosylase. In a further effort to understand the nature of Glc-I-P stimulation two experimental approaches were taken: (a) Glc-r-P was incubated with glycogen and extract for 60 min, the reaction mixture digested at too ° in 20% KOH, and glycogen precipitated twice with x.25 volumes of ethanol. Table IV shows that addition of glycogen thus obtained in place of the commercial preparation resulted in Biochim. Biophys. Acta, 73 (1963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
571
T A B L E IV ISOLATED GLYCOGEN AS PRIMER OF UDPG-GLYCOGEN TRANSGLUCOSYLASE C o m p l e t e s y s t e m , i n c u b a t i o n , c h r o m a t o g r a p h y a n d r a d i o a c t i v i t y d e t e r m i n a t i o n as in T a b l e I [l*C] #~ose incorporated (ml~moles/mg protein)
Incubalion mixture
I 2 3
" Incubation E D T A , i /*mole; I n c u b a t i o n period ** I n c u b a t i o n a d d i t i o n of K O H ,
Complete O m i t glycogen + tit 0 of t h e glycogen f r o m i n c u b a t i o n I" O m i t glycogen + t / t 0 of t h e glycogen f r o m i n c u b a t i o n I I ' "
2 i2 258 195
I c o n t a i n e d (in 2.o ml): Tris buffer (pH 7.5), 5 0 / , m o l e s ; MgCI,, 5 # m o l e s ; glycogen, io m g ; G l c - I - P , 20 # m o l e s a n d t i s s u e e x t r a c t (1.55 m g protein). 60 m i n . G l y c o g e n o b t a i n e d as described in t h e t e x t . II is identical to I n c u b a t i o n I b u t G l c - I - P a d d e d a t 6o m i n i m m e d i a t e l y before
a slight stimulation of [14C]glucoseincorporation but the magnitude of the effect cannot be compared to that of Glc-I-P (Table II). (b) In a series of experiments, the results of which are given in Table V, the extract was preincubated with Glc-I-P for 60 rain, before addition of UDp[14C]glucose. Orthophosphate determination showed that during this period of time approximately half of ¢]he added Glc-I-P had been converted to glycogen. Incorporation of E14C]TABLE V PREINCUBATION
W I T H GLC-I-P AND THE ACTIVITY OF UDPG-GLYCOGEN
TRANSGLUCOSYLASE C o n t e n t s of i n c u b a t i o n m i x t u r e (in 0.2 m l ) : Tris buffer (pH 7-5), 5 #*moles; MgC1v 0. 5 # m o l e ; E D T A , o. i # m o l e ; glycogen, i.o m g ; UDP[X'C]glucose, o. 5 / , m o l e a d d e d w h e r e s h o w n ( c o n t a i n i n g a p p r o x . 20 ooo c o u n t s / m i n on paper) ; G l c - I - P as s h o w n a n d t i s s u e e x t r a c t (92 jug protein). A f t e r I2O m i n i n c u b a t i o n t h e r e a c t i o n m i x t u r e s c o n t a i n i n g UDP[t~C]gluc0se were t r e a t e d as in T a b l e I, I n E x p t . I, a f t e r 6o m i n i n c u b a t i o n t h e t u b e s were placed in a boiling w a t e r b a t h for i m i n cooled, c e n t r i f u g e d a n d t h e s u p e r n a t a n t solution a n a l y z e d for o r t h o p h o s p h a t e .
Expt.
I 2 3
4
5
Add/tionsat. zero time
2 #moles Glc-I-P None 2/*moles G l c - I - P
Additions at 60 rain
None
Net total orthophospkate at 60 rain (#moles)
{x~CJglucose incorporated (ra#motes/mg protein
i.o 3
--
UDPEl*C]glucose
--
218
UDP[t*C]glucose
--
249
I #mole Glc-I-P + UDp[xtC]glucose
--
7o9
2 #moles Glc-I-P + UDP[l*C]glucose
--
i258
2 #moles Glc-I-P + UDP[X4C]glucose
__
None
2/,moles Glc-I-P
l°32
B i o e h i m . B i o p h y s . A c t a , 73 (1963) 565-573
572
A. VARDANIS
glucose from U D P G during the second 6o-min interval of incubation was again slightly higher than the control, but failed to approach the degree of stimulation obtained when Glc-I-P was added together with the UDp[14C]glucose. It was still feasible, however, that a more efficient primer was produced for a comparatively short period of time within the initial 60 min. This possibility is discounted by the results of Expt. 5 (Table V). I t is evident that availability of Glc-I-P is the critical factor in stimulation, rather than a particular size of primer outer branches.
