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Structural changes in microsomal suspensions
.~CHIVES
OF
BIOCHEMISTRY
Structural
AND
BIOPHYSICS
Changes
118,
Department
of Pharmacology,
State
(1967)
in Microsomal
V. Interactions JOSEPH
649-658
with
Suspensions
Nucleotides’
D. ROBINSON
tTniversity
of Sew York, I’ork 13210
Received
September
Upstate
Xedical
Center,
Syracuse,
New
26, 1966
Brain microsomes incubated with 3 mM ATP or cytidine triphosphate (CTP) exhibited diminished changes in light scattering during incubation in a hypotonic medillm; this difference was abolished on subsequent addition of the sulfhydryl reagent, p-chloromercuribenzoate (pCMB). Incubation with 3 mM ADP had little efYect on the spontaneous changes, but significantly increased changes in light scattering induced by pCMB, thereby demonstrating a second type of structural change. Preincubation of the microsomes with pCMB prevented all these changes, and greatly reduced the binding of ADP to the membranes. These two types of changes in light scattering could be correlated with changes in permeability to sucrose: ATP, but not ADP, decreased sucrose permeability; on the other hand, preincubation with ADP, but not ATP, increased sucrose permeability during subsequent incubation with pCMB. ADP, and to a lesser extent ATP, reduced the content of reactive sulfhydryl groups in the microsomal preparation, suggesting that the structural changes involved a nucleotide-membrane-sulfhydryl system in which ADP reduced the content of reactive sulfhydryl groups while sensitizing the system to sulfhydryl reagents. Possible relevance to the transport ATPase is suggested in which the substrate of the enzyme, ATP, induces one structural change, whereas ADP, a product, induces a second structural state ill the enzyme-membrane complex.
taneous changes in turbidity that occurred during incubation in hypotonic media, whereas the subsequent additions of a sulfhydryl reagent, pCMB, abolished the difference in turbidity changes. The present communication is concerned with the specificity of this process and with the interrelationship between structural changes, as indicated by alterations in light scattering, membrane sulfhydryl groups, and membrane permeability. Recently, models for the membrane sodium pump, linked to an ATPase, have been proposed (4) in which cation transport is driven by changes in membrane conformat’ion. Evidence presented here indicates that ATP specifically induced one structural state -
A microsomal preparation isolated from brain provides a model membrane system with which alOerations in structural organizat’ion may be observed (l-3). Changes in turbidity of microsomes suspended in various media may be correlated with alterations in chemical composition and can provide information on permeability, conformation, and structural st’ate. In a previous study (3) it was found that ATP2 diminished the sponrat
1 This work was supported by U.S. Public Health Service grant NB-5430-02. 2 Abbreviations used: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP adenosine triphosphate; ATPase, adenosine, triphosphatase; CTP, cytidine triphosphate; DTNB, 5,5’-dithiobisnitrobenzoate; EDTA, ethylenediaminetetraacetate; GTP, g~lanosine triphosphate; ITP, inosine triphosphate; P, inorganic orthophosphate ; PP, pyrophosphat e;
pCMB, nucleic 649
p-chloromercnribenzoate; acid; and UTP, uridine
RNA, t,riphosphate.
ribo-
650
ROBINSON
in the membrane complex, as shown by alterations in optical density and 90” light scattering, and by diminished permeability to sucrose. On the other hand, ADP induced a second distinct structural state in the membrane, as shown by the same parameters. Thus the sub&ate for the enzymic reaction, ATP, induced one structural state in the enzyme-membrane complex, whereas a product of the reaction, ADP, induced a second st’at’e. METHODS Microsomes were prepared from rat brains as previously described (1). The standard incubation medium (1) contained, per milliliter, 0.1 ml of the microsomal stock suspension (about 1 mg of protein in 0.25 1~ sucrose) and 50 &moles of Tris-Cl, pH 7.3. Measurement of structural changes. Changes in structural organization in the microsomal suspension were followed during incubation by two related optical methods. With the first, turbidity changes were measured on microsomes incubated in spectrophotometer cuvettes by recording changes in optical density at 520 mp at intervals of l-6 minutes (1, 3). The effects on turbidity changes resulting from additions to the standard incubation mixture were compared in duplicate with controls incubated concurrently. Data are expressed both as absolute changes and as percentages of the changes o:curring in the controls incubated simultaneously. Witch the second method, the amount of light scattered at 90” to the incident beam was measured on an AmincoBowman spectrophotofluorometer. The wavelengt,h of the incident and of the scattered light was 520 rnM. In these experiments measuring 90” light, scattering, control and experimental incuba.tions, identical to those used in the turbidity were observed sequentially in measurement)s, randomized order. Chemical determinations. Protein was measured by the billret procedure and RNA by the orcinol method, as previously cited (5). The content of reactive sulfhydryl groups was measured with DTNB (1); the reagent was standardized with fresh crystalline glutathione. During these experiments a gross arithmetic error was discovered in the earlier calibration of DTNB (1); values reported here represent experiments yielding equivalent spectrophotometric changes correctly calculated. Nucleotidase activity was estimated by measuring the liberation of inorganic phosphate (6); values were corrected for any release of inorganic phosphate during the assay procedure.
Sucrose perrtreahilil~y, Challges ill perrncabilil~ of the microsomal membrane to PIICIYJS~ were recorded by measuring t,he space availahle to sucrose-l%, as previously described (3). ,liicrosomes were incltbated at 30” with sucrose-14C, glycerolJ%, or dextran-carboxyl-‘C; after incubation the microsomes were collected by centrifugation for 30 minutes at 80,000 g, and the radioactivit,y in the pellet and in the supernatant material was measured (3). The ratio of the intramicrosomal space accessible t,o sucrose to the total intramicrosomal space [calculated as described previously (3)] gave an indication of the permeabilit,y of the vesicldar membrane to sucrose. Binding of ADP. To measure binding of ADP to the microsomes, ADP+Y%, 30-33 mC/mmole, 97-99% ADP (Schwarz BioResearch Corp.) was used. About 0.25 pC (8 mpmoles) of ADPJrC was added to the standard incubation mixture, scaled to a final volume of 4.0 ml. In some experiments the microsomes were preincubat,ed at, 30” for 5 minut,es, with or without pCMB, before addition of ADPJ4C. After incubation at 30”, the reaction was stopped by centrifugation for 30 minutes at 80,000 g. To calculate the amount of ADP bound to the microsomes, the total radioactivity in the pellet and in the supernatant material was measured (3). The value for the supernatant material was taken as the total amount added to the incubation medium. To correct for ADP trapped in the int,erstices of the pellet, the extramicrosomal volume of the pellet was calculated in a similar fashion by measuring the radioactivity present in the pellet and in the supernatant material after incubations, in parallel and under the same conditions, with dextran-carboxylJ4C (3). The quantity of ADP bound was calculated with the formula: ( V’AIAU~-TIT)EX) C~np = ADP bound (,umoles), where V
ADPX ADP = ADPJ4C
v DEX C*op
in pellet in medium
DextranJ%
(cpm) (cpm/ml)
in pellet
= Dextran-14C
in medium
= concentration ml).
of ADP
(cpm) (cpm/ml) in medium
&moles/
Materials. Unlabeled nucleotides were purchased as sodium salts from Sigma Chemical Company, and were passed through a Chelex-100 chelating resin in the sodium form before use (3). Statistics. Data are presented, where approwith standard deviations. Statistical priate, significance was calculated with the t-test for paired observations; a value for p less than .05 was considered the criterion for significance.
