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Some regulatory properties of glycogen phosphorylase from Streptococcus salivarius
JLRCHIVES
OF
BIOCHEMISTRY
Some
.\ND
Regulatory
164,
BIOPHYSICS
Properties
306-313
of
Glycogen
Streptococcus TERRY
N. SPEARMAN,
Department
of Oral
RAMJI
Biology
(1973)
Received
from
salivarius
L. KHANDELWAL
(Biochemistry), Winnipeg,
Phosphorylase
Faculty of Dentistry, Canada RSE OWS December
AND
IAN University
R. HAMILTON’ of Manitoba,
16. 1971
Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) oi Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K, for AMP was 0.25 mM and was decreased to 0.125 mM in the presence of NaF. The K, for NaF was 0.49 M and was decreased to 0.40 M in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 m&r AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K,, values for glucose-l-P and glycogen in the direction of synthesis, but increased the T’ of the enzyme. When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P2 had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P* and ATP, both in the presence and absence of AMP + NaF.
The control of glycogen degradation in animal systems is now known to involve the regulation of phosphorylase (EC 2.4.1.1) through a complex sequence of enzymatic steps which are triggered by the hormoneinduced formation of cyclic adenosine 3’) 5’monophosphate (cyclic AMP) (l-4). The present state of the knowledge regarding the control of glycogen degradation in lower forms of life is, however, much less complete, although a number of recent studies have examined the properties of glycogen phos1 The author be directed.
to whom
reprint
requests
phorylase from various microorganisms (511). Wit’h the exception of the fungus, Neurospora crassa, which possessesa glycogen phosphorylase capable of existing in two interconvertible forms: one an AMP-independent “active” form and the other an inactive (AMP-dependent) form (lo), microbial phosphorylases appear to exist in only one active form. Like t’he N. crassa phosphorylase (8, lo), the enzyme in E. coli (6) was activated by AMP and inhibitfed by glucose-6-P and various nucleoside diphosphate glucose compounds. Glucose-6-P and
should 306
Copyright All rights
@ 1973 by ~4cademic Press, of reproduction in any form
Inc. reserved
Streptococcus salivarius
the gluccw nucleotides were also inhibitory for the glycogen phosphorylase from t’he fungus, Cryptococcus Zaurentii (5), as wcrc AMP, BDP and ATP. This nucleot’ide and sugar nurkotide inhibition could be reversed by l\Ig?- salts. The AMP-independent enzyme from Tetralzymena pyrifomis was inhibited by UDPG and ATP, but, was insensitive to glucose-&P (12). Yeast were also shown to contain an AMP-independent glycogen phosphorylase which \\-a~ inhibited by glucoses,glucose-6-P and UDI’G (11). Depending on the direction of assay, T\‘aF is either an activator (6, 7) or an inhibitor (8) of microbial phosphorylases. \Yc have, in a preceding paper (13), outlined the purification of gl?cogen phosphorylaw from Streptococcus saharius, an organism c2:llJ:l~Jle of synt’hcsizing and degrading massiw quantities of glycogen (14). In order to mow cl(Larly understand the factors \\-hich regulate the activity of this enzyme in vivo, V-C 11:~~~~ drttrmined the cffcct of various cellular nwt,abolit’es on t,he activity of the purified enzyme. X4TP:NIBLB
AND
METHODS
Enzyur purijication. The procedure for the purification of glycogen phosphorylase from the soluble frartioll of S. saliaarius (ATCC 25975) has been described previously (13). The 200.fold purified enzyme obtained after chromat,ography on Sephades G-200 was used throughout this work. E’nz,~nlt assay. Glycogen phosphorylasc a,‘tivity was assayed both in the direction of synthesis and in the dire&on of phosphorolysis. With the former assay, the reaction mixture contained 0.1 M $-glycerophosphate buffer (pH ti.O), 1 mu [14C]glucoae-l-P (1-2 X lo3 dpm/nmole); 0.5 mg glycogcn and enzyme in a final volume of 0.1 ml. l’hosphorolysis was assayed by radioactive method of shepherd ~1 al. (S) in a reaction mixture containing 0.5 mg glycogen, 50 mM [32P]phosphate (pH 7.0) and euzymc in a final volume of 0.1 ml. Variatiolls in the reaction mistlxes have been noted with t hc appropriate experiment,. Jlol~/~icr/s. All radioactive compounds (Ised in this strrdy were purchmed from New I~~ngland Nuclrar Corp. The glycogcn (oyster) used in this study was obtained from Fisher Scientific Co,, and was always treated before use with Norit A according to the method of Helmreich and Cori (15). All glycolytic intermediates and n\lcleot,ides werr obtained either from Boehringer nfannheim
Corp. or from chemicals were
307 Sigma Chemical of reagent grade.
Co.
All
other
RESULTS
EJect of A:lfP and NaF on enzyme activity. When assayed in the direction of synthesis with either glgcogen or dextrin as t’he primer, purified glycogen phosphorylase from S. salivarius was activated slightly by AMP and by NaF when incubated with these compounds alone (Table I). However, the act’ivation observed in the presence of these compounds combincld was greater than the sum of the activity increasesobtained in the prewncc of these compounds singly. The KaF cffcct was very specific since activation n-as not’ ohwrved &th KC1 or NaCl. It will be not’ed that t,hc fold activation by AMP + NaF was significantly greater with glycogen as the primer than with dcxt’rin. By incubating the basic reaction mixture wit,h increasing amounts of AMP in the prcscnce and absence of NaF, this latter compound could be shown to alter the Xrhaelis const#antfor ARIP (Fig. 1). The K, for AMP was 0.25 rnl\L when incubated &hout Sal:, but’ decreased to 0.12,T rnJI in the prcsenw of t’hix compound. This latter value was indrpcndcnt of the -\:a]’ concentration used in t,hcl rraction mixturrt, howcvw, tjhe maximal wlocit,y increasrd as the Xal’ concthntration was inTABLE
Additions to basic assay
I
Glycogen
Dextrin
-. Activity -~~~
None 13* + i1MP” 184 + NaF” 148 + AMP + 407 NaF -. __.G nmoles [‘%!]glucose 30 min. b AMP concentrat,ion c NaF concentration
Fold ac-, Activity tivation _~~ ( I 1.37 ~ 1.11 3.04
incorporated/mg is 1.0 m&r. is 0.1 M.