DISCUSSION Carbohydrate in insects is stored mainly in the form of glycogen, while the disaccharide trehalose m a y represent a more readily available source of energy. The presence of U D P G in insect tissues has already been reported 2s. This substance is, no doubt, connected with the biosynthesis of both glycogen and trehalose. Availability of Glc6 - P i n vitro seems to determine the extent of U D P G utilization b y one or the other of the two alternative routes 14 (see also Table I), in a system of two enzymes competing for a common substrate. As such, Glc-6-P has no apparent effect on the activity of UDPG-glycogen transglucosylase, similar to that observed with the m a m malian enzyme~, n. We have found that addition of GIc-i-P to our cockroach fat body enzyme results in a substantial stimulation of [x*C]glucose incorporation from U D P G into glycogen. Results available so far (Tables IV and V) indicate that this stimulation is not due to independent, phosphorylase mediated glycogen synthesis from Glc-x-P and provision of a more efficient primer for the transglucosylase reaction. Possibly, GIc-I-P is a true activator of UDPG-glycogen transglucosylase in insects and assumes the role played b y Glc-6-P in mammalian systems. This finding presents itself as a likely control mechanism of glycogen synthesis whereby accumulation of Glc-I-P would lead to its accelerated removal through the UDPG-pyrophosphorylase, UDPG-glycogen transghicosylase series of reactions. LARNER et al.8,12, ~ have recently presented evidence indicating that hormonal control of glycogen synthesis in mammals takes place through a shift in the Glc-6-P dependency of transglucosylase. Further work is now being pursued in our laboratory to determine the regulatory action of insect hormones connected with glycogen metabolism as related to Glc-I-P stimulation of UDPG-glycogen transglucosylase.
ACKNOWLEDGEMENTS The author wishes to thank Mr. L. CRAWFORD for his technical assistance. Thanks are also due to Drs. G. WHITE and R. M. KRUPKA for valuable criticism and m a n y interesting discussions. REFERENCES 1 L. F. LELOIRAND C. E. CARDINI,J. A~$*. Chem. Sot., 79 (1957) 6340. 2 L. F. LELOIR, J. M. OLAVARRIA,S. H. GOLD~-MB~-RGAND H. CARMINATTI,Arch. Biochem. Biophys., 81 (1959) 508. s C. VILLAR-PALASIAND J. LARUER, Biochim. Biophys. Acta, 3° (1958) 449. 4 p. W. ROB~INS,R. R. TRAUTAND F. LIPMANN,Proc. Natl. Acad. Sci. U.S., 45 (1959) 6. 5 B. M. BRECKEI~RIDGEAND E. j. CRAWFORD,J. Biol. Chem., 235 (196o) 3054. Biochim. Biophys. Acta, 73 (1963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
~73
6 L. F. LELOIR AND S. H. GOLDEMBERG,J. Biol. Chem., 235 (I96O) 919. D. K. BAsu AND B. K. BACHHAWAT,Biochim. Biophys. Acta, 5 ° (I961) I23. s M. RosEr,L-PEREZ XND J. LARNER, Biochemistry, r (1962) 769. 8 I. D. ALG.RANXTI AND E. CABIn, Biochim. Biophys. Acta, 43 (196o) I4X. 10 N. B. MADSEN, Biochim. Biophys. Acta, 5 ° ( I 9 6 I ) I94. lx R. KOEN:FELD XND D. H. BROWS, J. Biol. Chem., 237 (1962) I772. 1' M. ROSEI,L-PEREZ, C. VILLAR-PALASX AND J. LARNER, Biochemistry, I (I962) 763 . t8 D. L. FR~IDMAN AND J. LARr~ER, Biochim. Biophys. Acta, 64 (1962) 185. 14 j . C. TRI'¢ELLONI, Arch. Biochem. Biophys., 89 (196o) I49. ~5 R. HESS AND A. G. E. PEARSE, Enzymol. Biol. Clin., I (I961) 15. ~e C. S. HANES AND F. A. ISHERWOOD, Nature, I64 (1949) IIO7. ~ R. H. HORROCES, Nature, I64 (1949) 444. 18 C. H. FISKE AND Y. SUBBARow, J. Biol. Chem., 66 (1925) 375. x 0 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARE AND R. J. RANDALL, J. Biol. Chem., 193 (I951) 265. so D. J. CANDY AND B. A. KILBY, Biochem. J., 78 (I96I) 531. 11 H. SHIGEMATSU, Nippon Sanshigaku Zasshi, 25 (1956) 115. ~ R. H. KORNFELD, Ph.D. Thesis, W a s h i n g t o n University, Saint Louis, Miss., (1961). as F. G. CAJ~EY AND G. R. WYATt, Biochim. Biophys. Acta, 41 (196o) I78. ** C. VILLA1t-PALASI AND J. LARNER, Arch. Biochem. Biophys., 94 (1961) 436.