RESULTS
Effects of nude&ides on turbidity changes. During 4%minute incubations in t,he st’andard hypotonic medium the turbidity of microsomal suspensions decreased 5.8 ‘i: (Fig. 1; Table I). The addition of 3 m&I ATP or CT!? diminished t,his change in turbidity by half. No other mwleotides tested had a .90
r 20
40
60
MINUTES
FIG. 1. Etfects of nucleotides on tlwbidity changes. The control cuvette contained 0.1 ml of the microsomal stock suspension and 50 @moles of Tris-Cl, pH 7.3, in 1.0 ml. The other cuvettes contained in addition, as indicated, 3 rnM ATP or ADP. At the arrow pCMB was added to a final concentration of 0.1 miw.
significant, effect on the turbidity changes. In wmparison with control incubations, turbidity changes in media containing 3 mxr EDTA were diminished by only one-fifth. Interaction with sulfhydql l’eagents. Wllen 0.1 rnRr pCMB was added t’o the standard medium after a 45minute incubation, it, induced a furt,her decrease in turbidity. In incubations that) included 3 ~RI ATP, ADP, or CTP, this decrease in turbidity MU augmented significantly (Fig. 1; Table I). The greatest turbidit,y change with pC1\IB occurred in media containing ADP (significant,ly greater than with ATP or C’TP). Analogous results were obtained when 3 ml1 N-ethylmaleimide or 0.2 ml\r DTSB w:w substit’ut#ed for pC%IB. I’veincubation experiments. Since this drop in turbidit,y following the late addit,ion of pCi\IB l-o media containing ATP and CTP might represent, merely the abolition of t#he diminished turbidity changes induced by these nucleot’ides (Fig. I), experiments were performed in which short,er preincubaGon periods were used. Preincubat#ion of the microsomes with various nucleotides for 3 minutes before adding pCMB caused significant changes in turbidit,y, both at 10 and at, 45 minutes, only with ADP (Fig. %A; Table II). Turbidity changes in media containing XTP, CTP, or the &her nucleotides were not
TABLE EFFECTS
OF NUCLEOTIDES
I ON TERBIDITY
Spontaneous decrease in turbidity Addition
7’ of change in optical density
in 45 min
% of change from control
None
5.8
f
.7
-
ATP, 3 mu ADP, 3 mM AMP, 3 mM CTP, 3 rnM GTP, 3 m&f ITP, 3 mM UTP, 3mhx PP, 3 mM P, 3 rnM
3.2 4.9 5.4 3.3 4.F 4.9 4.9 5.8 5.7
f f f f f f rk f. f
.G .9 1.0 .7 .8 .7 .8 .7 .7
55 f 7 85 f 8 93 f 9 56 & G 81 & 9 84f8 SD f 9 97 f 8 100 f 7
a Turbidity changes were observed in suspensions of microsomes (50 mM Tris-Cl, pH 7.3, and about 1 mg/ml of the microsomal stock cated; after 45 minutes, pCMB was added to a final concentration bidity changes from 45 to G5 minutes were observed.
CH.\NGES* pCMB-induced
decrease in turbidity
7, of change in optical density f
.6
6.G f 7.0 f 5.9 rf 5.4 f 4.4 zt 3.7 f 4.2 f 4.7 f 4.9 f
A.3
.T .8 .9 .7 .7 .A .6 .8 .8
incubated suspension) of 0.1 mM,
% of change from control 154 zk 10 165 zk 7 117 f 11 12s* 8 101 f 8 88& 9 98f 7 108 & 9 1lBzt 9
in the standard medium with additions as indiand t.he subsequent tur-
652
ROBINSON
B
ATP ntrol ADP
20
40
20 MIN
40
UTES
Fro. 2. Effects of preincubation on nucleot’ide-microsome-pCMB interactions. In panel A, microsomes were preincubated for 5 minutes in the control medium, with or without added nucleotides as indicated. At zero time 0.1 mM pCMB was added to all cuvettes and the subsequent changes in optical density were recorded. In panel B, microsomes were preincubated for 5 minutes in the control medium which contained, in all cases, 0.1 rnbx pCMB; at zero time nncleotides were added, as indicated, to the cuvettes. TABLE EFFECTS
OF PREINCUBATION
WITH
II
NUCLEOTIDES
ON pCMB-INDUCED
Turbidity y. of change in optical density
ATP, ADP, AMP, CTP, CTP, ITP, UTP,
3 mw 3 mM 3 mM 3 mrvr 3 mM 3 mM 3 mM
5.8 5.7 6.8 5.7 4.2 4.6 4.2 4.1
f f f f f f f f
.6 .7 .4 .9 .9 .7 .8 .7
at % of change from control
100 f 117 f 97 * 72 f 79* 7G+ 7Gxt
8 G 11 12 9 9 8
a Microsomes were preincubated for 5 minutes in the standard nucleotide listed; then, @MB was added to a final concentration were observed during the 45-minute incubation period.
increased above controls preincubated without any nucleotide. However, preincubation of the microsomes for 5 minutes with 0.1 mM pCMB before adding the nucleotides resulted in no significant increase in turbidity changes with any nucleotide at either time (Fig. 2B; Table III). Experiments measuring light scattering at 90’. Although the preceding experiments provided statistically significant data indi-
CHANGES”
changes during incubation
at 10 min
Addition
None
TURBIDITY
Y. of change in optical density 8.4 8.3 10.0 7.5 8.0 8.1 7.0 7.6
f f f f f f f f
.8 .8 1.0 1.1 1.0 .9 1.1 .9
45
min 70 of change from control 99f 120f 92 f 96 f 96f 87 f 93f
8 G 11 11 9 10 8
medium containing in addition the of 0.1 m&r, and the turbidity changes
eating specific changes with ATP (and CTP) and with ADP, the measured changes were quite small. By measuring light scattering at 90”, an analogous but inherently more sensitive method, the preceding experiments were confirmed. Thus ATP, but not ADP, significantly decreased changes in light scattering during incubation in the standard medium (Fig. 3A) ; and after preincubation for 5 minutes before addition of pCMB, ADP, but not ATP, significantly increased the drop
TABLE: EFFECW
OF PREINCURAPION
WITH
pCMB
III
ox
NUCLEO.~IDE-INDUCED
Turbidity Addition
AT!?, ADP, ilMP, CTP, GTP, ITP, UT!?,
3 rnM 3 mM 3 rnbx 3 mM 3 mM 3 mM 3 m&f
a hiicrosomes were tionallg, in all cases, were observed during
during
at 10 min
-$ of change in optical density
None
changes
4.8
3~ 1.7
4.8 4.4 4.5 4.2 4.7 5.0 4.6
f f zt 4~ i f f
7” of change in optical density
-
ti.8
99f 91f 93* 88f cJ(i f 105 f 98 rk
CHINGEF
at 45 min
5:;. of change with control
1.9 1.9 I.0 1.8 1.8 2.2 1.7
TURBIDITY incubation
7 7 ci 0 li 10 0
preincubated for 5 minutes in the st,andard 0.1 rn>f pChlB; then the nucleotide listed the 45-minute incubation period.
It
2.3
0.7 f 0.0 * 5.6 f (i.2 f 0.5 f 7.1 * 6.7 i
2.2 2.4 2.5 2.1 2.2 2.6 2.1
incubation was added
“& of change from control
99 98i 84f 93f 96 105 102
medium and the
zt
7 8 9 7 f ti zkz 13 & 6
containing sdditurbidity changes
90r
A
B 60 -
60
5
IO
15
20
25
MINUTES
&a. 3. Changes in light scattering induced by nucleotides. Light scattering at 90” was measured at a wavelength of 520 rnp with a spectrofluorometer. In panel A, microsomes were incubated in the control medium, with or without 3 mM ADP or ATP as indicated. In panel B, microsomes were preincubated for 5 minutes in the control medium \vhich con tained, in all cases, 0.1 mM pCMB; at zero time nucleotides were added as indicated, and the subsequent changes in light scattering were measured.