25+ 329 246 441
Fold activation 1.26 0.97 1.74
protein/
30s
SPEARMAN,
KHANDELWAL
AND
HAMILTON
tivity with AMP or NaF, and with SaF + AMP. As indicated in Table II, the c+fcct observed with these compounds was almost completely reversible as indicated by the control values (column 1). While preincubation with AMP resulted in slightly increased activity (i.e., 0.65 to 0.71), the pre$rnce of NaF caused a slight decline (i.e., 0.65 to 0.57). This It-as generally the case al$o when the preincubated samples were incubated 4
8 AMP
12 (mM)-’
‘6
FIG. 1. Effect of varying concentrations of AMP on the activity of purified S. salivarius glycogen phosphorylase incubated with two concentrations of NaF and assayed in the direct,ion of glycogen synthesis.
creased. On the other hand, the K, for Na,F was 0.49 M in the absence of AMP and was reduced slightly to 0.40 M in the presence of this nucleotide (Fig. 2). As was the case for NaF, the concentration of AMP had no effect on the K, for NaF. These results indicate a cooperative effect of these two compounds on the activity of glycogen phosphorylase. It should also be noted from Fig. 1 that AMP activation was reduced at AMP concentrations higher than l-2 rnnr, particularly in the presence of NaF. Since AMP, or AMP + NaF, had no effect on the elution pattern of the enzyme from Sephadex G-200 (13), these compounds probably do not alter the molecular weight of the enzyme. Similar activation of the S. saliva&s glycogen phosphorylase by AMP and NaF was observed when the enzyme was assayed in the direction of phosphorolysis provided the AMP concentration did not exceed 2 rnRI (Fig. 3). At concentrations above 2 rnq AMP was a significant inhibitor of enzyme activity. In order to test whether the AMP and NaF effects in the direction of synthesis were reversible or irreversible, the enzyme was preincubated for 10 min under the following conditions : (a) no addition (control), (b) +l mnZ AMP, (c) +O.l 11 NaF, and (d) + 1 rn>r AMP and 0.1 ~1 NaF. After the incubation period, the enzyme in each case was dialyzed against Tris buffer (10 mM, pH 7.0 containing 5 mlzl mercaptoethanol) for 4 hr and was assayed for phosphorylase ac-
2
6 NaF
FIG. 2. Effect of varying NaF on the activity of purified cogen phosphorylase incubated centrations of AMP and assayed
concentratious S. salivnri,ts with three as in Fig. I.
20
li! z c B ‘0
I(
(Ml-’
of glycon-
A.
IO
0 /ImlIL 0 2 4 AMP (mM)
6
NaF (Ml FIG. 3. Effect of varying concentmbions AMP, NaF and AMP + NaF on the activity purified S. salivarim glycogen phosphorylase when assayed in the direction of phosphorolysis.
of of
TABLE EFFECT
OS
OF PREINCUBATIOS TIIE ACTIVITY
PHOSPHOR~L.\W: SI~;QUF:NTLY
PRCSICXCE ASSAYICD
;\dditions
II \VITH i\blP PUIUFIED
OF
FROM S. INCUFJATED WTH
NaF GLYCOGEN sdivarius SUB-
GLYCOGEN
AND
IX
011 Anussol: ok AMP AND NaF, IN THIS DIRECTION\OF SYNTHESIS to
i\dditions
to reaction
THE
AKD
mixture AMP + NaF
glycolytic intermcdiatcs arc known to affctct the activity of glycogcn pl~ospl~or~ln~~ from various sourws (6, 7, 11, 12). WV havc~ examinod the effect of t,hroc glycolytic’ intermediates, glwosc-G-p, I’-c~nolp.-ruvntc~ and fructose-l , G-l’, ) as \\-cl1 as glucose nucl -ADPglucoses, on thn activity of purifirtl S. snlivczri7~s glycogcn pl~osphorylnsc when assayed wit#h g!.lvcogScnin both dircct’ions in thl> prw cnw ~~~abkmcr of A1\IP and/or Sal:. In the dircxction of synt~lwsid (Table III)! the: :LCtiV$V
Il’OIlC
1
+ AMPa + N aF b + iZ&lP + NaF
yp”
1
0.57 0.57
;.;;
;
1:90 1.68 I
y;
3.80 3.66
0.58 O.GG
2.90 3.00
/
a AMP concentration is 1.0 mar. B NaF concentration is 0.1 nz. c nmoles of [‘%]glucose incorporation gl~cogen/30 min.
(Jf
the
pLn-ificd
8.
s&?im?‘i&~
1’111
!spho-
rvlaw in tlic‘ ubscncc~ of AMP and Sal: was siight,ly- inhibited by glucose (6.2 ’ ; ) and glucose:-6-P (12.2 7:’), but n-as activated l)y I~-cwolpyruvatc: (12-34 %) and ADPglucose (up to 170 7;‘). Only in the latter tn3b (3scs
into 5
with AMP or KaF, and with n’al: + AMP (columns 24). Thn presence of NaF in t,he preincubation medium, whether alone or in combinat’ion with AMP, caused a S-25 % loss in phosphorylase act’ivit’y compared to tho enzyme prrincubation in abscncc of either NaF or AMP. E$ect of AMP and NaF OS the Michaelis constants JOY glucose-l-l’ and glycoqen. The effect of AMP on the K, values for glucose-1-P and glycogen, and on the maximum velocity of glycogen phosphorylase from various sources, is specific and is dependent on the source of the isolated enzvme. With the enzyme from N. crassa {7), AMP changed t’he 17 of t’he enzyme but’ had no effect on the K, for glucose-1-P and glycogen, lvhereas both t’he K, values and tho V of the cnzymo from E. coli (6) were altcrcd by this nucleotide. In view of these reports, we tested the effect of AMP (1 rnhr), and AMP (1 m&r) combined Tvith T\TaF (0.1 11) on the K, values for glucose-l-P and glycogcn, and on the I’ of the purified phosphorylase from S. saliva&s. As shown in Figs. 4 and 5, both ARIP, and AMP + NaF increased the V of the enzyme, but had no cffcct on the Michaelis constants for glucase-1-P and glycogcn. Effect of glycolytic intermediates. Various
Km = 3.4 mM
0.2
06 GLUCOSE-I-P
10 ImM)-1
FIG. 4. Effect of AMP and AMP + KaF con the activit,y of purified 8. salivarills glycogen phosphorylase at varying concentrations of glttcose1-P when assayed as in Fig. 1.
0.04
0.12 GUCOGEN
020 hg/n’:-’
FIG. 5. Effect of AMP and AMP + SaF on the activity of purified S. saZivarius glycogen phosphorylase at varping concentrations of glycogen when assayed RS in Fig. 1.