Biochim. Biophys. Attic, 73 (1963) 565-573
505
BBA 12229
GLYCOGEN SYNTHESIS IN T H E INSECT FAT BODY* A. VARDANIS Agricultural Research Institute, Canada Department of Agriculture, University Sub Post O~ce, London, Ontario (Canada)
(Received January 4th, 1962)
SUMMARY A tissue extract prepared from the fat body of the american cockroach (Periplaneta american¢, L.) has been shown to contain UDPG-pyrophosphorylase (EC 2.7.7.9) and UDPG-glycogen transglucosylase (EC 2.4.1.11) activity. The latter enzyme is not stimulated by Glc-6-P. Addition of Glc-I-P to the system results in a substantial enhancement of [14C~glucose incorporation from UDPG. The present evidence suggests that Glc-:c-P is a true activator of UDPG-glycogen transglucosylase of the cockroach fat body. INTRODUCTION The enzyme UDPG-glycogen transglucosylase (UDP glucose: a-l,4-glucan a- 4glucosyl transferase, EC 2.4.1.11 ) is generally thought to be responsible for the synthesis of a-l, 4 bonds in glycogen. Since its discovery by LELOIR AND GARDINI1, it has been found in a variety of tissues 2-s and in microorganismsS, 1°. Glc-6-P is reported 1to stimulate the mammalian enzyme to a greater or lesser extent depending upon the tissue of origin and subsequent treatment of the preparation6,S, 11. The possible importance of Glc-6-P stimulation has recently been underscored by the findings of LARNER and his group 12 who have been able to distinguish between two species of the enzyme, one dependent and the other independent of Glc-6-P for activity, and have shown their interconversion through an ATP-mediated phosphorylation reaction 18. So far, a short note has appeared in the literature on the existence of UDPGglycogen transghicosylase in tissue extracts of the locust 14, and a histochemical demonstration of UDPG-mediated glycogen synthesis in muscle from the same insect is has been reported. The purpose of this paper is to present the results of a study of UDPG-glycogen transglucosylase in extracts of the fat body of the american cockroach (Periplaneta americana L.). MATERIALS AND METHODS Materials
The following substances used in this investigation were commercial prepa* Contribution No. 245. Biochim. Biophys. Acta, 73 (1963) 565-573
566
A. VARDANIS
rations: NADP, UDPG, UTP, Glc-6-P and muscle phosphorylase-a (EC2.4.I.I) (Sigma Chemical Co.); maltose (Nutritional Biochemicals Corp.); Glc-I-P (C. F. B5hringer p n d Srhne) ; glycogen (shellfish, Mann Research Laboratories) ; E14CJstarch (Atomic E n e r g y of Canada Ltd.); and sweet potato fl-amylase (EC3.2.I.2), (uamylase free (EC 3.2.1.1)) (California Corporation for Biochemical Research).
Preparation of extracts One month old male cockroaches bred in our laboratory were used. Fat bodies were dissected out, immersed in ice cold water (0.3 ml per fat body) and homogenized at o ° for approx. I min in a Potter-Elvehjem unit fitted with a Teflon pestle. The homogenate was spun at 2000 × g for IO min at 00-4 ° in the Servall refrigerated centrifuge, and the sediment was discarded. The supernate was quick-frozen in a mixture of acetone and solid COz and stored at --25 °. Under these conditions there was no loss of the UDPG-glycogen transglucosylase activity of the preparation, for periods of up to twenty days. Before use, the extract was allowed to thaw, it was centrifuged at 12 ooo x g for IO min and the supernate was removed and dialyzed against distilled water for 2 h in the cold. The dialysis step was omitted in the determination ofGlc-6-P dehydrogenase (EC I.I.I.49), phosphoglucomutase (EC 2.7.5.1 ) and U D P G pyrophosphorylase (EC 2.7.7.9) activities.