in light scattering (Fig. 33). The relat,ive magnitudes of t’he induced changes measured by light scattering and of those measured by optical density are not directly comparable. This is due, in part, to the light scattering data being recorded on a scale of transmitted light’, which is logarithmically,
not linearly, related to the optical density scale. Changes in membrane permeability. Permeability to sucrose, as measured by the ratio of the intramicrosomal space accessible to sucrose-14C to t’he intramicrosomal space accessible to glycerol-14C was determined after
654
ROB
incubation in the standard medium (3). The addition of 3 mM ATP, but not ITP or ADP, significantly decreased sucrose permeability (Table IV). When the microsomes were preincubat’ed for 6 minutes, with or without nucleotides, and then incubated for 45 minutes with 0.1 mM pCMB, 3 mM ADP, but not ATP, increased sucrose permeability (Table V). ZQect of pCMB on the binding of ADP. Data are presented asdescribed under Methods, for the volume of supernatant material containing as much labeled ADP (or dextran) as in the pellet, per gram of microsoma1 protein incubated. Subtracting the volume for dextran from that for ADP eliminated t.he volume of ADP trapped in the extramicrosomal space; the remainder represents the volume of supernatant material containing as much ADP as that’ bound to the pellet. Such calculations showed that preincubat’ion of the microsomes for 5 minut’es with 0.1 mnz pCMB reduced the binding of ADPJ4C during a subsequent 5-minute incubation, to 24 % of that in controls preincubated without pCMB (Table VI). When unlabeled ADP, 3 mM, was added together with ADP-14C; the amount of ADP-14C bound was reduced to 4 % of that bound in the absenceof unlabeled ADP; however, bhe actual dilution of ADP-14C was 15OO:l. In TABLE EFFECTS
OF
IV
NUCLEOTIDES
ON
SUCROSE
PERMEABILITY~ Addition
None ATP, ITP, ADP,
3 mM 3 mM 3 w
Ratio: sucrose space/ glycerol space
.36 .22 .31 .35
f f f f
.03 .04 .05 .03
Change control
from (70)
60 f 84*7 96 f
7 6
a Microsomes were incubated for 60 minutes at 30” in the standard medium, with additions as noted, in the presence of sucrose-W, glycerol-*4C, or dextran-carboxyl-1% (3). After centrifugation, radioactivity in pellet and supernatant material was measured, and spaces available to sucrose and to glycerol (both corrected for the dextran space) were calculated (3). In each experiment controls were incubated together with nucleotide-containing tubes.
INSON TABLE
V
EFFECTS OF PREINCUBATIOX WITH ON pCMB-INDUCED CH.~NGES
NUCLEOTIDE~ IN SFCROSE
PERMEABILITYO Addition
None ATP, ADP,
3 mM 3 mM
Ratio: sucmse space/ glycerol space
.79 z!z .05 .77 I!I .07 .92 f .05
Change control
97 f 116 f
from (70)
6 5
a Microsomes were preincubated for 5 minutes at 30” in the control medium, with additions as noted; at zero time 0.1 mM pCMB was added to all tubes, and they were then incubated for 45 minutes. Determination of sucrose and glycerol spaces was as in Table IV.
these experiments with 3 mM ADP, preincubation with pCMB caused no significant. change in binding of ADP-14C. On the other hand, when unlabeled AMP, 3 mM, was added together with ADP-14C, the amount of ADPJ4C bound was reduced only one-third; preincubation wit’h pCMB reduced the binding in these experiments 76 %. When the microsomeswere incubated with ADP-14C for the full 10 minutes (without preincubation), the amount of bound ADP14Cwas 88 % of that for the j-minute incubat,ion (Table VII). Addition of pCMB halfway through a lo-minute incubation reduced the amount of ADP bound only by half (Table VII). Content of reactive su(fhydryl groups. The content of reactive sulfhydryl groups, as measured by DTNB, was diminished in incubations with ATP (90 f 2 % of control) and ADP (77 f 4 % of control) (Fig. 4). EDTA, 3 mM, only reduced the sulfhydryl content 4 %.. Dissolution oj miwosomal structure during incubation. When the microsomal mixture was centrifuged for 30 minutes at 80,OOOg after incubation in the standard medium for one hour at 30”, and the resultant pellet was analyzed in comparison with unincubated microsomes, t’here was little loss in sedimentable protein and about a 20% loss in sedimentable RNA (Table VIII), in accord with earlier experiments (1). Addition of 0.1 mM pCMB to the medium had no effect on protein content, but doubled the loss of RNA. Neit,her ATP, ADP, nor EDTA altered t’he
NUCLEOTIDES
AND
BRAIN
TABLE EFFECT
OF PREIXCGBATION PrW3Ke of pCMB
Addition mith ADP-“C None None
+
ADP, 3 rnM
-
ADP,
3 rnM
+
AMP, AMP,
3 mM 3 mM
+
WITH
Equivalent Dextran 2.28 1.90 2.28 1.92 2.42
* i f * i
1.98 f
VI
pCMB
ON THE BINDIXG
volumes (ml/gram space
protein)
39.3 10.8 3.64 3.33 29.0 8.33
0.20
f i f i * *
OF ADP-10 Effect of pCMB (ADP bound as % bound without pCMB)
ADP bound (pmoles/gm protein)
ADP space
0.15 0.18 0.17 0.18 0.18
635
MICROSOMES
2.1 0.9 0.47 0.52 1.7 0.82
74 1s 2.7 2.8 53 13
f * f f i *
9 2 0.5 0.3 4 1
24f
4
105 *
17
24%
2
a In all experiments before addition of nucleotides t,he microsomes were preincubated for 5 minutes at 30°C with or without 0.1 mM pCMB. The mixtnre was then incubated for 5 minutes and the reaction was st.opped by centrifl\gation. Data were calculated as described under Methods.
EFFECTS
OF PREINCUBATION Equivalent
Addition after preincubation
Dextran
None
pCMB,
WITH
2.16 1.83
0.1 mM
f f
TABLE VII ADP ON PCMB-INDUCED
volumes (ml/gm protein)
space
34.4 17.8
f *
IN BINDINGS Effect of pCMB (ADP bound as 70 bound without pCMB)
ADP bound (Irmoles/gm protein)
ADP space
.19 .21
CHANGES
1.8 1.1
ti4 i 32 f
7 4
5054
a In all experiments the microsomes were preincubated for 5 minutes at 30% with ADP-1%. 0.1 mM pCMB was added to half the tubes and all were incubated for 5 minutes. TABLE CHIIIGGES
IN
VIII
PROTEIN :\ND RNA AFTER INCUBATION
Additions
CONTENT
Content in microsomes after incubation as % of that in unincubated stock suspension Protein
L U) a3 0 E i
None
pCMB, 0.1 mM ATP, 3 mM ATP and pCMB
IO-
ADP,
5-
L
I
I
1
I
20
1
40
3 m&I
ADP and pCMB H&l, , 0.1 rnM EDTA,
3 mM
Then
96 94 96 95 96 90 97 95
f f f i f f + i
RNA 2 2 2 3 2 3 2 3
82f 04 79f 63 80f 03 78 77f
5 7 4 f 11 4 Z!Z 9 f 10 6
f
MINUTES FIG.
4. Content
of reactive
sulfhydryl
groups.
The
content of reactive sulfhydryl groups, as measured by DTNB (l), is plotted for microsomes incubated in the control medium and in media containing, in addition, 3 mM ATP or ADP.