310
SPEARMAN, TABLE
KHANDELWALIAND
III
EFFECT OFVARIOUS ~IETABOLITES ON THE ACTIVITY OF PURIFIED GLYCOGEN PHOSPHORLYASE FROM S. salivarius INCUBBTED WITH GLYCOGEN IN THE PRESENCE OR ABSENCE OF AMP AND NaF, AND ASSAYED IN THE DIRECTION OF SYNTHESIS Additions
( Zoncentration k-4
Additions
to reaction mixture
None
None Glucose Glucose-6-P P-enolpyruvate Fructose-l,BP2 ADPglucose
2.5 5.0 1.5 3.0 1.5 3.0 1.5 3.0 0.5 1.5
-
AMP”
AMP t NaFb
1.98 1.92 1.87 1.88 1.81 2.35 2.39 2.01 2.00 1.74 1.37
4.41 4.14 3.20 3.72 3.57 4.92 4.96 4.46 4.22 3.13 1.39
0.54” 0.51 0.51 0.48 0.47 0.61 0.72 0.54 0.56 0.57 1.46
a AMP concentration is 1.0 mM. b NaF concentration is 0.1 M. c nmoles of [14C]glucose incorporation glycogen/30 min.
into
HAMILTON
latter compounds, inhibition by glucose was reduced (to 7 %), while the activation by P-enolpyruvate was increased significantly (56-187 %). E$ect of nucleosides and nucleotides. As demonstrated in Table I and Fig. 3, the purified S. saliva&s glycogen phosphorylase was activated by AMP and this activation was further augmented by NaF. To test whether AMP could be replaced by other adenine and guanine nucleosides and nucleotides, the effect of these compounds was examined in the presence and absence of AMP and/or NaF. When assayed in the direction of synthesis in the absence of AMP and NaF, the enzyme was activated by adenosine (24%), ATP (34%), GMP (43 %), cyclic AMP (91%) and ADP (195%), and was TABLE
Concentration bd
Additions
had no effect on enzyme activity. When the effect of t’hese compounds was tested in the presence of AMP, or AMP + NaF, all of these substances, except P-enolpyruvate and fructose-l, 6-Pz , inhibited the enzyme 368 %; inhibition was most severe with ADPglucose. Under these conditions, P-enolpyruvate again activated the enzyme 12 and 20 % at concentrations of 1.5 and 3.0 m&II, respectively. Fructose-l, 6-Pz again showed no effect. The above compounds had a somewhat different effect on the enzyme when assayed with glycogen in the direction of phosphorolysis. Under these conditions, and in the absence of AMP or NaF, the enzyme was inhibited by glucose (15-23 %) and ADPglucose (38-53 %), was unaffected by glucose-6-P, and was activated by P-enolpyruvate (11-56 %) and fructose-l, B-P2 (3-24 %) (Table IV). With the exception of glucose and P-enolpyruvate, similar results were obtained when the assays were carried out with NaF (0.1 AX) and AMP (2 mM). With these
IV
EFFECT OFVARIOUS METABOLITES ON THE ACTIVITY OF PURIFIED GLYCOGEN PHOSPHORYLASE FROMS. salivarius WHEN ASSAYEDWITHOYSTER GLYCOCJEN IN THE DIRECTION OFPHOSPHOROLYSIS IN THE PRESENCE AND ABSENCE OF AMP AND NAF~
Glucose-B-P P-enolpyruvate Fructose-l,
6-PZ
ADP-glucose
ATP
-
-
None Glucose
Cyclic
Additions to reaction mixture
AMP
2.5 5.0 1.5 3.0 1.5 3.0 1.5 3.0 0.5 1.5 0.5 1.0 2.0 3.0 4.0 1.25 5.0
-
None
AMP + NaFY
0.93* 0.72 0.79 0.91 0.90 1.04 1.46 0.96 1.16 0.67 0.44 0.76 0.69 0.76 0.76 1.13 0.94 1.67
1.31 1.33 1.22 1.41 1.36 3.13 3.78 1.48 1.62 1.07 0.73 1.36 1.34 1.37 1.16 1.17 1.49 2.52
5 AMP and NaF concentrations were 2.0 mM and 0.1 M, respectively. b nmoles of glucose-l-P formed/30 min.
Streptococcus
slightly inhihitcd by GTP and GDP (S %) (Table V). The activation of the enzyme by these compounds, hojvever, was less than the act.ivation by the same concent,ration of AMP (270 %). When AMP was a component of the reaction mixture, these compounds either did not show any effect or were slightly inhibitory (max 26 %). Similar result,s \vere obtained with the enzyme incubated with AMP and NaF, although slight activation of the enzyme was observed with adrnosine (12%). In the direction of phosphorolysis and in the absence of AMP and NaF, cyclic AMP activated the enzyme 21% at a concentration of 4 rnN[, but was inhibitory (to 26 %) at lower concenbrations (Table IV). In the presence of 2 ml,1 AMP and 0.1 M NaF, this cyclic nucleotide was slightly inhibitory above a concentration of 2 rn)I. ATP, on the other hand, activated the enzyme 79% at the 5 mnI level and this activation was increased to 97 % by the presence of AMP and XaF. This activation was not reversed by sat,urating levels of Mg*+ ions. TABLE
V
EFFECT OF THE NUCLEOSIDES AND NUCLEOTIDES (1 mM) OF ADENINE .&ND GU~NINE ON THE ACTIVITY OF PURIFIED GLYCOGEN PHOSPHORYLASE FROM S. salivarius INCUBATED WITH GLYCOGEN IN THE PRESENCE OR ABSENCE OF AMP AND NaF, AND ASSAYED IN THE DIRECTION OF SYNTHESIS Additions
Additions None
to reaction AMP”
mixture AMP + NaFb
None
0.14c
0.52
1.10
ATP ADP Cyclic AMP Adenosine
0.19 0.42 0.27 0.18
0.39 0.45 0.51 0.48
1.14 1.14 1.09 1.23
GTP GDP GMP Guanosine
0.13 0.13 0.20 0.15
0.45 0.44 0.49 0.49
1.07 0.96 1.04 1.11
a AMP concentration is 1.0 mM. b NaF concentration is 0.1 M. c nmoles of [W Jglucose incorpora.tion glycogen/30 min.
311
sulivarizis
I FIG. AMP(.4) purified incubated
3 AMP(mM)
5
I Cyclic
3
5
AM? (mM)
0. Effect of varying concentrations and cyclic AMP(B) on the activity S. salivu~i~ts glycogen phoaphorylase with the other component and SaF.