Preparation of UDP[X~C]glucose* E14C]starch was initially incubated with phosphorylase a. Subsequently U T P was added in excess, as well as a cell free extract of Xanthomonas phaseoli (utilized as a source of UDPG-pyrophosphorylase). The synthesized UDP[X~C]glucose was isolated b y chromatography (ethanol : i M ammonium acetate (7:3) and elution. The product was not contaminated with unreacted [14C]starch or [14C]-Glc-I-P.
Enzyme assays Incubation temperature was 22 ° throughout. UDPG-pyrophosphorylase activity was assayed spectrophotometrically. N A D P reduction was followed at 34o m/~, using U D P G as substrate, in the presence of endogenous phosphoglucomutase and Glc-6-P dehydrogenase. UDPG-glycogen transglucosylase activity was measured as follows: Reactions with UDPE14C]glucose were stopped b y immersing the tubes in a boiling water b a t h for I rain. The precipitated protein was spun down and an aliquot of the supernatant solution was spotted on W h a t m a n No. i paper. The papers were developed overnight in ethanol I 1 : I M ammonium acetate (7:3) in a descending system. Under these conditions glycogen did not move from the origin and it was thus possible to measure the extent of [l~C]glucose incorporation by counting the origin before and after chromatographic development 10. Radioactivity was estimated with a mica windowed Geiger-Miiller probe. Experiments showed that absorption of x4C b y W h a t m a n No. I paper was a constant percentage of the total, over a wide range of activities spotted. * The author is indebted to Dr. N. B. MADSEN, Department of Biochemistry, University of Alberta, for providing the particulars of this technique.
Biochim. Biophys. Aaa, 73 (I963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
507
Since all calculations were on the basis of radioactivity on paper, self absorption corrections; were unnecessary. Chromatographic sprays Glc-I-P was locatec~ by the technique of HANES-ISHERWOODre. Maltose was detected ~dth a benzidine spray as recommended by HORROCKSxT. Analytical methods Inorganic phosphate was determined by the method of FISKE AND SOBBAROW18. Protein concentration was estimated by the procedure of LOWRY et al. 19.
EXPERIMENTAL RESULTS
U D PG-pyrop hosphorylase This enzyme, required for the synthesis of the substrate of UDPG-glycogen transglucosylase, catalyzes the reaction: G l c - I - P + U T P ~- U D P G + P P t
Providing our tissue extract contained phosphoglucomutase and Glc-6-P dehydrogenase activities, we should be able to demonstrate Glc-I-P formation from UDPG and PPt through NADP reduction in the Glc-6-P dehydrogenase reaction. Preliminary experiments (Fig. I) showed that the two enzyme activities, required for the indirect assay of UDPG-pyrophosphorylase, were present. Subsequently (Fig. 2) NADPH 2 formation, dependent on the addition of both UDPG and PPt was shown. Extracts .of the cockroach fat body were thus found to contain the first enzyme involved in the incorporation of glucose from Glc-I-P to glycogen through UDPG.
0.~
.......'"'"" ...*" /" / :: /s - / .:/ /"
0.4
0.3
o.a
//
¢o .io
I. 0.1
.Q
<
!
!
5
10
Time (rain)
Fig. I. Glc-6-P d e h y d r o g e n a s e a n d p h o s p h o g l u c o m u t a s e a c t i v i t y o f f a t b o d y e x t r a c t . C u v e t t e c o n t e n t s (in 3 m l ) : g l y c y l - g l y c i n e buffer ( p H 7.8), 3 oo # m o l e s ; MgC1t, 3 o # m o l e s ; N A D P (added a t zero t i m e ) , o. 3 # m o l e ; e x t r a c t , o. 4 m l (i.2 m g protein) a n d s u b s t r a t e as s h o w n , 3 o # m o l e s (added in o.o 5 m l a t t h e t i m e i n d i c a t e d b y a r r o w ) . . . . . , Glc-6-P; . ., G l c - I - P ; , no substrate.