protein neither induced
or RNA
content
significantly, and the pCMBloss in RISA (Table VIII). Nucleotidase activity. Since the rates of
ATP nor ADP altered
met’abolism of the nucleotides might be reflected in the nat’ure and durat8ion of the specific structural changes, especially during preincubation experiments, nucleotidase activity was determined. All the nucleoside t’riphosphates were catabolized by t’he microsomal preparation in the standard medium without additional divalent cation (Fig. .i). Inorganic phosphat’e production was most rapid with ATP and ITP, slowest
656
ROBIPiSOiX
(Fig. 3A). The spoutjaueous decrease in turbidity occurring in hypotonic media may be attribut,ed, at least in part, to osmotic swelling (1, 3, 5, 7) ; the diminution of t)his process by ATP suggests either (a) stjruct,ural alteration in the membrane resulting in a change in the selective permeability of t,he membrane (1, 3), (b) gross change in vesicular conformat,ion altering light scatt,ering, or 0c aggregation of the microsomal elements. Possibly the first two processesmight occur concomitantjly. In this context,, experiments demonst’rating t,hat ATP reduced the perme60 20 40 ability of the vesicular membrane to sucrose MINUTES (Table IV) support t’he interpretat,ion of the light scattering experiments as indicating an FIG. 5. Nucleotidase activity. The liberation of inorganic phosphate from various nucleotides is alteration in membrane permeability. Ko other nucleotide tested induced these plotted against the duration of incubation at 30”. Microsomes were incubated in the control medium changes in optical density, light, scatt’ering, containing 3 mM ATP (O--O); ITP (A- - -A); and sucrose permeability, except CTP CTP (*ml ; UTP (A---A); GTP (o- - -0) ; (Table I). The specificity for the two nucleoor ADP (IJ- - -0). Each point is the average of tides suggestsan interaction with the mem4 experiments. brane through both t#he B-amino group on the purine or pyrimidine ring and the triwith GTP and UTP; with CTP the rate phosphate complex. Specificit’y for ATP and was intermediate, but total phosphate pro- CTP, to the exclusion of other nucleot8ides, duction equaled that from ITP by one hour. has been noOedwith the myosin ATPase (8) Nearly half the added ATP (3 m;M) was and the microsomal salt-activated BLTPase catabolized in the standard medium by 45 (9). minutes. In the presenceof 3 rnM MgCl, the These findings differ sharply from studies initial rate was increased about S-fold, and wit’h liver microsomes in which all nucleonearly all the ATP was split by 45 minutes. side triphosphates, as well as EDTA and The microsomal preparations also cata- PP, induced a marked decreasein light scatlyzed the release of inorganic phosphate tering (10) t’hat was correlated with a loss in from ADP (Fig. 5). Phosphate liberation sediment,able RNA (5). The lack of nucleofrom ADP was diminished by monovalent tide specificity, the equivalent effects of cations, in marked contrast to ATP. EDTA and PP, and the ant,agonism by The initial rate of nucleotidase activity, Mg++ (10) indicat’ed that in t’he liver prepain all cases, was diminished about half in ration divalent cation chelation was inthe presence of 0.1 mM pCMB. volved (5, 10). By contrast, wit,h t.he brain preparation the effect’ of ATP was (a) to DISCUSSION diminish (not increase) the changes in light These experiments indicate that specific scattering (Fig. l), (b) was not’ antagonized nucleotides interact with the microsomes to by added MgClz (3), (c) was not reproduced alter the structural organization of the mem- by other nucleotides (except CTP) nor by brane fragments, as manifested by changes PP or EDTA (a more potent chelating in light scattering and in membrane permeability, and that these int’eractions may be agent), and (d) did not cause a loss in sedimentable RNA (Table VIII). A partial modified by sulfhydryl-binding compounds. explanation for this difference was the nearly Thus ATP significantly diminished the total absence of membrane-bound ribosomes spontaneous changes in optical density (Fig. 1; Table I) and in 90’ light scattering in the brain preparation, as shown in electron ATP
~~CI,IZOTIDES
AXD
micrographs (1~. Kleinfeld and J. D. Robinson, unpublished observations). A second distinct st,ructural alt,eration could bc induced by ADP, as shown by cxperiments in which microsomes were preincubaM with the nucleot,ide before incubat,ion with pCMB. After a s-minute preincubation with various nucleotidcs, during which lit’tle catabolism occurred (Fig. 5) and lit,tle cliff erencesin light, scattering could be not’cd (Figs. 1 and S), subsequent,incubation wit,h pCMB caused increased changes in opt,icnl density (Fig. 2; Table II) and in 90” light scattering (Fig. 3B) only with ADP. Such changes may be interpreted in terms of (a) alteration in premeabilit’y, (b) gross alt,eration in conformation, or (c) microsomnl dissolut,ion. Alt’hough pCMB did cause some dissolution, as indicated by loss of sediment!able RiSA (Table VIII), ADP did not induce further measurable lossesof prot,ein or RSA either alone or with pCMB (Table VIII). Finally, experiment,s demonstrating that preincubation with ADP increased the permeability of the vesicular membrane t)o sucrose during subsequent incubation wit,h pCMB (Table V), whereas preincubation w&h ATP did not, support the interpretation of t’he light’ scntt’ering experiment,s as indicating a second structural change. All these changes were intimately related to the st,at)e of the membrane sulfhydryl groups as shown by experiments with pCR4B (and confirmed wit’h N-ethylmaleimide and DTNB). Brief preincubation with pCMB prevented any changes in light, scat’tering by any nucleotide (Fig. 2; Table IIIj, and addition of pCMB to microsomesincubated wit’h ATP or CTP abolished the alterations in light, scat,tering induced by these nucleotides (I;ig. 1; Table I). Preincubat,ing the microsomcswith pCMF markedly reduced t)he binding of ADP-14C during subsequent incubation (Table VI), in accord with the observations that preincubatsion with pCMB blocked the effects of ADP on turbidity changes. The binding of labeled ADP appeared to be, in part, specific since unlabeled ADP almost eliminated t’he binding of ADP-14C, whereas unlabeled AMP had little effect (Table VI). The ap-
BRAIN
MICROSOMES
6.57
parent, binding of ADP-14C in the presence of a GOO-fold excessof unlabeled ADP may be a measure of t,he binding of labeled impurities in t,he preparat’ion rather than t#he binding of ADP-14C itself; this could explain why pChIB did not reduce t>he binding of radioact,ive material in experiments with 3 ml,1ADP. The preparat’ions of ADP-14C used cont,ained l-3 (7 labeled impurities; however, no differences between preparations were observed. II’urthermore, the effect of the act’ive nucleotides was not t,o increase the reactivity of t’he membrane sulfhydryl groups, but rather to reduce the cont’ent of reactive sulfhydryl groups (Fig. 4). ADP, which reduced sulfhydryl reactivity more, potentiated the effect,s of pClUB on light scat)tering and sucrose permeability. The sit)uation is reminiscent)of the interaction of nucleotides wit,h myosin: at low concentrat’ions of pCMR the myosin ATPase is activated for ATP and CTP (but nob for ITP), but at higher concentrations it is inhibited (8). The interaction with myosin has been attributed not to binding of nucleotides through sulfhydryl groups but t,o an allosteric effect on the protein (11, 12). A similar ADEmembranesulfhydryl system seems probable here, in which ADP renders a fraction of the sulfhydry1 groups unreactive while sensitizing a portion of the complex t’o sulfhydryl reagents. [For discussions of the changes in content of react)ive sulfhydryl groups during incubation in various media see references (I) and (2).] Since ATP (and CTP) induced one structural stat’e in the membrane syst)em while ADP induced a second distinct strurtural alteration, possible relevance t,o the membrane transport ATPase is suggested. Models for cation transport employing conformational changes in the membrane and involving t,he Na+-K+-stimulated ATPase have been proposed (4), and several interesting correlates exist, between t,he Nn+-K+-stimulated ATPase and the structural changes. The nucleotidase act,ivity present in many crude microsomal preparabions has a high act’ivity with ITP (e.g., Fig. 5), but exhibits salt,-stimulated activity only with ATP or CTP and not with ITP or other nucleobides
ROBINSON
(9) ; furthermore, the nucleotidase activity, which is sensitive to sulfhydryl reagents, may be protected by preincubation with ATP but not ITP (CTP was not tried) (13). If the nucleotide-induced changes were related to ATPase activity, then these changes might be modified by cations that alter ATPase activity. However, divalent and monovalent cations themselves induce changes in t,he light-scattering properties of the preparation, and it was impossible to demonstrate any specific effect on the ATPinduced turbidity changes with MgCl, , NaCl, KCI, NaCl + KCl, or ouabain (3). Quite recently experiments with the postmitochondrial supernate from crayfish nerve have been reported (14) which show that, in accord with the above experiments, ATP caused an increase in turbidity of the suspension. The authors interpret these findings in terms of an actomyosin-like protein within the axon membrane that, under the influence of ATP, controls permeability and impulse conduction. ACKNOWLEDGMENT The
Swiderski
proficient is gratefully
assistance
of Mr. acknowledged.
Frank
E.
REFERENCES 1. ROBIXSO;~, J. D., Arch. 110, 475 (1965). 2. ROBINSON, J. Il., Arch. 112, 170 (1965). 3. ROBINSON, J. I>., Arch. 113, 526 (1966). 4. OPIT, L. J., AND CHARNOCK, 471 (1965); (1966).
JIIRDETZKY,
Biochem.
Biophys.
Biochem.
Biophys.
Biochem.
Biophys.
J. S., Nature O.,
Lvature
211,
208, 969
5. ROBIXSON, J. Il., Arch. Biochem. Biophys. 106, 207 (1964). 6. LOWRY, 0. H., AND LOPEZ, J. A., J. Biol. Chem. 162, 421 (1946). 7. TEDESCHI, H., JAMES, J. M., AND ANTHONY, W., J. Cell Biol. 18, 503 (1963). 8. BLUM, J. J., Arch. Biochem. Biophys. 87, 104 (1960). 9. NAKAO, T., TASIXIMA, Y., NAGANO, K., AND NAKAO, M., Biochem. Biophys. Res. Commm. 19, 755 (1965). 10. PACKER, L., AND RAHMAX, M. M., Texas Repts. Biol. Med. 20, 414 (1962). 11. MORALES, M. F., AND HOTTA, K., J. Biol. Chem. 236, 1979 (1960). D. R., Science 149, 1374 (1965). 12. KOMINZ, 13. SKOU, J. C., .~ND HILBERG, C., B&him. Biophys. Acta 110, 359 (1965). 14. BOWLER, K., AND DUNCAN, C. J., Nature 211, 642 (1966).