of of
The relationship between thr AMP and cyclic AMP concentrations on the activity of purified S. salivarius glycogen phosphorylase in the dir&ion of svnthesis is illustrated more completely in Fig. 6. It en11 1~ seen that, as a function of t#he AMP concentration (Fig. GA), 1 mi\l cyclic AMP had no additional effect on the enzyme compnrrtl to the activity with AMP alone (i.r.. control). Cyclic AMP, as seen in Table T‘, activated t,he enzyme only in the absence of AMP. Furthermore, 5 mRL cyclic AJIP was inhibitory, albeit, slightly. This inhibition is more clearly observed \yhcn the values are plott8cd as a function of the cyclic AMP concentration (Fig. GH). In the presence of 1 and 5 m&I AMP, increasing levels of q-clic ARIP resulted in a slight but steady decline in activit.y, which, under these conditions, was &ill greater than tho act,ivit!- with cyclic ARIP alone. Tha influence of XaF on varying levels of both AMP and cyclic AMP should also be noted. The combination of AMP + NaF produced twice the activation of the S. salivarius phosphorylase, and at. :I Iolver AlIP level (Fig. GA), than did the combination of cyclic ARlP + XaF (Fig. AB). DISCUSSION
into
The activity of glycogen phosphorylase in animal systems is controlled by (a) the conversion of the AMP-independent ‘iactive” form to the AMP-dependent “inactive” form and (1,) by the activation of this 1at.k form by A3IP (18). As mcnt.ioned previously, the
312
SPEARMAN,
KHANDELWAL
only report of int’crconvertible forms of glycogen phosphorylase in microorganisms has come from the eukaryotic fungus, N. crassa (IO). Other microbial eukaryotic forms (5, 11, 12), as well as all of the prokaryotic organisms reported to date (6, 9) appear to contain phosphorylases which are active in the absence of AMP, but which may or may not be activated by this nucleotide. All attempt,s to demonstrate two forms of glycogen phosphorylase in S. saliva&s, similar to that in mammalian systems, were unsuccessful (unpublished results) which, along with the previous studies mentioned above, suggests that “sophisticated” interconvertible phosphorylases probably do not exist in prokaryotic cells or that the conversion between two forms has different regulatory properties than mammalian systems. The combined activation of purified S. salivarius glucogen phosphorylase by AMP and NaF (Table I) suggests a complex mechanism of enzyme action. The fact that the K, values for AMP and NaF were decreased in the presence of the other compound, as well as the much greater activation of the enzyme when AMP and NaF were combined, indicates cooperative effects either between separate AMP and NaF binding &es, or at a single site binding both compounds. The K, values for glucose-l-p and glycogen were not altered by AMP, or A?tIP + NaF (Figs. 4 and 5), which is similar t’o the results obtained with glycogen phosphorylase from N. crassa (7), but in direct contrast to the evidence obtained with the E. coli enzyme (6). In this latter case, 5 mnr AMP increased the K, for glucose-l-P from 1 rnI\r to 2.9 rnM, while decreasing the K, for glycogen from 6.7 mg/ml to 4.6 mg/ml. The effects observed with NaF arc difficult to extrapolate to physiological conditions where this compound at low concentrations will act, as an inhibitor of glucose metabolism by S. saliva&s (17, 18). A number of salts which may exist in vivo produce activating effects similar to those produced by KaF. For example, the purified S. salivarius glycogen phosphorylase was activated by the sulfate salts of sodium and ammonium (0.1 ~lr) in the presence of AXIP to the same extent
AND
HAMILTOX
(i.e., four- to sixfold) as observed with XaF. Similar results have also been observed with the E. coli enzyme (6). The absence of a pronounced effect of cyclic AMP on the S. salivarim glycogen phosphorylase activity clearly separates this enzyme from t.hose functioning in animal cells (19). While cyclic AMP could be shown to activate the S. salivarius enzyme in the absence of AMP (Table IV and Fig. 6), the presence of AMP modified this effect as it did for the other nuclcosides and nucleotides tested (Table V). Thus, tho activation of the purified S. salivarim phosphorylase by ARIP probably constitutes one of the major controlling mechanisms in this organism for the degradation of glycogen. Consequent,ly, in a cell with a low ‘Lencrgy charge” (i.e., high ADP + AMP) (20), phosphorylase and glycogen degradation would be activated, while the synthesis of glycogen would be decreased through the inhibition of ADP-glucose pyrophosphorylase by these nuclcotidcs (21). Furthermore, the activating effect of AMP would be augmented under these conditions by P-enolpyruvate, which has been shown to stimulate enzyme activit,y when assayed in both directions (Tables III and IV). Since glucose is transported into cells of S. salivarius via the P-enolpyruvate phosphotransferase system, the cellular concentration of P-enolpyruvatc is low during glucose metabolism but increases dramatically after the exhaustion of this exogenous carbon source as the organism approaches a condition of low energy charge (22). The increased level of this metabolite would then serve to activate glycogen phosphorylase to ensure the continued flow of carbon through the glycolytic pathway. On the other hand, under conditions of exogenous glucose metabolism when the energy chargo is high, the cttllular concentrations of AMP and P-enolpyruvatn would be low but the level of ADP-glucose would be high since glycogen synt.hesis is proceeding at a rapid rate (14). The elevat’ed level of this metabolite in the cell would consequently result in the inhibition of glycogen degradation through it’s action on glycogcn phosphorylase. Less readily understood in this situation, however, is the role played by AT!?,
14.
I~.GVIII,TON,
1.
II.
J.
A.,
(1!)1i8)
,I. illicmbiol.
Can.
14, (i5.
1.
~IiTHEILLAh‘l~,
1’:.
w.,
:\S,>
~~OI,lSCJS,
2.
Diabclcs 18, 797. BUTCHER, 11. W., llour~~s, .J. G., AXD ~uTEIEItl,.lN~,
cd;.
.4.
(1969)
.2dzlan.
3.
s.
I~TTOM,
H.UIUM.\X,
15.
vc’.
I<.,
h’UW,
J. I’.
I).
(19X))
Llr/van.
.lX\Iu
fJIcKl!!S-
E’ntymc
VILLM-P.\LASI,
5.
S~~IWARTZ,
.lnn. ,I. 6.
CH~;N,
C.,
IZev.
Biochem. M.,
C.
39,
IAI~NLI:,
J.
fi39.
HOE’STJNG, br. (lN7j
.\SU
(1970)
EM.
2, 132.
Kiochern.
i2ioc~hcm.
.lsu
p.,
.ISD
8iophy.s.
SI,;GP:I,,
I.
127,
175.
1-T.
(I%%)
.lrch.
~~OBISON.
KIIANI~I~:LWAL,
I.
11. 144, 5YCi. G.
S~.WIII~:I~L~ND,
A.,
(1971)
It.
,lrch.
~C.TcIII:II,
1L.
1%;.W. (1968) dnn.
20.
chcrn. 37, 11!). ATICINBON, I). W. (1968)
21.
Pn~,:rss,
Regul.
8, 205. 4.
Biophys. 19.
I$.,
KZN.UX.I, IhtlII,TON,
(l!)(i8)
Reqd. 6, 357.
Enzyrnc
h~.ZPER,
18. G. A.,
Biochernisfr~
L., \SD f3iochcub. w.,
.\Nl,
h’cv. Nio7, 4030.
J. (1969) in Clurcrlt Topics of Cellular IIegrdation (EIorwker, U. I,., and 15. IL Slatltman, etls), Vol. 1, p. 125, Academic Press, Xcw York. 22. K.INMSI, J. A., ANL, HAMILTON, I. Ii. (1971) ilrch. Uiochem. Hiophys. 146, 167.