Biochlm. Biophys. Acta, 73 (I963) 565-573
568
A. VARDANIS
//// 1/
0.3
0~ 0 . 2 ~1'
~o o.1 .a
NADP
o
o
I
5
I
I
10 15 Time (rain)
I
20
/
25
Fig. 2. U D P G - p y r o p h o s p h o r y l a s e a c t i v i t y of f a t b o d y extract. C u v e t t e c o n t e n t s (in 3 ml) : g l y c y l glycine buffer (pH 7.8), 3 o o / , m o l e s ; MgCll, 30 p m o l e s ; U D P G , 3o/*moles; s o d i u m p y r o p h o s p h a t e , 3 ° / , m o l e s ; e x t r a c t , o. 4 m l (1.2 m g protein) a n d N A D P , o. 3 / * m o l e (added a t zero time). C u r v e I, all c o m p o n e n t s p r e i n c u b a t e d for io m i n before t h e a d d i t i o n o f N A D P , C u r v e s 2 a n d 3, as a b o v e w i t h either U D P G ( ) (Curve 2) or s o d i u m p y r o p h o s p h a t e ( . . . . ) (Curve 3) o m i t t e d u n t i l t h e t i m e i n d i c a t e d b y arrow.
UDPG-glycogen transglucosylase Evidence for the presence of this enzyme was obtained by incubating the extract with UDP[14C]glucose and measuring the extent of incorporation of [14C]glucose into glycogen (see MATERIALSAND METHODS). Table I gives the results. It can be seen that Ex4C]glucose incorporation was dependent upon the addition of primer glycogen and that the activity of the extract was virtually destroyed by heating. Addition of Glc6-P, a known stimulator of the mammalian enzymed,s, resulted in decreased radioTABLE I UDPG-GLYCOGEN TRANSGLUCOSYLASE ACTIVITY AND THE E F F E C T O F GLC-6-P T h e c o m p l e t e i n c u b a t i o n m i x t u r e c o n t a i n e d (in 0.2 ml) : Tris buffer (pH 7.5), 5 / , m o l e s ; E D T A , o.i /,mole; glycogen, I.O m g ; UDP[llC]glucose, o. 5 pmole, a d d e d l a s t a t zero t i m e ( c o n t a i n i n g a p p r o x . 2o ooo c o u n t s / m i n on paper) a n d e x t r a c t (15o/~g protein). A f t e r 60 m i n t h e t u b e s were placed in a boiling w a t e r b a t h for I m i n , cooled, c e n t r i f u g e d a n d o.o5 ml of t h e s u p e r n a t a n t solution s u b j e c t e d to p a p e r c h r o m a t o g r a p h y . I n c o r p o r a t i o n o f [xIC]glucose into p o l y s a c c h a r i d e w a s d e t e r m i n e d as e x p l a i n e d in t h e t e x t . [xICJglu.Cos¢
i 2 3 4 5 6 7
Incubation mixture
incorporated (m/~moles/mg t~,omn)
Complete O m i t glycogen Omit extract As in 3 + h e a t e d e x t r a c t * C o m p l e t e + o . 5 / , m o l e Glc-6-P C o m p l e t e + i . o / * m o l e Glc-6-P C o m p l e t e + 2 . o / , m o l e s Glc-6-P
2i i 13 o 8 2oo lO 3 lO2
* E x t r a c t t r e a t e d a t i o o ° for I min.
Biochim. Biophys. Aaa, 73 (1963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
509
activity in the synthesized glycogen. This effect is probably due to competition of two enzymes for a common substrate, since UDPG and Glc-6-P are known to undergo the reaction: UDPG + Glc-6-P -+ Trehalose phosphate + UDP in the insect fat body ~°. The results agree well with those of TRIVELLON114who has stated that under the same conditions [nC]trehalose formation was detected by paper chromatography. It appears that Glc-6-P at the level of I #mole saturates trehalose phosphate synthetase (EC 2.4.1.15) as far as competition for available UDPG is concerned. The absence of additional effects at higher levels of Glc-6-P indicates that UDPG-glycogen transglucosylase as such, is insensitive to Glc-6-P. The effect of Glc-z-P on UDPG-glycogen transglucosylase
UDP[:t4C]glucose could theoretically give rise to [14C]glycogen through the UDPGpyrophosp]aorylase, glycogen phosphorylase series of reactions. This was not probable in our incubation mixtures since it was already shown (Fig. 2) that the presence of a high concentration of pyrophosphate was necessary for the formation of Glc-I-P from UDPG, Nevertheless, as an additional safeguard against the above possibility, incubations wffh added, non-radioactive Glc-I-P were carried out. It was reasoned that if phosphorylase was responsible for the observed glycogen synthesis, addition of Glc-I-P should reduce glycogen radioactivity by isotopic dilution. Table I I gives the results obtained in these experiments. Unexpectedly, the TABLE II EFFECTOF GLC-I-P ON UDPG-GLYCOGENTRANSGLUCOSYLACEACTIVITY Complete system, incubation, chromatography and radioactivity determination as in Table I. [~'C]glueose