OF
BIOCHEMISTRY
Structural
AND
BIOPHYSICS
Changes
118,
Department
of Pharmacology,
State
(1967)
in Microsomal
V. Interactions JOSEPH
649-658
with
Suspensions
Nucleotides’
D. ROBINSON
tTniversity
of Sew York, I’ork 13210
Received
September
Upstate
Xedical
Center,
Syracuse,
New
26, 1966
Brain microsomes incubated with 3 mM ATP or cytidine triphosphate (CTP) exhibited diminished changes in light scattering during incubation in a hypotonic medillm; this difference was abolished on subsequent addition of the sulfhydryl reagent, p-chloromercuribenzoate (pCMB). Incubation with 3 mM ADP had little efYect on the spontaneous changes, but significantly increased changes in light scattering induced by pCMB, thereby demonstrating a second type of structural change. Preincubation of the microsomes with pCMB prevented all these changes, and greatly reduced the binding of ADP to the membranes. These two types of changes in light scattering could be correlated with changes in permeability to sucrose: ATP, but not ADP, decreased sucrose permeability; on the other hand, preincubation with ADP, but not ATP, increased sucrose permeability during subsequent incubation with pCMB. ADP, and to a lesser extent ATP, reduced the content of reactive sulfhydryl groups in the microsomal preparation, suggesting that the structural changes involved a nucleotide-membrane-sulfhydryl system in which ADP reduced the content of reactive sulfhydryl groups while sensitizing the system to sulfhydryl reagents. Possible relevance to the transport ATPase is suggested in which the substrate of the enzyme, ATP, induces one structural change, whereas ADP, a product, induces a second structural state ill the enzyme-membrane complex.
taneous changes in turbidity that occurred during incubation in hypotonic media, whereas the subsequent additions of a sulfhydryl reagent, pCMB, abolished the difference in turbidity changes. The present communication is concerned with the specificity of this process and with the interrelationship between structural changes, as indicated by alterations in light scattering, membrane sulfhydryl groups, and membrane permeability. Recently, models for the membrane sodium pump, linked to an ATPase, have been proposed (4) in which cation transport is driven by changes in membrane conformat’ion. Evidence presented here indicates that ATP specifically induced one structural state -
A microsomal preparation isolated from brain provides a model membrane system with which alOerations in structural organizat’ion may be observed (l-3). Changes in turbidity of microsomes suspended in various media may be correlated with alterations in chemical composition and can provide information on permeability, conformation, and structural st’ate. In a previous study (3) it was found that ATP2 diminished the sponrat
1 This work was supported by U.S. Public Health Service grant NB-5430-02. 2 Abbreviations used: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP adenosine triphosphate; ATPase, adenosine, triphosphatase; CTP, cytidine triphosphate; DTNB, 5,5’-dithiobisnitrobenzoate; EDTA, ethylenediaminetetraacetate; GTP, g~lanosine triphosphate; ITP, inosine triphosphate; P, inorganic orthophosphate ; PP, pyrophosphat e;
pCMB, nucleic 649
p-chloromercnribenzoate; acid; and UTP, uridine
RNA, t,riphosphate.
ribo-
650
ROBINSON
in the membrane complex, as shown by alterations in optical density and 90” light scattering, and by diminished permeability to sucrose. On the other hand, ADP induced a second distinct structural state in the membrane, as shown by the same parameters. Thus the sub&ate for the enzymic reaction, ATP, induced one structural state in the enzyme-membrane complex, whereas a product of the reaction, ADP, induced a second st’at’e. METHODS Microsomes were prepared from rat brains as previously described (1). The standard incubation medium (1) contained, per milliliter, 0.1 ml of the microsomal stock suspension (about 1 mg of protein in 0.25 1~ sucrose) and 50 &moles of Tris-Cl, pH 7.3. Measurement of structural changes. Changes in structural organization in the microsomal suspension were followed during incubation by two related optical methods. With the first, turbidity changes were measured on microsomes incubated in spectrophotometer cuvettes by recording changes in optical density at 520 mp at intervals of l-6 minutes (1, 3). The effects on turbidity changes resulting from additions to the standard incubation mixture were compared in duplicate with controls incubated concurrently. Data are expressed both as absolute changes and as percentages of the changes o:curring in the controls incubated simultaneously. Witch the second method, the amount of light scattered at 90” to the incident beam was measured on an AmincoBowman spectrophotofluorometer. The wavelengt,h of the incident and of the scattered light was 520 rnM. In these experiments measuring 90” light, scattering, control and experimental incuba.tions, identical to those used in the turbidity were observed sequentially in measurement)s, randomized order. Chemical determinations. Protein was measured by the billret procedure and RNA by the orcinol method, as previously cited (5). The content of reactive sulfhydryl groups was measured with DTNB (1); the reagent was standardized with fresh crystalline glutathione. During these experiments a gross arithmetic error was discovered in the earlier calibration of DTNB (1); values reported here represent experiments yielding equivalent spectrophotometric changes correctly calculated. Nucleotidase activity was estimated by measuring the liberation of inorganic phosphate (6); values were corrected for any release of inorganic phosphate during the assay procedure.
Sucrose perrtreahilil~y, Challges ill perrncabilil~ of the microsomal membrane to PIICIYJS~ were recorded by measuring t,he space availahle to sucrose-l%, as previously described (3). ,liicrosomes were incltbated at 30” with sucrose-14C, glycerolJ%, or dextran-carboxyl-‘C; after incubation the microsomes were collected by centrifugation for 30 minutes at 80,000 g, and the radioactivit,y in the pellet and in the supernatant material was measured (3). The ratio of the intramicrosomal space accessible t,o sucrose to the total intramicrosomal space [calculated as described previously (3)] gave an indication of the permeabilit,y of the vesicldar membrane to sucrose. Binding of ADP. To measure binding of ADP to the microsomes, ADP+Y%, 30-33 mC/mmole, 97-99% ADP (Schwarz BioResearch Corp.) was used. About 0.25 pC (8 mpmoles) of ADPJrC was added to the standard incubation mixture, scaled to a final volume of 4.0 ml. In some experiments the microsomes were preincubat,ed at, 30” for 5 minut,es, with or without pCMB, before addition of ADPJ4C. After incubation at 30”, the reaction was stopped by centrifugation for 30 minutes at 80,000 g. To calculate the amount of ADP bound to the microsomes, the total radioactivity in the pellet and in the supernatant material was measured (3). The value for the supernatant material was taken as the total amount added to the incubation medium. To correct for ADP trapped in the int,erstices of the pellet, the extramicrosomal volume of the pellet was calculated in a similar fashion by measuring the radioactivity present in the pellet and in the supernatant material after incubations, in parallel and under the same conditions, with dextran-carboxylJ4C (3). The quantity of ADP bound was calculated with the formula: ( V’AIAU~-TIT)EX) C~np = ADP bound (,umoles), where V
ADPX ADP = ADPJ4C
v DEX C*op
in pellet in medium
DextranJ%
(cpm) (cpm/ml)
in pellet
= Dextran-14C
in medium
= concentration ml).
of ADP
(cpm) (cpm/ml) in medium
&moles/
Materials. Unlabeled nucleotides were purchased as sodium salts from Sigma Chemical Company, and were passed through a Chelex-100 chelating resin in the sodium form before use (3). Statistics. Data are presented, where approwith standard deviations. Statistical priate, significance was calculated with the t-test for paired observations; a value for p less than .05 was considered the criterion for significance.