OF
BIOCHEMISTRY
Some
.\ND
Regulatory
164,
BIOPHYSICS
Properties
306-313
of
Glycogen
Streptococcus TERRY
N. SPEARMAN,
Department
of Oral
RAMJI
Biology
(1973)
Received
from
salivarius
L. KHANDELWAL
(Biochemistry), Winnipeg,
Phosphorylase
Faculty of Dentistry, Canada RSE OWS December
AND
IAN University
R. HAMILTON’ of Manitoba,
16. 1971
Purified (200-fold) glycogen phosphorylase (EC 2.4.1.1) oi Streptococcus salivarius was activated by AMP and NaF when assayed both in the direction of synthesis and in the direction of phosphorolysis. Activation by NaF + AMP was greater than the sum of their individual effects. In the direction of synthesis, the K, for AMP was 0.25 mM and was decreased to 0.125 mM in the presence of NaF. The K, for NaF was 0.49 M and was decreased to 0.40 M in the presence of AMP. Glycogen phosphorolysis was similarly affected by AMP and NaF, except that above a concentration of 2 m&r AMP was inhibitory. The effects of AMP and NaF were reversible since preincubation with these compounds, followed by dialysis, restored activity almost to the control values although some inhibition of enzyme activity was noted with the samples preincubated with NaF. The presence of both NaF and AMP had no effect on the K,, values for glucose-l-P and glycogen in the direction of synthesis, but increased the T’ of the enzyme. When assayed in the absence of AMP and NaF in the direction of synthesis, the enzyme was slightly inhibited by glucose and glucose-6-P, and activated by P-enolpyruvate and ADP-glucose. In the presence of AMP and NaF, the enzyme was inhibited by glucose, glucose-6-P and ADP-glucose, but was activated by P-enolpyruvate. Fructose-1,6-P2 had no effect on the enzyme. The enzyme was further activated in the absence of AMP and NaF by adenosine, ATP, GMP, cyclic AMP and ADP, and was slightly inhibited by GTP and GDP. In the presence of AMP and NaF, however, these compounds, with the exception of adenosine, either did not show any effect or were slightly inhibitory. Adenosine was slightly stimulatory with NaF + AMP, but not with AMP alone. In the direction of phosphorolysis, the enzyme was inhibited by glucose and ADP-glucose, and activated by P-enolpyruvate, fructose-1,6-P* and ATP, both in the presence and absence of AMP + NaF.
The control of glycogen degradation in animal systems is now known to involve the regulation of phosphorylase (EC 2.4.1.1) through a complex sequence of enzymatic steps which are triggered by the hormoneinduced formation of cyclic adenosine 3’) 5’monophosphate (cyclic AMP) (l-4). The present state of the knowledge regarding the control of glycogen degradation in lower forms of life is, however, much less complete, although a number of recent studies have examined the properties of glycogen phos1 The author be directed.
to whom
reprint
requests
phorylase from various microorganisms (511). Wit’h the exception of the fungus, Neurospora crassa, which possessesa glycogen phosphorylase capable of existing in two interconvertible forms: one an AMP-independent “active” form and the other an inactive (AMP-dependent) form (lo), microbial phosphorylases appear to exist in only one active form. Like t’he N. crassa phosphorylase (8, lo), the enzyme in E. coli (6) was activated by AMP and inhibitfed by glucose-6-P and various nucleoside diphosphate glucose compounds. Glucose-6-P and
should 306
Copyright All rights
@ 1973 by ~4cademic Press, of reproduction in any form
Inc. reserved
Streptococcus salivarius
the gluccw nucleotides were also inhibitory for the glycogen phosphorylase from t’he fungus, Cryptococcus Zaurentii (5), as wcrc AMP, BDP and ATP. This nucleot’ide and sugar nurkotide inhibition could be reversed by l\Ig?- salts. The AMP-independent enzyme from Tetralzymena pyrifomis was inhibited by UDPG and ATP, but, was insensitive to glucose-&P (12). Yeast were also shown to contain an AMP-independent glycogen phosphorylase which \\-a~ inhibited by glucoses,glucose-6-P and UDI’G (11). Depending on the direction of assay, T\‘aF is either an activator (6, 7) or an inhibitor (8) of microbial phosphorylases. \Yc have, in a preceding paper (13), outlined the purification of gl?cogen phosphorylaw from Streptococcus saharius, an organism c2:llJ:l~Jle of synt’hcsizing and degrading massiw quantities of glycogen (14). In order to mow cl(Larly understand the factors \\-hich regulate the activity of this enzyme in vivo, V-C 11:~~~~ drttrmined the cffcct of various cellular nwt,abolit’es on t,he activity of the purified enzyme. X4TP:NIBLB
AND
METHODS
Enzyur purijication. The procedure for the purification of glycogen phosphorylase from the soluble frartioll of S. saliaarius (ATCC 25975) has been described previously (13). The 200.fold purified enzyme obtained after chromat,ography on Sephades G-200 was used throughout this work. E’nz,~nlt assay. Glycogen phosphorylasc a,‘tivity was assayed both in the direction of synthesis and in the dire&on of phosphorolysis. With the former assay, the reaction mixture contained 0.1 M $-glycerophosphate buffer (pH ti.O), 1 mu [14C]glucoae-l-P (1-2 X lo3 dpm/nmole); 0.5 mg glycogcn and enzyme in a final volume of 0.1 ml. l’hosphorolysis was assayed by radioactive method of shepherd ~1 al. (S) in a reaction mixture containing 0.5 mg glycogen, 50 mM [32P]phosphate (pH 7.0) and euzymc in a final volume of 0.1 ml. Variatiolls in the reaction mistlxes have been noted with t hc appropriate experiment,. Jlol~/~icr/s. All radioactive compounds (Ised in this strrdy were purchmed from New I~~ngland Nuclrar Corp. The glycogcn (oyster) used in this study was obtained from Fisher Scientific Co,, and was always treated before use with Norit A according to the method of Helmreich and Cori (15). All glycolytic intermediates and n\lcleot,ides werr obtained either from Boehringer nfannheim
Corp. or from chemicals were
307 Sigma Chemical of reagent grade.
Co.
All
other
RESULTS
EJect of A:lfP and NaF on enzyme activity. When assayed in the direction of synthesis with either glgcogen or dextrin as t’he primer, purified glycogen phosphorylase from S. salivarius was activated slightly by AMP and by NaF when incubated with these compounds alone (Table I). However, the act’ivation observed in the presence of these compounds combincld was greater than the sum of the activity increasesobtained in the prewncc of these compounds singly. The KaF cffcct was very specific since activation n-as not’ ohwrved &th KC1 or NaCl. It will be not’ed that t,hc fold activation by AMP + NaF was significantly greater with glycogen as the primer than with dcxt’rin. By incubating the basic reaction mixture wit,h increasing amounts of AMP in the prcscnce and absence of NaF, this latter compound could be shown to alter the Xrhaelis const#antfor ARIP (Fig. 1). The K, for AMP was 0.25 rnl\L when incubated &hout Sal:, but’ decreased to 0.12,T rnJI in the prcsenw of t’hix compound. This latter value was indrpcndcnt of the -\:a]’ concentration used in t,hcl rraction mixturrt, howcvw, tjhe maximal wlocit,y increasrd as the Xal’ concthntration was inTABLE
Additions to basic assay
I
Glycogen
Dextrin
-. Activity -~~~
None 13* + i1MP” 184 + NaF” 148 + AMP + 407 NaF -. __.G nmoles [‘%!]glucose 30 min. b AMP concentrat,ion c NaF concentration
Fold ac-, Activity tivation _~~ ( I 1.37 ~ 1.11 3.04
incorporated/mg is 1.0 m&r. is 0.1 M.