Incubationmixture
I 2 3 4 5 6 7
Complete Omit glycogen Complete + i/*mole Glc-I-P Complete + 2/*moles Glc-I-P As in 2 + 2/*moles Glc-I-P As in 4 + 2o/,moles NaF Complete + I mg glycogen
incor1~orated (mr, moles/rag tn'otein)
16i 3 527 690 171 7o8 18o
addition of Glc-I-P effected a large stimulation of UDPG conversion. Although a stimulation was observed in the absence of added glycogen the bulk of the radioactivity recovered was dependent on the addition of primer. The degree of stimulation was not influenced b y high concentrations of NaF which is known to inhibit phosphoglucomutase in fat-body homogenates 21. This finding together with the ineffectiveness of Glc-6-P (Table I) indicates that stimulation is due to Glc-I-P, rather than any of its catabolic metabolites. It was at first essential to show that the radioactivity on the basis of which Biochim. Biophys. Acta. 7:t (io6a~ ~6~-v,.,
570
A. VARDANIS
[14C]glucose incorporation was measured, consisted solely of [14C]glycogen. This was done in the following manner:Radioactivity at the origin from experiments with and without Glc-i-P, was eluted with a small volume of water. The eluates were incubated with either muscle phosphorylase a (at pH 7.0 and 30 °) or r-amylase (at pH 4.8 and 22°). Reactions were stopped by heating and the supernates were spotted on Whatman No. r paper and developed in ethanol: i M ammonium acetate (7:3), containing o.I °/o EDTA, for 20 h. In all cases 95% of the iadioactivity applied travelled away from the origin as a single spot, coinciding with either GIc-I-P (Re o.4 I) or maltose (Re 0.70 ) in the phosphorylase and amylase incubations respectively. In an effort to understand the mechanism of stimulation by Glc-I-P we considered the possibility that Glc-I-P can independently synthesize glycogen through phosphorylase and thus provide more primer (i.e. additional outer branches assuming the "brancher" enzyme is present) for UDPG-glycogen transglucosylase. On the basis of results of Expt. 7, Table II and those reported in Table IV this explanation does not seem likely. Alternatively, a more efficient primer could be produced from Glcx-P through extension of primer outer branches. KORNFELDzz working with muscle transglucosylase has shown that the optimal outer chain length of the primer ranges from about xo-24 glucose units; with longer chains the reaction is inhibited. Table III
TABLE III PHOSPHORYLASE
ACTIVITY
OF THE FAT BODY EXTRACT
The complete incubation mixture contained (in 0.4 ml) : Tris buffer (pH 7.5), i0/tmoles; MgCI,, i /~mole; EDTA, o.z/tmoles; glycogen, 2 mg; Glc-I-P, 4/tmoles; NaF, 4 °/~moles and extract (260/tg protein). After the appropriate incubation time the tubes were placed in a boiling water bath for I min, cooled, centrifuged and the supernatant solution analyzed for orthophosphate. Incubation time (rain) Incubation mixture
30 60 Ne~ total P~ .found
(~moles)
I 2 3 4
Complete Omit glycogen Omit glycogen and NaF Omit NaF
I.O3 0.35 o.3z I.I6
1.8I o.7I -
-
--