RESULTS
Effects of nude&ides on turbidity changes. During 4%minute incubations in t,he st’andard hypotonic medium the turbidity of microsomal suspensions decreased 5.8 ‘i: (Fig. 1; Table I). The addition of 3 m&I ATP or CT!? diminished t,his change in turbidity by half. No other mwleotides tested had a .90
r 20
40
60
MINUTES
FIG. 1. Etfects of nucleotides on tlwbidity changes. The control cuvette contained 0.1 ml of the microsomal stock suspension and 50 @moles of Tris-Cl, pH 7.3, in 1.0 ml. The other cuvettes contained in addition, as indicated, 3 rnM ATP or ADP. At the arrow pCMB was added to a final concentration of 0.1 miw.
significant, effect on the turbidity changes. In wmparison with control incubations, turbidity changes in media containing 3 mxr EDTA were diminished by only one-fifth. Interaction with sulfhydql l’eagents. Wllen 0.1 rnRr pCMB was added t’o the standard medium after a 45minute incubation, it, induced a furt,her decrease in turbidity. In incubations that) included 3 ~RI ATP, ADP, or CTP, this decrease in turbidity MU augmented significantly (Fig. 1; Table I). The greatest turbidit,y change with pC1\IB occurred in media containing ADP (significant,ly greater than with ATP or C’TP). Analogous results were obtained when 3 ml1 N-ethylmaleimide or 0.2 ml\r DTSB w:w substit’ut#ed for pC%IB. I’veincubation experiments. Since this drop in turbidit,y following the late addit,ion of pCi\IB l-o media containing ATP and CTP might represent, merely the abolition of t#he diminished turbidity changes induced by these nucleot’ides (Fig. I), experiments were performed in which short,er preincubaGon periods were used. Preincubat#ion of the microsomes with various nucleotides for 3 minutes before adding pCMB caused significant changes in turbidit,y, both at 10 and at, 45 minutes, only with ADP (Fig. %A; Table II). Turbidity changes in media containing XTP, CTP, or the &her nucleotides were not
TABLE EFFECTS
OF NUCLEOTIDES
I ON TERBIDITY
Spontaneous decrease in turbidity Addition
7’ of change in optical density
in 45 min
% of change from control
None
5.8
f
.7
-
ATP, 3 mu ADP, 3 mM AMP, 3 mM CTP, 3 rnM GTP, 3 m&f ITP, 3 mM UTP, 3mhx PP, 3 mM P, 3 rnM
3.2 4.9 5.4 3.3 4.F 4.9 4.9 5.8 5.7
f f f f f f rk f. f
.G .9 1.0 .7 .8 .7 .8 .7 .7
55 f 7 85 f 8 93 f 9 56 & G 81 & 9 84f8 SD f 9 97 f 8 100 f 7
a Turbidity changes were observed in suspensions of microsomes (50 mM Tris-Cl, pH 7.3, and about 1 mg/ml of the microsomal stock cated; after 45 minutes, pCMB was added to a final concentration bidity changes from 45 to G5 minutes were observed.
CH.\NGES* pCMB-induced
decrease in turbidity
7, of change in optical density f
.6
6.G f 7.0 f 5.9 rf 5.4 f 4.4 zt 3.7 f 4.2 f 4.7 f 4.9 f
A.3
.T .8 .9 .7 .7 .A .6 .8 .8
incubated suspension) of 0.1 mM,
% of change from control 154 zk 10 165 zk 7 117 f 11 12s* 8 101 f 8 88& 9 98f 7 108 & 9 1lBzt 9
in the standard medium with additions as indiand t.he subsequent tur-
652
ROBINSON
B
ATP ntrol ADP
20
40
20 MIN
40
UTES
Fro. 2. Effects of preincubation on nucleot’ide-microsome-pCMB interactions. In panel A, microsomes were preincubated for 5 minutes in the control medium, with or without added nucleotides as indicated. At zero time 0.1 mM pCMB was added to all cuvettes and the subsequent changes in optical density were recorded. In panel B, microsomes were preincubated for 5 minutes in the control medium which contained, in all cases, 0.1 rnbx pCMB; at zero time nncleotides were added, as indicated, to the cuvettes. TABLE EFFECTS
OF PREINCUBATION
WITH
II
NUCLEOTIDES
ON pCMB-INDUCED
Turbidity y. of change in optical density
ATP, ADP, AMP, CTP, CTP, ITP, UTP,
3 mw 3 mM 3 mM 3 mrvr 3 mM 3 mM 3 mM
5.8 5.7 6.8 5.7 4.2 4.6 4.2 4.1
f f f f f f f f
.6 .7 .4 .9 .9 .7 .8 .7
at % of change from control
100 f 117 f 97 * 72 f 79* 7G+ 7Gxt
8 G 11 12 9 9 8
a Microsomes were preincubated for 5 minutes in the standard nucleotide listed; then, @MB was added to a final concentration were observed during the 45-minute incubation period.
increased above controls preincubated without any nucleotide. However, preincubation of the microsomes for 5 minutes with 0.1 mM pCMB before adding the nucleotides resulted in no significant increase in turbidity changes with any nucleotide at either time (Fig. 2B; Table III). Experiments measuring light scattering at 90’. Although the preceding experiments provided statistically significant data indi-
CHANGES”
changes during incubation
at 10 min
Addition
None
TURBIDITY
Y. of change in optical density 8.4 8.3 10.0 7.5 8.0 8.1 7.0 7.6
f f f f f f f f
.8 .8 1.0 1.1 1.0 .9 1.1 .9
45
min 70 of change from control 99f 120f 92 f 96 f 96f 87 f 93f
8 G 11 11 9 10 8
medium containing in addition the of 0.1 m&r, and the turbidity changes
eating specific changes with ATP (and CTP) and with ADP, the measured changes were quite small. By measuring light scattering at 90”, an analogous but inherently more sensitive method, the preceding experiments were confirmed. Thus ATP, but not ADP, significantly decreased changes in light scattering during incubation in the standard medium (Fig. 3A) ; and after preincubation for 5 minutes before addition of pCMB, ADP, but not ATP, significantly increased the drop
TABLE: EFFECW
OF PREINCURAPION
WITH
pCMB
III
ox
NUCLEO.~IDE-INDUCED
Turbidity Addition
AT!?, ADP, ilMP, CTP, GTP, ITP, UT!?,
3 rnM 3 mM 3 rnbx 3 mM 3 mM 3 mM 3 m&f
a hiicrosomes were tionallg, in all cases, were observed during
during
at 10 min
-$ of change in optical density
None
changes
4.8
3~ 1.7
4.8 4.4 4.5 4.2 4.7 5.0 4.6
f f zt 4~ i f f
7” of change in optical density
-
ti.8
99f 91f 93* 88f cJ(i f 105 f 98 rk
CHINGEF
at 45 min
5:;. of change with control
1.9 1.9 I.0 1.8 1.8 2.2 1.7
TURBIDITY incubation
7 7 ci 0 li 10 0
preincubated for 5 minutes in the st,andard 0.1 rn>f pChlB; then the nucleotide listed the 45-minute incubation period.
It
2.3
0.7 f 0.0 * 5.6 f (i.2 f 0.5 f 7.1 * 6.7 i
2.2 2.4 2.5 2.1 2.2 2.6 2.1
incubation was added
“& of change from control
99 98i 84f 93f 96 105 102
medium and the
zt
7 8 9 7 f ti zkz 13 & 6
containing sdditurbidity changes
90r
A
B 60 -
60
5
IO
15
20
25
MINUTES
&a. 3. Changes in light scattering induced by nucleotides. Light scattering at 90” was measured at a wavelength of 520 rnp with a spectrofluorometer. In panel A, microsomes were incubated in the control medium, with or without 3 mM ADP or ATP as indicated. In panel B, microsomes were preincubated for 5 minutes in the control medium \vhich con tained, in all cases, 0.1 mM pCMB; at zero time nucleotides were added as indicated, and the subsequent changes in light scattering were measured.