25+ 329 246 441
Fold activation 1.26 0.97 1.74
protein/
30s
SPEARMAN,
KHANDELWAL
AND
HAMILTON
tivity with AMP or NaF, and with SaF + AMP. As indicated in Table II, the c+fcct observed with these compounds was almost completely reversible as indicated by the control values (column 1). While preincubation with AMP resulted in slightly increased activity (i.e., 0.65 to 0.71), the pre$rnce of NaF caused a slight decline (i.e., 0.65 to 0.57). This It-as generally the case al$o when the preincubated samples were incubated 4
8 AMP
12 (mM)-’
‘6
FIG. 1. Effect of varying concentrations of AMP on the activity of purified S. salivarius glycogen phosphorylase incubated with two concentrations of NaF and assayed in the direct,ion of glycogen synthesis.
creased. On the other hand, the K, for Na,F was 0.49 M in the absence of AMP and was reduced slightly to 0.40 M in the presence of this nucleotide (Fig. 2). As was the case for NaF, the concentration of AMP had no effect on the K, for NaF. These results indicate a cooperative effect of these two compounds on the activity of glycogen phosphorylase. It should also be noted from Fig. 1 that AMP activation was reduced at AMP concentrations higher than l-2 rnnr, particularly in the presence of NaF. Since AMP, or AMP + NaF, had no effect on the elution pattern of the enzyme from Sephadex G-200 (13), these compounds probably do not alter the molecular weight of the enzyme. Similar activation of the S. saliva&s glycogen phosphorylase by AMP and NaF was observed when the enzyme was assayed in the direction of phosphorolysis provided the AMP concentration did not exceed 2 rnRI (Fig. 3). At concentrations above 2 rnq AMP was a significant inhibitor of enzyme activity. In order to test whether the AMP and NaF effects in the direction of synthesis were reversible or irreversible, the enzyme was preincubated for 10 min under the following conditions : (a) no addition (control), (b) +l mnZ AMP, (c) +O.l 11 NaF, and (d) + 1 rn>r AMP and 0.1 ~1 NaF. After the incubation period, the enzyme in each case was dialyzed against Tris buffer (10 mM, pH 7.0 containing 5 mlzl mercaptoethanol) for 4 hr and was assayed for phosphorylase ac-
2
6 NaF
FIG. 2. Effect of varying NaF on the activity of purified cogen phosphorylase incubated centrations of AMP and assayed
concentratious S. salivnri,ts with three as in Fig. I.
20
li! z c B ‘0
I(
(Ml-’
of glycon-
A.
IO
0 /ImlIL 0 2 4 AMP (mM)
6
NaF (Ml FIG. 3. Effect of varying concentmbions AMP, NaF and AMP + NaF on the activity purified S. salivarim glycogen phosphorylase when assayed in the direction of phosphorolysis.
of of
TABLE EFFECT
OS
OF PREINCUBATIOS TIIE ACTIVITY
PHOSPHOR~L.\W: SI~;QUF:NTLY
PRCSICXCE ASSAYICD
;\dditions
II \VITH i\blP PUIUFIED
OF
FROM S. INCUFJATED WTH
NaF GLYCOGEN sdivarius SUB-
GLYCOGEN
AND
IX
011 Anussol: ok AMP AND NaF, IN THIS DIRECTION\OF SYNTHESIS to
i\dditions
to reaction
THE
AKD
mixture AMP + NaF
glycolytic intermcdiatcs arc known to affctct the activity of glycogcn pl~ospl~or~ln~~ from various sourws (6, 7, 11, 12). WV havc~ examinod the effect of t,hroc glycolytic’ intermediates, glwosc-G-p, I’-c~nolp.-ruvntc~ and fructose-l , G-l’, ) as \\-cl1 as glucose nucl -ADPglucoses, on thn activity of purifirtl S. snlivczri7~s glycogcn pl~osphorylnsc when assayed wit#h g!.lvcogScnin both dircct’ions in thl> prw cnw ~~~abkmcr of A1\IP and/or Sal:. In the dircxction of synt~lwsid (Table III)! the: :LCtiV$V
Il’OIlC
1
+ AMPa + N aF b + iZ&lP + NaF
yp”
1
0.57 0.57
;.;;
;
1:90 1.68 I
y;
3.80 3.66
0.58 O.GG
2.90 3.00
/
a AMP concentration is 1.0 mar. B NaF concentration is 0.1 nz. c nmoles of [‘%]glucose incorporation gl~cogen/30 min.
(Jf
the
pLn-ificd
8.
s&?im?‘i&~
1’111
!spho-
rvlaw in tlic‘ ubscncc~ of AMP and Sal: was siight,ly- inhibited by glucose (6.2 ’ ; ) and glucose:-6-P (12.2 7:’), but n-as activated l)y I~-cwolpyruvatc: (12-34 %) and ADPglucose (up to 170 7;‘). Only in the latter tn3b (3scs
into 5
with AMP or KaF, and with n’al: + AMP (columns 24). Thn presence of NaF in t,he preincubation medium, whether alone or in combinat’ion with AMP, caused a S-25 % loss in phosphorylase act’ivit’y compared to tho enzyme prrincubation in abscncc of either NaF or AMP. E$ect of AMP and NaF OS the Michaelis constants JOY glucose-l-l’ and glycoqen. The effect of AMP on the K, values for glucose-1-P and glycogen, and on the maximum velocity of glycogen phosphorylase from various sources, is specific and is dependent on the source of the isolated enzvme. With the enzyme from N. crassa {7), AMP changed t’he 17 of t’he enzyme but’ had no effect on the K, for glucose-1-P and glycogen, lvhereas both t’he K, values and tho V of the cnzymo from E. coli (6) were altcrcd by this nucleotide. In view of these reports, we tested the effect of AMP (1 rnhr), and AMP (1 m&r) combined Tvith T\TaF (0.1 11) on the K, values for glucose-l-P and glycogcn, and on the I’ of the purified phosphorylase from S. saliva&s. As shown in Figs. 4 and 5, both ARIP, and AMP + NaF increased the V of the enzyme, but had no cffcct on the Michaelis constants for glucase-1-P and glycogcn. Effect of glycolytic intermediates. Various
Km = 3.4 mM
0.2
06 GLUCOSE-I-P
10 ImM)-1
FIG. 4. Effect of AMP and AMP + KaF con the activit,y of purified 8. salivarills glycogen phosphorylase at varying concentrations of glttcose1-P when assayed as in Fig. 1.
0.04
0.12 GUCOGEN
020 hg/n’:-’
FIG. 5. Effect of AMP and AMP + SaF on the activity of purified S. saZivarius glycogen phosphorylase at varping concentrations of glycogen when assayed RS in Fig. 1.