shows that in our tissue extract, and, under the conditions of the transglucosylase assay, there was considerable phosphorylase activity. Unfortunately, activity was not dependent on the presence of NaF, indicating that hydrolysis of Glc-I-P by phosphatase and conversion of phosphorylase a to phosphorylase b did not take place to any great extent under these conditions. Otherwise, at this stage, we might have been able to dissociate independent glycogen synthesis from Glc-r-P activation of the transglucosylase. In a further effort to understand the nature of Glc-I-P stimulation two experimental approaches were taken: (a) Glc-r-P was incubated with glycogen and extract for 60 min, the reaction mixture digested at too ° in 20% KOH, and glycogen precipitated twice with x.25 volumes of ethanol. Table IV shows that addition of glycogen thus obtained in place of the commercial preparation resulted in Biochim. Biophys. Acta, 73 (1963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
571
T A B L E IV ISOLATED GLYCOGEN AS PRIMER OF UDPG-GLYCOGEN TRANSGLUCOSYLASE C o m p l e t e s y s t e m , i n c u b a t i o n , c h r o m a t o g r a p h y a n d r a d i o a c t i v i t y d e t e r m i n a t i o n as in T a b l e I [l*C] #~ose incorporated (ml~moles/mg protein)
Incubalion mixture
I 2 3
" Incubation E D T A , i /*mole; I n c u b a t i o n period ** I n c u b a t i o n a d d i t i o n of K O H ,
Complete O m i t glycogen + tit 0 of t h e glycogen f r o m i n c u b a t i o n I" O m i t glycogen + t / t 0 of t h e glycogen f r o m i n c u b a t i o n I I ' "
2 i2 258 195
I c o n t a i n e d (in 2.o ml): Tris buffer (pH 7.5), 5 0 / , m o l e s ; MgCI,, 5 # m o l e s ; glycogen, io m g ; G l c - I - P , 20 # m o l e s a n d t i s s u e e x t r a c t (1.55 m g protein). 60 m i n . G l y c o g e n o b t a i n e d as described in t h e t e x t . II is identical to I n c u b a t i o n I b u t G l c - I - P a d d e d a t 6o m i n i m m e d i a t e l y before
a slight stimulation of [14C]glucoseincorporation but the magnitude of the effect cannot be compared to that of Glc-I-P (Table II). (b) In a series of experiments, the results of which are given in Table V, the extract was preincubated with Glc-I-P for 60 rain, before addition of UDp[14C]glucose. Orthophosphate determination showed that during this period of time approximately half of ¢]he added Glc-I-P had been converted to glycogen. Incorporation of E14C]TABLE V PREINCUBATION
W I T H GLC-I-P AND THE ACTIVITY OF UDPG-GLYCOGEN
TRANSGLUCOSYLASE C o n t e n t s of i n c u b a t i o n m i x t u r e (in 0.2 m l ) : Tris buffer (pH 7-5), 5 #*moles; MgC1v 0. 5 # m o l e ; E D T A , o. i # m o l e ; glycogen, i.o m g ; UDP[X'C]glucose, o. 5 / , m o l e a d d e d w h e r e s h o w n ( c o n t a i n i n g a p p r o x . 20 ooo c o u n t s / m i n on paper) ; G l c - I - P as s h o w n a n d t i s s u e e x t r a c t (92 jug protein). A f t e r I2O m i n i n c u b a t i o n t h e r e a c t i o n m i x t u r e s c o n t a i n i n g UDP[t~C]gluc0se were t r e a t e d as in T a b l e I, I n E x p t . I, a f t e r 6o m i n i n c u b a t i o n t h e t u b e s were placed in a boiling w a t e r b a t h for i m i n cooled, c e n t r i f u g e d a n d t h e s u p e r n a t a n t solution a n a l y z e d for o r t h o p h o s p h a t e .
Expt.
I 2 3
4
5
Add/tionsat. zero time
2 #moles Glc-I-P None 2/*moles G l c - I - P
Additions at 60 rain
None
Net total orthophospkate at 60 rain (#moles)
{x~CJglucose incorporated (ra#motes/mg protein
i.o 3
--
UDPEl*C]glucose
--
218
UDP[t*C]glucose
--
249
I #mole Glc-I-P + UDp[xtC]glucose
--
7o9
2 #moles Glc-I-P + UDP[l*C]glucose
--
i258
2 #moles Glc-I-P + UDP[X4C]glucose
__
None
2/,moles Glc-I-P
l°32
B i o e h i m . B i o p h y s . A c t a , 73 (1963) 565-573
572
A. VARDANIS
glucose from U D P G during the second 6o-min interval of incubation was again slightly higher than the control, but failed to approach the degree of stimulation obtained when Glc-I-P was added together with the UDp[14C]glucose. It was still feasible, however, that a more efficient primer was produced for a comparatively short period of time within the initial 60 min. This possibility is discounted by the results of Expt. 5 (Table V). I t is evident that availability of Glc-I-P is the critical factor in stimulation, rather than a particular size of primer outer branches.