in light scattering (Fig. 33). The relat,ive magnitudes of t’he induced changes measured by light scattering and of those measured by optical density are not directly comparable. This is due, in part, to the light scattering data being recorded on a scale of transmitted light’, which is logarithmically,
not linearly, related to the optical density scale. Changes in membrane permeability. Permeability to sucrose, as measured by the ratio of the intramicrosomal space accessible to sucrose-14C to t’he intramicrosomal space accessible to glycerol-14C was determined after
654
ROB
incubation in the standard medium (3). The addition of 3 mM ATP, but not ITP or ADP, significantly decreased sucrose permeability (Table IV). When the microsomes were preincubat’ed for 6 minutes, with or without nucleotides, and then incubated for 45 minutes with 0.1 mM pCMB, 3 mM ADP, but not ATP, increased sucrose permeability (Table V). ZQect of pCMB on the binding of ADP. Data are presented asdescribed under Methods, for the volume of supernatant material containing as much labeled ADP (or dextran) as in the pellet, per gram of microsoma1 protein incubated. Subtracting the volume for dextran from that for ADP eliminated t.he volume of ADP trapped in the extramicrosomal space; the remainder represents the volume of supernatant material containing as much ADP as that’ bound to the pellet. Such calculations showed that preincubat’ion of the microsomes for 5 minut’es with 0.1 mnz pCMB reduced the binding of ADPJ4C during a subsequent 5-minute incubation, to 24 % of that in controls preincubated without pCMB (Table VI). When unlabeled ADP, 3 mM, was added together with ADP-14C; the amount of ADP-14C bound was reduced to 4 % of that bound in the absenceof unlabeled ADP; however, bhe actual dilution of ADP-14C was 15OO:l. In TABLE EFFECTS
OF
IV
NUCLEOTIDES
ON
SUCROSE
PERMEABILITY~ Addition
None ATP, ITP, ADP,
3 mM 3 mM 3 w
Ratio: sucrose space/ glycerol space
.36 .22 .31 .35
f f f f
.03 .04 .05 .03
Change control
from (70)
60 f 84*7 96 f
7 6
a Microsomes were incubated for 60 minutes at 30” in the standard medium, with additions as noted, in the presence of sucrose-W, glycerol-*4C, or dextran-carboxyl-1% (3). After centrifugation, radioactivity in pellet and supernatant material was measured, and spaces available to sucrose and to glycerol (both corrected for the dextran space) were calculated (3). In each experiment controls were incubated together with nucleotide-containing tubes.
INSON TABLE
V
EFFECTS OF PREINCUBATIOX WITH ON pCMB-INDUCED CH.~NGES
NUCLEOTIDE~ IN SFCROSE
PERMEABILITYO Addition
None ATP, ADP,
3 mM 3 mM
Ratio: sucmse space/ glycerol space
.79 z!z .05 .77 I!I .07 .92 f .05
Change control
97 f 116 f
from (70)
6 5
a Microsomes were preincubated for 5 minutes at 30” in the control medium, with additions as noted; at zero time 0.1 mM pCMB was added to all tubes, and they were then incubated for 45 minutes. Determination of sucrose and glycerol spaces was as in Table IV.
these experiments with 3 mM ADP, preincubation with pCMB caused no significant. change in binding of ADP-14C. On the other hand, when unlabeled AMP, 3 mM, was added together with ADP-14C, the amount of ADPJ4C bound was reduced only one-third; preincubation wit’h pCMB reduced the binding in these experiments 76 %. When the microsomeswere incubated with ADP-14C for the full 10 minutes (without preincubation), the amount of bound ADP14Cwas 88 % of that for the j-minute incubat,ion (Table VII). Addition of pCMB halfway through a lo-minute incubation reduced the amount of ADP bound only by half (Table VII). Content of reactive su(fhydryl groups. The content of reactive sulfhydryl groups, as measured by DTNB, was diminished in incubations with ATP (90 f 2 % of control) and ADP (77 f 4 % of control) (Fig. 4). EDTA, 3 mM, only reduced the sulfhydryl content 4 %.. Dissolution oj miwosomal structure during incubation. When the microsomal mixture was centrifuged for 30 minutes at 80,OOOg after incubation in the standard medium for one hour at 30”, and the resultant pellet was analyzed in comparison with unincubated microsomes, t’here was little loss in sedimentable protein and about a 20% loss in sedimentable RNA (Table VIII), in accord with earlier experiments (1). Addition of 0.1 mM pCMB to the medium had no effect on protein content, but doubled the loss of RNA. Neit,her ATP, ADP, nor EDTA altered t’he
NUCLEOTIDES
AND
BRAIN
TABLE EFFECT
OF PREIXCGBATION PrW3Ke of pCMB
Addition mith ADP-“C None None
+
ADP, 3 rnM
-
ADP,
3 rnM
+
AMP, AMP,
3 mM 3 mM
+
WITH
Equivalent Dextran 2.28 1.90 2.28 1.92 2.42
* i f * i
1.98 f
VI
pCMB
ON THE BINDIXG
volumes (ml/gram space
protein)
39.3 10.8 3.64 3.33 29.0 8.33
0.20
f i f i * *
OF ADP-10 Effect of pCMB (ADP bound as % bound without pCMB)
ADP bound (pmoles/gm protein)
ADP space
0.15 0.18 0.17 0.18 0.18
635
MICROSOMES
2.1 0.9 0.47 0.52 1.7 0.82
74 1s 2.7 2.8 53 13
f * f f i *
9 2 0.5 0.3 4 1
24f
4
105 *
17
24%
2
a In all experiments before addition of nucleotides t,he microsomes were preincubated for 5 minutes at 30°C with or without 0.1 mM pCMB. The mixtnre was then incubated for 5 minutes and the reaction was st.opped by centrifl\gation. Data were calculated as described under Methods.
EFFECTS
OF PREINCUBATION Equivalent
Addition after preincubation
Dextran
None
pCMB,
WITH
2.16 1.83
0.1 mM
f f
TABLE VII ADP ON PCMB-INDUCED
volumes (ml/gm protein)
space
34.4 17.8
f *
IN BINDINGS Effect of pCMB (ADP bound as 70 bound without pCMB)
ADP bound (Irmoles/gm protein)
ADP space
.19 .21
CHANGES
1.8 1.1
ti4 i 32 f
7 4
5054
a In all experiments the microsomes were preincubated for 5 minutes at 30% with ADP-1%. 0.1 mM pCMB was added to half the tubes and all were incubated for 5 minutes. TABLE CHIIIGGES
IN
VIII
PROTEIN :\ND RNA AFTER INCUBATION
Additions
CONTENT
Content in microsomes after incubation as % of that in unincubated stock suspension Protein
L U) a3 0 E i
None
pCMB, 0.1 mM ATP, 3 mM ATP and pCMB
IO-
ADP,
5-
L
I
I
1
I
20
1
40
3 m&I
ADP and pCMB H&l, , 0.1 rnM EDTA,
3 mM
Then
96 94 96 95 96 90 97 95
f f f i f f + i
RNA 2 2 2 3 2 3 2 3
82f 04 79f 63 80f 03 78 77f
5 7 4 f 11 4 Z!Z 9 f 10 6
f
MINUTES FIG.
4. Content
of reactive
sulfhydryl
groups.
The
content of reactive sulfhydryl groups, as measured by DTNB (l), is plotted for microsomes incubated in the control medium and in media containing, in addition, 3 mM ATP or ADP.