310
SPEARMAN, TABLE
KHANDELWALIAND
III
EFFECT OFVARIOUS ~IETABOLITES ON THE ACTIVITY OF PURIFIED GLYCOGEN PHOSPHORLYASE FROM S. salivarius INCUBBTED WITH GLYCOGEN IN THE PRESENCE OR ABSENCE OF AMP AND NaF, AND ASSAYED IN THE DIRECTION OF SYNTHESIS Additions
( Zoncentration k-4
Additions
to reaction mixture
None
None Glucose Glucose-6-P P-enolpyruvate Fructose-l,BP2 ADPglucose
2.5 5.0 1.5 3.0 1.5 3.0 1.5 3.0 0.5 1.5
-
AMP”
AMP t NaFb
1.98 1.92 1.87 1.88 1.81 2.35 2.39 2.01 2.00 1.74 1.37
4.41 4.14 3.20 3.72 3.57 4.92 4.96 4.46 4.22 3.13 1.39
0.54” 0.51 0.51 0.48 0.47 0.61 0.72 0.54 0.56 0.57 1.46
a AMP concentration is 1.0 mM. b NaF concentration is 0.1 M. c nmoles of [14C]glucose incorporation glycogen/30 min.
into
HAMILTON
latter compounds, inhibition by glucose was reduced (to 7 %), while the activation by P-enolpyruvate was increased significantly (56-187 %). E$ect of nucleosides and nucleotides. As demonstrated in Table I and Fig. 3, the purified S. saliva&s glycogen phosphorylase was activated by AMP and this activation was further augmented by NaF. To test whether AMP could be replaced by other adenine and guanine nucleosides and nucleotides, the effect of these compounds was examined in the presence and absence of AMP and/or NaF. When assayed in the direction of synthesis in the absence of AMP and NaF, the enzyme was activated by adenosine (24%), ATP (34%), GMP (43 %), cyclic AMP (91%) and ADP (195%), and was TABLE
Concentration bd
Additions
had no effect on enzyme activity. When the effect of t’hese compounds was tested in the presence of AMP, or AMP + NaF, all of these substances, except P-enolpyruvate and fructose-l, 6-Pz , inhibited the enzyme 368 %; inhibition was most severe with ADPglucose. Under these conditions, P-enolpyruvate again activated the enzyme 12 and 20 % at concentrations of 1.5 and 3.0 m&II, respectively. Fructose-l, 6-Pz again showed no effect. The above compounds had a somewhat different effect on the enzyme when assayed with glycogen in the direction of phosphorolysis. Under these conditions, and in the absence of AMP or NaF, the enzyme was inhibited by glucose (15-23 %) and ADPglucose (38-53 %), was unaffected by glucose-6-P, and was activated by P-enolpyruvate (11-56 %) and fructose-l, B-P2 (3-24 %) (Table IV). With the exception of glucose and P-enolpyruvate, similar results were obtained when the assays were carried out with NaF (0.1 AX) and AMP (2 mM). With these
IV
EFFECT OFVARIOUS METABOLITES ON THE ACTIVITY OF PURIFIED GLYCOGEN PHOSPHORYLASE FROMS. salivarius WHEN ASSAYEDWITHOYSTER GLYCOCJEN IN THE DIRECTION OFPHOSPHOROLYSIS IN THE PRESENCE AND ABSENCE OF AMP AND NAF~
Glucose-B-P P-enolpyruvate Fructose-l,
6-PZ
ADP-glucose
ATP
-
-
None Glucose
Cyclic
Additions to reaction mixture
AMP
2.5 5.0 1.5 3.0 1.5 3.0 1.5 3.0 0.5 1.5 0.5 1.0 2.0 3.0 4.0 1.25 5.0
-
None
AMP + NaFY
0.93* 0.72 0.79 0.91 0.90 1.04 1.46 0.96 1.16 0.67 0.44 0.76 0.69 0.76 0.76 1.13 0.94 1.67
1.31 1.33 1.22 1.41 1.36 3.13 3.78 1.48 1.62 1.07 0.73 1.36 1.34 1.37 1.16 1.17 1.49 2.52
5 AMP and NaF concentrations were 2.0 mM and 0.1 M, respectively. b nmoles of glucose-l-P formed/30 min.
Streptococcus
slightly inhihitcd by GTP and GDP (S %) (Table V). The activation of the enzyme by these compounds, hojvever, was less than the act.ivation by the same concent,ration of AMP (270 %). When AMP was a component of the reaction mixture, these compounds either did not show any effect or were slightly inhibitory (max 26 %). Similar result,s \vere obtained with the enzyme incubated with AMP and NaF, although slight activation of the enzyme was observed with adrnosine (12%). In the direction of phosphorolysis and in the absence of AMP and NaF, cyclic AMP activated the enzyme 21% at a concentration of 4 rnN[, but was inhibitory (to 26 %) at lower concenbrations (Table IV). In the presence of 2 ml,1 AMP and 0.1 M NaF, this cyclic nucleotide was slightly inhibitory above a concentration of 2 rn)I. ATP, on the other hand, activated the enzyme 79% at the 5 mnI level and this activation was increased to 97 % by the presence of AMP and XaF. This activation was not reversed by sat,urating levels of Mg*+ ions. TABLE
V
EFFECT OF THE NUCLEOSIDES AND NUCLEOTIDES (1 mM) OF ADENINE .&ND GU~NINE ON THE ACTIVITY OF PURIFIED GLYCOGEN PHOSPHORYLASE FROM S. salivarius INCUBATED WITH GLYCOGEN IN THE PRESENCE OR ABSENCE OF AMP AND NaF, AND ASSAYED IN THE DIRECTION OF SYNTHESIS Additions
Additions None
to reaction AMP”
mixture AMP + NaFb
None
0.14c
0.52
1.10
ATP ADP Cyclic AMP Adenosine
0.19 0.42 0.27 0.18
0.39 0.45 0.51 0.48
1.14 1.14 1.09 1.23
GTP GDP GMP Guanosine
0.13 0.13 0.20 0.15
0.45 0.44 0.49 0.49
1.07 0.96 1.04 1.11
a AMP concentration is 1.0 mM. b NaF concentration is 0.1 M. c nmoles of [W Jglucose incorpora.tion glycogen/30 min.