DISCUSSION Carbohydrate in insects is stored mainly in the form of glycogen, while the disaccharide trehalose m a y represent a more readily available source of energy. The presence of U D P G in insect tissues has already been reported 2s. This substance is, no doubt, connected with the biosynthesis of both glycogen and trehalose. Availability of Glc6 - P i n vitro seems to determine the extent of U D P G utilization b y one or the other of the two alternative routes 14 (see also Table I), in a system of two enzymes competing for a common substrate. As such, Glc-6-P has no apparent effect on the activity of UDPG-glycogen transglucosylase, similar to that observed with the m a m malian enzyme~, n. We have found that addition of GIc-i-P to our cockroach fat body enzyme results in a substantial stimulation of [x*C]glucose incorporation from U D P G into glycogen. Results available so far (Tables IV and V) indicate that this stimulation is not due to independent, phosphorylase mediated glycogen synthesis from Glc-x-P and provision of a more efficient primer for the transglucosylase reaction. Possibly, GIc-I-P is a true activator of UDPG-glycogen transglucosylase in insects and assumes the role played b y Glc-6-P in mammalian systems. This finding presents itself as a likely control mechanism of glycogen synthesis whereby accumulation of Glc-I-P would lead to its accelerated removal through the UDPG-pyrophosphorylase, UDPG-glycogen transghicosylase series of reactions. LARNER et al.8,12, ~ have recently presented evidence indicating that hormonal control of glycogen synthesis in mammals takes place through a shift in the Glc-6-P dependency of transglucosylase. Further work is now being pursued in our laboratory to determine the regulatory action of insect hormones connected with glycogen metabolism as related to Glc-I-P stimulation of UDPG-glycogen transglucosylase.
ACKNOWLEDGEMENTS The author wishes to thank Mr. L. CRAWFORD for his technical assistance. Thanks are also due to Drs. G. WHITE and R. M. KRUPKA for valuable criticism and m a n y interesting discussions. REFERENCES 1 L. F. LELOIRAND C. E. CARDINI,J. A~$*. Chem. Sot., 79 (1957) 6340. 2 L. F. LELOIR, J. M. OLAVARRIA,S. H. GOLD~-MB~-RGAND H. CARMINATTI,Arch. Biochem. Biophys., 81 (1959) 508. s C. VILLAR-PALASIAND J. LARUER, Biochim. Biophys. Acta, 3° (1958) 449. 4 p. W. ROB~INS,R. R. TRAUTAND F. LIPMANN,Proc. Natl. Acad. Sci. U.S., 45 (1959) 6. 5 B. M. BRECKEI~RIDGEAND E. j. CRAWFORD,J. Biol. Chem., 235 (196o) 3054. Biochim. Biophys. Acta, 73 (1963) 565-573
GLYCOGEN SYNTHESIS IN THE INSECT FAT BODY
~73
6 L. F. LELOIR AND S. H. GOLDEMBERG,J. Biol. Chem., 235 (I96O) 919. D. K. BAsu AND B. K. BACHHAWAT,Biochim. Biophys. Acta, 5 ° (I961) I23. s M. RosEr,L-PEREZ XND J. LARNER, Biochemistry, r (1962) 769. 8 I. D. ALG.RANXTI AND E. CABIn, Biochim. Biophys. Acta, 43 (196o) I4X. 10 N. B. MADSEN, Biochim. Biophys. Acta, 5 ° ( I 9 6 I ) I94. lx R. KOEN:FELD XND D. H. BROWS, J. Biol. Chem., 237 (1962) I772. 1' M. ROSEI,L-PEREZ, C. VILLAR-PALASX AND J. LARNER, Biochemistry, I (I962) 763 . t8 D. L. FR~IDMAN AND J. LARr~ER, Biochim. Biophys. Acta, 64 (1962) 185. 14 j . C. TRI'¢ELLONI, Arch. Biochem. Biophys., 89 (196o) I49. ~5 R. HESS AND A. G. E. PEARSE, Enzymol. Biol. Clin., I (I961) 15. ~e C. S. HANES AND F. A. ISHERWOOD, Nature, I64 (1949) IIO7. ~ R. H. HORROCES, Nature, I64 (1949) 444. 18 C. H. FISKE AND Y. SUBBARow, J. Biol. Chem., 66 (1925) 375. x 0 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARE AND R. J. RANDALL, J. Biol. Chem., 193 (I951) 265. so D. J. CANDY AND B. A. KILBY, Biochem. J., 78 (I96I) 531. 11 H. SHIGEMATSU, Nippon Sanshigaku Zasshi, 25 (1956) 115. ~ R. H. KORNFELD, Ph.D. Thesis, W a s h i n g t o n University, Saint Louis, Miss., (1961). as F. G. CAJ~EY AND G. R. WYATt, Biochim. Biophys. Acta, 41 (196o) I78. ** C. VILLA1t-PALASI AND J. LARNER, Arch. Biochem. Biophys., 94 (1961) 436.
Biochim. Biophys. Attic, 73 (1963) 565-573