protein neither induced
or RNA
content
significantly, and the pCMBloss in RISA (Table VIII). Nucleotidase activity. Since the rates of
ATP nor ADP altered
met’abolism of the nucleotides might be reflected in the nat’ure and durat8ion of the specific structural changes, especially during preincubation experiments, nucleotidase activity was determined. All the nucleoside t’riphosphates were catabolized by t’he microsomal preparation in the standard medium without additional divalent cation (Fig. .i). Inorganic phosphat’e production was most rapid with ATP and ITP, slowest
656
ROBIPiSOiX
(Fig. 3A). The spoutjaueous decrease in turbidity occurring in hypotonic media may be attribut,ed, at least in part, to osmotic swelling (1, 3, 5, 7) ; the diminution of t)his process by ATP suggests either (a) stjruct,ural alteration in the membrane resulting in a change in the selective permeability of t,he membrane (1, 3), (b) gross change in vesicular conformat,ion altering light scatt,ering, or 0c aggregation of the microsomal elements. Possibly the first two processesmight occur concomitantjly. In this context,, experiments demonst’rating t,hat ATP reduced the perme60 20 40 ability of the vesicular membrane to sucrose MINUTES (Table IV) support t’he interpretat,ion of the light scattering experiments as indicating an FIG. 5. Nucleotidase activity. The liberation of inorganic phosphate from various nucleotides is alteration in membrane permeability. Ko other nucleotide tested induced these plotted against the duration of incubation at 30”. Microsomes were incubated in the control medium changes in optical density, light, scatt’ering, containing 3 mM ATP (O--O); ITP (A- - -A); and sucrose permeability, except CTP CTP (*ml ; UTP (A---A); GTP (o- - -0) ; (Table I). The specificity for the two nucleoor ADP (IJ- - -0). Each point is the average of tides suggestsan interaction with the mem4 experiments. brane through both t#he B-amino group on the purine or pyrimidine ring and the triwith GTP and UTP; with CTP the rate phosphate complex. Specificit’y for ATP and was intermediate, but total phosphate pro- CTP, to the exclusion of other nucleot8ides, duction equaled that from ITP by one hour. has been noOedwith the myosin ATPase (8) Nearly half the added ATP (3 m;M) was and the microsomal salt-activated BLTPase catabolized in the standard medium by 45 (9). minutes. In the presenceof 3 rnM MgCl, the These findings differ sharply from studies initial rate was increased about S-fold, and wit’h liver microsomes in which all nucleonearly all the ATP was split by 45 minutes. side triphosphates, as well as EDTA and The microsomal preparations also cata- PP, induced a marked decreasein light scatlyzed the release of inorganic phosphate tering (10) t’hat was correlated with a loss in from ADP (Fig. 5). Phosphate liberation sediment,able RNA (5). The lack of nucleofrom ADP was diminished by monovalent tide specificity, the equivalent effects of cations, in marked contrast to ATP. EDTA and PP, and the ant,agonism by The initial rate of nucleotidase activity, Mg++ (10) indicat’ed that in t’he liver prepain all cases, was diminished about half in ration divalent cation chelation was inthe presence of 0.1 mM pCMB. volved (5, 10). By contrast, wit,h t.he brain preparation the effect’ of ATP was (a) to DISCUSSION diminish (not increase) the changes in light These experiments indicate that specific scattering (Fig. l), (b) was not’ antagonized nucleotides interact with the microsomes to by added MgClz (3), (c) was not reproduced alter the structural organization of the mem- by other nucleotides (except CTP) nor by brane fragments, as manifested by changes PP or EDTA (a more potent chelating in light scattering and in membrane permeability, and that these int’eractions may be agent), and (d) did not cause a loss in sedimentable RNA (Table VIII). A partial modified by sulfhydryl-binding compounds. explanation for this difference was the nearly Thus ATP significantly diminished the total absence of membrane-bound ribosomes spontaneous changes in optical density (Fig. 1; Table I) and in 90’ light scattering in the brain preparation, as shown in electron ATP
~~CI,IZOTIDES
AXD
micrographs (1~. Kleinfeld and J. D. Robinson, unpublished observations). A second distinct st,ructural alt,eration could bc induced by ADP, as shown by cxperiments in which microsomes were preincubaM with the nucleot,ide before incubat,ion with pCMB. After a s-minute preincubation with various nucleotidcs, during which lit’tle catabolism occurred (Fig. 5) and lit,tle cliff erencesin light, scattering could be not’cd (Figs. 1 and S), subsequent,incubation wit,h pCMB caused increased changes in opt,icnl density (Fig. 2; Table II) and in 90” light scattering (Fig. 3B) only with ADP. Such changes may be interpreted in terms of (a) alteration in premeabilit’y, (b) gross alt,eration in conformation, or (c) microsomnl dissolut,ion. Alt’hough pCMB did cause some dissolution, as indicated by loss of sediment!able RiSA (Table VIII), ADP did not induce further measurable lossesof prot,ein or RSA either alone or with pCMB (Table VIII). Finally, experiment,s demonstrating that preincubation with ADP increased the permeability of the vesicular membrane t)o sucrose during subsequent incubation wit,h pCMB (Table V), whereas preincubation w&h ATP did not, support the interpretation of t’he light’ scntt’ering experiment,s as indicating a second structural change. All these changes were intimately related to the st,at)e of the membrane sulfhydryl groups as shown by experiments with pCR4B (and confirmed wit’h N-ethylmaleimide and DTNB). Brief preincubation with pCMB prevented any changes in light, scat’tering by any nucleotide (Fig. 2; Table IIIj, and addition of pCMB to microsomesincubated wit’h ATP or CTP abolished the alterations in light, scat,tering induced by these nucleotides (I;ig. 1; Table I). Preincubat,ing the microsomcswith pCMF markedly reduced t)he binding of ADP-14C during subsequent incubation (Table VI), in accord with the observations that preincubatsion with pCMB blocked the effects of ADP on turbidity changes. The binding of labeled ADP appeared to be, in part, specific since unlabeled ADP almost eliminated t’he binding of ADP-14C, whereas unlabeled AMP had little effect (Table VI). The ap-
BRAIN
MICROSOMES
6.57
parent, binding of ADP-14C in the presence of a GOO-fold excessof unlabeled ADP may be a measure of t,he binding of labeled impurities in t,he preparat’ion rather than t#he binding of ADP-14C itself; this could explain why pChIB did not reduce t>he binding of radioact,ive material in experiments with 3 ml,1ADP. The preparat’ions of ADP-14C used cont,ained l-3 (7 labeled impurities; however, no differences between preparations were observed. II’urthermore, the effect of the act’ive nucleotides was not t,o increase the reactivity of t’he membrane sulfhydryl groups, but rather to reduce the cont’ent of reactive sulfhydryl groups (Fig. 4). ADP, which reduced sulfhydryl reactivity more, potentiated the effect,s of pClUB on light scat)tering and sucrose permeability. The sit)uation is reminiscent)of the interaction of nucleotides wit,h myosin: at low concentrat’ions of pCMR the myosin ATPase is activated for ATP and CTP (but nob for ITP), but at higher concentrations it is inhibited (8). The interaction with myosin has been attributed not to binding of nucleotides through sulfhydryl groups but t,o an allosteric effect on the protein (11, 12). A similar ADEmembranesulfhydryl system seems probable here, in which ADP renders a fraction of the sulfhydry1 groups unreactive while sensitizing a portion of the complex t’o sulfhydryl reagents. [For discussions of the changes in content of react)ive sulfhydryl groups during incubation in various media see references (I) and (2).] Since ATP (and CTP) induced one structural stat’e in the membrane syst)em while ADP induced a second distinct strurtural alteration, possible relevance t,o the membrane transport ATPase is suggested. Models for cation transport employing conformational changes in the membrane and involving t,he Na+-K+-stimulated ATPase have been proposed (4), and several interesting correlates exist, between t,he Nn+-K+-stimulated ATPase and the structural changes. The nucleotidase act,ivity present in many crude microsomal preparabions has a high act’ivity with ITP (e.g., Fig. 5), but exhibits salt,-stimulated activity only with ATP or CTP and not with ITP or other nucleobides
ROBINSON
(9) ; furthermore, the nucleotidase activity, which is sensitive to sulfhydryl reagents, may be protected by preincubation with ATP but not ITP (CTP was not tried) (13). If the nucleotide-induced changes were related to ATPase activity, then these changes might be modified by cations that alter ATPase activity. However, divalent and monovalent cations themselves induce changes in t,he light-scattering properties of the preparation, and it was impossible to demonstrate any specific effect on the ATPinduced turbidity changes with MgCl, , NaCl, KCI, NaCl + KCl, or ouabain (3). Quite recently experiments with the postmitochondrial supernate from crayfish nerve have been reported (14) which show that, in accord with the above experiments, ATP caused an increase in turbidity of the suspension. The authors interpret these findings in terms of an actomyosin-like protein within the axon membrane that, under the influence of ATP, controls permeability and impulse conduction. ACKNOWLEDGMENT The
Swiderski
proficient is gratefully
assistance
of Mr. acknowledged.
Frank
E.
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5. ROBIXSON, J. Il., Arch. Biochem. Biophys. 106, 207 (1964). 6. LOWRY, 0. H., AND LOPEZ, J. A., J. Biol. Chem. 162, 421 (1946). 7. TEDESCHI, H., JAMES, J. M., AND ANTHONY, W., J. Cell Biol. 18, 503 (1963). 8. BLUM, J. J., Arch. Biochem. Biophys. 87, 104 (1960). 9. NAKAO, T., TASIXIMA, Y., NAGANO, K., AND NAKAO, M., Biochem. Biophys. Res. Commm. 19, 755 (1965). 10. PACKER, L., AND RAHMAX, M. M., Texas Repts. Biol. Med. 20, 414 (1962). 11. MORALES, M. F., AND HOTTA, K., J. Biol. Chem. 236, 1979 (1960). D. R., Science 149, 1374 (1965). 12. KOMINZ, 13. SKOU, J. C., .~ND HILBERG, C., B&him. Biophys. Acta 110, 359 (1965). 14. BOWLER, K., AND DUNCAN, C. J., Nature 211, 642 (1966).