311
sulivarizis
I FIG. AMP(.4) purified incubated
3 AMP(mM)
5
I Cyclic
3
5
AM? (mM)
0. Effect of varying concentrations and cyclic AMP(B) on the activity S. salivu~i~ts glycogen phoaphorylase with the other component and SaF.
of of
The relationship between thr AMP and cyclic AMP concentrations on the activity of purified S. salivarius glycogen phosphorylase in the dir&ion of svnthesis is illustrated more completely in Fig. 6. It en11 1~ seen that, as a function of t#he AMP concentration (Fig. GA), 1 mi\l cyclic AMP had no additional effect on the enzyme compnrrtl to the activity with AMP alone (i.r.. control). Cyclic AMP, as seen in Table T‘, activated t,he enzyme only in the absence of AMP. Furthermore, 5 mRL cyclic AJIP was inhibitory, albeit, slightly. This inhibition is more clearly observed \yhcn the values are plott8cd as a function of the cyclic AMP concentration (Fig. GH). In the presence of 1 and 5 m&I AMP, increasing levels of q-clic ARIP resulted in a slight but steady decline in activit.y, which, under these conditions, was &ill greater than tho act,ivit!- with cyclic ARIP alone. Tha influence of XaF on varying levels of both AMP and cyclic AMP should also be noted. The combination of AMP + NaF produced twice the activation of the S. salivarius phosphorylase, and at. :I Iolver AlIP level (Fig. GA), than did the combination of cyclic ARlP + XaF (Fig. AB). DISCUSSION
into
The activity of glycogen phosphorylase in animal systems is controlled by (a) the conversion of the AMP-independent ‘iactive” form to the AMP-dependent “inactive” form and (1,) by the activation of this 1at.k form by A3IP (18). As mcnt.ioned previously, the
312
SPEARMAN,
KHANDELWAL
only report of int’crconvertible forms of glycogen phosphorylase in microorganisms has come from the eukaryotic fungus, N. crassa (IO). Other microbial eukaryotic forms (5, 11, 12), as well as all of the prokaryotic organisms reported to date (6, 9) appear to contain phosphorylases which are active in the absence of AMP, but which may or may not be activated by this nucleotide. All attempt,s to demonstrate two forms of glycogen phosphorylase in S. saliva&s, similar to that in mammalian systems, were unsuccessful (unpublished results) which, along with the previous studies mentioned above, suggests that “sophisticated” interconvertible phosphorylases probably do not exist in prokaryotic cells or that the conversion between two forms has different regulatory properties than mammalian systems. The combined activation of purified S. salivarius glucogen phosphorylase by AMP and NaF (Table I) suggests a complex mechanism of enzyme action. The fact that the K, values for AMP and NaF were decreased in the presence of the other compound, as well as the much greater activation of the enzyme when AMP and NaF were combined, indicates cooperative effects either between separate AMP and NaF binding &es, or at a single site binding both compounds. The K, values for glucose-l-p and glycogen were not altered by AMP, or A?tIP + NaF (Figs. 4 and 5), which is similar t’o the results obtained with glycogen phosphorylase from N. crassa (7), but in direct contrast to the evidence obtained with the E. coli enzyme (6). In this latter case, 5 mnr AMP increased the K, for glucose-l-P from 1 rnI\r to 2.9 rnM, while decreasing the K, for glycogen from 6.7 mg/ml to 4.6 mg/ml. The effects observed with NaF arc difficult to extrapolate to physiological conditions where this compound at low concentrations will act, as an inhibitor of glucose metabolism by S. saliva&s (17, 18). A number of salts which may exist in vivo produce activating effects similar to those produced by KaF. For example, the purified S. salivarius glycogen phosphorylase was activated by the sulfate salts of sodium and ammonium (0.1 ~lr) in the presence of AXIP to the same extent
AND
HAMILTOX
(i.e., four- to sixfold) as observed with XaF. Similar results have also been observed with the E. coli enzyme (6). The absence of a pronounced effect of cyclic AMP on the S. salivarim glycogen phosphorylase activity clearly separates this enzyme from t.hose functioning in animal cells (19). While cyclic AMP could be shown to activate the S. salivarius enzyme in the absence of AMP (Table IV and Fig. 6), the presence of AMP modified this effect as it did for the other nuclcosides and nucleotides tested (Table V). Thus, tho activation of the purified S. salivarim phosphorylase by ARIP probably constitutes one of the major controlling mechanisms in this organism for the degradation of glycogen. Consequent,ly, in a cell with a low ‘Lencrgy charge” (i.e., high ADP + AMP) (20), phosphorylase and glycogen degradation would be activated, while the synthesis of glycogen would be decreased through the inhibition of ADP-glucose pyrophosphorylase by these nuclcotidcs (21). Furthermore, the activating effect of AMP would be augmented under these conditions by P-enolpyruvate, which has been shown to stimulate enzyme activit,y when assayed in both directions (Tables III and IV). Since glucose is transported into cells of S. salivarius via the P-enolpyruvate phosphotransferase system, the cellular concentration of P-enolpyruvatc is low during glucose metabolism but increases dramatically after the exhaustion of this exogenous carbon source as the organism approaches a condition of low energy charge (22). The increased level of this metabolite would then serve to activate glycogen phosphorylase to ensure the continued flow of carbon through the glycolytic pathway. On the other hand, under conditions of exogenous glucose metabolism when the energy chargo is high, the cttllular concentrations of AMP and P-enolpyruvatn would be low but the level of ADP-glucose would be high since glycogen synt.hesis is proceeding at a rapid rate (14). The elevat’ed level of this metabolite in the cell would consequently result in the inhibition of glycogen degradation through it’s action on glycogcn phosphorylase. Less readily understood in this situation, however, is the role played by AT!?,
14.
I~.GVIII,TON,
1.
II.
J.
A.,
(1!)1i8)
,I. illicmbiol.
Can.
14, (i5.
1.
~IiTHEILLAh‘l~,
1’:.
w.,
:\S,>
~~OI,lSCJS,
2.
Diabclcs 18, 797. BUTCHER, 11. W., llour~~s, .J. G., AXD ~uTEIEItl,.lN~,
cd;.
.4.
(1969)
.2dzlan.
3.
s.
I~TTOM,
H.UIUM.\X,
15.
vc’.
I<.,
h’UW,
J. I’.
I).
(19X))
Llr/van.
.lX\Iu
fJIcKl!!S-
E’ntymc
VILLM-P.\LASI,
5.
S~~IWARTZ,
.lnn. ,I. 6.
CH~;N,
C.,
IZev.
Biochem. M.,
C.
39,
IAI~NLI:,
J.
fi39.
HOE’STJNG, br. (lN7j
.\SU
(1970)
EM.
2, 132.
Kiochern.
i2ioc~hcm.
.lsu
p.,
.ISD
8iophy.s.
SI,;GP:I,,
I.
127,
175.
1-T.
(I%%)
.lrch.
~~OBISON.
KIIANI~I~:LWAL,
I.
11. 144, 5YCi. G.
S~.WIII~:I~L~ND,
A.,
(1971)
It.
,lrch.
~C.TcIII:II,
1L.
1%;.W. (1968) dnn.
20.
chcrn. 37, 11!). ATICINBON, I). W. (1968)
21.
Pn~,:rss,
Regul.
8, 205. 4.
Biophys. 19.
I$.,
KZN.UX.I, IhtlII,TON,
(l!)(i8)
Reqd. 6, 357.
Enzyrnc
h~.ZPER,
18. G. A.,
Biochernisfr~
L., \SD f3iochcub. w.,
.\Nl,
h’cv. Nio7, 4030.
J. (1969) in Clurcrlt Topics of Cellular IIegrdation (EIorwker, U. I,., and 15. IL Slatltman, etls), Vol. 1, p. 125, Academic Press, Xcw York. 22. K.INMSI, J. A., ANL, HAMILTON, I. Ii. (1971) ilrch. Uiochem. Hiophys. 146, 167.